Bone marrow mesenchymal stem cells (BM-MSCs) are a vital component of regenerative medicine due to their ability to differentiate into various cell types and modulate immune responses. Their therapeutic potential has led to extensive research on their biological properties, mechanisms of action, and clinical applications.

Understanding BM-MSCs requires examining their microenvironment, distinguishing characteristics, isolation techniques, differentiation pathways, and how they compare to other stem cell types.

BM-MSCs originate from the mesodermal germ layer during embryonic development and persist into adulthood for tissue maintenance and repair. Within the bone marrow, they coexist with hematopoietic stem cells (HSCs) and other stromal components, contributing to the marrow niches structure and function. Their distribution is not uniform, with higher concentrations in trabecular-rich regions such as the iliac crest, femur, and sternum. These sites provide a supportive environment where BM-MSCs interact with the extracellular matrix, soluble factors, and neighboring cells to regulate proliferation and differentiation.

The bone marrow microenvironment is a specialized niche that governs BM-MSC behavior through biochemical and mechanical cues. It consists of an extracellular matrix composed of collagen, fibronectin, and laminin, which provides structural support and modulates adhesion. Oxygen tension in the marrow is lower than in peripheral tissues, with hypoxic conditions (1% to 7% oxygen) helping maintain BM-MSC quiescence and stemness. Hypoxia-inducible factors (HIFs) mediate responses to low oxygen levels, promoting genes involved in self-renewal and metabolic adaptation.

Cellular interactions further shape BM-MSC function. Crosstalk with endothelial cells, osteoblasts, and pericytes influences their role in supporting hematopoiesis and tissue homeostasis. Endothelial cells secrete vascular endothelial growth factor (VEGF), enhancing BM-MSC survival and migration. Osteoblasts provide osteogenic signals that prime BM-MSCs for differentiation into bone-forming cells. Pericytes, which share similarities with BM-MSCs, contribute to vascular stability and regulate stem cell fate.

BM-MSCs are defined by a unique set of surface markers that distinguish them from other stromal and hematopoietic populations. Unlike HSCs, BM-MSCs lack CD34, CD45, and CD14, which are associated with blood cell lineages. Instead, they express CD73, CD90, and CD105, as established by the International Society for Cell and Gene Therapy (ISCT). These markers facilitate identification, isolation, and functional characterization.

CD73, also known as ecto-5-nucleotidase, catalyzes the conversion of extracellular AMP into adenosine, modulating microenvironmental signals. CD90, or Thy-1, is a glycoprotein involved in cell-cell and cell-matrix interactions, influencing BM-MSC proliferation and differentiation. CD105, or endoglin, serves as a co-receptor for transforming growth factor-beta (TGF-), maintaining BM-MSC multipotency and guiding lineage commitment.

Additional markers refine BM-MSC characterization. CD146, a pericyte-associated marker, is linked to heightened clonogenic potential. STRO-1, an early mesenchymal progenitor marker, correlates with enhanced osteogenic differentiation but diminishes with cell expansion. CD271, or low-affinity nerve growth factor receptor (LNGFR), has been proposed for isolating highly pure BM-MSC populations with superior regenerative properties.

Isolating and expanding BM-MSCs are critical for research and clinical applications. Various techniques selectively extract BM-MSCs while minimizing contamination from hematopoietic and other stromal cells.

Density gradient centrifugation separates mononuclear cells from other bone marrow components based on cell density. Ficoll-Paque and Percoll are commonly used media that enrich BM-MSCs by allowing lower-density mononuclear cells to form a distinct layer after centrifugation. This method is simple and cost-effective but does not exclusively isolate BM-MSCs, as the mononuclear fraction contains hematopoietic cells. To improve purity, plastic adherence-based selection is often employed, where BM-MSCs attach to tissue culture plastic while non-adherent cells are removed. However, this approach has limitations, including variability in yield and potential contamination.

Fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) isolate BM-MSCs based on surface marker expression. FACS uses fluorescently labeled antibodies targeting BM-MSC markers such as CD73, CD90, and CD105, allowing high-purity selection through laser-based detection. MACS employs magnetic beads conjugated to antibodies, enabling rapid and scalable cell separation. While FACS provides greater resolution, it requires specialized equipment and is time-intensive. MACS, though less precise, is more accessible and suitable for large-scale cell enrichment.

Enzymatic digestion methods use proteolytic enzymes such as collagenase and trypsin to break down the extracellular matrix and release BM-MSCs. Collagenase digestion is commonly used to degrade collagen-rich structures while preserving viability. Trypsin, often combined with other enzymes, aids in cell detachment. While enzymatic dissociation enhances cell recovery, excessive enzyme exposure can compromise viability and surface marker integrity. This method is often combined with culture-based selection for further enrichment.

BM-MSCs can differentiate into osteoblasts, chondrocytes, and adipocytes. This process is governed by transcription factors and environmental cues that guide lineage commitment. The surrounding microenvironment, including mechanical forces and biochemical signals, influences differentiation outcomes.

Osteogenic differentiation is driven by RUNX2, which activates genes responsible for bone matrix deposition. Calcium, phosphate, and bone morphogenetic proteins (BMPs) reinforce osteogenesis by enhancing mineralization. Chondrogenic differentiation is regulated by SOX9, which promotes cartilage-specific proteins such as aggrecan and type II collagen. Hypoxic conditions sustain chondrocyte-like characteristics. Adipogenic differentiation is controlled by PPAR and C/EBP, which drive lipid accumulation and adipocyte-specific gene expression.

BM-MSC differentiation is regulated by signaling pathways that govern self-renewal, proliferation, and lineage commitment. The Notch, Wnt, and BMP pathways play key roles in directing fate decisions.

The Notch pathway influences BM-MSC proliferation and differentiation through cell-to-cell communication. Activation occurs when Notch ligands bind to receptors, triggering cleavage and release of the Notch intracellular domain (NICD). NICD translocates to the nucleus and modulates gene expression. Notch signaling maintains BM-MSCs in an undifferentiated state by suppressing osteogenic and adipogenic differentiation while promoting chondrogenesis. Sustained Notch activation enhances cartilage formation by upregulating SOX9, while inhibition facilitates osteogenesis by relieving suppression on RUNX2.

The Wnt signaling cascade affects BM-MSC fate through canonical and non-canonical pathways. In the canonical pathway, Wnt ligands bind to Frizzled receptors, stabilizing -catenin, which activates osteogenic genes. This pathway promotes bone formation by enhancing RUNX2 expression and matrix mineralization. The non-canonical pathway, independent of -catenin, regulates cytoskeletal organization and migration. Canonical Wnt signaling favors osteogenesis while inhibiting adipogenesis by suppressing PPAR, maintaining a balance in BM-MSC differentiation.

Bone morphogenetic proteins (BMPs) regulate BM-MSC differentiation, particularly in bone and cartilage formation. BMP ligands bind to receptors, triggering SMAD phosphorylation and transcriptional regulation. BMP2 and BMP7 induce osteogenesis by upregulating RUNX2 and enhancing extracellular matrix deposition. BMP signaling also synergizes with SOX9 to promote chondrogenesis. While BMPs favor skeletal differentiation, excessive signaling can lead to aberrant ossification.

BM-MSCs differ from other stem cell populations in differentiation potential, immunomodulatory effects, and tissue origin. Unlike HSCs, which primarily generate blood cells, BM-MSCs contribute to mesodermal-derived tissues such as bone, cartilage, and adipose. Their ability to differentiate into multiple skeletal and connective tissue types makes them valuable for regenerative applications.

Compared to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), BM-MSCs have a more restricted differentiation capacity, as they do not generate cells from all three germ layers. However, this reduces the risk of teratoma formation, a concern with pluripotent stem cell-based therapies. BM-MSCs are also more accessible and ethically uncontroversial, as they can be harvested from adult bone marrow. Their immunomodulatory properties further distinguish them, as they modulate immune responses through cytokine secretion and direct interactions, making them useful in inflammatory and autoimmune conditions.

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Bone Marrow Mesenchymal Stem Cells: Key Insights and Functions

Stem cells that give rise to other blood cells

Hematopoietic stem cells (HSCs) are the stem cells[1] that give rise to other blood cells. This process is called haematopoiesis.[2] In vertebrates, the first definitive HSCs arise from the ventral endothelial wall of the embryonic aorta within the (midgestational) aorta-gonad-mesonephros region, through a process known as endothelial-to-hematopoietic transition.[3][4] In adults, haematopoiesis occurs in the red bone marrow, in the core of most bones. The red bone marrow is derived from the layer of the embryo called the mesoderm.

Haematopoiesis is the process by which all mature blood cells are produced. It must balance enormous production needs (the average person produces more than 500 billion blood cells every day) with the need to regulate the number of each blood cell type in the circulation. In vertebrates, the vast majority of hematopoiesis occurs in the bone marrow and is derived from a limited number of hematopoietic stem cells that are multipotent and capable of extensive self-renewal.

Hematopoietic stem cells give rise to different types of blood cells, in lines called myeloid and lymphoid. Myeloid and lymphoid lineages both are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets. Lymphoid cells include T cells, B cells, natural killer cells, and innate lymphoid cells.

The definition of hematopoietic stem cell has developed since they were first discovered in 1961.[5] The hematopoietic tissue contains cells with long-term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors. Hematopoietic stem cells constitute 1:10,000 of cells in myeloid tissue.

HSC transplants are used in the treatment of cancers and other immune system disorders[6] due to their regenerative properties.[7]

They are round, non-adherent, with a rounded nucleus and low cytoplasm-to-nucleus ratio. In shape, hematopoietic stem cells resemble lymphocytes.

The very first hematopoietic stem cells during (mouse and human) embryonic development are found in aorta-gonad-mesonephros region and the vitelline and umbilical arteries.[8][9][10] Slightly later, HSCs are also found in the placenta, yolk sac, embryonic head, and fetal liver.[3][11]

Stem and progenitor cells can be taken from the pelvis, at the iliac crest, using a needle and syringe.[12] The cells can be removed as liquid (to perform a smear to look at the cell morphology) or they can be removed via a core biopsy (to maintain the architecture or relationship of the cells to each other and to the bone).[citation needed]

A colony-forming unit is a subtype of HSC. (This sense of the term is different from colony-forming units of microbes, which is a cell counting unit.) There are various kinds of HSC colony-forming units:

The above CFUs are based on the lineage. Another CFU, the colony-forming unitspleen (CFU-S), was the basis of an in vivo clonal colony formation, which depends on the ability of infused bone marrow cells to give rise to clones of maturing hematopoietic cells in the spleens of irradiated mice after 8 to 12 days. It was used extensively in early studies, but is now considered to measure more mature progenitor or transit-amplifying cells rather than stem cells[citation needed].

Since hematopoietic stem cells cannot be isolated as a pure population, it is not possible to identify them in a microscope.[citation needed] Hematopoietic stem cells can be identified or isolated by the use of flow cytometry where the combination of several different cell surface markers (particularly CD34) are used to separate the rare hematopoietic stem cells from the surrounding blood cells. Hematopoietic stem cells lack expression of mature blood cell markers and are thus called Lin-. Lack of expression of lineage markers is used in combination with detection of several positive cell-surface markers to isolate hematopoietic stem cells. In addition, hematopoietic stem cells are characterised by their small size and low staining with vital dyes such as rhodamine 123 (rhodamine lo) or Hoechst 33342 (side population).

Hematopoietic stem cells are essential to haematopoiesis, the formation of the cells within blood. Hematopoietic stem cells can replenish all blood cell types (i.e., are multipotent) and self-renew. A small number of hematopoietic stem cells can expand to generate a very large number of daughter hematopoietic stem cells. This phenomenon is used in bone marrow transplantation,[13] when a small number of hematopoietic stem cells reconstitute the hematopoietic system. This process indicates that, subsequent to bone marrow transplantation, symmetrical cell divisions into two daughter hematopoietic stem cells must occur.

Stem cell self-renewal is thought to occur in the stem cell niche in the bone marrow, and it is reasonable to assume that key signals present in this niche will be important in self-renewal.[2] There is much interest in the environmental and molecular requirements for HSC self-renewal, as understanding the ability of HSC to replenish themselves will eventually allow the generation of expanded populations of HSC in vitro that can be used therapeutically.

Hematopoietic stem cells, like all adult stem cells, mostly exist in a state of quiescence, or reversible growth arrest. The altered metabolism of quiescent HSCs helps the cells survive for extended periods of time in the hypoxic bone marrow environment.[14] When provoked by cell death or damage, Hematopoietic stem cells exit quiescence and begin actively dividing again. The transition from dormancy to propagation and back is regulated by the MEK/ERK pathway and PI3K/AKT/mTOR pathway.[15] Dysregulation of these transitions can lead to stem cell exhaustion, or the gradual loss of active Hematopoietic stem cells in the blood system.[15]

Hematopoietic stem cells have a higher potential than other immature blood cells to pass the bone marrow barrier, and, thus, may travel in the blood from the bone marrow in one bone to another bone. If they settle in the thymus, they may develop into T cells. In the case of fetuses and other extramedullary hematopoiesis. Hematopoietic stem cells may also settle in the liver or spleen and develop.

This enables Hematopoietic stem cells to be harvested directly from the blood.

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood.[16][17][13] It may be autologous (the patient's own stem cells are used), allogeneic (the stem cells come from a donor) or syngeneic (from an identical twin).[16][17]

It is most often performed for patients with certain cancers of the blood or bone marrow, such as multiple myeloma or leukemia.[17] In these cases, the recipient's immune system is usually destroyed with radiation or chemotherapy before the transplantation. Infection and graft-versus-host disease are major complications of allogeneic HSCT.[17]

In order to harvest stem cells from the circulating peripheral blood, blood donors are injected with a cytokine, such as granulocyte-colony stimulating factor (G-CSF), that induces cells to leave the bone marrow and circulate in the blood vessels.[18]In mammalian embryology, the first definitive Hematopoietic stem cells are detected in the AGM (aorta-gonad-mesonephros), and then massively expanded in the fetal liver prior to colonising the bone marrow before birth.[11]

Hematopoietic stem cell transplantation remains a dangerous procedure with many possible complications; it is reserved for patients with life-threatening diseases. As survival following the procedure has increased, its use has expanded beyond cancer to autoimmune diseases[19][20] and hereditary skeletal dysplasias; notably malignant infantile osteopetrosis[21][22] and mucopolysaccharidosis.[23]

Stem cells can be used to regenerate different types of tissues. HCT is an established as therapy for chronic myeloid leukemia, acute lymphatic leukemia, aplastic anemia, and hemoglobinopathies, in addition to acute myeloid leukemia and primary immune deficiencies. Hematopoietic system regeneration is typically achieved within 24 weeks post-chemo- or irradiation therapy and HCT. HSCs are being clinically tested for their use in non-hematopoietic tissue regeneration.[24]

DNA strand breaks accumulate in long term hematopoietic stem cells during aging.[25] This accumulation is associated with a broad attenuation of DNA repair and response pathways that depends on HSC quiescence.[25] Non-homologous end joining (NHEJ) is a pathway that repairs double-strand breaks in DNA. NHEJ is referred to as "non-homologous" because the break ends are directly ligated without the need for a homologous template. The NHEJ pathway depends on several proteins including ligase 4, DNA polymerase mu and NHEJ factor 1 (NHEJ1, also known as Cernunnos or XLF).

DNA ligase 4 (Lig4) has a highly specific role in the repair of double-strand breaks by NHEJ. Lig4 deficiency in the mouse causes a progressive loss of hematopoietic stem cells during aging.[26] Deficiency of lig4 in pluripotent stem cells results in accumulation of DNA double-strand breaks and enhanced apoptosis.[27]

In polymerase mu mutant mice, hematopoietic cell development is defective in several peripheral and bone marrow cell populations with about a 40% decrease in bone marrow cell number that includes several hematopoietic lineages.[28] Expansion potential of hematopoietic progenitor cells is also reduced. These characteristics correlate with reduced ability to repair double-strand breaks in hematopoietic tissue.

Deficiency of NHEJ factor 1 in mice leads to premature aging of hematopoietic stem cells as indicated by several lines of evidence including evidence that long-term repopulation is defective and worsens over time.[29] Using a human induced pluripotent stem cell model of NHEJ1 deficiency, it was shown that NHEJ1 has an important role in promoting survival of the primitive hematopoietic progenitors.[30] These NHEJ1 deficient cells possess a weak NHEJ1-mediated repair capacity that is apparently incapable of coping with DNA damages induced by physiological stress, normal metabolism, and ionizing radiation.[30]

The sensitivity of hematopoietic stem cells to Lig4, DNA polymerase mu and NHEJ1 deficiency suggests that NHEJ is a key determinant of the ability of stem cells to maintain themselves against physiological stress over time.[26] Rossi et al.[31] found that endogenous DNA damage accumulates with age even in wild type Hematopoietic stem cells, and suggested that DNA damage accrual may be an important physiological mechanism of stem cell aging.

A study shows the clonal diversity of hematopoietic stem cells gets drastically reduced around age 70 , substantiating a novel theory of ageing which could enable healthy aging.[32][33] Of note, the shift in clonal diversity during aging was previously reported in 2008[34] for the murine system by the Christa Muller-Sieburg laboratory in San Diego, California.

A cobblestone area-forming cell (CAFC) assay is a cell culture-based empirical assay. When plated onto a confluent culture of stromal feeder layer,[35] a fraction of hematopoietic stem cells creep between the gaps (even though the stromal cells are touching each other) and eventually settle between the stromal cells and the substratum (here the dish surface) or trapped in the cellular processes between the stromal cells. Emperipolesis is the in vivo phenomenon in which one cell is completely engulfed into another (e.g. thymocytes into thymic nurse cells); on the other hand, when in vitro, lymphoid lineage cells creep beneath nurse-like cells, the process is called pseudoemperipolesis. This similar phenomenon is more commonly known in the HSC field by the cell culture terminology cobble stone area-forming cells (CAFC), which means areas or clusters of cells look dull cobblestone-like under phase contrast microscopy, compared to the other hematopoietic stem cells, which are refractile. This happens because the cells that are floating loosely on top of the stromal cells are spherical and thus refractile. However, the cells that creep beneath the stromal cells are flattened and, thus, not refractile. The mechanism of pseudoemperipolesis is only recently coming to light. It may be mediated by interaction through CXCR4 (CD184) the receptor for CXC Chemokines (e.g., SDF1) and 41 integrins.[36]

Hematopoietic stem cells (HSC) cannot be easily observed directly, and, therefore, their behaviors need to be inferred indirectly. Clonal studies are likely the closest technique for single cell in vivo studies of HSC. Here, sophisticated experimental and statistical methods are used to ascertain that, with a high probability, a single HSC is contained in a transplant administered to a lethally irradiated host. The clonal expansion of this stem cell can then be observed over time by monitoring the percent donor-type cells in blood as the host is reconstituted. The resulting time series is defined as the repopulation kinetic of the HSC.

The reconstitution kinetics are very heterogeneous. However, using symbolic dynamics, one can show that they fall into a limited number of classes.[37] To prove this, several hundred experimental repopulation kinetics from clonal Thy-1lo SCA-1+ lin(B220, CD4, CD8, Gr-1, Mac-1 and Ter-119)[38] c-kit+ HSC were translated into symbolic sequences by assigning the symbols "+", "-", "~" whenever two successive measurements of the percent donor-type cells have a positive, negative, or unchanged slope, respectively. By using the Hamming distance, the repopulation patterns were subjected to cluster analysis yielding 16 distinct groups of kinetics. To finish the empirical proof, the Laplace add-one approach was used to determine that the probability of finding kinetics not contained in these 16 groups is very small. By corollary, this result shows that the hematopoietic stem cell compartment is also heterogeneous by dynamical criteria.

It was originally believed that all hematopoietic stem cells were alike in their self-renewal and differentiation abilities. This view was first challenged by the 2002 discovery by the Muller-Sieburg group in San Diego, who illustrated that different stem cells can show distinct repopulation patterns that are epigenetically predetermined intrinsic properties of clonal Thy-1lo Sca-1+ lin c-kit+ HSC.[39][40][41] The results of these clonal studies led to the notion of lineage bias. Using the ratio = L / M {displaystyle rho =L/M} of lymphoid (L) to myeloid (M) cells in blood as a quantitative marker, the stem cell compartment can be split into three categories of HSC. Balanced (Bala) hematopoietic stem cells repopulate peripheral white blood cells in the same ratio of myeloid to lymphoid cells as seen in unmanipulated mice (on average about 15% myeloid and 85% lymphoid cells, or 3 10). Myeloid-biased (My-bi) hematopoietic stem cells give rise to very few lymphocytes resulting in ratios 0 < < 3, while lymphoid-biased (Ly-bi) hematopoietic stem cells generate very few myeloid cells, which results in lymphoid-to-myeloid ratios of > 10. All three types are normal types of HSC, and they do not represent stages of differentiation. Rather, these are three classes of HSC, each with an epigenetically fixed differentiation program. These studies also showed that lineage bias is not stochastically regulated or dependent on differences in environmental influence. My-bi HSC self-renew longer than balanced or Ly-bi HSC. The myeloid bias results from reduced responsiveness to the lymphopoetin interleukin 7 (IL-7).[40]

Subsequently, other groups confirmed and highlighted the original findings.[42] For example, the Eaves group confirmed in 2007 that repopulation kinetics, long-term self-renewal capacity, and My-bi and Ly-bi are stably inherited intrinsic HSC properties.[43] In 2010, the Goodell group provided additional insights about the molecular basis of lineage bias in side population (SP) SCA-1+ lin c-kit+ HSC.[44] As previously shown for IL-7 signaling, it was found that a member of the transforming growth factor family (TGF-beta) induces and inhibits the proliferation of My-bi and Ly-bi HSC, respectively.

From Greek haimato-, combining form of haima 'blood', and from the Latinized form of Greek poietikos 'capable of making, creative, productive', from poiein 'to make, create'.[45]

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Hematopoietic stem cell - Wikipedia

SOMERVILLE, Mass., May 17, 2025 (GLOBE NEWSWIRE) -- Tessera Therapeutics, the biotechnology company pioneering a new approach in genetic medicine known as Gene Writing™, is presenting updates across its in vivo genetic medicine programs for AATD, PKU, and SCD, as well as advances in in vivo T cell therapies. These data were shared across four oral presentations and three poster presentations at the American Society of Gene and Cell Therapy (ASGCT) Annual Meeting taking place in New Orleans, Louisiana, May 13 – 17, 2025.

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Tessera Therapeutics Features New Preclinical Data Demonstrating Progress Across its In Vivo Gene Writing™ Programs and Delivery Platform at the...

Abstract

Induced pluripotent stem (iPS) cells are laboratory-produced cells that combine the biological advantages of somatic adult and stem cells for cell-based therapy. The reprogramming of cells, such as fibroblasts, to an embryonic stem cell-like state is done by the ectopic expression of transcription factors responsible for generating embryonic stem cell properties. These primary factors are octamer-binding transcription factor 4 (Oct3/4), sex-determining region Y-box 2 (Sox2), Krppel-like factor 4 (Klf4), and the proto-oncogene protein homolog of avian myelocytomatosis (c-Myc). The somatic cells can be easily obtained from the patient who will be subjected to cellular therapy and be reprogrammed to acquire the necessary high plasticity of embryonic stem cells. These cells have no ethical limitations involved, as in the case of embryonic stem cells, and display minimal immunological rejection risks after transplant. Currently, several clinical trials are in progress, most of them in phase I or II. Still, some inherent risks, such as chromosomal instability, insertional tumors, and teratoma formation, must be overcome to reach full clinical translation. However, with the clinical trials and extensive basic research studying the biology of these cells, a promising future for human cell-based therapies using iPS cells seems to be increasingly clear and close.

Keywords: induced pluripotent stem cells, regeneration, cellular therapy, stem cells, muscular dystrophy

Stem cells can be classified as totipotent, pluripotent, or multipotent cells according to their biological source and the capacity to differentiate into other cell types. Totipotent stem cells are found very early in embryonal development and can differentiate into all cell types in the organism, as well as into extraembryonic tissues. Pluripotent cells can be isolated from blastocysts or the umbilical cord immediately after birth, and are also able to differentiate into all tissue cells, except extraembryonic structures. However, certain disadvantages must be observed when considering these stem cells in regenerative medicine. These include the high risk of rejection and ethical issues when the isolation is performed from embryos. On the other hand, due to their high plasticity, pluripotent stem cells are considered ideal to obtaining the multiple cell types required after stem cell-based therapies.

Multipotent stem cells are isolated from adult tissues and have no ethical issues involved. These include hematopoietic, mesenchymal, and neural stem cells. Multipotent stem cells can be isolated from the patients subjected to treatment, with no risk of rejection, and be expanded in vitro for transplant. However, these cells display reduced plasticity, as they can only differentiate into specialized cell types present in specific tissues or organs, their main disadvantage. The ideal cellular population best suited for stem cell-based therapies should combine the high plasticity of embryonic stem cells and the convenient isolation from patients under treatment. To this end, induced pluripotent stem (iPS) cells were generated using embryonic or adult somatic cells. The somatic cells are subjected to the ectopic expression of transcription factors that induce the stem cell-like properties and the high plasticity required for cell therapy. Therefore, iPS cells can potentially revolutionize the field of regenerative medicine and provide new tools for stem cell research.

In the nineties, it was demonstrated that somatic cells could be reprogrammed to an undifferentiated state by transferring their nuclear content into unfertilized oocytes [1]. These results showed that cellular differentiation is reversible. Later, the resetting of a somatic epigenotype to a totipotent state was successfully achieved when adult thymocytes were fused with embryonic stem cells [2]. These and other pioneering studies [3] paved the way for the Nobel prize-awarded paper published by Takahashi and Yamanaka [4], who hypothesized that factors that play important roles in the maintenance of embryonic stem cell identity also play pivotal roles in the induction of pluripotency in somatic cells. In this study, mouse embryonic and adult fibroblasts were genetically reprogrammed to a pluripotent state, and the authors coined the term iPS cells. These cells were generated by using a retrovirus-based gene transfer system carrying the octamer-binding transcription factor 4 (Oct3/4), sex determining region Y-box 2 (Sox2), Krppel-like factor 4 (Klf4), and c-Myc transcription factors, all involved in pluripotency maintenance in embryonic stem cells [4].

IPS cell technology brings great promise to medicine, such as personalized cell therapy, disease modeling, and new drug development and screening. However, some challenges must be circumvented, such as reprogramming efficiency and the risks associated with chromosomal instability, insertional tumor development, and teratoma formation. In this context, here, we review the literature, present the main methods of cell reprogramming, and show some initial results of clinical tests. Besides, we discuss the possibility of applying iPS cells in the treatment of muscular dystrophies.

Various delivery methods have been used to insert reprogramming factors into somatic cells. These approaches can be divided into integrative, which involves the insertion of exogenous genetic material into the host genome, and non-integrative methods. The integrative systems include the use of viral vectors (lentivirus, retrovirus, and inducible or excisable retro or lentivirus) and non-viral vectors (linear or plasmid DNA fragments and transposons). Likewise, non-integrative systems include viral (Sendai virus and adenovirus) and non-viral vectors (episomal DNA vectors, RNAs, human artificial chromosomes (HAC), proteins, and small molecule compounds) [5,6] (Figure 1 and Table 1).

Somatic Cells Reprogramming Methods. The methods used to produce iPS cells can be classified into integrative viral, such as retrovirus (a), lentivirus (b), or inducible retro or lentivirus (c); and integrative non-viral, such as linear or circular DNA fragments (d) or transposons (e). In regards to non-integrative methods, they can also be separated as viral, such as adenovirus (f) or Sendai virus (g). Non-integrating non-viral methods are episomal DNA (h), RNAs (i), human artificial chromosome (HAC) (j), proteins (k), or small molecules (compounds) (l). The red DNA represents epigenetic inserted sequences for cellular reprogramming.

Comparison of multiple reprogramming techniques.

The expression of primarily just four transcription factors (c-Myc, Klf4, Oct4, and Sox2) is sufficient to reprogram somatic cells into a pluripotent state. The discovery of those factors related to the embryonic stem cell phenotype allowed the production of embryonic stem-like cells first from mouse embryonic and adult murine fibroblasts [4] and then from adult human dermal fibroblasts [7,8]. The Oct4 seems to be the most important reprogramming factor, whereas Klf4 and c-Myc can be replaced by Nanog and Lin28, for example [9].

The first experiments achieved the conversion of somatic cells into iPS cells using retroviral or lentiviral transduction of the transcription factors. However, these vectors become integrated into the cell genome and represent a risk of insertional mutagenesis [10]. Moreover, they may leave residual transgene sequences as part of the host genome, leading to unpredictable alterations in the phenotype of downstream applications. To reduce multiple proviral integrations of the transcription factors and to increase the efficiency of the retrovirus- or lentivirus-based reprogramming process, polycistronic RNA viral vectors were created. These constructs allowed the expression of all reprogramming factors driven by a single promoter, reducing the number of genomic insertions [11]. Once the integration of the reprogramming factors is achieved, it is also essential to control the extent of expression. To this end, the use of excisable Cre-loxP technology for site-specific recombination and inducible tetracycline- or doxycycline-based vector systems allowed greater control of inserted genes expression, reducing inefficient silencing and uncontrolled reactivation [5].

It is important to highlight that other factors have been described as being able to induce cellular pluripotency and self-renewal. Besides, several types of somatic cells have also been subjected to in vitro reprogramming, such as pancreatic cells, neural stem cells, stomach and liver cells, mature B lymphocytes, melanocytes, adipose stem cells, and keratinocytes. These results are summarized in the review published by Oldole and Fakoya [5].

The integrative non-viral technologies used to obtain iPS cells are based on the transference of DNA sequences using liposomes or electroporation [5], for example. It was possible to reprogram both mouse and human fibroblasts using a single multiprotein expression vector comprising the coding sequences of c-Myc, Klf4, Oct4, and Sox2 linked with 2A peptide [24]. When this single vector-based reprogramming system was combined with a piggyBac transposon, the authors successfully established reprogrammed human cell lines from embryonic fibroblasts with sustained pluripotency markers expression. PiggyBac is a mobile genetic element that includes a transposase enzyme that mediates gene transfer by targeted insertion and excision in the DNA. Moreover, Woltjen and collaborators showed the efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PiggyBac transposition. The authors also showed that the individual PiggyBac insertions could be removed from the iPS cell lines [15], being completely excised from its integration site in the original DNA sequence [25], which is a significant advantage.

The integrative methods for random or site-specific DNA insertion can affect normal cell function and physiology, including the transformation for tumorigenic cells, proliferation, and apoptosis control. Therefore, non-integrating viral vectors were constructed to generate iPS cells, the most promising of which is the Sendai virus, a negative-strand RNA virus [26]. The Sendai virus has the advantage of being an RNA virus that does not enter the nucleus and can produce large amounts of proteins [27]. Adenoviruses are also non-integrating viruses that appear to be excellent expression vehicles to generate iPS cells. They show DNA demethylation (a characteristic of reprogrammed cells), express endogenous pluripotency genes, and can generate multiple cells and tissues. However, the reprogramming efficiency of adenoviral vectors is only 0.001%0.0001% in mouse [28] and 0.0002% in human cells [29], several orders of magnitude lower, when compared to lentiviruses or retroviruses [5]. The use of viruses, even in non-integrating systems, requires refined steps to exclude reprogrammed cells with active replicating viruses. Moreover, viral vectors may elicit an innate and adaptive immune response against viral antigens after the transplant to patients. In this case, the transplanted cells would become the target of molecular and cellular cytotoxic pathways, directly compromising the engraftment and therapy success.

Non-integrating non-viral systems include the transient expression of reprogramming factors inserted as combined episomal minicircles or plasmids. These contain the complementary DNA (cDNA) of Oct3/4, Sox2, and Klf4 and another plasmid containing the c-Myc cDNA, for example. This technique resulted in iPS cells with no evidence of plasmid integration [16], suggesting that episomal plasmids may be the best option for clinical translation. This technique has already been used in the autologous induced stem cell-derived retinal treatment for macular degeneration [30]. Moreover, minicircle vectors are also used as a method for cellular reprogramming and consist of minimal vectors containing only the eukaryotic promoter and the cDNA(s) that will be expressed. This technique was able to reprogram human adipose stromal cells, but the reprogramming efficiency is substantially lower (~0.005%) when compared to lentiviral-based techniques, for example [31].

HACs are also non-integrative systems for gene delivery with the main advantage of being able to transfer multiple genes and large sequences, which can be combined with sequences that increase therapy security and expression control. The authors constructed two different HACs, and the reprogramming of mouse embryonic fibroblasts into iPS cells was better achieved when the artificial chromosome also encoded a p53-knockdown cassette. The iPS cells were uniformly generated, and a built-in safeguard system was included, consisting of a reintroduced HAC encoding the Herpes Simplex virus thymidine kinase, which allowed the targeted elimination of reprogrammed cells by ganciclovir treatment [19].

Another promising strategy focusing on non-integrative non-viral reprogramming methods for iPS cell generation is through RNA molecules, such as micro-RNAs. These sequences are small endogenous non-coding RNAs that play important post-transcriptional regulatory roles [32]. They also repress gene expression through translational inhibition or by promoting the degradation of mRNAs [33]. One study showed that normal human hair follicles could be reprogramed into human iPS cells via doxycycline-inducible pTet-On-tTS vectors inserted by electroporation. These constructs contained pre-microRNA members of the miR-302 cluster, including pre-miR-302a, 302b, 302c, and 302d [34]. Although the reprogramming efficiency was not reported in this study, it is known that iPS cells induced by micro-RNAs have a reprogramming efficiency above 10% and also have the lowest tumorigenicity rate. Although this approach has not yet been used in any clinical test, it may help in future developments in regenerative medicine [33]. More recently, micro-RNAs were used in combination with other reprogramming methods to increase reprogramming efficiency [5].

Another promising transgene-free approach is the direct mRNA transfection of synthetic modified coding sequences of the Yamanaka factors (c-Myc, Klf4, Oct4, and Sox2). This is a non-integrating method that can reprogram multiple human cell types to pluripotency very efficiently, avoiding the antiviral immune response. The authors further showed that the same technology efficiently directed the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells [35]. The method of the direct delivery of synthetically transcribed mRNAs triggered somatic cell reprogramming with higher efficiency when compared to retroviruses [35]. These mRNAs are commercially available, and the authors used cationic lipid delivery vehicles for transfection in cell culture for seven days [27]. Similar alternatives are emerging as the cellular introduction of all reprogramming factors via a single synthetic polycistronic RNA replicon that requires single transfection [36]. In this case, the transfection of adult fibroblasts resulted in an efficient generation of iPS cells with the expression of all stem cell markers tested, consistent global gene expression profile, and in vivo pluripotency for all three germ layers.

Transgene-free cellular reprogramming has also been achieved using recombinant proteins. In this case, the generation of stable iPS cells was possible by directly delivering the four reprogramming proteins fused with a cell-penetrating peptide [22]. However, it has been technically challenging to synthesize large amounts of bioactive proteins that can cross the plasma membrane. This problem associated with low efficiency shows that much remains to be done for the use of recombinant proteins as a viable method. Two research groups were able to make enough bioactive proteins in an E. coli expression system and to reprogram mouse [37] and human fibroblasts [22]. More recently, Weltner and collaborators also used Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 nuclease (CRISPR-Cas9)-based gene activation (CRISPRa) for reprogramming human skin fibroblasts into iPS cells [38]. CRISPR/Cas9 is a genome-editing tool powered by the design principle of the guide RNA that targets Cas9 to the desired DNA locus and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks [39].

Another system for cellular reprogramming to generate iPS cells was the use of small-molecule compounds, which was developed by Hou and collaborators [23]. These authors used a combination of seven small molecules, but the efficiency achieved was only 0.2%. Small molecules have some advantages such as structural versatility, reasonable cost, easy handling, and no immune response. They can boost the application of iPS cells in disease therapy and drug screening. Some of these chemical compounds are valproic acid, trichostatin A (TSA), and 5-azacytidine, all capable of enhancing iPS cell generation [40]. One of the main advantages is that small (chemical) molecules can stimulate endogenous human cells to make tissue repair and regeneration in vivo, with no ectopic expression of factors. On the other hand, the method is time-consuming, and there is still a risk of genetic instability [6] to be overcome in future studies.

Despite all developments in the field of iPS cells, viral vector-based methods remain most popular among researchers [41]. Still, non-integrating non-viral self-excising vectors are more likely to be clinically applicable. To select an iPS cell reprogramming method, it is essential to maximize the capacity of cellular expansion in vitro, validate the detection and removal of incompletely differentiated cells, and search for genomic and epigenetic alterations. Probably, different somatic cell types will require different reprogramming methods to differentiate into the required terminal cell type in vivo.

Regardless of the reprogramming method, the risk of teratoma formation is inherent to iPS cells, as residual undifferentiated pluripotent cells retain very high plasticity. Although this risk has been reduced by highly sensitive methods for detecting remaining undifferentiated cells, teratoma formation cannot be ruled out [42]. Besides, c-Myc, one of the factors used for cellular reprogramming, is a well-known proto-oncogene, and its reactivation can give rise to transgene-driven tumor formation [43].

IPS cells can differentiate into cells from any of the three primary germ layers [44], with great potential for clinical applications. Neurodegenerative disorders, for example, and diseases in which in vitro differentiation and transplant protocols have been established using conventional embryonic stem cells, are areas of immediate interest for iPS-based cell therapy. IPS cell lines can be generated in virtually unlimited numbers from patients affected by diseases of known or unknown causes. These cells can differentiate in vitro into the disease-affected cell type and offer an opportunity to gain insight into the disease mechanism to identify novel disease-specific drugs. In Table 2, we show examples of iPS cells generated from patients with sporadic or genetic diseases.

Examples of terminally differentiated cells generated from induced pluripotent stem (iPS) cells.

Some drugs that are in clinical trials were derived from iPS cell studies. For example, cardiomyocyte-derived iPS cells obtained from patients with type-2 long QT syndrome were used to test the efficacy and potency of new and existing drugs [51]. In regenerative medicine, iPS cells can be used for tissue repair or replacement of injured tissues after cell transplantation. Early trials using iPS cell transplantation focused on age-related macular degeneration, and this is a refractory ocular disease that causes severe deterioration in the central vision due to senescence in the retinal pigment epithelium (RPE). Preclinical studies showed good results in various animal models and corroborated the first clinical trial that began in 2014 [54]. Kamao and collaborators generated human iPS cells derived from RPE (hiPSC-RPE) cells that met clinical use requirements, including cellular quality and quantity, reproducibility, and safety. After the transplant, autologous non-human primate iPSC-RPE cell sheets showed no immune rejection or tumor formation [55]. Then, in the clinical trial using iPS cells, the cells were generated from skin fibroblasts obtained from patients with advanced neovascular age-related macular degeneration and were differentiated into RPE cells. In this test, autologous iPS cell-based therapy did not cause any significant adverse event [30]. However, the test with the second patient was discontinued due to genetic aberrations detected in the autologous iPS cells. With the rapid progress of genomic technologies, genetic aberrations in iPS cells will probably be reduced to a minimal level, with technological advances also focusing on automated closed culture systems [56].

Recent advances in genome editing technology have made it possible to repair genetic mutations in iPS cell lines derived from patients. Special attention has recently been focused on organoids derived from iPS cells, which are three-dimensional cellular structures mimicking part of the organization and functions of organs or tissues. Organoids were generated for various organs from both mouse and human stem cells, generating intestinal, renal, brain, and retinal structures, as well as liver organoid-like tissues, named liver buds [57]. Therefore, iPS cells-derived organoids can also be useful for drug testing and in vitro studies based on more complex cell models.

Moreover, iPS cells derived from cancer cells (cancer-iPS cells) can be a novel strategy for studying cancer. Primary cancer cells have been reprogrammed into iPS cells or at least to a pluripotent state, allowing the study and elucidation of some of the molecular mechanisms associated with cancer progression [58].

The possibility of using iPS cells in the treatment of various diseases has brought hope regarding their potential to treat an increasing number of conditions. As iPS cells can be differentiated into all different cell types, new prospects for studying diseases and developing treatments by regenerative medicine and drug screening have emerged. Therefore, a large number of clinical and preclinical trials are being carried out [59] to treat human diseases using iPS cells.

The reprogramming of somatic cells was demonstrated using different animal species, including mouse, rat [60], dog [61], a variety of non-human primate species [62], pig [63], horse [64], cow [65], goat [66], and sheep [67]. However, once the goal of pre-clinical trials is the clinical use of iPS cells, a number of these trials are being conducted using human iPS cells. For specific applications, however, human cells are expected to be rejected by the animal hosts, and immunosuppressive protocols are required for long-term observation. On the other hand, immunomodulating drugs may affect the disease phenotype, and careful planning of every step is necessary. Any stem-cell-based clinical trial must follow all precedents already established for the evaluation of small biological molecules or human tissue remodeling and must be safe and effective. The production of cells must be carried out in facilities that follow the current Good Manufacturing Practices (GMP) and have stringent quality control for reagents with well-defined product release and potency assays. GMP is a set of conditions that define the principles and details of the manufacturing process, quality control, evaluations, and documentation for a particular product. Moreover, the best delivery system of iPS cells must be evaluated for each disease, which can be the use of intravascular catheters or surgical injection, for example.

Human-derived iPS cell lines successfully repopulated the murine cirrhotic liver tissue with hepatic cells at various differentiation stages. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to those of proteins secreted by human primary hepatocytes [68]. In other preclinical studies, iPS cells were generated using adult dsRed mouse dermal fibroblasts via retroviral induction, following transplantation into the eye of immune-compromised retinal degenerative mice. After thirty-three days of differentiation, a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and photoreceptor markers. Therefore, adult fibroblast-derived iPS cells are a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease [69]. IPS cells were also generated from nonobese diabetic mouse embryonic fibroblasts or nonobese diabetic mouse pancreas-derived epithelial cells and differentiated into functional pancreatic beta cells. The differentiated cells expressed diverse pancreatic beta-cell markers and released insulin in response to glucose and KCl stimulation. Moreover, the engrafted cells responded to glucose levels by secreting insulin, thereby normalizing blood glucose levels, showing that these cells may be an important tool to help in the treatment of diabetic patients [70]. Human cardiomyocytes derived from iPS cells are another source of cells capable of inducing myocardial regeneration for the recovery of cardiac function. These cells were established using human dermal fibroblasts transfected with a retrovirus carrying the conventional factors Oct3/4, Sox2, Klf4, and c-Myc. When the iPS cells were transplanted into the myocardial infarcted area in a porcine model of ischemic cardiomyopathy, the activation of WNT signaling pathways induced cardiomyogenic differentiation. It was also observed that the transplanted cells significantly improved cardiac function and attenuated left ventricular remodeling [71]. In another study, dopaminergic neurons derived from protein-induced human iPS cells exhibited gene expression, physiology, and electrophysiological properties similar to the dopaminergic neurons found in the midbrain. The transplantation of these cells significantly rescued the motor deficits of rats with striatal lesions, an experimental model of Parkinsons disease [72]. Moreover, after stroke-induced brain damage, adult human fibroblast-derived iPS cells were transplanted into the cortical lesion and, one week after the transplantation, there was the initial recovery of the forepaw movements. Moreover, engrafted cells exhibited electrophysiological properties of mature neurons and received synaptic input from host neurons [73].

In October 2018, 2.4 million iPS cells reprogrammed into dopaminergic precursor cell neurons were implanted into the brain of a patient in his 50s. In the three-hour procedure, the team deposited the cells into twelve sites, known to be centers of dopamine activity. The patient showed no significant adverse effects [74]. The first allogeneic clinical trial using iPS cells derived from mesenchymal stem cells for the treatment of graft-versus-host disease has also been reported, and no treatment-related serious adverse effects were observed [75]. Other clinical studies using iPS cells are being conducted in patients with heart failure [76,77]. Moreover, other tests have been approved for neural precursor cells for spinal cord injuries [78] and corneal epithelial cell sheets for corneal epithelial stem cell deficiency [79]. Thus, ongoing clinical tests provide a better understanding of clinical aspects involving immunosuppressants and fundamental elements such as genomic data that will pave the way for therapies using iPS cells.

The iPS cells have the potential to revolutionize the field of neurodegenerative diseases, which are characterized by the progressive deterioration of neuronal function. Therefore, multiple capacities are affected, leading to cognitive impairment, memory deficits, deficiency in motor function, loss of sensitivity, dysfunction of the autonomous brain system, changes in perception, and mood [80]. Among neurodegenerative diseases, Alzheimers disease is the most prevalent form of dementia, characterized by the accumulation of amyloid-beta (A) plaques and Tau-laden neurofibrillary tangles. Tau is a microtubule-associated protein found in the axons of the nerve cells, and these aggregates and tangles are the histopathological hallmarks of the disease [81]. The dysfunction and degeneration of neurons indeed underlie much of the observed decline in cognitive function, but various other types of non-neuronal cells are increasingly being implicated in the disease progression [82]. Therefore, iPS cells are emerging as an invaluable tool to better modeling the complex interactions that occur between multiple cell types in vivo. 3D and co-culture systems of iPS-derived cells in vitro hold promise to better understand the relevance of multiple cell types and the pathomechanisms that underlie the disease progression. Therefore, iPS cells have been generated from patients and healthy donors to study multiple genetic mutations in neurons, astrocytes, oligodendrocytes, microglia, pericytes, and vascular endothelial cells [83]. Moreover, a mutant Tau model derived from iPS cells was generated and showed several phenotypes associated with this neurodegenerative disease, including the pathogenic accumulation of Tau for drug screening [84]. Choi et al., 2014 showed a 3D culture model based on iPS cells that histopathologically reproduces the hallmarks of Alzheimers disease, including a robust extracellular deposition of A. This model was sensitive to drugs, which reversed the pathological phenotype [85]. The use of neural models derived from iPS cells can validate molecular mechanisms identified in the disease models in rodents, for example, and play an important role in the discovery and screening of new drugs [86].

Parkinsons disease is another important disease; being the second most common neurodegenerative disorder, it affects 2% to 3% of the population over 65 years of age. Characteristic features of Parkinsons disease include neuronal loss in specific areas of the substantia nigra and widespread intracellular protein (-synuclein) accumulation [87]. Due to the loss of dopaminergic neurons in localized regions of the brain, the use of human cells for therapeutic purposes has been studied with special attention. These assessments include iPS cells, whose good results supported the deployment of some studies that are already in the clinical phase. Pre-clinical studies have shown the efficient generation of iPS cells-derived dopaminergic motor neurons from non-human primates. Then, these cells were efficiently transplanted into a model of Parkinsons disease in rats [88]. Several new protocols have improved the efficiency of obtaining dopaminergic neurons from iPS cells for the study and modeling of Parkinsons disease [89]. The iPS cells used in some studies were mainly from patients carrying mutations in synuclein alpha, leucine-rich repeat kinase 2, PTEN-induced putative kinase 1, parkin RBR E3 ubiquitin-protein ligase, cytoplasmic protein sorting 35, and variants in glucosidase beta acid [90]. Although improvements are still needed, iPS cells make it possible to develop patient-specific disease models using disease-relevant cell types. Interestingly, using a human iPS cells-derived model of Parkinsons disease, it was found that the myocyte enhancer factor 2C-peroxisome proliferator-activated receptor- coactivator-1 (MEF2C-PGC1) pathway may be a new therapeutic target for Parkinsons disease. The data from this study provided mechanistic insight into geneenvironmental interaction in the pathogenesis of the disease [91]. Thus, it is important to develop models of neurodegenerative diseases using iPS cells because they involve a complex interplay of genetic alterations, transcriptional feedback, and endogenous control by transcription factors. Probably, the combination of different experimental approaches, using cellular systems and animal models, will increase the successful translation to the clinical practice [92].

In a successful pre-clinical study, the authors demonstrated that human dopaminergic neurons generated from iPS cells, and transplanted into a primate model of Parkinsons disease, established connections with the host monkey brain cells with no tumor formation after two years [93]. Immediately after the successful animal experiments, the Japanese research group implanted reprogrammed stem cells into the brain of a patient with Parkinsons disease for the first time in 2018 (as NEWS Reported by Nature https://www.nature.com/articles/d41586-018-07407-9).

Recently, extracellular vesicles/exosomes derived from iPS cells of different lineages were involved in neurological diseases. Extracellular vesicles are lipid-enclosed structures with a diameter of 301000 nm, carrying transmembrane and cytosolic proteins. Exosomes are a subset of extracellular vesicles, with a diameter ranging between 30 and 200 nm. Functionally, they play an important role in intercellular communications, immune modulation, senescence, proliferation, and differentiation in various biological processes, and are vital in maintaining tissue homeostasis [94]. On the other hand, and as cited before, abnormal protein aggregation has been implicated in many neurodegenerative processes that lead to human neurological disorders. Recent reports suggested that exosomes combine these two important characteristics, as they are involved in the intercellular transfer of macromolecules, such as proteins and RNAs, and seem to play an important role in the aggregate transmission among neurons [95]. The authors showed that extracellular vesicles from iPS cells carry proteins and mRNA that can induce or maintain pluripotency, which can be used in regenerative strategies for neural tissue [96]. If this is true, extracellular vesicles/exosomes derived from corrected iPS cells, which do not accumulate protein aggregates, may be safer for human treatment than iPS cells themselves [94]. The infusion of neuronal exosomes into the brains of a murine model of Alzheimers disease decreased the A peptide and amyloid depositions [97]. Moreover, exosomes obtained from stem cells were able to rescue dopaminergic neurons from apoptosis [98]. The authors showed that extracellular vesicles from mesenchymal stem cells, when injected into a mouse model of Alzheimers disease, reduced the A plaque burden and the number of dystrophic neurites in the cortex and hippocampus [99]. Extracellular vesicles were also derived from human iPS neural stem cells and used for stroke treatment [100]. The results using extracellular vesicles/exosomes obtained from iPS cells point to a promising future in the treatment of neurodegenerative diseases.

Muscular dystrophies (MD) are a group of genetic diseases that lead to skeletal muscle wasting and may affect many organs (multisystem) [101]. The terminal pathology often shows muscle fibers necrosis and muscle tissue replacement by fibrotic or adipose tissues. Currently, there is no cure for MD, and the available treatments are palliative or of limited effectiveness [102]. The most frequent and one of the most severe forms of all MD is the Duchenne muscular dystrophy (DMD), a muscle pathology caused by the lack of the protein dystrophin. In this case, previous cell-based therapies did not show satisfactory results after myoblast transplantation [103]. Myoblasts are the progeny of muscle satellite cells (SC), the main stem cell population found in adult skeletal muscles. Quiescent SCs are triggered to reenter into the cell cycle mainly by muscle damage, and the SC-derived myoblasts proliferate and fuse to form new multinucleated myofibers [101]. In most myoblast-based therapies, allogeneic cells were obtained from muscle biopsies from healthy donors, resulting in transplanted cell rejection by the immune system, with low surviving rates, poor dispersion, and differentiation [103,104,105]. With the advances of iPS cell technology, some of these issues are being addressed (Figure 2).

iPS cells in Duchene muscular dystrophy cell therapy. The somatic cells derived from specific patients with Duchenne muscular dystrophy (DMD) can be reprogrammed into iPS cells with reprogramming factors. These cells are then genetically corrected to express the protein dystrophin for the autologous muscular injection of muscle-committed cells.

One of the main problems in the application of stem cell therapy in muscle diseases is to obtain large numbers of cells for sufficient engraftment, and the use of iPS cells may overcome this obstacle. For this purpose, Darabi and colleagues [106] applied the conditional expression of Pax7 to iPS cells, a transcription factor that plays a role in SC proliferation. Then, Pax7+ iPS cells were obtained on a larger scale for transplant into a mouse dystrophic muscle, which showed dystrophin+ fibers with superior strength [106]. Moreover, the authors genetically restored the dystrophin expression in autologous iPS cells derived from DMD patients. For this, three corrective methods were used, which were exon knock-in, exon skipping, and frameshifting, and the exon knock-in was the most effective approach [107]. The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is a technology that has also emerged as an approach capable of targeting the mutated dystrophin gene, aiming to rescue its expression in vitro in iPS cells derived from selected patients [108].

Moreover, using CRISPR-Cas9 technology with single guide RNA, dystrophin expression was restored by exon skipping through myoediting in iPS cells. The genetic alterations observed in the multiple patients included large deletions, point mutations, or duplications within the DMD gene. The corrected iPS cells efficiently restored the expression of dystrophin and the corresponding mechanical contraction force in derived cardiomyocytes [109]. In summary, several methods of gene editing have been applied for the correction of the DMD gene to allow the transplantation of genetically corrected autologous iPS cells. Of these, the CRISPR-Cas9 system, in particular, has passed multiple proof-of-principle tests and is now being used in pre-clinical trials (Figure 2).

Reprogrammed fibroblast- and myoblast-derived iPS cells were also obtained from patients with limb-girdle muscular dystrophy type 2D (LGMD2D). This disease is a sarcoglycanopathy caused by mutations in the SCGA gene, which provides instructions for making the alpha component of the sarcoglycan protein complex. This multiprotein complex plays a role in the anchoring of the dystrophin-glycoprotein complex (DGC) to the extracellular matrix and helps to maintain muscle fiber membrane integrity. The iPS cells were expanded and genetically corrected in vitro with a lentiviral vector carrying the human gene encoding the -sarcoglycan. Finally, the transplantation of mouse iPS cells into -sarcoglycan-null immunodeficient mice, an experimental model of the disease, resulted in the amelioration of the dystrophic phenotype [110]. This transplant also showed that iPS cells restored the compartment of SC, an essential checkpoint for sustained muscle regeneration.

Recently, Perepelina and collaborators generated iPS cells from EmeryDreifuss muscle dystrophy associated with the genetic variant LMNAp.Arg527Pro. Patient-specific peripheral blood mononuclear cells were reprogrammed using the Sendai virus system, and the authors comment that this is a useful tool to study laminopathies in vitro [111]. Moreover, using three-dimensional (3D) tissue engineering techniques, artificial skeletal muscle tissue was generated using iPS cells from patients with Duchenne, limb-girdle, or congenital muscular dystrophies [112]. In this way, artificial muscles recapitulated characteristics of human skeletal muscle tissue, providing an invaluable tool to study pathological mechanisms, drug testing, cell therapy, and the development of tissue replacement protocols.

The use of iPS cells still has many challenges ahead before they can be clinically used in the supportive treatment of patients with MD. Among these, we can cite the injection of iPS cells (or muscle-committed iPS-derived cells) into large muscles, the immunological recognition of proteins expressed only after the genetic correction, the capacity of cellular dispersion through the muscle, the number of therapeutic interventions needed to replenish cellular muscle populations, the ability to produce corrected SC for sustained muscle recovery, and the control of transplanted cells death.

To address these and other limitations, we propose that autologous iPS cells be submitted to multiple treatments aiming to improve cellular engraftment and clinical use. Besides the genetic correction of underlying pathological mutations, these cells can be further treated in culture to boost cell proliferation, long-term survival, dispersion in the muscle, differentiation into muscle fibers, and others. We proposed before the use of multiple combined in vitro treatments for adoptively transferred myoblasts for cell-based therapy, and these are summarized in [101]. These treatments include vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF), Wnt7a, Ursolic acid, and extracellular matrix components. Moreover, the recipient muscle to be injected with the corrected and boosted iPS cells can also be treated to favor the engraftment. These treatments include actinin receptor type 2B inhibitor, IL-6, JAK/STAT 3 inhibitor, growth factors, the coinjection of other supportive cell types, such as macrophages and fibroblasts, and others.

We believe that the correct choice for the ideal combination of the cell type to be reprogrammed into iPS cells, the technical procedure for genetic correction, the in vitro treatments to boost iPS cells, and the in vivo preparation of recipients muscles, hold the key for a more successful application of iPS cells in clinical translation. However, we believe that systemic treatments consisting of the injection of cells will not lead to individual muscle damage and strength improvement. The transplanted cells do not express the required repertoire of molecules necessary for endothelial transmigration. Probably, selected individual and more affected muscles are more likely to benefit from cellular-based therapies, followed by treatments that can increase injected cell dispersion within the muscle.

Currently, publicprivate partnership consortia are providing resources to form iPS cell banks for clinical and research purposes. These banks have coordinated standards to meet international criteria for quality-controlled repositories of iPS cells. Although the use of iPS cells for autologous therapy seems more appropriate, having allogeneic banks of iPS cells already generated and tested would reduce the time needed to start treatment, decrease costs, and increase the chances of recovery of treated individuals [113]. Thus, although many technical challenges must still be overcome, the technology of iPS cells has already taken a marked leap in clinical management and in vitro models to study and treat diseases.

D.G.B.: manuscript preparation and review; S.I.H.: manuscript review and preparation of figure; C.M.C.: manuscript preparation; L.A.A.: manuscript review and figure preparation; A.H.-P.: manuscript and figure preparation and review. All authors have read and agreed to the published version of the manuscript.

This work was funded by CNPq (Conselho Nacional de Desenvolvimento Cientfico e Tecnolgico) grant numbers 407711/2012-0 and 421803/2017-7 and Fundao Oswaldo Cruz.

The authors declare no conflict of interest.

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Induced Pluripotent Stem Cells: Hope in the Treatment of Diseases ...

What are possible side effects of stem cell therapy ? Patients often reach out to ask about such risks They usually refer to unproven stem cell clinics.

Todays post addresses the scope of stem cell therapy side effects and risks based on available hard data. Its also important to discuss possible unknown risks.

Stem cell risks at unproven clinics | Why do stem cells pose risks | Tumors| Impact of lab growth | Infections | Blood clots | MSCs |Other risks | Intranasal stem cells and exosomes | References

In this post I am focusing on the risks primarily associated with unproven stem cell clinics. Not for established methods like bone marrow transplantation, which have their own risks including the shared one of infection.

Recent publications in journals have helped clarify risks. This literature includes a study by my UC Davis colleague Gerhard Bauer and a special report by The Pew Charitable Trust. Gerhards paper presents the types of side effects that appear more common after people go to stem cell clinics. After closely following this area for a decade I was familiar with many of the examples of problems.

One of the highest profile examples of bad outcomes was the case where three people lost their vision due to stem cells injected by a clinic.

I have included a YouTube video below on stem cell therapy side effects as well.

Why do stem cells pose risks?

One major reason is that stem cells are uniquely powerful cells.

By definition they can both make more of themselves and turn into at least one other kind of specialized cells. This latter attribute is called potency and the process of becoming other cells is called differentiation. The ability to make more of themselves is called self-renewal.

The most powerful stem cells are totipotent stem cells that can literally make any kind of differentiated cell. The fertilized human egg is the best example of a cell having totipotency. The first few cell divisions after that retain the totipotency. Next in the power lineup are pluripotent stem cells including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). These cells are not directly used in therapies.

Adult stem cells are multipotent, which means they can make just a few types of specialized cells.

What is the best type of stem cell? The best type of stem cell depends on the condition that is trying to be treated and may not be the most powerful.

In any case, the power of stem cells is one reason they also pose risks along with mishandling that can cause infections. Stem cells are not always easy to control and misdirected power can do harm.

Let me explain and start with the side effect that seems most concerning to most people but is probably the rarest. Tumor formation.

If someone injects a patient with stem cells, its possible that the self-renewal power of stem cells just wont get shut off. In that scenario, the stem cells could drive the formation of a tumor.

Why wouldnt a transplanted stem cell always eventually hit the brakes on self-renewal? It could be that the stem cell has one or more mutations. For any stem cells grown in a lab, within the population of millions of cells in a dish, there are going to be at least a few with mutations that crop up. Thats just the way it goes with growing cells in a lab. The longer you grow them the more mutations they will have on average.

Even stem cells not grown in the lab have the same spectrum of mutations as the person they were isolated from. It may seem odd to think about, but we all have some mutations.

Research suggests it takes more than one cell with cancer-causing potential to make a tumor in experiments in the lab, but in actual people, we just dont know. Many cancers may arise from one stem cell gone awry. If a clinic injects 100 or 500 million cells, a one-in-a-million rate of potentially dangerous cells means that 100-500 such risky cells end up in the patient. The risk of getting an actual tumor may still be low but I wouldnt take those odds.

The encouraging news here is that the incidence of tumors in stem cell clinic customers, particularly in the U.S., appears extremely low.

The odds of getting a tumor are far lower for cells never grown in a lab but its still possible. Oddly, receiving someone elses stem cells (we call this allogeneic) might pose a lower cancer risk because your immune system is going to see those cells as foreign from the start. Itll reject them. Still, an immunocompromised state could play a role.

Some stem cells, especially those with mutations, might be able to somewhat fly under the radar of the immune system to some extent. This could allow them to grow into a tumor.

The Pew report does a nice job of summarizing risks and there are several reports of tumors.

The possibility of infections after stem cell injections is another risk that is often discussed. Infections from injections of stem cells or other biologics are probably the most common type of side effect. Bacteria can sometimes already be in the product that is injected. Or germs can be introduced by poor injection or preparation methods by the one doing the procedure.

The distributor Liveyon had a product contaminated with bacteria that sickened at least a dozen people who were hospitalized. Some of them ended up in the ICU. A few may even have permanent issues.

Infection risk usually does not arise from the cells themselves.

Another risk is the potential for blood clots.

In the case of adipose biologics life SVF, they mostly consist of a mixture of a dozen or so other kinds of cells found in fat. Fat cells just live in fat so they arent supposed to be floating around in your blood. As a result, after IV injection, many fat cells are thought to get killed right away by the immune system or the microenvironment. While in the blood, fat and other stromal-type cells, whether dead or alive, may catalyze clot formation, which is dangerous.

Some of these cells end up landing in the lungs. There many cells are probably being killed and theres also risk of blood clot formation.

Unproven clinics mainly sell MSCs.

MSCs could have some powerful uses in medicine. I can already see a few rigorous clinical trials that look exciting.

However, the way some unproven clinics use MSCs can be highly risky.

Such cells just shouldnt be injected willy-nilly into dozens of places in patients including into peoples eyes. Further, what are called MSCs by some unproven clinics may also not meet even basic lab standards and may not have the potential of other MSC preps. Some such clinic preps are likely just fibroblasts or mostly dead cells.

MSCs produced in a rigorous manner in clean labs by experienced teams are likely to be a far superior product than that typically made by just any strip mall clinic. I dont endorse any cell therapy clinic selling MSCs at this time, but some are doing far better than others. They do research and publish papers.

Properly conducted injections of unmodified, high quality MSC-type cells or marrow cells into joints or for other orthopedic conditions by qualified providers in theory should pose almost zero risk of pulmonary emboli or cancer. Clinics using excellent procedures and cell products also should pose a very low risk of infection, a risk more similar to getting medical procedures in general even unrelated to stem cells.

Overall, Im not sure I believe such MSCs even from the best clinics can provide lasting benefit for diverse orthopedic conditions, but the overall risks associated with them should be quite low there relatively speaking.

Patients have also asked me about other potential risks of cell injections.

I wrote a post about possible graft versus host disease in stem cell recipients. This would only happen in people receiving someone elses stem cells and probably only with IV administration. Its not clear if GvHD is something that happens to patients after going to clinics selling allogeneic cells. With no immunosuppression, it should be highly unlikely.

Beyond outright tumor formation, it is also possible that stem cells may become an undesired or even dangerous tissue type that isnt technically an actual tumor. The example that comes to mind is the practice mentioned earlier of some clinics injecting fat cells into peoples eyes. What seems to have happened in some cases is that the mesenchymal cells (MSCs) or other cells like fibroblasts that were injected turned into scar tissue, which caused retinal detachment.

In addition, we have seen indications that patients getting IV infusions of stem cells might be at some risk for heart attacks. Perhaps via clot formation. For example, read this piece: Cellular Performance Institute death.

One of the challenges is that it can be difficult to figure out if heart attacks or other outcomes were linked to the actual stem cell procedures or just incidental. Many patients getting stem cells may already be at higher risks for these issues. In any particular case, one can ask: was the cell infusion linked to the death? Im not sure we could ever know. Such outcomes should be carefully tracked and analyzed. One challenge is that adverse events at hundreds of unproven clinics may never be reported or otherwise come to light.

Specific routes of administration may pose unique risks as well. For instance, intranasal stem cells are getting popular with some unproven clinics and could lead to cells or other material ending up in the brain. Intranasal delivery of stem cells could have real promise such as for treating brain conditions, but you need rigorous clinical data to back that up. You need to work with the FDA and send them data. Clinics without such data are already selling the procedure.

Other products in the regenerative sphere that are not stem cells may be risky as well for various reasons. For instance, an exosome product harmed quite a few people in Nebraska. Some problems may relate to product contamination. Here again, exosomes may have promise for some conditions but should not be sold already as therapies at this time.

Finally, stem cells and other cell therapies also pose unknown risks because of their newness and power.

We also just dont have long-term follow-up data to have a clear sense of all major risks after people go to clinics.

In general, so much depends on collecting good data before trying to make money form vulnerable patients.

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Stem cell therapy side effects & risks at clinics - The Niche

1. Introduction

Stem cells are unspecialized cells and are the essential building blocks of all organisms. They can differentiate into any specialized cell within an organism [1]. In this capacity, stem cells possess the ability to self-renewal, in addition to differentiating into all cells within tissues and ultimately organ systems [2, 3, 4]. Stem cells exist from conception and remain functional through adulthood, with many regulatory factors responsible for their specialization. As stem cells mature, differentiation becomes more limited which is referred to as commitment to a specific lineage. This means a unipotent stem cell is restricted in differentiation compared to a pluripotential stem cell (PSC) that can produce a variety of lineage specific cells. Thus, PSCs are more restricted when compared to a totipotent stem cell (TSC) [5, 6].

TSCs are capable of cell division with the ability to differentiate into mature cells comprising all the physiological systems associated with an intact and complete organism [6]. TSCs have unlimited potential to fully differentiate. This property allows TSCs to form both embryonic and extra-embryonic structures such as the placenta and the tissues associated with pregnancy [7, 8]. An example of a TSC is the zygote that forms after a sperm fertilizes an egg. TSCs will form a blastocyst which develops the inner cell mass (ICM). The ICM contains a unique population of stem cells known as embryonic stem cells (ESCs). ESCs are capable of remaining pluripotent in vitro [9, 10]. ESCs form the three germ layers associated with developmental biology, i.e., ectoderm, mesoderm, and endoderm [10], thus providing the core foundation of an organism through each germ layer by providing all the anatomical and physiological systems of the organism [11].

Pluripotential stem cells (PSCs) form structures associated with only the germ layers [11]. Another example of stem cells possessing pluripotency was achieved following the reprogramming capability to produce induced pluripotent stem cells (iPSCs) [12]. iPSC pluripotency is a continuum, starting from totipotent cells to cells possessing less potency as in multi-, oligo- or unipotent cells. The independence of iPSCs allows for using improved methods that are more promising for therapeutic stem cell use now and for future applications as defined in regenerative medicine [13].

Within their respective lineages, multipotent stem cells can generate more specialized cells. It differentiates blood cell development to form a variety of diverse cells such as erythrocytes, leukocytes, and thrombocytes [14]. A myeloid stem cell is an example where a stem cell may differentiate into different types of leukocytes, e.g., white blood cells such as granulocytes or monocytes, but never erythrocytes or platelets [15].

As mentioned above, during embryogenesis, stem cells form aggregates referred to as germ layers [16]. Once hESCs differentiate into a specific germ layer, they become multipotent stem cells and can only differentiate according to their respective layer. Pluripotent stem cells are present throughout the life of any organism existing as undifferentiated cells [17]. Regulatory signals influence stem cell specialization to create specific tissues that are produced via physical contact between cells through the microenvironment/stroma or as stimulators in the form of cytokines, interleukins, and/or tissue factors secreted by surrounding tissues. These factors from internal sources are controlled via the presence of the genome, i.e., genes, thus DNA acting through transcription translation reactions [11]. Stem cells provide a mechanism designed to function as the bodys internal repair system. For as long as an organism remains functional, its stem cells will continue to provide differentiation pathways to replace more mature cell lineages. This is the repair and replenishment aspect of stem cell vitality [11, 18].

The growth and development of an organism depends on the presence of stem cells. Overall, somatic stem cells such as ESCs can be distinguished based upon their characteristic lineage line of development. ESCs can be derived without isolating them from the inner cell mass; however, their growth potential is limited [11]. ESCs can be propagated in vitro using tissue culture conditions indefinitely without restriction if their growth requirements are maintained [19, 20]. ESCs can be propagated in culture with appropriate culture medium containing essential nutrients [19]. Passage of ESCs is an adequate method of sub-culturing stem cells to propagate their numbers over time. Because ESCs are totipotent, they can differentiate into every cell type required in any organ cell system [21]. However, because totipotent stem cells demonstrate immortality, ethical restrictions restrict the procurement of these cells. The origin of these totipotent stem cells is from the ICM of the blastocyst present in embryos. Thus, the procedure to obtain them destroys the viability of that embryo from further development. However, most ESCs are derived from fertilized eggs in an in vitro clinic rather than from eggs harvested from pregnant women [22].

Among the many stem cell types that exist are as follows:

Hematopoietic stem cells have the potential to differentiate into many types of blood cells, e.g., erythrocytes, leukocytes, and thrombocytes.

Mesenchymal stem cells are found in multiple types of tissues. They can differentiate into multiple lineages such as bone, adipose, vascular, and cartilage tissue. They can be harvested from sources including but not limited to the umbilical cord, bone marrow, and endometrial polyps [23].

Neural stem cells develop into glial or neuronal cells such as nerve cells, oligodendrocytes, and astrocytes. These cells have been used in treatments regarding Parkinsons disease through transplants [24].

Skin stem cells (SSCs) consist of several types that are separated into their own niches including hair follicle stem cells, melanocyte stem cells, and dermal stem cells. SSCs have greater potential to be used for stem cell therapies and treatments since these cells can differentiate into more cell lineages [25].

Human ESCs are involved in whole-body development and can eventually become pluripotent, multipotent, and unipotent stem cells. Compared to adult somatic stem cells, they also have a quicker proliferation time and greater range of differentiation causing them to be more ideal and preferred in therapies [26].

Stem cells can also be taken from the placenta. Placental fetal mesenchymal stem cells can differentiate into a wide variety of cells and are abundant, not requiring invasive procedures to procure. They are not surrounded with ethical issues that ESCs have since the placenta is usually considered medical waste after birth, making it favorable for use as treatment. They can produce ectodermal, endodermal, and mesodermal lineages in vitro and contain the same cell markers as ESCs, making them very similar. Placental stem cells are pluripotent and have low immunogenicity which allows them to be ideal for therapies and treatments [27].

Differentiation was thought to be restricted and non-reversible. However, after several major experiments through cloning, even differentiated cells can be reprogrammed or induced to be pluripotent. Two major cloning-related discoveries were made in 1962 and 1987. The first was done by John Gurdon who cloned frogs through the process of somatic nuclear cell transfer (SNCT) into an enucleated frog egg [28]. This showed that the nucleus of a specialized somatic cell could be reverted and develop cells that could eventually produce an entirely new organism [29]. The specialized somatic cell became pluripotent which, before this experiment, was thought to be impossible [30, 31]. This technique was famously used successfully in the cloning of Dolly, the sheep [28]. The 1987 experiment focused on gene expression. The forced expression of one gene, known as myogenic differentiation 1 (Myod1), could cause fibroblasts to turn into myoblasts [32]. This was another example of transforming cells, but this was done through programming the cell in the DNA.

These discoveries provided the turning point in stem cell research by advancing the therapeutic application of stem cells when a Japanese team of scientists showed adult multipotent stem cells could be reverted into a pluripotent state. These cells functioned like ESCs but did not need to be acquired from embryos. This discovery created a process to avoid endangering the life of a fetus to obtain ESCs. The determining factor in the process using murine fibroblasts was incorporating a retrovirus-mediated transduction system containing four transcription factors found in ESCs known as Oct-3/4, Sox2, KLF4, and c-Myc [17]. These factors induced the fibroblasts to become pluripotent. The newly formed reprogrammed stem cells were named induced pluripotent stem cells (iPSCs). A later study succeeded using human cells [33]. This technological breakthrough created a new line of research in stem cell biology that coincided with the generation of iPSC cell lines. Importantly, as mentioned, iPSCs can be made biocompatible with any patient, thus dramatically improving the therapeutic potential of this newly created cellular therapy [13]. ESCs are still the only naturally occurring pluripotent cells, but from these experiments, terminally differentiated cells can be induced into pluripotency to become iPSCs. Still, reprogramming cells comes with risks to cellular development due to histone alteration. However, an experiment was done by sequencing DNA from murine iPSCs and confirmed that although mutations were introduced, reprogramming cells could create iPSCs that were not seriously genetically affected or produce ill-functioning cells [11, 34].

As these cells are manufactured, controlling the quality of iPSC lines is necessary for use as treatments. Ways that they are controlled for their quality are as follows (Table 1) [35]:

Different ways that stem cells can be verified and tested during growth to ensure their quality and viability.

A common source for iPSCs includes fibroblasts. Especially in treatments, taking the patients own fibroblasts for the treatment has shown to be beneficial as the autologous cells do not risk being rejected. However, at first, they were the only source that could be used, and obtaining these cells required a biopsy. Thus, further research was conducted to enhance the techniques efficiency. Other cell types have also been reprogrammed, but fibroblasts are still preferred since their stimulation can be fast and controlled [36, 37].

Stem cells are only potentially useful if they can be differentiated into specific lineages. If not, they can form a teratoma in vivo. However, this condition can be regulated; therefore, if the process can be controlled, it allows clinicians and researchers to improve their therapeutic use when using specific signaling pathways for differentiation. In regenerative medicine, it is important to ensure that these cells will then differentiate in a timely and efficient manner. Directed differentiation exists to push the ESCs to differentiate. As cells develop, they send out signals within their surroundings [38]. Messages from the extracellular environment can also control the differentiation of stem cells which has been shown in in vitro cultures [39]. This can be done easily in in vitro cultures by controlling the conditions in culture. However, replicating such environments in vivo, has been challenging, requiring strict culture conditions [11].

For hESC treatments to be used on patients, the therapies must be culture-free, meaning the stem cells are not contaminated with any feeder or animal cell components [40]. The FDA requires this pertaining to procurement and storage of any type of stem cells contemplated for human use [41]. A difficulty in procuring these treatments is that great amounts of these cells used for treatment must be cultivated in the absence of feeder cells.

Directed differentiation protocols replicate the development of the ICM during embryogenesis. Pluripotent stem cells differentiate into derived progenitors from each of the three germ layers, just as is observed in vivo. Specific molecules act as growth factors to induce stem cells to become specific progenitor cells eventually to develop into a specific cell type. Growth factors function as important regulatory molecules that affect germ layer development in vivo; examples include bone morphogenic proteins (BMP) [42, 43], fibroblast growth factors (FGFs) [44], transcription factors of the Wnt family [45], or transforming growth factors-beta (TGF). How each factor influences germ cell differentiation is unclear and research is ongoing.

The concentration levels and duration of action of a targeted signaling molecule such as a growth factor produces a variety of outcomes. However, the high cost of recombinant molecules currently restricts their routine use in therapy limiting their clinical application. A more promising approach is to focus on using small molecules, thereby activating or deactivating specific signaling pathways [46]. These methods are effective in improving reprogramming efficiency by helping to generate cells that are compatible with the target tissue type. Also, they offer a more cost-effective and non-immunogenic therapy method [47]. Endogenously generated small molecules, e.g., retinoic acid is effective for patterning nervous system development in vivo. It functions effectively in embryonic development where it is used in vitro in culture systems to induce the differentiation of somatic cells [48, 49]. These cells can also induce retinal cell formation when hESCs are used [50]. Through the control of biochemical signals and the environment as important factors can be essential to achieve optimal hESC differentiation when culturing stem cells.

Culture systems have been regulated by multiple agencies around the world including the Food and Drug Administration (FDA) and the European Medicine Agency (EMA). Initially, animal-derived products were utilized, however, that introduced possible animal pathogens. Some stem cell lines derived from embryos and human feeder cell lines have been established which include stem cell-derived cardiac progenitors and mesenchymal stem cells. Xeno-free culture systems also include the development of human foreskin fibroblasts (HFFs) [11, 51, 52, 53].

Stem cells hold immense promise as an important therapeutic option for the future of medicine. Beyond their crucial role in regenerative medicine, stem cell research has demonstrated their intricate processes when involved in growth development. In stem cells, DNA is loosely organized, allowing genes to remain active. Differentiated cells differ in that these cells deactivate certain genes and activate others that are essential to the signals that the cell receives. This process is reversible, demonstrating that pluripotency can be induced through specific gene modifications. Several core transcription factors including Oct3/4, (SRY)-box 2, and Nanog genes have been found to keep these cells pluripotent [17, 54]. Nuclear transcription factors Oct3/4 and Sox2 are crucial for producing iPSCs [54].

Presently, various therapies using stem cells are offered as treatments for conditions like spinal cord injuries, heart failure, retinal and macular degeneration, tendon ruptures, and type 1 diabetes [52, 55, 56, 57, 58]. Stem cell research improves our understanding of stem cell physiology, potentially leading to new treatments for presently untreatable diseases. Many of which are dermatological disorders which were previously thought to have no good solution. This chapter focuses on the application of stem cells treating various dermatological disorders and compliments recent reviews on the same topic [11, 59].

Stem cell therapy has not been actively used as a solution for restoring hair growth, but current results are promising. One study used harvested autologous adipose-derived stromal vascular cells through injected into the scalp of 20 patients with alopecia areata (AA) [60]. At three and six months of follow-up, all patients produced statistically significant hair growth. Adipose-derived stem cell conditioned medium (ADS-CM) contains growth factors essential for hair follicle regrowth such as basic fibroblast growth factor, hepatocyte growth factor, platelet-derived growth factor, vascular endothelial growth factor, and transforming growth factor-beta (TGF-) [61]. Another study isolated human adult stem cells by centrifuging human hair follicles obtained through punch biopsy and injected them into the scalps of 11 androgenetic alopecia (AGA) patients resulting in an increase in hair density and count compared to baseline and placebo [62]. In a larger study with 140 AGA patients, autologous cellular micrografts containing HFSCs were used as a treatment. Within one session, over two-thirds of the patients showed positive results while there was significant increase in their regrowth and thickness [63, 64].

A study randomly assigned 40 patients (20 with AGA and 20 with AA) to receive either autologous bone marrow-derived mononuclear cells or autologous follicular stem cell injections into the scalp, found significant improvement in hair loss with no significant difference between the two preparations [65]. An investigation introduced a novel stem cell method, termed stem cell educator therapy in which patients mononuclear cells are separated from whole blood and allowed to interact with human cord bloodderived multipotent stem cells, thus educating these stem cells after returning them to patients [61]. In nine patients with severe AA, all but one experienced improved hair regrowth of varying degrees. Two patients (one with alopecia totalis and one with patchy AA) experienced complete hair regrowth at 12weeks without relapse after two years. A combination of platelet-rich plasma and stem cell technology also showed promising results [61].

Numerous murine studies have demonstrated the progression of allergies in atopic dermatitis (AD) can be inhibited by using umbilical cord blood mesenchymal stem cells (UCB-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), or adipose-derived mesenchymal stem cells (AD-MSCs) [66, 67, 68, 69]. It is important to consider the type of stem cell used, the number of cells transplanted, the preconditioning of the cell preparation, the therapys relevant targets, and the route and frequency of administration. One example highlighting the complexity of stem cell-based therapy was shown in a study where human UCB-MSCs were pre-treated with mast cell granules [68]. This pre-treatment method enhanced their therapeutic effectiveness, as evidenced by the reduced signs of AD in a NC/Nga mouse model. It was found that hUCB-MSCs primed with mast cell granules were more effective in suppressing the activation of mast cells and B lymphocytes compared to nave MSCs, both in vitro and in vivo [70].

Despite promising results from murine studies in AD, only a few clinical trials have been conducted. In one study, a single subcutaneous administration of hUCB-MSCs was given to 34 adult participants with moderate-to-severe AD [66]. The improvement in AD symptoms was measured using the eczema area and severity index (EASI) score. Treatments for both low and high doses of hUCB-MSCs showed symptom improvement. In the higher dose group, six out of 11 subjects experienced a 50% reduction in EASI score, with no reported side effects. Additionally, typical biomarkers of AD, such as serum IgE levels and the number of eosinophils, decreased after treatment.

A later clinical trial had the injection of clonal mesenchymal stem cells (MSCs) into five patients with atopic dermatitis (AD) who had not responded to conventional treatments [71]. Patients received either one or two cycles of MSC treatment. Effective treatment was evaluated using cytokine biomarkers (CCL-17, CCL-22, IL-13, IL-18, IL-22, and IgE) and EASI scores. Results showed four out of five patients achieved more than a 50% reduction in EASI scores after one treatment cycle. Additionally, significant decreases in IL-13 and IL-22 levels were observed with other biomarkers showing decreasing trends during the studies.

In a more recent phase 1 clinical trial published in 2024, 20 subjects were treated intravenously with human clonal MSCs, given a low dose of cells in Arm 1 and a higher dose in Arm 2. There was an overall improvement for both arms, and the difference in dosage did not make a statistically significant effect. A phase 2 trial proceeded and was randomized, double-blind, and placebo controlled. In this, 72 subjects were tested. The half given the treatment were given the high dosage of hcMSCs originally tested in phase 1. Compared to the placebo group, the treated group had a statistically significant improvement response [72]. These findings suggest MSC administration might help normalize the immune system in AD patients. However, further studies are needed to understand the long-term mechanisms and effects of MSC treatment in this context.

Dermatomyositis remains a mystery with its exact etiology still unknown. Research using stem cells to treat the disease is limited with few studies and case reports available. One report detailed successful autologous stem cell transplants for two patients with juvenile dermatomyositis who had not responded to initial treatments [73]. In the first patient, the procedure involved transferring CD3/CD19-depleted mobilized peripheral blood mononuclear cells (PBMCs), which included 7.5106/kg CD34+ stem cells and 2.9104/kgT cells. Following a 26-month follow-up period, significant improvements were observed. The Childhood Myositis Assessment Scale (CMAS) score increased from 6 to 51and the manual muscle testing (MMT) score rose from 61 to 150. These results demonstrated a substantial improvement in symptoms with the patient regaining the ability to walk and showing significant reductions in inflammatory reactions after the autologous stem cell transplant.

In the second patient, a similar response was observed. The patient was treated with CD3/CD19-depleted autologous PBMC graft (7.51106/kg CD34+; 1.6104/kg CD3+). After three months of treatment, the patient had less muscle pain and contractures, and she began also regained the ability to walk [73].

An uncontrolled study in which 10 patients received allogenic mesenchymal stem cell therapy was reported where one or two MSC infusions were given to patients depending on whether they had disease recurrence within a short time after initial treatment. Out of the 10 patients, eight showed significant clinical improvement, with their symptoms improving after MSC therapy [74]. However, further research is required to evaluate the long-term effects of MSC treatment in patients with dermatomyositis.

Epidermolysis bullosa (EB) is a genetic condition that currently has no treatment, but stem cell therapy is one cell-based therapy under investigation that may be able to correct the skin and its underlying genetic component. Autologous or allogenic stem cells are options that can be used, with mesenchymal stem cell therapy showing potential; therefore, they may be more useful in alleviating some symptoms when tested in additional studies.

One study followed two patients with severe generalized recessive dystrophic epidermolysis bullosa (EB) treated with intradermal administration of allogenic mesenchymal stem cells from bone marrow showed complete healing of ulcers around the treated site by 12weeks [75]. Type VII collagen was detected along the basement membrane zone and the dermal-epidermal junction was continuous in the treated site 1week after treatment. Unfortunately, the clinical effect lasted for only 4months in both patients.

In the case of junctional EB treated with primary cultured keratinocytes, it showed normal morphology and the absence of spontaneous and induced blisters or erosions at 21months of follow-up [76]. Studies using BMSCs to treat recessive dystrophic EB have also shown promise [77, 78]. One study investigated 10 recessive dystrophic EB children treated with intravenous allogeneic bone marrow-derived mesenchymal stem cells and found that the procedure was well tolerated with minimal side effects over the nine-month period [79]. However, skin biopsies performed at the two-month time point showed no increase in type VII collagen and no new anchoring fibrils. While the initial clinical improvement was favorable, it was not maintained over time due to insufficient production of durable proteins like collagen and laminins. The current evidence for stem cell therapy in treating EB is limited because few patients have been treated. This underscores the need for additional research to assess the therapys effectiveness and the balance of its risks and benefits [80].

Despite significant progress in understanding psoriasis pathogenesis in recent years, it remains unclear what is the exact etiology. Current research suggests that dysfunction in certain types of stem cells might be a primary cause of the inflammatory response dysregulation in psoriasis [81]. This hypothesis came after observing long-term remission in psoriasis patients who underwent hematopoietic stem cell therapy [82, 83]. Conversely, there have been reports of acquired psoriasis in patients who received bone marrow transplants from donors with psoriasis, indicating a significant role of hematopoietic stem cells in disease pathogenesis [84, 85]. MSCs have also shown success in treatment likely due to their engraftment, paracrine, or immunomodulatory effects [86]. However, the availability of cost-effective and safe alternatives limits the use of stem cell transplantation as a practical option for treating psoriasis.

Scleromyxedema is a chronic fibro-mucinous disorder that can result in respiratory complications. A study conducted on five patients who underwent high-dose chemotherapy followed by stem cell rescue led to durable remission in most cases, although it did not cure the disease [87]. Another study showed scleromyxedema was successfully treated with chemotherapy and autologous stem cell transplantation [88]. The patient achieved complete recovery within six months and remained in remission for 3years post-transplantation. In a 2022 report, a male patient underwent an autologous hematopoietic stem cell (HSC) transplant after previous therapies failed to improve his symptoms. Improvements were seen in the patients skin, but the renal and pulmonary complications required the use of steroids and plasmapheresis. Unfortunately, the patient contracted SARS-CoV-2 virus and died [89]. More studies still need to be done to determine if stem cell therapy might be useful alone or combined with other therapies to treat scleromyxedema.

Systemic sclerosis (SSc) is an autoimmune disease characterized by excess collagen in the internal organs and skin, causing ulcers and organ damage. HSC therapy and MSC therapy have been tested and found to improve pain, blood flow, lung function, among other symptoms of the disease [90]. Autologous hematopoietic stem cell therapy is preferred over allogeneic therapy due to its lower treatment-related mortality and absence of graft-vs.-host disease [91].

Stem cell therapy has been extensively studied in three randomized controlled trials: the American Scleroderma Stem Cell versus Immune Suppression Trial (ASSIST, phase 2, 19 patients), the Autologous Stem Cell Transplantation International Scleroderma Trial (ASTIS, phase 3, 156 patients), and the Scleroderma Cyclophosphamide or Transplantation study (SCOT, phase 3, 75 patients), with several pilot and case studies [92, 93, 94]. These studies have demonstrated autologous hematopoietic stem cell therapy is an effective and safe treatment for systemic sclerosis. However, patients with severe major organ involvement (pulmonary, cardiac, or renal) or serious comorbidities were excluded from all three trials due to contraindications [59].

MSC therapy has the ability to suppress innate and adaptive immunity and can differentiate into a wide variety of tissues, making it seem like an ideal choice for SSc [95]. However, if donors are not carefully chosen, there is the chance that collagen production can be increased, thus this therapy can worsen symptoms [96]. This research suggests that autologous MSCs from patients that have advanced stage SSc should not be used for treatment. On the other hand, allogenic MSC therapy has lived up closer to the promises of stem cell therapy. Allogenic MSCs were administered intravenously in a female patient, where her skin condition improved, reducing the appearance of ulcers and her pain score [95]. In a clinical trial, combining MSC therapy with plasmapheresis was shown to improve lung function and skin thickness shown in improved modified Rodnan Skin Scores. The current research suggests that MSC therapy may be most effective when paired with another therapeutic option, but research still needs to be done to explore this.

Stem cell therapy has been found to be more effective than conventional immunosuppressive drugs and is currently the only disease-modifying strategy that improves long-term survival, prevents organ deterioration, enhances skin and pulmonary function, and improves overall quality of life.

The European Society for Blood and Marrow Transplantation (ESBMT) and the British Society of Blood and Marrow Transplantation (BSBMT) classify autologous hematopoietic stem cell therapy in severe resistant cases as a clinical option, requiring a risk-benefit assessment [97, 98]. Guidelines from the American Society for Blood and Marrow Transplantation (ASBMT) categorize this therapy as standard of care, rare indication for children (indicating it is an option for individual patients after careful risk-benefit evaluation) and developmental for adults [98]. Patients with acute onset rapidly progressive disease refractory to conventional therapy and mild initial organ damage carry a better prognosis after HSC therapy. Patients with long standing conditions, indolent course and/or irreversible organ damage are contraindications to this therapy [99]. Thus, the challenge is to identify patients who are likely to be benefitted with HSC therapy.

HSC therapy has been tested in patients with refractory systemic lupus erythematosus (SLE). Many observational studies and clinical trials have been aimed at assessing the effectiveness and safety of this transplant approach [100, 101, 102]. In a long-term follow-up of a female patient who underwent allogenic BM-HSC treatment, her systemic lupus erythematosus disease activity index (SLEDAI) score was found to improve, pain improved, and engraftment remained functional [103]. Collectively, these reports show HSCs to be beneficial for patients with a shorter duration of refractory disease suggesting that earlier intervention might lead to better outcomes [104].

The therapeutic potential of MSCs has been investigated for various autoimmune diseases including SLE [105]. In a recent study, six refractory SLE patients were treated with an intravenous infusion of MSCs. Five of the patients reached the threshold for improvement, achieving an SLE Responder Index (SRI) of 4 [106]. In a separate long-term follow-up study done in 2021, 81 patients were treated with allogenic BM-MSC and/or UC-MSCs. After 5years, 37 patients had achieved clinical remission. MSC therapy has been shown to improve patient survival and reduce the severity of the disease as it has been shown to be safe and effective in treatments [107]. MSCs have been shown to alleviate SLE severity, improve renal function, decrease autoantibody production, upregulate peripheral T-cells, and restore balance between Th1- and Th2-related cytokines [108]. These collective immunomodulatory and regenerative properties position MSCs as a promising treatment for SLE.

Steroid topical treatment is the first line of therapy for vitiligo, but when it proves ineffective, surgical options may be viewed next [109, 110]. Cellular grafts using autologous non-cultured outer root sheath hair follicle cell suspension (NCORSHFS) have been tested as a method to treat vitiligo [111]. This method utilizes the regenerative capacity of hair follicle melanocytes, as they can repigment areas where vitiligo has caused depigmentation by allowing melanocyte precursors to proliferate into the areas that lack melanocytes, making them preferable over epidermal melanocytes for cell-based vitiligo treatments. One study reported NCORSHFS achieved an average repigmentation rate of 65.7%, with more than 75% repigmentation observed in nine out of 14 patients [112]. Another study investigated factors affecting therapeutic outcomes in 30 patients with 60 target lesions treated with NCORSHFS [111]. They found that 35% of the lesions achieved repigmentation greater than 75%. The study showed patients who achieved optimal repigmentation had significantly higher numbers of transplanted melanocytes and hair follicle stem cells. Also, the absence of dermal inflammation was a significant predictor of successful repigmentation. These results emphasize the importance of specific cellular components, and a favorable dermal environment is necessary for the effective treatment of vitiligo with NCORSHFS.

Another promising stem cell treatment for vitiligo is multilineage-differentiating stress-enduring (MUSE) cells [113]. In three-dimensional skin culture models, ex vivo studies have identified factors that encourage MUSE cells to differentiate into melanocytes. The melanocytes are integrated into the epidermis, promoting melanogenesis. However, the impact of MUSE cells in vivo remains to be determined [114].

Chronic or non-healing skin wounds present an ongoing challenge in advanced wound care. Current wound healing treatments remain insufficient. Stem cell therapy has emerged as a promising new approach for wound healing using MSCs [115]. MSCs are an attractive cell type for cell-based therapy due to their ease of isolation, vast differentiation potential, and immunomodulatory effects during transplantation. MSCs are known to play a key role in the wound healing process making them an obvious candidate for clinical use. When introduced into the wound bed, MSCs have been shown to promote fibroblast migration, stimulate extracellular matrix (ECM) deposition, facilitate wound closure, initiate re-epithelialization, enhance angiogenesis, and mitigate inflammation in preclinical animal models. MSC efficacy and safety use for the treatment of chronic wounds was further confirmed by several clinical studies involving human subjects which yielded similar positive results with no adverse side effects [116]. However, while MSCs appear to be a promising resource for chronic wound care, additional studies are needed to determine optimal cell source and route of delivery before this treatment can be recommended for clinical use.

MSCs for the treatment of chronic wounds has proven to be feasible, effective, and safe, reported through preclinical and clinical trials [117]. MSCs stimulate the healing process in chronic wounds through several biological and molecular mechanisms. One of the primary roles of MSCs is to promote the directional migration of fibroblast cells to the injury site where they can localize in the wound bed [115, 118]. Once localized fibroblasts facilitate wound closure and synthesize the necessary components of the ECM such as collagen. MSCs can also downregulate MMP-1, a type of collagenase primarily responsible for ECM degradation. MSCs function to preserve ECM and maintain dermal structure. MSC-treated wounds have increased elastin levels which provides recovering tissue with resiliency that is not typically seen in normal wound healing [116]. MSCs play a role in the re-epithelialization process by activating the proliferation, differentiation, and migration of keratinocytes that support the formation of a multi-layered and well-differentiated epidermis [117, 119].

MSCs are believed to stimulate the development of new hair follicles and sweat glands, which suggests these stem cells are capable of not only accelerating wound healing but also improving the quality of wound healing. MSCs use for chronic wounds supports angiogenesis by upregulating VEGF and Ang-1 increasing microvessels throughout the wound bed [120]. This allows the nutrient and oxygen transport to developing cells enhancing their longevity. Also, MSCs help to modulate the wound environment and in turn support proper healing by mitigating inflammation at the site of injury. Importantly, MSCs decrease infiltration of inflammatory cells and pro-inflammatory cytokines and initiate the polarization of M1 macrophages to anti-inflammatory M2 macrophages. MSCs also downregulate ICAM1, a protein involved in inflammation, and upregulate superoxide dismutase, an enzyme which breaks down harmful superoxide radicals [118, 121]. By supporting wound healing MSCs by optimizing the healing environment can produce efficient wound closure.

Several clinical trials in human subjects have generated positive results when MSCs were applied to chronic or non-healing wounds [122]. No adverse side effects have been observed which confirms the safety and feasibility of this cellular therapy for human application. However, further research is needed to determine the best cell source and route of delivery before this procedure can be recommended for human use clinically.

MSCs can be isolated from various tissue types including bone marrow, adipose tissue, cord blood, and placenta. MSCs demonstrate unique properties. Several comparative studies have reported MSCs as the most promise for cell therapy due to their abundance and ease of isolation as well as their regenerative and immunomodulatory properties [123]. How these MSCs are delivered into the wound is the critical question. MSCs can be delivered locally to the wound bed via injection, topical application, or incorporation into a 3D scaffold to avoid issues related to low engraftment efficiency observed following IV injection [124, 125]. Investigating local delivery methods, MSCs seeded into a biomaterial scaffold appears to hold promise as it allows for the localization of the cells into the wound bed and provides donor cells with protection and structure [126, 127]. Following additional research, the application of MSCs for chronic or non-healing wounds could provide a major development in advanced wound care.

Epidermal stem cells have potential to regenerate the epidermis and differentiate under appropriate stimuli into various skin cell types and tissues [128]. This property can be used to initiate and accelerate healing of chronic non-healing wounds. MSCs promote wound healing by decreasing inflammation, promoting angiogenesis, and decreasing scarring [129]. One study successfully applied human MSCs to non-healing and acute wounds using a specialized fibrin spray system [130]. Another study demonstrated the efficacy of stem cell therapy in diabetic foot ulcers [131].

Read more:
Stem Cell Use to Treat Dermatological Disorders - IntechOpen

Abstract

Exosomes are tiny extracellular vesicles secreted by most cell types, which are filled with proteins, lipids, and nucleic acids (non-coding RNAs, mRNA, DNA), can be released by donor cells to subsequently modulate the function of recipient cells. Skin photoaging is the premature aging of the skin structures over time due to repeated exposure to ultraviolet (UV)which is evidenced by dyspigmentation, telangiectasias, roughness, rhytides, elastosis, and precancerous changes. Exosomes are associated with aging-related processes including, oxidative stress, inflammation, and senescence. Anti-aging features of exosomes have been implicated in various in vitro and pre-clinical studies. Stem cell-derived exosomes can restore skin physiological function and regenerate or rejuvenate damaged skin tissue through various mechanisms such as decreased expression of matrix metalloproteinase (MMP), increased collagen and elastin production, and modulation of intracellular signaling pathways as well as, intercellular communication. All these evidences are promising for the therapeutic potential of exosomes in skin photoaging. This review aims to investigate the molecular mechanisms and the effects of exosomes in photoaging.

Keywords: Skin photoaging, UV-induced signaling, Stem cell, Exosome

The harmful effects of ultraviolet (UV) irradiation on the skin, the largest organ in the body, have resulted in an increased demand for sun-damaged skin care products. Photoaging is the premature aging of human skin due to continuousexposure to UV radiationleads to significant alterations including, irregular pigmentation, telangiectasias, roughness, deep wrinkles, dryness, rhytides, elastosis, and precancerous lesions. Moreover, photoaged skin is associated with cellular and extracellular changes. These changes include high epidermal thickness, disorganization of collagen fibers, accumulation of dystrophic elastic fibers, cell genomic instability, as well as diminished viability, and morphological changes of keratinocytes and human dermal fibroblasts, all of which contribute to the pathogenesis of skin photodamage [1, 2].

Exosomes are nano-sized vesicles that serve as a subgroup of vesiclesinvolved in cell-to-cell communication, containing bioactive ingredients such as lipids, proteins, and nucleic acids for cell-to-cell communications. Exosomes can be easily endocytosed and transfer their contents to recipient cells. Exosome therapy as a cell-free therapeutic intervention is correlated with lower risks of tumorigenicity and immunogenicity, reduced potential for uncontrolled cell differentiation and cell proliferation compared to stem cell therapy. Exosomes also show promise as vehiclesfor drug or gene delivery [3]. A large number of studies have demonstrated the therapeutic implications of stem cell-derived exosomes (including those derived from bone marrow mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, adipose-derived stem cells, and pluripotent stem cells) in age-related diseases, tissue regeneration, wound healing, and dermatological conditions [4]. The biological functions of exosomes have mostly been investigated in preclinical studies. For example, exosomal mmu-miR-291a-3p could exert anti- senescence effect in human dermal fibroblasts, through TGF- receptor 2 signaling pathway and promote skin wound healing in aged mice [5]. Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs)-derived exosomes which contain a high concentration of growth factors such as epithelial growth factor (EGF), have been found toincrease collagen production and migration ability of normal fibroblasts. These stem cells-derived exosomes penetrate into the epidermis of skin samples in a time-dependent manner and increase collagen I and elastin while decreasing MMP1 expression [6]. Because of the similarity between the molecular mechanism of aging and photoaging, these findings hold promise for the potential use of exosomes in anti-photoaging-related cosmetics or therapeutics for skin rejuvenation and regeneration.. The cosmetic and therapeutic benefits of exosomes for skin care are mediated through their immunomodulatory function, reduction of oxidative stress, decreasing senescence, and stimulation of extracellular matrix (ECM) components production. The aim of this review is to provide an overview of the molecular mechanism of UV-induced skin aging and to highlight the efficiency of exosomes in skin photoaging.

Photoaging is one of the most common skin defects. In the recent years, many studies have been conducted to understand the underlying mechanisms of skin aging. It has been discovered that a multitude of signaling pathways and molecules are involved in regulating this process [7]. In the subsequent section, we will provide an overview of the current understanding of the mechanisms involved in photoaging.

Many inflammatory pathways activated in response to UV radiation contribute to the generation of reactive oxygen species (ROS) and the degradation of collagen and elastin, which are two proteins responsible for skin elasticity and firmness. Interleukin-1 alpha (IL-1) and interleukin-1 beta (IL-1) are proinflammatory cytokines that are suggested to play a role in the photoaging process. In response to UV radiation, these cytokines are produced and contribute to the inflammation and damage caused by ROS. IL-1 and IL-1 can facilitate the breakdown of collagen and elastin by upregulating the expression of matrix metalloproteinases (MMPs), enzymes responsible for the degradation of these proteins [8]. Similarly,cytokine, like IL-6, can contribute to the breakdown of collagen and elastin by increasing the expression of MMPs. Additionally, IL-6 can promote the formation of senescent cells, which are damaged cells that have stopped dividing and can contribute to the aging process [9]. In addition, Toll-like receptors (TLRs), a type of receptor found in the body's immune system [10] are triggered by UV radiation, resulting in a cascade of inflammatory responses in the skin and finally leading to signs of aging [8]. TLR4 signaling pathway may contribute to the increased amount of IL-6 and IL-8 in the senescent skin cells following UV exposure [11]. UV radiation can induce expression of COX-2, which can lead to inflammation and skin damage in the context of photoaging. UV light-induced MAPK pathway can eventually promote COX-2 production [12, 13]. Other pro-inflammatory cytokines, such as TNF- and IL-1, can also enhance COX-2 synthesis [14]. Moreover, a recent study argued suppression of COX-2 can decrease the UV-induced consequences, underscoring the importance of this protein in photoaging [15].

UV radiation causes the production of ROS in skin cells, leading tooxidative stress. This stress causes damage tocellular components such as lipids, proteins, and DNA, which can lead to cellular dysfunction and ultimately contribute to the signs of photoaging, such as wrinkles, age spots, and loss of skin elasticity. The Nrf2/ARE pathway is a key regulator of the cellular response to oxidative stress, and it plays an important role in protecting skin cells from the damaging effects of UV radiation in photoaging. Under normal conditions, NF-E2-related factor-2 (Nrf2) is sequestered in the cytoplasm by its inhibitor protein, Keap1. However, in response to oxidative stress, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it binds to the antioxidant response element (ARE) in the promoter region of genes that encode antioxidant and detoxification enzymes [1618]. This leads to the activation of these genes and the subsequent synthesis of antioxidant and detoxification enzymes, that help neutralize ROS and prevent oxidative damage [19]. It was shown that upregulation of antioxidant enzymes' expression levels in human skin fibroblasts (HSF) via modulation of the KEAP1-Nrf2/ARE signaling pathway enhances cell antioxidant capacity and reduces UVA-induced ROS and lipid oxidation product malondialdehyde (MDA) [20]. Peroxisomes and peroxisomal enzymes also play a crucial role in regulating the levels of ROS. Investigators indicated the efficiency of catalase and superoxide dismutase in photoaging progression collapses significantly [21].

UV can cause various types of DNA damage, including the formation of pyrimidine dimers (such as thymine dimers), which distort the DNA structure and interfere with normal replication and transcription processes. Moreover, it can lead to the generation of reactive oxygen species and indirectly cause nuclear DNA damage. Base-excision repair is responsible for repairing this type of damage, while UVB radiation directly damages DNA and is repaired through nucleotide excision repair [22]. As individuals age, the efficiency of various DNA repair mechanisms, including NER, BER, double-strand break repair, and mismatch repair, declines [23]. This results in a gradual accumulation of DNA damage over time, particularly in intrinsic aging, which can give rise to aging-related traits. UV exposure can exacerbate this process by causing more DNA damage. Concerning photoaging, prolonged exposure to UV radiation can lead to the accumulation of photoproducts in the skin, surpassing its DNA repair capacity [24]. Moreover, evidence suggeststhat UV-induced telomere mutations, shortening, and telomerase dysfunction might facilitate photoaging and cell death progression [23, 25].

In photoaging, the accumulation of DNA damage can trigger the persistent activation of the p53 pathway, which can contribute to the loss of skin elasticity and the development of wrinkles. Additionally, the ATM/ATR pathway is involved in the response to DNA damage. It activates DNA repair mechanisms and can induce cell cycle arrest to facilitate DNA repair. These pathways can also induce apoptosis if the damage is too severe or if the repair mechanisms are overwhelmed [26, 27]. Poly(ADP-ribose) polymerase-1 (PARP-1) is a well-studied nuclear enzyme that belongs to the PARP superfamily. PARP-1 functions as a sensor for DNA damage.. Upon detecting DNA damage, PARP-1 utilizes NAD+as a substrate to add mono-ADP-ribose or poly(ADP-ribose) (PAR) to various acceptor proteins, including PARP-1 itself. Subsequently, activated PARP-1 can induce DNA repair through a base excision repair [28, 29]. However, high UV exposure can also lead to excessive activation of PARP-1 and therefore lead to depletion of the cellular stores of NAD+and ATP, which can contribute to cell death [30].

One of the important mechanisms implicated in thephotoaging of the skin tissue is programmed cell death or apoptosis. It has been shown that there are a vast number of mechanisms underlying this process during photoaging, and many of them still remain unclear. The cascade begins with the dysregulation of crucial apoptosis-related proteins, including Bax, Bcl-xL, PARP, and caspases. [31]. One study discovered that induced deregulation in apoptotic genes, such as p53, caspase-8 and 3, Bax, and Bcl-2 can interestingly enhance anti-photoaging effects by preventing UVB-induced apoptosis [32]. Furthermore, UV might induce upregulation of MAPK pathway-related genes in the chemokine signaling pathwayresulting in oxidative stress and necrotic cell death [33]. On the other hand, it is shown that UV exposure can directly and indirectly (via induced ROS production) activate the mechanism of neutrophil extracellular traps (NET or netosis) which is an immune programmed cell death pathway in that neutrophils release their DNA and sacrifice themselves. Therefore, UV-induced netosis is suggested as a novel pathway that contributes to photoaging progression [34].

Extracellular matrix (ECM) degradation is one of the main hallmarks of photoaging. Exposure to UV radiation can cause damage to the ECM by inducing the production of MMPs, which are enzymes responsible for breaking down collagen and elastin [35]. The stimulation of the MAPK pathway is the primary regulator of UVR-induced MMP upregulation. In addition, ROS generation is essential for UVR-induced MAPK-mediated signal transduction [36]. The UV-dependent MAPK induction results in MMP-1 overexpression followed by type I collagen (COL-1) degradation [37]. Moreover, another study suggested that inhibition of ERK and p38 protects against UVB-induced photoaging by promoting COL-1 accumulation [38].

In the skin, TGF signaling inhibits keratinocyte development and acts as a profibrotic agent in the dermis. In photoaging, chronic UV exposure triggers the TGF1/SMAD3 signaling pathway and leads to metalloproteinase-induced collagen breakdown and photo inflammation. UV irradiation also induces gene alterations in TGF pathway components such as TGFRI, TGFRII, SMAD2, and SMAD4 [39]. Furthermore, several studies support the idea that increased pro-collagen production through TGF-/Smad pathways, and the expression suppression of MMPs by blocking MAPKs, AP-1, and NF-B pathways could exhibit anti-photoaging effects [4043].

Autophagy is a cellular process that involves the degradation and recycling of damaged or dysfunctional cellular components [44]. In the context of photoaging, studies showed that UV exposure can both induce and inhibit autophagy in a context-dependent manner.. Autophagy plays a complex role, with both protective and harmful effects [45, 46]. On one hand, autophagy can help to remove damaged proteins and organelles and can promote cell survival in response to oxidative stress and DNA damage caused by UV radiation. Autophagy can also help to maintain cellular energy homeostasis, which can be disrupted in response to UV radiation [47]. Specifically, exposure to UVB radiation leads to the direct and rapid activation of three proteins including AMPK, UVRAG, and p53, which in turn activate autophagy [45, 48, 49].

Autophagy can be inhibited by UV radiation and subsequent pro-inflammatory signals such as TNF-, IL-1, and IL-6 [50]. This inhibition of autophagy can contribute to the accumulation of damaged proteins and organelles, leading tocellular dysfunction and development of photoaging [51].

Chronic exposure to UVA irradiation decreases the expression of Bach2 (BTB and CNC homology 1, basic leucine zipper transcription factor 2) in skin fibroblasts,which increasesthe expression of cell senescence-related genes and enhances UVA-induced photoaging. Conversely, overexpression of Bach2 can decrease the expression of cell senescence-related genes. Bach2 plays a critical role in suppressing UVA-induced cell senescence via autophagy by modulating the expression of autophagy-related genes and directly interacting with autophagy-related proteins. The precise molecular mechanism underlying the connection between Bach2 and autophagy remains unknown, and further studies are necessary to elucidate this signaling pathway [52]. Also, another more recent study revealed that autophagy inhibition can result in higher photodamage in fibroblasts. It was shown that colony-stimulating factor 2(CSF2) can enhance autophagy while decreasing the expression level of MMP-1 and MMP-3. The negative correlation between autophagy and mentioned MMPs supports the importance of autophagy in anti-photoaging response. Moreover, the expression of AKT can influence the activation of autophagy, which is overexpressed along with the JAK2/STAT3 pathway and may contribute to several severe UV-induced consequences [46]. Collectively, the impact of autophagy during photoaging depends on its balance with apoptosis induction, while more studies are needed to investigate the impact of autophagy in photoaging.

Heat shock protein 27 (HSP27), a member of heat shock protein family, has been implicated in various cellular processes, including stress response, apoptosis, and cytoskeletal organization [53]. HSP27 has been shown to interact with several proteins involved in the regulation of oxidative stress, apoptosis, and aging, such as Bcl-2, p53, p21, and p16 after UV exposure [54]. Reduction in HSP27 expression has been associated with increased levels of MMP-1 and MMP-3, along with the downregulation of type I collagen [55]. Furthermore, the suppression of HSP27 expression can partially enhance apoptosis through further activation of p65 and caspase-3 [56]. These interactions can modulate the balance between cell survival and death, ECM degradation, and oxidative stress response in response to UV radiation.

Skin-associated adipose tissue, consisting of dermal (DWAT) and subcutaneous (SWAT) adipocytes, is critical in skin photoaging. In particular, DWAT, located in the reticular dermis of the skin, serves as a unique layer of adipocytes that can extend into the upper dermis and create a "fat bridge" between the skin surface and subcutaneous fat, linking the area directly exposed to UV radiation with the deeper fat layer [57, 58]. However, the turnover rate of DWAT adipocytes exceeds that of SWAT, and long-term excessive exposure to UV radiation can lead to DWAT depletion and skin fibrosis due to adipocyte-myofibroblast transition [59, 60]. This transition results in the replacement of fibrosis with DWAT volume, causing an uneven skin structure and the formation of skin folds [61]. UV radiation induces the activation of the TGF- signaling pathway, which contributes to the conversion of adipocytes to myofibroblasts, resulting in the depletion of DWAT [62].

In addition to DWAT, SWAT also plays a crucial role in skin photoaging [63, 64]. Proinflammatory chemokines (IL-6 and IL-8) deregulation and their regulatory pathways (JAK pathway) due to UV-induction can lead to SWAT depletion and thinning of connective tissue, resulting in skin atrophy and wrinkle formation [65]. Moreover, chronic UV radiation inhibits the differentiation of preadipocytes and reduces the accumulation of triglycerides in mature adipocytes due to the decrease in lipid synthesis, including acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), sterol regulatory element binding proteins (SREBPs), and peroxisome proliferator-activated receptors (PPAR) expression [66]. The decrease in both DWAT and SWAT contributes to the overall deterioration of skin structure and function in photoaging.

Exosomes are a subclass of extracellular vesicles with a size less than<150nm in diameter that facilitates intercellular communication [67]. Exosome biogenesis begins with formation of early endosomes through the invagination of plasma membrane which later generates multivesicular bodies (MVBs) containing Intraluminal Vesicles (ILV) (Fig.1). During maturation of early endosomes to late endosomes or MVBs, the cargoes are incorporated into ILVs. ILVs are formed through the (endosomal sorting complex required for transport) ESCRT-regulated mechanism. The ESCRT is a family of proteins consist of ESCRT-0, -I, -II, -III, and Vps4which are essential for vesicle budding, cargo sorting, and the formation of ILVs [68]. Recent evidence showed there is a second mechanism for exosome formationand cargo sorting in an ESCRT-independent manner which involves proteins such as tetraspanin [69]. The MVBs can fuse with the plasma membrane to release ILVs, which are called exosomes, to the extracellular environment. Exosomes include various proteins that participate in the formation and secretion of vesicles (Rab GTPase), proteins, major histocompatibility complex (MHC) proteins (MHC I and MHC II), tetraspanin family, heat shock proteins, and cytoskeleton proteins. Exosomes may carry other cell-specific proteins which their presence depends on pathophysiological conditions [68, 70].

Exosome are small membrane vesicles that are formed by internalization of plasma membrane and formation of early endosomes. The early endosomes transform to late endosomes through maturation, then late endosomes, which termed as multivesicular bodies (MVBs), undergo inward membrane budding intraluminal vesicles (ILVs). MVBs fusion with the plasma membrane leads to release ILVs, or exosomes, into the extracellular space. Exosomes contain various biomolecules depends on the cell type of origin. Lipids, proteins and nucleic acids are the common molecular constituents of the majority of exosomes [67]. Exosomes are also rich in cytokines, growth factor and antioxidant

The release of exosome is regulated by the SNARE proteins, RABs, and other Ras GTPase proteins. Rab GTPases is the member of Ras superfamily of GTPases and is responsible for the formation, membrane fusion, and secretion of vesicles. There are four Rab GTPase proteins including RAB7, RAB11, RAB27, and RAB35,whichare involved in the formation and release of exosome. SNARE proteins mediate the fusion exosome with the plasma membrane or the membrane of organelles [71]. After fusion with plasma membrane, exosomes are released into the extracellular environment and deliver signals to recipient cells through different mechanisms. They can directly merge with the cell membrane and release their contents, interact with cell surface receptors through exosomal surface proteins, or undergoes endocytic uptake [70].

Cells can release exosomes with different sizes, contents, and functional effects on the target cells. At present, different methods are used to separate distinct subpopulations of exosomes. Among them, ultracentrifugation is the most common method that can separate exosomes based on their size and density. Other methods such as polymer precipitation, size-exclusion chromatography, and immunoaffinity are also used to isolate exosomes [72]. The isolated exosomes are then characterizedby analyzing the exosomal markers. Exosomes contain two types of protein. The first group is the common proteins including tetraspanin family (CD9, CD63, CD81), cytoskeletal proteins (actin, tubulin), heat shock proteins (HSP70, HSP90), and the presence of exosome can be confirmed by identification of these proteins. Other specific proteins are varying depending on the cell of origin, for example exosomes derived from malignant tumors contain tumor antigens, which can be used to determine the origin of exosome, related disease and response to the specific treatment [73]. Besides proteins, exosomes contain lipids, mRNA, and other small RNA such as miRNA and other non-coding RNAs. Exosomes have the ability to transfer their genetic contents into the recipient cells and modify different cellular functions. Moreover, they have the potential to be used as diagnostic biomarkers or therapeutic tools for different pathologies [67].

Some studies have indicated that different cells including stem cells and non-stem cells can release exosomes and exert therapeutic effect against photoaging (Table1), which will be discussed in the next sections.

In vitro and in vivo studies have proved the therapeutic potential of exosomes in amelioration ofskinphotoaging

HSF cells, Kunming mice

UVB-irradiated mice

Human umbilical cord mesenchymal stem cells (HucMSCs) are mesenchymal stem cells that are collected from the different parts of the human umbilical cord. These cells possess the ability to self-renew and differentiate into multiple cell types, including osteoblasts, chondrocytes, and adipocytes. HucMSCs exhibit immunomodulatory, anti-inflammatory, and anti-oxidative properties, making them promising candidates for cell therapy and regenerative medicine [83].

Recent studies have investigated the effects of HucMSC-derived exosomes on mitigating the harmful consequences of UV exposure on the skin. Specifically, researchers focused on the role of 143-3, a protein found in HucMSC exosomes, and its interaction with SIRT1. The study demonstrated that HucMSC exosomes containing 143-3 could effectively protect skin cells from UV-induced damage by reducing oxidative stress and inflammation by mediating the SIRT1 pathway [74]. Moreover, these exosomes can enhance the proliferation and migration of HaCaT keratinocytes while inhibiting UVB-induced damage. The findings also show that these exosomes can reduce apoptosis and senescence, increase collagen type I expression, and decrease matrix metalloproteinase (MMP1) expression in photo-aged skin cells [84, 85].

The process of adipocyte development from mesenchymal cells is a multifaceted series of events, both transcriptional and non-transcriptional, that takes place throughout the lifespan of humans. Cells with preadipocyte traits can be derived from adipose tissue in adult individuals and can be grown in vitro. These cells can then be encouraged to differentiate into adipocytes [86].

The role of exosomes derived from adipose tissue-derived stem cells (ADSCs) in preventing photoaging has been extensively studied. Studies indicate that these exosomes effectively inhibit UVB-induced cellular DNA damage through ROS downregulation. Moreover, they can also significantly prevent MMP-1, MMP-3, and COL-3 overexpression and, therefore, protect the ECM integrity. These exosomes may also regulate Nrf2 and MAPK/AP-1 and activate TGF-/Smad pathways upstream of the latter ones [87, 88].

Furthermore, studies have also shown that miR-1246, a highly prevalent nucleic acid in ADSC-derived exosomes, inhibits the MAPK/AP-1 signaling pathway to reduce MMP-1 production and activates the TGF-/Smad pathway, resulting in enhanced pro-collagen type I secretion and an anti-inflammatory impact. In-vivo experiments on Kunming mice demonstrated that miR-1246 might protect against UVB-induced skin photoaging by inhibiting the production of wrinkles, epidermal thickening, and collagen fiber loss. Together, these findings suggest that exosomes derived from ADSCs, particularly miR-1246, play a vital role in the treatment of photoaging by regulating various signaling pathways [75]. Moreover, lncRNA H19, a reach component of ADSC-derived exosomes, shows MMP inhibition and COL-1 production effect on UVB-irradiated mice. It can also sponge miR-138 to target SIRT1, therefore mediating SIRT1 expression and its anti-photoaging impact [76].

Bone marrow mesenchymal stem cells (BM-MSCs) are a type of adult stem cells that have great therapeutic potential in regenerative medicine. Exosomes secreted by BM-MSCs have emerged as a crucial component of their paracrine signaling mechanisms. BM-MSC-derived exosomes contain a variety of bioactive molecules, such as growth factors, cytokines, and miRNAs, that can promote tissue repair and regeneration in various injury and disease models [89].

It is shown that BM-MSCs can mitigate UV-induced oxidative stress and inflammation in a dose-dependent manner and increase cell viability in human dermal fibroblasts (HDFs). BMSCs-exosomes also reduced the expression of MMP-1 and MMP-3 while promoting the expression of COL-1 by reversing MAPK/AP-1 pathway [90]. Moreover, miR-29b-3p, which is found in BM-MSCs-derived exosomes, can participate in reversion of UVB-induced HDF migration suppression, oxidative stress increase, and apoptosis promotion. It is suggested that mentioned miRNA can target MMP-2 and thus prevent COL-1 degradation [77].

Induced pluripotent stem cells (iPSCs) are a type of stem cells that are generated by reprogramming adult cells, such as skin cells, to an embryonic-like state. iPSCs have the ability to differentiate into virtually any cell type in the body and have significant potential for regenerative medicine and drug discovery. iPSCs were first successfully created in 2006 by reprogramming human skin cells using a combination of four transcription factors, including Oct4, Sox2, Klf4, and c-Myc. This discovery was a significant breakthrough in the field of stem cell research and has led to a greater understanding of cellular reprogramming and its potential applications in the future [9193].

It was observed that exosomes derived from human iPSCs (iPSCs-Exo) promoted the proliferation and migration of HDFs under normal conditions. Upon UVB irradiation, HDFs were damaged and overexpressed matrix-degrading enzymes (MMP-1/3), but pretreatment with iPSCs-Exo inhibited these damages. iPSCs-Exo also increased the expression of collagen type I in photo-aged HDFs. Furthermore, iPSCs-Exo significantly reduced the expression of SA--Gal and MMP-1/3 and restored the expression of COL-1 senescent HDFs [78]. SA--Gal is known to be a switch that shifts cells toward senescence fate and is known as an aging marker [94]. Therefore, these results suggest that iPSCs-Exo may have therapeutic potential in the treatment of skin aging.

Human dermal fibroblasts (HDFs) are the main cells in skin derived from MSCs, which play a critical role in extracellular matrix (ECM) remodeling and providing integrity and elasticity to the skin. In the process of skin aging, HDFs proliferation is declined, with decreased collagen production and increased MMPs, resulting in the degradation of the ECM. All of these processes lead to loss of integrity and elasticity and the formation of wrinkles.

Exosomes secreted by human dermal fibroblast cell UVB-irradiated human dermal fibroblasts (UVB-HDFs) are associated with skin photoaging. The analysis of miRNA expression profiling showed the number of dysregulated miRNAs in extracellular vesicles (EVs) derived from UVB-irradiated HDF. Upon UVB-irradiation, expression of miRNA-22-5p was significantly increased in HDF cells and their derived EVs, and can be transferred to other HDFs cells. further analysis showed that miRNA-22-5p upregulation promotes photoaging by targeting growth differentiation factor 11 (GDF11), a protein that protects HDF cells from photoaging [79]. In another study, exosomes derived from three-dimensional (3D) aggregation of HDF cells or spheroid induced collagen synthesis and reduced inflammation in a photoaged skin of mice model. It was hypothesis that miR-133a and miR-223 were upregulated and miR-196a was downregulated in the exosome derived from 3D cultured HDF spheroids, which might inhibit MMP expression, enhance collagen restoring and replacing and activate TGF- signal pathway. Thus 3D HDF-XOs can be used as an effective approach to prevent skin photoaging [80].

Human umbilical vein endothelial cell(HUVEC) is a model cell line to study endothelial cells and can be derived from umbilical cords. Recently, Ellistasari et al. have conducted an in vitro study to investigate the effect of exosomes derived from HUVEC cells in attenuating skin photoaging. They observed that Exo-HUVEC can markedly increase cell proliferation and collagen synthesis in UVB-irradiated fibroblasts, Moreover, Exo-HUVEC can decrease MMP expression which leads to inhibiting collagen degradation in the photoaged cell line model. This source of exosome has the potential efficiency to prevent and treat skin photoaging [81]. Exosome sources are not limited to animal cells. Interestingly, natural exosomes, that originate from plants or other organisms, contain more bioactive molecules than those derived from animal cells. In the study by Han et al. exosome-like nanovesicles derived from a medicinal mushroom, Phellinus linteus (PL), has been shown to have anti-aging and anticancer effects. The fungi exosome-like nanovesicles (FELNVs) can protect skin from UV-induced photoaging. It was shown that fungal EVs are enriched with different miRNAs including miR-CM1-5, and among them miR-CM1 could protect HaCaT cells from UV-induced damage. MiR-CM1 exerts a protective effect through reduction of aging-related markers such as SA--Gal, ROS level, MMP1, and COL1A2 expression. Mical2 was known as a direct target of miR-CM1 which is involved in the regulation of age-related processes [82].

In recent years, exosomes have been exploited as a novel candidate for treatment of many diseases including central nervous system disorders, cardiovascular diseases, and cancer. Under the pathophysiological condition, biological components of exosomes are changed, reflecting the alteration in the cell functions. The alteration in the exosomal components can be served as diagnostic and prognostic biomarkers in many diseases from cancer to aging [95]. Exosomes can be extracted from cell culture, tissues, and biological fluids including plasma, serum, urine, etc. [96]. Exosomes can act locally or transported to distant tissues via body fluids and modulate the function of target cells [97].

Mesenchymal stem cells are multipotent stem cells that that possess a the high ability to release exosome and can be extracted from bone marrow, umbilical cord, and adipose tissue [98]. Exosome therapy as a cell-free strategy offers severaladvantages of small size, no risk of tumorigenicity, and long-term storage making it a potentially safer and more effective alternative to stem cell therapy [3]. Also, exosomes show great promises as the drug delivery carrier due to high stability, biocompatibility, and low immunogenicity compared to virus-based delivery and other non-viral methods. However, there are still some challenges for the application of exosomes in clinics such as low yield of isolation [72].

Preclinical investigations showed that exosomes may have a therapeutic role in aging and other age-related diseases [99]. Cellular aging is due to various biological changes including, epigenetic alteration, genomic instability, senescence, oxidative stress, mitochondrial decline, and dysregulation of intracellular communication [100]. Some studies have demonstrated the therapeutic potential of exosome in preclinical models of age-related diseases such as Alzheimers, Type 2 diabetes (T2DM), osteoarthritis, chronic kidney disease, etc. [99].

Exosomes have many beneficial effects for skin care as they contain various biological molecules that can help to promote skin repair and regeneration [101]. Previous studies have demonstrated that exosomes and other EVs have therapeutic benefits in skin defects such as wound and aging. Most of these studies on the potential use of exosomes in skin repair have been conducted in animal models. For example, it was found that bioengineered exosomes loaded with miRNA-542-3p, derived from bone marrow MSCs (BMMSCs), could promote cell proliferation, collagen synthesis, and wound closure in mice models. Currently, the clinical applicability of exosome-based therapy is limited to skin wound repair [102]. To date, there is no clinical trial has been conducted on exosome in photoaging.

Exosomes are able to deliver various bioactive compounds into the skin cells, which can effectively delay skin aging and inhibit photoaging signatures. These nanovesicles would be artificially engineered with desired biological molecules [4, 103]. Exosomes can be delivered to skin through various invasive and non-invasive methods. In the non-invasive treatment exosomes are incorporated into topical creams, serums, oils, and masks to cover and protect skin [104]. Exosomes can also be incorporated into bioactive polymeric materials like hydrogel, allowing for sustained release, pH maintenance, and enhanced regenerative potential [105]. Local injection is the invasive type of treatment in which anti-aging molecules are injected into the inner layer of skin to enhance therapeutic effects and overcome skin barrier. Subdermal injection of ADSCs has been demonstrated to be effective in reducing anti-photogaing effects through ECM remodeling and neoelastogenesis (Fig.2) [106]. Since MSCs-derived exosomes represent biological activity corresponding to these stem cells, similar and even more effective therapeutic outcome is expected in exosome-based therapeutic protocols. Local injection provides more effective skin treatment compared to topical products due to skipping skin barrier [104]. The stability of exosomes is critical both before and after injection. Exosome lyophilization is often used to increase stability and maintain the activity of biological molecules. This method involves in dehydration and drying of exosome under vacuum condition at low temperature, resulting in their longer storage without loss of activity [107]. Systemic treatment is another method previously used to deliver exosomes through intravenous injection. It has been shown that topical application of exosome combined with intravenous injection effectively accelerates non-diabetic wound healing [108]. Exosomes stimulate collagen production in photoaged skin and reduce the appearance of pigmentation [4]. Moreover,photoaging is associated with a greater risk of malignant tumors like melanoma [109]. Thus, treatment of skin photoaging has important clinical significance and exosome-based therapy could be a helpful method not only in cosmetic application but also in skin cancer prevention.

Exosomes derived from different types of stem cells can play an important role in reducing photoaging by entering the target cells and transferring their contents. UV radiation induce generation of reactive oxygen species (ROS), leading to DNA damage, activation of inflammatory pathway, production of matrix metalloproteinases (MMPs) and degradation of collagen fibers. Skin photoaging is characterized by structural change, appearance of wrinkles and pigmentation (Reviewed in [7]). Exosomes derived from stem cells can be served as novel treatment option for skin repair and regeneration. Administering exosomes in the form of lyophilized injection may be one of the effective approaches to repair photo-damaged skin

Photoaging is a prominent manifestation of skin aging characterized by the appearance of mottled pigmentation, fine lines, and wrinkles. The main molecular mechanisms of photoaging are accumulation of reactive oxygen species, cellular senescence, inflammation, and collagen degradation. Targeting these pathways through novel therapeutics is an intriguing area of study in regenerative medicine. Exosomes are able to regulate multiple cellular processes due to their important role in cellular communication. In the last years, exosomes have emerged as a novel therapeutic option for treatment of many diseases. This review aims to summarize the current findings on the roles of exosomes, particularly those derived from stem cells, in the context of skin photoaging. While most studies investigating the use of exosomes in treating skin defects have been conducted at the preclinical level, additional research is needed to evaluate the therapeutic potentials and clinical values of exosomes in the field of skin treatment medicine.

None.

Reactive oxygen species

Interleukin

Matrix metalloproteinase

Toll-like receptor

Mitogen-activated protein kinase

Cyclooxygenase-2

Tumor necrosis factor-alpha

Nuclear factor kappa B

IB kinase

Inhibitory kappa B

NF-E2-related factor-2

Antioxidant response element

Deacetylase silent information regulator 1

PAR coactivator-1

Human skin fibroblast

Malondialdehyde

Poly(ADP-ribose) polymerase-1

Neutrophil extracellular traps

Extracellular signal-regulated kinase

C-Jun amino-terminal kinase

Collagen

AMP-activated protein kinase

UV radiation resistance-associated gene

Tuberous sclerosis complex

BTB and CNC homology 1, basic leucine zipper transcription factor 2

Broad complex, tramtrack, bric-a-brac/poxvirus, and zinc finger

Heat shock protein

Dermal white adipose tissue

Subcutaneous white adipose tissue

Acetyl-CoA carboxylase

Fatty acid synthase

Stearoyl-CoA desaturase

Sterol regulatory element binding proteins

Peroxisome proliferator-activated receptor

A.HN., N.M. and M.HS. wrote the manuscript. MH.S. conceived the original idea and drafted the manuscript. All listed authors read and approved the final manuscript.

None.

Not applicable.

The authors declare no competing interests.

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Exosomes in skin photoaging: biological functions and therapeutic ...

Abstract

Induced pluripotent stem (iPS) cells, are a type of pluripotent stem cell derived from adult somatic cells. They have been reprogrammed through inducing genes and factors to be pluripotent. iPS cells are similar to embryonic stem (ES) cells in many aspects. This review summarizes the recent progresses in iPS cell reprogramming and iPS cell based therapy, and describe patient specific iPS cells as a disease model at length in the light of the literature. This review also analyzes and discusses the problems and considerations of iPS cell therapy in the clinical perspective for the treatment of disease.

Keywords: Cellular therapy, disease model, embryonic stem cells, induced pluripotent stem cells, reprogramm.

Induced pluripotent stem (iPS) cells, are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed to an embryonic stem (ES) cell-like state through the forced expression of genes and factors important for maintaining the defining properties of ES cells.

Mouse iPS cells from mouse fibroblasts were first reported in 2006 by the Yamanaka lab at Kyoto University [1]. Human iPS cells were first independently produced by Yamanakas and Thomsons groups from human fibroblasts in late 2007 [2, 3]. iPS cells are similar to ES cells in many aspects, including the expression of ES cell markers, chromatin methylation patterns, embryoid body formation, teratoma formation, viable chimera formation, pluripotency and the ability to contribute to many different tissues in vitro.

The breakthrough discovery of iPS cells allow researchers to obtain pluripotent stem cells without the controversial use of embryos, providing a novel and powerful method to "de-differentiate" cells whose developmental fates had been traditionally assumed to be determined. Furthermore, tissues derived from iPS cells will be a nearly identical match to the cell donor, which is an important factor in research of disease modeling and drug screening. It is expected that iPS cells will help researchers learn how to reprogram cells to repair damaged tissues in the human body.

The purpose of this paper is to summarize the recent progresses in iPS cell development and iPS cell-based therapy, and describe patient specific iPS cells as a disease model, analyze the problems and considerations of iPS therapy in the clinical treatment of disease.

The methods of reprogramming somatic cells into iPS cells are summarized in Table 1. It was first demonstrated that genomic integration and high expression of four factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28 by virus, can reprogram fibroblast cells into iPS cells [1-3]. Later, it was shown that iPS cells can be generated from fibroblasts by viral integration of Oct4/Sox2/Klf4 without c-Myc [4]. Although these iPS cells showed reduced tumorigenicity in chimeras and progeny mice, the reprogramming process is much slower, and efficiency is substantially reduced. These studies suggest that the ectopic expression of these three transcription factors (Oct4/Klf4/Sox2) is required for reprogramming of somatic cells in iPS cells.

Various growth factors and chemical compounds have recently been found to improve the induction efficiency of iPS cells. Shi et al., [5] demonstrated that small molecules, able to compensate for Sox2, could successfully reprogram mouse embryonic fibroblasts (MEF) into iPS cells. They combined Oct4/Klf4 transduction with BIX-01294 and BayK8644s and derived MEF into iPS cells. Huangfu et al., [6, 7] reported that 5-azacytidine, DNA methyltransferase inhibitor, and valproic acid, a histone deacetylase inhibitor, improved reprogramming of MEF by more than 100 folds. Valproic acid enables efficient reprogramming of primary human fibroblasts with only Oct4 and Sox2.

Kim et al. showed that mouse neural stem cells, expressing high endogenous levels of Sox2, can be reprogrammed into iPS cells by transduction Oct4 together with either Klf4 or c-Myc [19]. This suggests that endogenous expression of transcription factors, that maintaining stemness, have a role in the reprogramming process of pluripotency. More recently, Tsai et al., [20] demonstrated that mouse iPS cells could be generated from the skin hair follicle papilla (DP) cell with Oct4 alone since the skin hair follicle papilla cells expressed endogenously three of the four reprogramming factors: Sox2, c-Myc, and Klf4. They showed that reprogramming could be achieved after 3 weeks with efficiency similar to other cell types reprogrammed with four factors, comparable to ES cells.

Retroviruses are being extensively used to reprogram somatic cells into iPS cells. They are effective for integrating exogenous genes into the genome of somatic cells to produce both mouse and human iPS cells. However, retroviral vectors may have significant risks that could limit their use in patients. Permanent genetic alterations, due to multiple retroviral insertions, may cause retrovirus-mediated gene therapy as seen in treatment of severe combined immunodeficiency [25]. Second, although retroviral vectors are silenced during reprogramming [26], this silencing may not be permanent, and reactivation of transgenes may occur upon the differentiation of iPS cells. Third, expression of exogenous reprogramming factors could occur. This may trigger the expression of oncogenes that stimulate cancer growth and alter the properties of the cells. Fourth, the c-Myc over-expression may cause tumor development after transplantation of iPS derived cells. Okita et al. [10] reported that the chimeras and progeny derived from iPS cells frequently showed tumor formation. They found that the retroviral expression of c-Myc was reactivated in these tumors. Therefore, it would be desirable to produce iPS cells with minimal, or free of, genomic integration. Several new strategies have been recently developed to address this issue (Table 1).

Stadtfeld et al. [16] used an adenoviral vector to transduce mouse fibroblasts and hepatocytes, and generated mouse iPS cells at an efficiency of about 0.0005%. Fusaki et al. [22] used Sendai virus to efficiently generate iPS cells from human skin fibroblasts without genome integration. Okita et al. [27] repeatedly transfected MEF with two plasmids, one carrying the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other carrying the c-Myc cDNA. This generated iPS cells without evidence of plasmid integration. Using a polycistronic plasmid co-expressing Oct4, Sox2, Klf4, and c-Myc, Gonzalez et al., [28] reprogrammed MEF into iPS cells without genomic integration. Yu et al. [29] demonstrated that oriP/EBNA1 (EpsteinBarr nuclear antigen-1)-based episomal vectors could be used to generate human iPS cells free of exogenous gene integration. The reprogramming efficiency was about 36 colonies/1 million somatic cells. Narsinh et al., [21] derived human iPS cells via transfection of human adipocyte stromal cells with a nonviral minicircle DNA by repeated transfection. This produced hiPS cells colonies from an adipose tissue sample in about 4 weeks.

When iPS cells generated from either plasmid transfection or episomes were carefully analyzed to identify random vector integration, it was possible to have vector fragments integrated somewhere. Thus, reprogramming strategies entirely free of DNA-based vectors are being sought. In April 2009, it was shown that iPS cells could be generated using recombinant cell-penetrating reprogramming proteins [30]. Zhou et al. [30] purified Oct4, Sox2, Klf4 and c-Myc proteins, and incorporated poly-arginine peptide tags. It allows the penetration of the recombinant reprogramming proteins through the plasma membrane of MEF. Three iPS cell clones were successfully generated from 5x 104 MEFs after four rounds of protein supplementation and subsequent culture of 2328 days in the presence of valproic acid.

A similar approach has also been demonstrated to be able to generate human iPS cells from neonatal fibroblasts [31]. Kim et al. over-expressed reprogramming factor proteins in HEK293 cells. Whole cell proteins of the transduced HEK293 were extracted and used to culture fibroblast six times within the first week. After eight weeks, five cell lines had been established at a yield of 0.001%, which is one-tenth of viral reprogramming efficiency. Strikingly, Warren et al., [24] demonstrated that human iPS cells can be derived using synthetic mRNA expressing Oct3/4, Klf4, Sox2 and c-Myc. This method efficiently reprogrammed fibroblast into iPS cells without genome integration.

Strenuous efforts are being made to improve the reprogramming efficiency and to establish iPS cells with either substantially fewer or no genetic alterations. Besides reprogramming vectors and factors, the reprogramming efficiency is also affected by the origin of iPS cells.

A number of somatic cells have been successfully reprogrammed into iPS cells (Table 2). Besides mouse and human somatic cells, iPS cells from other species have been successfully generated (Table 3).

The origin of iPS cells has an impact on choice of reprogramming factors, reprogramming and differentiation efficiencies. The endogenous expression of transcription factors may facilitate the reprogramming procedure [19]. Mouse neural stem cells express higher endogenous levels of Sox2 and c-Myc than ES cells. Thus, two transcription factors, exogenous Oct4 together with either Klf4 or c-Myc, are sufficient to generate iPS cells from neural stem cells [19]. Ahmed et al. [14] demonstrated that mouse skeletal myoblasts endogenously expressed Sox2, Klf4, and c-Myc and can be easily reprogrammed to iPS cells.

It is possible that iPS cells may demonstrate memory of parental source and therefore have low differentiation efficiency into other tissue cells. Kim et al. [32] showed that iPS cells reprogrammed from peripheral blood cells could efficiently differentiate into the hematopoietic lineage cells. It was found, however, that these cells showed very low differentiation efficiency into neural cells. Similarly, Bar-Nur et al. found that human cell-derived iPS cells have the epigenetic memory and may differentiate more readily into insulin producing cells [33]. iPS cells from different origins show similar gene expression patterns in the undifferentiated state. Therefore, the memory could be epigenetic and are not directly related to the pluripotent status.

The cell source of iPS cells can also affect the safety of the established iPS cells. Miura et al. [54] compared the safety of neural differentiation of mouse iPS cells derived from various tissues including MEFs, tail-tip fibroblasts, hepatocyte and stomach. Tumorigenicity was examined. iPS cells that reprogrammed from tail-tip fibroblasts showed many undifferentiated pluripotent cells after three weeks of in vitro differentiation into the neural sphere. These cells developed teratoma after transplantation into an immune-deficient mouse brain. The possible mechanism of this phenomenon may be attributable to epigenetic memory and/or genomic stability. Pre-evaluated, non-tumorigenic and safe mouse iPS cells have been reported by Tsuji et al. [55]. Safe iPS cells were transplanted into non-obese diabetic/severe combined immunodeficiency mouse brain, and found to produce electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro.

The cell source of iPS cells is important for patients as well. It is important to carefully evaluate clinically available sources. Human iPS cells have been successfully generated from adipocyte derived stem cells [35], amniocytes [36], peripheral blood [38], cord blood [39], dental pulp cells [40], oral mucosa [41], and skin fibroblasts (Table 2). The properties and safety of these iPS cells should be carefully examined before they can be used for treatment.

Shimada et al. [17] demonstrated that combination of chemical inhibitors including A83-01, CHIR99021, PD0325901, sodium butyrate, and Y-27632 under conditions of physiological hypoxia human iPS cells can be rapidly generated from adipocyte stem cells via retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Miyoshi et al., [42] generated human iPS cells from cells isolated from oral mucosa via the retroviral gene transfer of Oct4, Sox2, c-Myc, and Klf4. Reprogrammed cells showed ES-like morphology and expressed undifferentiated markers. Yan et al., [40] demonstrated that dental tissue-derived mesenchymal-like stem cells can easily be reprogrammed into iPS cells at relatively higher rates as compared to human fibroblasts. Human peripheral blood cells have also been successfully reprogrammed into iPS cells [38]. Anchan et al. [36] described a system that can efficiently derive iPS cells from human amniocytes, while maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are autologous to a single donor. Takenaka et al. [39] derived human iPS cells from cord blood. They demonstrated that repression of p53 expression increased the reprogramming efficiency by 100-fold.

All of the human iPS cells described here are indistinguishable from human ES cells with respect to morphology, expression of cell surface antigens and pluripotency-associated transcription factors, DNA methylation status at pluripotent cell-specific genes and the capacity to differentiate in vitro and in teratomas. The ability to reprogram cells from human somatic cells or blood will allow investigating the mechanisms of the specific human diseases.

The iPS cell technology provides an opportunity to generate cells with characteristics of ES cells, including pluripotency and potentially unlimited self-renewal. Studies have reported a directed differentiation of iPS cells into a variety of functional cell types in vitro, and cell therapy effects of implanted iPS cells have been demonstrated in several animal models of disease.

A few studies have demonstrated the regenerative potential of iPS cells for three cardiac cells: cardiomyocytes, endothelial cells, and smooth muscle cells in vitro and in vivo. Mauritz [56] and Zhang [57] independently demonstrated the ability of mouse and human iPS cells to differentiate into functional cardiomyocytes in vitro through embryonic body formation. Rufaihah [58], et al. derived endothelial cells from human iPS cells, and showed that transplantation of these endothelial cells resulted in increased capillary density in a mouse model of peripheral arterial disease. Nelson et al. [59] demonstrated for the first time the efficacy of iPS cells to treat acute myocardial infarction. They showed that iPS cells derived from MEF could restore post-ischemic contractile performance, ventricular wall thickness, and electrical stability while achieving in situ regeneration of cardiac, smooth muscle, and endothelial tissue. Ahmed et al. [14] demonstrated that beating cardiomyocyte-like cells can be differentiated from iPS cells in vitro. The beating cells expressed early and late cardiac-specific markers. In vivo studies showed extensive survival of iPS and iPS-derived cardiomyocytes in mouse hearts after transplantation in a mouse experimental model of acute myocardial infarction. The iPs derived cardiomyocyte transplantation attenuated infarct size and improved cardiac function without tumorgenesis, while tumors were observed in the direct iPS cell transplantation animals.

Strategies to enhance the purity of iPS derived cardiomyocytes and to exclude the presence of undifferentiated iPS are required. Implantation of pre-differentiation or guided differentiation of iPS would be a safer and more effective approach for transplantation. Selection of cardiomyocytes from iPS cells, based on signal-regulatory protein alpha (SIRPA) or combined with vascular cell adhesion protein-1 (VCAM-1), has been reported. Dubois et al. [60] first demonstrated that SIRPA was a marker specifically expressed on cardiomyocytes derived from human ES cells and human iPS cells. Cell sorting with an antibody against SIRPA could enrich cardiac precursors and cardiomyocytes up to 98% troponin T+ cells from human ESC or iPS cell differentiation cultures. Elliott et al. [61] adopted a cardiac-specific reporter gene system (NKX2-5eGFP/w) and identified that VCAM-1 and SIRPA were cell-surface markers of cardiac lineage during differentiation of human ES cells.

Regeneration of functional cells from human stem cells represents the most promising approach for treatment of type 1 diabetes mellitus (T1DM). This may also benefit the patients with type 2 diabetes mellitus (T2DM) who need exogenous insulin. At present, technology for reprogramming human somatic cell into iPS cells brings a remarkable breakthrough in the generation of insulin-producing cells.

Human ES cells can be directed to become fully developed cells and it is expected that iPS cells could also be similarly differentiated. Stem cell based approaches could also be used for modulation of the immune system in T1DM, or to address the problems of obesity and insulin resistance in T2DM.

Tateishi et al., [62] demonstrated that insulin-producing islet-like clusters (ILCs) can be generated from the human iPS cells under feeder-free conditions. The iPS cell derived ILCs not only contain C-peptide positive and glucagon-positive cells but also release C-peptide upon glucose stimulation. Similarly, Zhang et al., [63] reported a highly efficient approach to induce human ES and iPS cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. These cells produce insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these cells co-expressed mature cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet beta cells in vivo.

Alipo et al. [64] used mouse skin derived iPS cells for differentiation into -like cells that were similar to the endogenous insulin-secreting cells in mice. These -like cells were able to secrete insulin in response to glucose and to correct a hyperglycemic phenotype in mouse models of both T1DM and T2DM after iPS cell transplant. A long-term correction of hyperglycemia could be achieved as determined by hemoglobin A1c levels. These results are encouraging and suggest that induced pluripotency is a viable alternative to directing iPS cell differentiation into insulin secreting cells, which has great potential clinical applications in the treatment of T1DM and T2 DM.

Although significant progress has been made in differentiating pluripotent stem cells to -cells, several hurdles remain to be overcome. It is noted in several studies that the general efficiency of in vitro iPS cell differentiation into functional insulin-producing -like cells is low. Thus, it is highly essential to develop a safe, efficient, and easily scalable differentiation protocol before its clinical application. In addition, it is also important that insulin-producing b-like cells generated from the differentiation of iPS cells have an identical phenotype resembling that of adult human pancreatic cells in vivo.

Currently, the methodology of neural differentiation has been well established in human ES cells and shown that these methods can also be applied to iPS cells. Chambers et al. [65] demonstrated that the synergistic action of Noggin and SB431542 is sufficient to induce rapid and complete neural conversion of human ES and iPS cells under adherent culture conditions. Swistowsk et al. [66] used a completely defined (xenofree) system, that has efficiently differentiated human ES cells into dopaminergic neurons, to differentiate iPS cells. They showed that the process of differentiation into committed neural stem cells (NSCs) and subsequently into dopaminergic neurons was similar to human ES cells. Importantly, iPS cell derived dopaminergic neurons were functional as they survived and improved behavioral deficits in 6-hydroxydopamine-leasioned rats after transplantation. Lee et al. [67] provided detailed protocols for the step-wise differentiation of human iPS and human ES into neuroectodermal and neural crest cells using either the MS5 co-culture system or a defined culture system (Noggin with a small-molecule SB431542), NSB system. The average time required for generating purified human NSC precursors will be 25 weeks. The success of deriving neurons from human iPS cells provides a study model of normal development and impact of genetic disease during neural crest development.

Wernig et al., [68] showed that iPS cells can give rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease (PD) upon transplantation into the adult brain. This study highlights the therapeutic potential of directly reprogrammed fibroblasts for neural cell replacement in the animal model of Parkinsons disease.

Tsuji et al., [55] used pre-evaluated iPS cells derived for treatment of spinal cord injury. These cells differentiated into all three neural lineages, participated in remyelination and induced the axonal regrowth of host 5HT+ serotonergic fibers, promoting locomotor function recovery without forming teratomas or other tumors. This study suggests that iPS derived neural stem/progenitor cells may be a promising cell source for treatment of spinal cord injury.

Hargus et al., [69] demonstrated proof of principle of survival and functional effects of neurons derived from iPS cells reprogrammed from patients with PD. iPS cells from patients with Parkinsons disease were differentiated into dopaminergic neurons that could be transplanted without signs of neuro-degeneration into the adult rodent striatum. These cells survived and showed arborization, and mediated functional effects in an animal model of Parkinsons disease. This study suggests that disease specific iPS cells can be generated from patients with PD, which be used to study the PD development and in vitro drug screen for treatment of PD.

Reprogramming technology is being applied to derive patient specific iPS cell lines, which carry the identical genetic information as their patient donor cells. This is particularly interesting to understand the underlying disease mechanism and provide a cellular and molecular platform for developing novel treatment strategy.

Human iPS cells derived from somatic cells, containing the genotype responsible for the human disease, hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. The differentiated cells from reprogrammed patient specific human iPS cells retain disease-related phenotypes to be an in vitro model of pathogenesis (Table 4). This provides an innovative way to explore the molecular mechanisms of diseases.

Disease Modeling Using Human iPS Cells

Recent studies have reported the derivation and differentiation of disease-specific human iPS cells, including autosomal recessive disease (spinal muscular atrophy) [70], cardiac disease [71-75], blood disorders [13, 76], diabetes [77], neurodegenerative diseases (amyotrophic lateral sclerosis [78], Huntingtons disease [79]), and autonomic nervous system disorder (Familial Dysautonomia) [80]. Patient-specific cells make patient-specific disease modeling possible wherein the initiation and progression of this poorly understood disease can be studied.

Human iPS cells have been reprogrammed from spinal muscular atrophy, an autosomal recessive disease. Ebert et al., [70] generated iPS cells from skin fibroblast taken from a patient with spinal muscular atrophy. These cells expanded robustly in culture, maintained the disease genotype and generated motor neurons that showed selective deficits compared to those derived from the patients' unaffected relative. This is the first study to show that human iPS cells can be used to model the specific pathology seen in a genetically inherited disease. Thus, it represents a promising resource to study disease mechanisms, screen new drug compounds and develop new therapies.

Similarly, three other groups reported their findings on the use of iPS cells derived cardiomyocytes (iPSCMs) as disease models for LQTS type-2 (LQTS2). Itzhaki et al., [72] obtained dermal fibroblasts from a patient with LQTS2 harboring the KCNH2 gene mutation and showed that action potential duration was prolonged and repolarization velocity reduced in LQTS2 iPS-CMs compared with normal cardiomyocytes. They showed that Ikr was significantly reduced in iPS-CMs derived from LQTS2. They also tested the potential therapeutic effects of nifedipine and the KATP channel opener pinacidil (which augments the outward potassium current) and demonstrated that they shortened the action potential duration and abolished early after depolarization. Similarly, Lahti et al., [73] demonstrated a more pronounced inverse correlation between the beating rate and repolarization time of LQTS2 disease derived iPS-CMs compared with normal control cells. Prolonged action potential is present in LQT2-specific cardiomyocytes derived from a mutation. Matsa et al., [74] also successfully generated iPS-CMs from a patient with LQTS2 with a known KCNH2 mutation. iPS-CMs with LQTS2 displayed prolonged action potential durations on patch clamp analysis and prolonged corrected field potential durations on microelectrode array mapping. Furthermore, they demonstrated that the KATP channel opener nicorandil and PD-118057, a type 2 IKr channel enhancer attenuate channel closing.

LQTS3 has been recapitulated in mouse iPS cells [75]. Malan et al. [75] generated disease-specific iPS cells from a mouse model of a human LQTS3. Patch-clamp measurements of LQTS 3-specific cardiomyocytes showed the biophysical effects of the mutation on the Na+ current, withfaster recovery from inactivation and larger late currents than observed in normal control cells. Moreover, LQTS3-specific cardiomyocytes had prolonged action potential durations and early after depolarizations at low pacing rates, both of which are classic features of the LQTS3 mutation.

Human iPS cells have been used to recapitulate diseases of blood disorder. Ye et al. [13] demonstrated that human iPS cells derived from periphery blood CD34+ cells of patients with myeloproliferative disorders, have the JAK2-V617F mutation in blood cells. Though the derived iPS cells contained the mutation, they appeared normal in phenotypes, karyotype, and pluripotency. After hematopoietic differentiation, the iPS cell-derived hematopoietic progenitor (CD34+/CD45+) cells showed the increased erythropoiesis and expression of specific genes, recapitulating features of the primary CD34+ cells of the corresponding patient from whom the iPS cells were derived. This study highlights that iPS cells reprogrammed from somatic cells from patients with blood disease provide a prospective hematopoiesis model for investigating myeloproliferative disorders.

Raya et al., [76] reported that somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency after correction of the genetic defect. They demonstrated that corrected Fanconi-anaemia specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal. This study offers proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

Maehr et al., [77] demonstrated that human iPS cells can be generated from patients with T1DM by reprogramming their adult fibroblasts. These cells are pluripotent and differentiate into three lineage cells, including insulin-producing cells. These cells provide a platform to assess the interaction between cells and immunocytes in vitro, which mimic the pathological phenotype of T1DM. This will lead to better understanding of the mechanism of T1DM and developing effective cell replacement therapeutic strategy.

Lee et al., [80] reported the derivation of human iPS cells from patient with Familial Dysautonomia, an inherited disorder that affects the development and function of nerves throughout the body. They demonstrated that these iPS cells can differentiate into all three germ layers cells. However gene expression analysis demonstrated tissue-specific mis-splicing of IKBKAP in vitro, while neural crest precursors showed low levels of normal IKBKAP transcript. Transcriptome analysis and cell-based assays revealed marked defects in neurogenic differentiation and migration behavior. All these recaptured familial Dysautonomia pathogenesis, suggesting disease specificity of the with familial Dysautonomia human iPS cells. Furthermore, they validated candidate drugs in reversing and ameliorating neuronal differentiation and migration. This study illustrates the promise of disease specific iPS cells for gaining new insights into human disease pathogenesis and treatment.

Human iPS cells derived reprogrammed from patients with inherited neurodegenerative diseases, amyotrophic lateral sclerosis [78] and Huntingtons disease 79, have also been reported. Dimos et al., [78] showed that they generated iPS cells from a patient with a familial form of amyotrophic lateral sclerosis. These patient-specific iPS cells possess the properties of ES cells and were reprogrammed successfully to differentiate into motor neurons. Zhang et al., [79] derived iPS cells from fibroblasts of patient with Huntingtons disease. They demonstrated that striatal neurons and neuronal precursors derived from these iPS cells contained the same CAG repeat expansion as the mutation in the patient from whom the iPS cell line was established. This suggests that neuronal progenitor cells derived from Huntingtons disease cell model have endogenous CAG repeat expansion that is suitable for mechanistic studies and drug screenings.

Disease specific somatic cells derived from patient-specific human iPS cells will generate a wealth of information and data that can be used for genetically analyzing the disease. The genetic information from disease specific-iPS cells will allow early and more accurate prediction and diagnosis of disease and disease progression. Further, disease specific iPS cells can be used for drug screening, which in turn correct the genetic defects of disease specific iPS cells.

iPS cells appear to have the greatest promise without ethical and immunologic concerns incurred by the use of human ES cells. They are pluripotent and have high replicative capability. Furthermore, human iPS cells have the potential to generate all tissues of the human body and provide researchers with patient and disease specific cells, which can recapitulate the disease in vitro. However, much remains to be done to use these cells for clinical therapy. A better understanding of epigenetic alterations and transcriptional activity associated with the induction of pluripotency and following differentiation is required for efficient generation of therapeutic cells. Long-term safety data must be obtained to use human iPS cell based cell therapy for treatment of disease.

These works were supported by NIH grants HL95077, HL67828, and UO1-100407.

The authors confirm that this article content has no conflicts of interest.

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Induced Pluripotent Stem Cells and Their Potential for Basic and ...

Abstract

Despite considerable advances in medicine, cardiovascular disease is still rising; with ischemic heart disease being the leading cause of death and disability worldwide. Thus extensive efforts are continuing to establish effective therapeutic modalities that would improve both quality of life and survival in this patient population. Novel therapies are now being investigated not only to protect the myocardium against ischemia-reperfusion injury but also regenerate the heart. Stem cell therapy, such as potential use of human mesenchymal stem cells, induced pluripotent stem cells and their exosomes will make it possible not only to address molecular mechanisms of cardiac conditioning, but also develop new therapies for ischemic heart disease.

Despite all the studies and progress made over the last 15 years on the use of stem cell therapy for cardiovascular disease, the efforts are still in their infancy. While the expectations have been high, the findings indicate that most of the clinical trials are generally small and the results are inconclusive. Because of many negative findings, there is certain pessimism that cardiac cell therapy is likely to yield any meaningful results over the next decade or so. Similar to other new technologies, early failures are not unusual and they may be followed by impressive success. Nevertheless, there has been considerable attention to safety by the clinical investigators since the adverse events of stem cell therapy have been impressively rare. In summary, while the regenerative biology might not help the cardiovascular patient in the near term, it is destined to do so over the next several decades.

Cardiovascular disease is the leading global cause of death, accounting for over 17 million deaths per year. The number of cardiovascular deaths is expected to grow to more than 23 million by 2030, according to a report from the American Heart Association.1 In 2011 nearly 787,000 people died from heart disease, stroke and other cardiovascular diseases in the United States. Two new approaches have been identified that have the potential of added benefits to the current therapeutic strategies. The first focuses on enhancing the heart/myocardiums tolerance to ischemia-reperfusion injury using cardiac conditioning that will be covered here only briefly as a historical background. The second approach is to create an environment within the heart muscle that will result in repair of the damaged myocardium; a topic of this review.

Considerable experimental evidence obtained in multiple models and species has demonstrated that all forms of myocardial ischemic conditioning (pre-conditioning, per-conditioning, post-conditioning and remote preconditioning) induce very potent cardioprotection in animal models.25 In healthy, young hearts, many of these conditioning methods can significantly increase the hearts resistance against ischemia and reperfusion injury. However, essentially none of these forms of myocardial ischemic conditioning have been effective in patients. Remote ischemic pre-conditioning using transient arm ischemiareperfusion did not improve clinical outcomes in the ERICCA study, with 1,612 patients undergoing elective on-pump coronary artery bypass grafting.6 Additionally, upper-limb remote ischemic preconditioning performed in 1,385 patients did not show any significant benefit among patients undergoing elective cardiac surgery.7 Therefore, these large multicenter trials have not only proved that ischemic conditioning was unsuccessful in cardiac surgeries; they also failed to confirm the presence of initial cardioprotection by ischemic conditioning-induced reduction of cardiac troponin release,8, 9 which is a standard diagnostic indicator of myocardial injury. The lack of clinical success most likely is due to underlying risk factors that interfere with cardiac conditioning, along with the use of cardioprotective agents that activate the endogenous cardioprotective mechanisms. Future preclinical validation of drug targets and cardiac conditioning will need to focus more on comorbid animal models (such as age, diabetes, and hypertension) and choosing the relevant endpoints for assessing the efficacy of cardioprotective procedures to have a successful, clinical translation.

While the existing therapies for the ischemic heart disease lower the early mortality rates, prevent additional damage to the heart muscle, and reduce the risk of further heart attacks, most of the patients are likely to have worse quality of life including frequent hospitalizations. Therefore, there is an ultimate need for a treatment to improve the clinical conditions by either replacing the damaged heart cells and/or improve cardiac performance. Thus, the cardiac tissue regeneration with the application of stem cells, or their exosomes, may be an effective therapeutic option.10 Stem cells, both adult and embryonic stem cells (ESCs) have the ability to self-replicate and transform into an array of specialized cells. Stem cells are becoming the most important tool in regenerative medicine since these cells have the potential to differentiate into cardiomyocytes. It would, therefore, be useful to find out if the differentiated cells can restore and improve cardiac function safely and effectively.

The purpose of this review is to present the current state of knowledge of potential use of human stem cells, induced human pluripotent stem cells (hiPSCs), and stem cell-derived exosomes as a cell based therapeutic strategy for the treatment of the damaged heart. These stem cells also provide feasibility to address fundamental research questions directly relevant to human health, including their challenges, limitations, and potential, along with future prospects. Human induced pluripotent stem cell technology, in particular, patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs) recently has enabled modeling of human diseases, offering a unique opportunity to investigate potential disease-causing genetic variants in their natural environment.

Although there are many different kinds of stem cells, in this review we will include only those that have been used for most current cardiac regeneration studies.

Embryonic stem cells are obtained from the inner cell mass of the blastocyst that forms three to five days after an egg cell is fertilized by a sperm. They can give rise to every cell type in the fully formed body, but not the placenta and umbilical cord.

Tissue-specific stem cells (also referred to as somatic or adult stem cells) are more specialized than embryonic stem cells. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found.

Mesenchymal stem cells are multipotent stromal cells which can be isolated from the bone marrow. They are non-hematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as bone cells, cartilage cells, muscle cells and fat cells.

Induced pluripotent stem cells, or iPS cells, are cells taken from any tissue (usually skin or blood) and are genetically modified to behave like an embryonic stem cell. They are pluripotent, which means that they have the ability to form all adult cell types.

Umbilical cord blood stem cells are collected from the umbilical cord at birth and they can produce all of the blood cells in the body.

There is great potential with the recent advances in stem cell research and hiPSC-CMs, as these cells express the same ion channels and signaling pathways as primary human cardiomyocytes, can be cultured for a long time and are available in sufficient quantity. In addition, hiPSCs derived from diseased patients may be able to provide new forms of treatment of ischemic heart disease due to their potential for repairing damaged cardiac tissue, as shown in the Wus laboratory.11 Apart from their more direct role of tissue regeneration, stem cells may also have a clinical impact by secreting multiple growth factors and cytokines. Trophic mediators secreted by stem cells improve cardiac function by a combination of various mechanisms such as attenuating tissue injury, inhibiting fibrotic remodeling, promoting angiogenesis, mobilizing host tissue stem cells, and reducing inflammation. The cardioprotective panel of stem cell secreted factors are considerable and include, but not limited to bFGF/FGF-2, IL-1, IL-10, PDGF, VEGF, HGF, IGF-1, SDF-1, thymosin-4, Wnt5a, Ang-1 and Ang-2, MIP-1, EPO and PDGF.1218 FGF-2 reduces ischemia-induced myocardial apoptosis, cell death and arrhythmias, and stimulates increased expression of anti-apoptotic Bcl-2.19, 20 HGF, bFGF, Ang-1 and -2, and VEGF secreted by BMMSCs lead to augmented vascular density and blood flow in the ischemic heart2123, whereas SDF-1, IGF-1, HGF facilitate circulating progenitor cell recruitment to injury sites thereby promoting repair and regeneration.2427 Stem cells also secrete ECM components including collagens, TGF-, matrix metalloproteinases (MMPs) and tissue-derived inhibitors (TIMPs) that inhibit fibrosis.2830

Therefore, the use of the right mediator may contribute to a better outcome in cell therapy. Many stem cell types have been used in regenerative cardiac research, including bone marrow-derived cells, myoblasts, endogenous cardiac stem cells, umbilical cord-derived mesenchymal stem cells and embryonic cells. However, an exciting new milestone in the field of regenerative and precision medicine was the development of hiPSCs. The therapeutic potential of hiPSCs is considerable, as they are patient-specific stem cells that do not face the immunologic barrier, in contrast to embryonic stem cells. Furthermore, there are sources of tissue to be reprogrammed into hiPSCs that are easily accessible, such as the donors skin, fat, or blood. Their use may avoid common legal and ethical problems that arise from the use of embryonic stem cells; they can differentiate into functional cardiomyocytes and they are now one of the most promising cell sources for cardiac regenerative therapy.

Pluripotent stem cells (PSCs) have been derived by explanting cells from embryos at different stages of development under various growth conditions. PSCs can be classified into two distinct states, naive and primed, which are believed to represent successive snapshots of pluripotency as embryonic development proceeds.31, 32 Nave pluripotent stem cells can be maintained in vitro by supplying leukocyte inhibitory factor combined with inhibition of mitogen-activated protein kinase/extracellular regulated kinase and glycogen synthase kinase 3 signaling, and are characterized by two active X chromosomes. Primed pluripotent stem cells are dependent on fibroblast growth factor 2 signaling and transforming growth factor- signaling, and display inactivation of one X chromosome.31 Human embryonic stem cells and induced PSCs (iPSCs) are considered to share some properties of nave mouse embryonic stem cells.33 Nave human iPSCs can be derived by reversion of primed iPSCs into a state that resembles nave mouse ESCs.34

Fibroblasts are the most commonly used primary somatic cell type for the generation of iPSCs. Fibroblasts can be reprogrammed to stable self-renewing iPSCs which resemble ESCs by enforced expression of a cocktail of transcription factors consisting of octamer-binding protein (Oct4), SRY-box containing gene 2 (Sox2), Kruppel-like factor 4 (Klf4), c-myelocytomatosis oncogene (c-Myc), Lin28, and Nanog gene.35, 36 iPSCs can be generated, expanded, and then differentiated into any cell types including endothelial cells (ECs) and cardiomyocytes for in vitro studies or, ultimately, cell therapy.37, 38

In recent years, it has been shown that somatic cells can be directly converted to cardiomyocytes, although the efficacy is extremely low. Transgenic expression of three cardiac-specific transcription factors (Gata4, Mef2c, and Tbx5) resulted in the trans-differentiation of fibroblasts into contracting cardiomyocytes referred to as induced cardiomyocytes (iCMs). In addition, other reports have also shown that direct reprogramming of somatic cells to iCMs is also feasible using various small molecules and microRNAs (miRNAs), such as Hand2, Mesp1, Myocardin, ESRRG, miR-1, and miR-133.3942 Subsequently, alternative approaches have succeeded in generating human iCMs with gene expression profiles and functional characteristics similar to those detected in ESC-CMs.43

Due to the aforementioned advances in iPSC-derived CMs, it is now possible to generate an unlimited quantity of a patients own heart cells. This new model allows researchers to study and understand the molecular and cellular mechanisms of inherited cardiomyopathies, channelopathies, as well as model acquired heart diseases. Although additional studies are needed to test their safety and efficacy, these heart cells may be also used for regenerative medicine applications following myocardial infarction.

It was shown that hiPSCs may lose their pluripotency when transplanted into a border zone of infarcted cardiac tissue, and engraft into native myocardium where they only partially differentiate into cardiac myocytes. In Yans study, they reported that iPS cell transplantation in the infarcted diabetic db/db and nondiabetic mice resulted in an increase in vascular smooth muscle and endothelial cells in the infarcted heart, leading to a significantly improved cardiac function (Figure 1).44

iPS cell transplantation in the infarcted diabetic db/db and nondiabetic mice resulted in an increase in vascular smooth muscle and endothelial cells in the infarcted heart, leading to a significantly improved cardiac function. Photomicrographs show anti-CD-31 in red (A, panels a, e, i), anti-red fluorescence protein (RFP) in green (A, panels b, f, j) and total nuclei stained with diamidino-phenylindole (DAPI) in blue (A, panels c, g, k). Merged images are shown in A, panels d, h, i. Scale bar=100 m. Panel B shows quantitative analysis of total endothelial cells generation from transplanted iPS cells in both C57BL/6 and db/db mouse hearts two weeks post-MI,*P < 0.001 vs MI.44

Another study demonstrated that iPSC derived progenitor cells differentiated into a cardiomyocyte phenotype and developed contracting areas in mice heart tissue. Beneficial remodeling and improved ventricular function were observed despite the lack of well-aligned mature donor cardiomyocytes.45

In regards to safety, an important obstacle to the clinical use of hiPSCs for the regenerative purposes is their great heterogeneity in terms of plasticity and epigenetic landscape. There is a potential that allogeneic hiPSC transplantation into the heart may cause in situ tumorigenesis.46 In addition, the heterogeneity of the cardiac cells produced from pluripotent hiPSCs administration and their random implantation is likely to cause cardiac arrhythmias. One of the main limitations of the hiPSC-derived cardiomyocytes is that they are embryonic in nature as compared to adult cells. Many laboratories are still trying to make these myocytes more mature and to make lineage-specific cells so as to obtain a pure population of atrial cells, nodal cells, or ventricular cells. iPSC-derived cardiomyocytes exhibit an immaturity of the sarcoplasmic reticulum, and a -adrenergic response that is significantly different from native ventricular tissue of a comparable age. Once the cells are mature, it is also likely that investigators will be able to test the effects of various drugs using hiPSC-CMs from a diverse population of patients with different sexes, ethnicities, and cardiovascular diseases.

We are utilizing a model of the patient-specific hiPSCs differentiated into cardiac lineage in order to delineate the environmental and cellular mechanisms responsible for impaired cardioprotection in diabetes. The advantage of this approach is that the effect of cardioprotection can be evaluated in human cells, thereby capturing the complex physiologic interactions at the patient-specific myocyte level. Our results indicate that iPSC-derived cardiomyocytes are not only a viable model to investigate the underlying mechanisms of anesthetic cardioprotection,47 but they also respond similarly to human myocytes48 and human embryonic stem-cell-derived cardiomyocytes.49 Isoflurane preconditioning protected hiPSC-derived cardiomyocytes from oxidative stress-induced lactate dehydrogenase release and mitochondrial permeability transition pore opening at normal glucose concentrations (Figure 2).50

Isoflurane delayed mitochondrial permeability transition pore (mPTP) opening and protected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from oxidative stress in 5 mM and 11 mM glucose. mPTP opening was induced by photoexcitation-generated oxidative stress. Isoflurane delayed mPTP opening in iPSC-CMs in the presence of 5 mM and 11 mM glucose concentrations (A). Isoflurane did not delay mPTP opening in the presence of 25 mM glucose concentrations (A). *P < 0.001 versus control, n=18 cells/group. H2O2-induced oxidative stress increased lactate dehydrogenase (LDH) release from iPSC-CMs in 5 mM, 11 mM and 25 mM glucose concentrations (B). In iPSC-CMs, isoflurane reduced stress-induced LDH release in 5 mM and 11 mM glucose, but not in 25 mM glucose (B). *P < 0.05 versus stress, n=3 experiments/group. Ctrl = Control; Iso = Isoflurane treatment; and Stress = H2O2 + 2-Deoxyglucose.50

Anesthetic preconditioning also protects cardiomyocytes indirectly through its action on other cell types in the heart, such as endothelial cells,51 or by modulation of inflammatory response. However, hyperglycemia undermines the effectiveness of anesthetic-induced cardioprotection by dysregulating cellular signaling.47 In addition, this study demonstrated that the cardioprotective effects of isoflurane in elevated glucose conditions can be restored by scavenging reactive oxygen species or inhibiting mitochondrial fission. These findings may contribute to further understanding and guidance for restoring pharmacological cardioprotection in hyperglycemic patients. Cardiomyocytes derived from healthy donors and patients with a particular disease (such as diabetes) open new possibilities of studying genotype and phenotype related pathologies in a human-relevant model. Such diseases were nearly impossible to investigate in the past due to the lack of human cardiac cells available for experimental investigation.

Some preclinical studies provide evidence that bone marrow stem cells contribute to cardiac function and reverse remodeling after ischemic damage52 acting both locally53 and remotely.54 In studies to date, bone marrow stem cells have been either infused55 or injected56 in areas that were undergoing revascularization. In preclinical studies, Bollis group reported that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy.57, 58 In their study on mice with a myocardial infarction received one or three doses of cardiac mesenchymal cells through the percutaneous infusion into the left ventricular cavity, 14 days apart. The single-dose group showed improved left ventricular ejection fraction after the 1st infusion but not after the 2nd and 3rd-vehicle infusion. In contrast, in the multiple-dose group, left ventricular ejection fraction improved after each cardiac mesenchymal cell-infusion. The multiple-dose group also exhibited less collagen in the non-infarcted region vs. the single-dose group. Engraftment and differentiation of cardiac mesenchymal cells were negligible in both groups, indicating paracrine effects.58 There appear to be at least three dominant mechanisms that underlie the cardiac reparative response: reduction in tissue fibrosis, neovascularization, and neomyogenesis.54, 59

Human ESC-CMs isolated from embryoid bodies have also been used for replacing myocardial scar tissue with new, functional cardiac cells, and therefore achieving actual myocardial regeneration. The ESC-CMs behave structurally and functionally like cardiomyocytes, expressing characteristic morphology, cell marker and transcription factor expression, sarcomeric organization and electrophysiological properties, including spontaneous action potentials and beating activity.60 Mouse and human cardiac-committed ESCs have been transplanted into small and large animal models of acute and old MI. Although these studies have demonstrated durable in vivo engraftment, proliferation and differentiation of ESC-CMs, as well as electromechanical integration with host cardiomyocytes61, they have not universally shown improvement in myocardial remodeling and function. While the reported benefits can thus be attributed to a potential synergy62 between the favorable environment created by the revascularization of the region and the mesenchymal stem cells, the precise delineation of each contribution, however, remains unknown. In addition, so far there is no evidence that critical number of new cells is regenerated or injected stem cells survive following the transplant. Animal studies have shown that only 1% of the stem cells injected into the heart tissue are detectable after 1 month. Nevertheless, in one of the recent systemic reviews and meta-analysis studies of preclinical work, cardiac stem cells treatment resulted in significant improvement of ejection fraction compared with placebo.63 In addition, there was a reduction in the magnitude of effect in large compared with small animal models.

A cell-based therapy could offer additional clinical benefits for post-ischemic heart by improving revascularization along with structural and functional properties.12, 64, 65 There are several limitations for effective stem cell therapy, but the major problems deal with their delivery, type of cells to be used, limited retention of the cells in the heart and the risk of immune rejection. Direct injection of stem cells into the heart muscle results in significant cell death and washout resulting in majority of cells being removed from the heart soon after the injection. Many preclinical studies have reported that intravenously administered MSCs for acute myocardial infarction attenuate the progressive deterioration in LV function and adverse remodeling in mice with large infarcts, and in ischemic cardiomyopathy, they improve LV function.63 Moreover, the cardiac phenotype of human embryonic stem cell-derived cardiac myocytes and human induced pluripotent stem cell-derived cardiac myocytes salvages the injured myocardium better than undifferentiated stem cells through their differential paracrine effects.66

In the clinical studies, the investigators have used mainly two approaches of cell administration: intramyocardial delivery and intracoronary injection. Direct cardiac muscle injections can be performed either surgically or using percutaneous endocardial injection catheters, while coronary injection of stem cells can be done using an antegrade intracoronary artery injection or a retrograde sinus injection. The antegrade intracoronary artery injection is more attractive because it is the least invasive but some microvascular plugging can occur as a result of stem cell injection leading to microinfactions when the cells injected are too large for the capillary bed. Since the stem cells also need to cross the capillary wall, this approach has been found to be less effective as compared to intramyocardial delivery. Although the cell type, dosage, concentration, and delivery modalities are important considerations for regenerative cell therapy clinical trials, the available data are inconclusive and additional early phase studies will be needed before proceeding to pivotal clinical trials.67

The stem cells derived from the bone marrow of the healthy donors have been used in majority of clinical trials as briefly summarized in Table 1. So far, the clinical trials for cardiac regeneration have mainly used cell-based therapies, including bone marrow-derived cells, mesenchymal stem cells and cardiac progenitor cells. While the listed studies have met safety end points either with autologous or allogeneic cell sources68, the effect on cardiac function has been somewhat disappointing. One of the largest multicenter clinical trials using bone-marrow cells given via intracoronary injection for myocardial infarct patients, failed to reinforce the notion that these therapies are efficacious since it did not meet its primary goal.69 A recently published Cochrane review of bone-marrow trials for heart attack patients also found no benefits for various primary goals such as mortality, morbidity, life quality and LVEF.70 An additional Cochrane review using bone-marrow-derived stem/progenitor cells as a treatment for chronic ischemic heart disease and congestive heart failure identified low-quality evidence of reduction in mortality and improvement of LVEF.71

Selected list of landmark clinical trials using mostly bone marrow-derived mesenchymal stem cells conducted to treat acute myocardial infarction and heart failure.

The limited clinical success of stem-cell injections for the treatment of myocardial infarction or heart failure has been mainly attributed to the low retention and survival of injected cells. One of the clinical trials for treatment of heart failure resulting from ischemic heart disease used autologous c-kit(+) cardiac stem cells and produced a significant improvement in both global (Figure 3) and regional LV function (Figure 4), a reduction in infarct size, and an increase in viable tissue that persisted at least 1 year after cardiac infusion (SCIPIO trial).72 Another study, also involving small number of patients, used intracoronary administration of autologous cardiosphere-derived cells and the treatment led to a decreased scar size, increased viable myocardium, and improved regional function of infarcted myocardium at 1 year post-injection (CADUCEUS trial) (Figure 5).73

Administration of Cardiac Stem Cells (CSC) in Patients with Ischemic Cardiomyopathy. Panel A: The mean baseline LVEF in the eight treated patients who were included in the cardiac magnetic resonance analysis was 27.5% at baseline (4 months after CABG surgery and before CSC infusion), and increased markedly to 35.1% (P=0.004, n=8) at 4 months and 41.2% (P=0.013, n=5) at 12 months after CSC infusion. Panel B: Change in LVEF at 4 months and 12 months after CSC infusion (absolute EF units). Data are means SEMs. 72

Panel A: Regional EF at baseline and 4 and 12 months after CSC infusion in the infarct-related regions. Panel B: Change in regional EF in the infarct-related regions at 4 and 12 months after CSC infusion (absolute EF units). Panel C: Regional EF in the dyskinetic segments of the infarct-related regions at baseline and 4 and 12 months after CSC infusion. Panel D: Change in regional EF in the dyskinetic segments of the infarct-related regions at 4 and 12 months after CSC infusion (absolute EF units). Panel E: Regional EF in the least functional segment of the infarct-related regions at baseline and 4 and 12 months after CSC infusion. Panel F: Change in regional EF in the least functional segment of the infarct-related regions at 4 and 12 months after CSC infusion (absolute EF units). Data are means SEMs. 72

(A) Representative matched, delayed contrast-enhanced magnetic resonance images and their corresponding cine short-axis images (at end-diastole [ED] and end-systole [ES]) at baseline and 1 year. In the pseudocolored, delayed contrast-enhanced images, infarct scar tissue, as determined by the full width half maximum method, appears pink. Each cardiac slice was divided into 6 segments and the infarcted segments were visually identified from delayed contrast enhanced images. Scar size (percentage of infarcted tissue per segment) and systolic thickening were calculated for each individual infarcted segment at baseline and 1 year. Endocardial (red) and epicardial (green) contours of the left ventricle are shown. In the CDC-treated patient (top row), scar decreased, viable mass increased and regional systolic function improved over the period of 1 year in the treated infarcted segments (highlighted by arrows). In contrast, no major changes in scar mass, viable myocardial mass or regional systolic function were observed in the control patient (bottom row). (B) Scatterplots of the changes in the percentage of infarcted tissue and the changes in systolic thickening for every infarcted segment of treated and control patients. ED = end-diastole. 73

Umbilical cord blood has been demonstrated as a very useful and rich source of stem and progenitor cells, capable of restoring blood formation and immunological functions after transplantation. Cord blood stem cells are currently used to treat a range of blood disorders and immune system conditions such as leukemia, anemia and autoimmune diseases. These stem cells are used largely in the treatment of children but have also started being used in adults following chemotherapy treatment. Another type of cell that can also be collected from umbilical cord blood is mesenchymal stromal cells. These cells can grow into bone, cartilage and other types of tissues and are being used in many research studies to see if patients could benefit from these cells too. The fact that cord blood can be frozen and stored for later use led, in 1991, to the establishment of the first cord blood bank from voluntary donors in New York. To date, there are over 54 public cord blood banks in different parts of the world with more than 350,000 units frozen and ready to be used.74 Indeed cord blood transplantation is being used as an alternative to bone marrow transplantation, and more than 14,000 transplants have been documented. Cord blood stem cell treatments differ from bone marrow stem cell treatments in three key areas: increased tolerance of the human leukocyte antigen-mismatching, decreased risk of graft-versus-host disease, and enhanced proliferation ability.75 Recent results of the RIMECARD study by Bartolucci et al. in human subjects using umbilical cord-derived MSCs as potential heart failure therapy are quite encouraging.76 The patients had stable heart failure (HF), with reduced ejection fraction of less than 40. Although the sample size was small (15 controls and 15 HF patients treated with UC-MSCs) to establish either safety or efficacy, the echocardiographic and cardiac MRI evaluations demonstrated improvements in ejection fraction, starting at 3 months, and persisting through 12 months. The patients treated with placebo did not improve in either left ventricular ejection fraction or clinical functional class. As indicated by the authors, it is tempting to speculate that the robust paracrine secretion of various factors, including hepatocyte growth factor, might play an important role in mediating the therapeutic effects of the UC-MSCs.

The main disadvantage of cord blood use is that the volume collected is fixed and relatively small. Therefore, the number of stem cells available for transplantation is low compared to the number of cells that can be collected in customizable bone marrow or peripheral blood stem cell harvests. Nevertheless, there are many opportunities for further development of this technology such as the cord blood selection algorithms that are currently heavily weighted toward maximizing cell doses at the expense of the human leukocyte antigen-matching.77

Beside the stem cell injection therapy, currently there are non-cardiogenic and cardiogenic stem-cell tissue patches, for the repair of myocardial infarction. Recent studies have utilized non-cardiogenic tissue patches made of skeletal myoblasts7881, bone marrow-derived stem cells82, 83, or endothelial progenitor cells84 for the repair of damaged heart. Compared with the injection of a cell suspension, the implantation of tissue sheets composed of skeletal myoblasts has been proven more advantageous for the treatment of myocardial infarction in rats78, 85, and dilated cardiomyopathy in hamsters.86 Moreover several cardiogenic cardiac tissue-engineering methodologies have been developed for use with primary neonatal cardiomyocytes. These include: injection of a mixture of bioactive hydrogels and cells followed by cell-hydrogel polymerization in situ87 and the epicardial implantation of a tissue-engineered cardiac patch.88, 89

Generally, implantation of the engineered myoblast sheets over an infarction site yielded improved neovascularization, attenuated left ventricular dilatation, decreased fibrosis, improved fractional shortening, and prolonged animal survival compared to the delivery of the same number of myoblasts by cell injection.85 In addition, the bone marrow-derived, spatially arranged SMC-endothelial progenitor bi-level cell sheet interactions between SMCs and endothelial progenitor cells augment mature neovascularization, limit adverse remodeling, and improve ventricular function after myocardial infarction.90 In diabetic patients treatment of diabetes mellitus-induced cardiomyopathy with tissue-engineered smooth muscle cell-endothelial progenitor cell bi-level cell sheets prevented cardiac dysfunction and microvascular disease associated with diabetes mellitus-induced cardiomyopathy.91 As indicated before, the main disadvantage of injecting the cell-suspensions directly into the heart muscle compare to engineered heart tissue technique is that most of the injected cells are washed out of the heart or do not survive the injection. This is inefficient and can also be dangerous if some cells have not yet fully developed into myocardial cells and are therefore still pluripotent. These cells could survive in other parts of the body and form tumors. The advantage of the tissue patches is that significantly fewer of the stem cell-derived heart cells are required and fewer cells undergo apoptosis. Some of the major drawbacks currently encountered with regeneration using tissue patches, include the problems with electrical continuity and patch vascularization. Using a similar tissue-engineering strategy, Shimko et al. formed cardiac constructs using pure differentiated cardiomyocytes derived by genetic selection from D3 mouse ESCs with a neomycin-resistance gene driven by the -MHC promoter.92 They found that 10% cyclic stretch at rate of 13 Hz for 3 days increased the expression of cardiac markers such as -cardiac actin, -MHC and Mef-2c, but the resulting cardiac tissues were noncontractile. Immunostaining showed that pure cardiomyocytes were present, but they had disorganized sarcomeres and a relatively rounded appearance.92

Recently, in a study published by Nummi et al., they reported that during on-pump coronary artery bypass graft surgery, part of the right atrial appendage can be excised upon insertion of the right atrial cannula of the heart-lung machine and the removed tissue can be easily cut into micrografts for transplantation.93 Appendage tissue is harvested during cannulation of the right atrium, and therefore, no additional procedure is needed. Isolation of the cells and preparing the matrix for transplantation is done simultaneously with the coronary artery bypass graft operation in the operating room, so the perfusion time and the aorta clamp time are not increased. After the bypass anastomoses, the atrial appendage sheet is placed on the myocardium with three to four sutures allowing the myocardium to contract without interference. They believe that atrial appendage-derived cells therapy administered during CABG surgery will have an impact on patient treatment in the future.93

While some of the outcomes of these trials have been modest at best, it is now evident that the success of future cardiac cell therapies will be highly dependent on the ability to overcome the problem of low retention and survival of implanted cells.94 Potential approaches to address this issue include: coinjecting cells with bioactive, in situ polymerizable hydrogels87, preconditioning cells with hypoxia or prosurvival factors95, genetic engineering of cells to enhance their angiogenic and/or antiapoptotic action96 and the epicardial implantation of a preassembled tissue-engineered patches.27, 85, 97 In particular, tissue patch implantation, although surgically more complex than cell or cell/hydrogel injections, may support long-term survival of transplanted cells and exert a more efficient structural and functional cardiac tissue reconstruction at the infarct site.98

The adult human heart lacks sufficient ability to replenish the damaged cardiac muscles since the rate of cardiomyocyte renewal activity is less than 1% per year. The mechanical and electrical engraftment of injected cardiomyocytes is largely not feasible at the scale that would be necessary for cardiac improvement. On the other hand, the human heart contains large population of fibroblasts that could be used for direct reprograming. As such, direct fibroblasts reprogramming in vivo has emerged as a possible approach for cardiac regeneration. With considerably better understanding of the various molecular mechanisms, direct fibroblast reprogramming has improved considerably but still lacks sufficient efficacy using human cells (Figure 6).

There are various novel treatment options that have been tested for the heart failure due to ischemic heart disease or genetic disorders. Previous clinical trials have employed various adult stem cell and progenitor cell populations to test their efficacy for therapeutic applications. Additional approaches are being explored, including implantation of in vitro constructed cell sheets of engineered heart muscles (EHMs) as well as direct in vivo reprogramming of cardiac fibroblasts in the scar region to cardiomyocytes. The regenerative capacity of adult stem and progenitor cell populations is also being evaluated along with administration of exosomes and small vesicles secreted by the stem cells.36

As indicated earlier, the survival of transplanted stem cells is dismal and the beneficial effects of stem cell therapies is not due to their differentiation into new cardiomyocytes but instead because they are the temporary source of the exosomal growth factors. Therefore, despite the stem cells early demise, there are some limited cardiac benefits from this treatment, including decreased cardiomyocyte apoptosis, reduced fibrosis, enhanced neovascularization and improved left ventricular ejection fraction. It is for that reason why the exosome therapy recapitulates the benefits of stem cell therapy,99 and many studies have shown that the activation of cardioprotective pathways obtained by stem cell therapy can be reproduced by the injection of exosomes produced by the stem cells.100 An additional benefit of using exosomes for cardioprotection and regeneration is the lack of tumor-forming potential of exosomes. However, the underlying mechanisms of stem cells or hiPSC-derived exosome therapy are still unclear. Numerous scientific investigations have identified recent applications of exosomes in the development of molecular diagnostics, drug delivery systems and therapeutic agents.

Exosomes are small membrane vesicles (30100 nm) of endocytic origin that are secreted by most cells after being formed in the cellular multivesicular bodies. The fusion of multivesicular bodies into the plasma membrane leads to the release of their intraluminal vesicles as exosomes. Once released in the extracellular environment, their cargo of functional molecules can be taken up by recipient cells via several mechanisms including fusion with the plasma membrane, phagocytosis and endocytosis. The formation and release can be upregulated through different steps based on environmental stimuli such as stress or hypoxia. There are two main mechanisms responsible for exosome release. First, there is a constitutive mechanism that is mediated by specific proteins involved in membrane trafficking, such as RAB heterotrimeric G-proteins and protein kinase D. Second, there exists an inducible mechanism that can be activated by several stimuli including increased intracellular Ca2+ and DNA damage. Studies have used different approaches to also increase the angiogenic potential of exosomes released by the stem cells.101 Exosome release with a basal angiogenic potential can be substantially increased in vitro using stress conditions that mimic organ injury, such as hypoxia, irradiation, or drug treatments. Changes in exosomal composition facilitate angiogenesis and tissue repair most likely via enhanced level of growth factors and cytokines.

Exosomes contain various molecular constituents of their cell of origin, including proteins and RNA. The cargo of mRNA and miRNA in exosomes was first discovered at the University of Gothenburg in Sweden.102 In that study, the differences in cellular and exosomal mRNA and miRNA content were described, as well as the functionality of the exosomal mRNA cargo. Exosomes facilitate cell-cell communication to the recipient cell membrane and deliver effectors including transcription factors, oncogenes, small and large non-coding regulatory RNAs (such as microRNAs) and mRNAs into recipient cells and can be used for cardiac protection and repair. Exosomes have also been shown to carry double-stranded DNA. Exosomes can be derived from many different types of stem cells including umbilical cord, cardiosphere-derived cells, cardiac stem cells, embryonic, induced pluripotent, mesenchymal and endothelial progenitor cells. They can carry and deliver mRNAs, miRNAs and proteins to the injured heart muscle and play a significant role in resident cardiac stem cell activity, cardiomyocyte proliferation, beneficial cardiac remodeling, apoptosis reduction, angiogenesis, anti-inflammatory response and a decrease in infarct size. The advantages for effective exosome therapy include the cell free component, log-term stability and low or no immune response. Some of the limitations include the necessity of repeated injections, target cell selection and the random packing of the exosome cargo.

Some preliminary reports have demonstrated that exosomes released from cardiac progenitor cells can improve cardiac function in the damaged heart.103, 104 It has been proposed that exosomes released from transplanted cardiomyocytes are involved in metabolic events in target cells by facilitating an array of metabolism-related processes, including modulation of gene expression. Moreover, exosomes secreted from the hiPSC-derived cardiomyocytes exert protective effects by transferring endogenous molecules to salvage injured neighboring cells by regulating angiogenesis, apoptosis, fibrosis, and inflammation. It has been shown that ischemic preconditioned hearts promote exosome release and help spread cardioprotective signals within the myocardium.105, 106 Also, the administration of mesenchymal stromal cell-secreted exosomes demonstrated improved cardiac function in the acute myocardial infarction mouse model. Mesenchymal stem cell-derived exosomes increased adenosine triphosphate levels, reduced oxidative stress, and activated the PI3K/Akt pathway to enhance cardiomyocyte viability after ischemia-reperfusion (I/R) injury.107 Recently, it was shown that ischemic preconditioning of mesenchymal stromal cells increased levels of miR-21, miR-22, miR-199a-3p, miR-210, and miR-24 in exosomes released by the cells, and the administration of mesenchymal stromal cell-ischemic preconditioning exosomes resulted in a reduction of cardiac fibrosis and apoptosis compared with the hearts treated with control exosomes. Stem cell-derived exosomes possess the ability to modulate cardiomyocyte survival and confer protection against angiotensin II-induced hypertrophy by activating PI3K/Akt/eNOS pathways via RNA enriched within the exosomes. Additionally, it has been shown that exosome treatment increased levels of adenosine triphosphate and NADH, decreased oxidative stress, increased phosphorylated-Akt and phosphorylated- glycogen synthase kinase 3, and reduced phosphorylated-c-JNK in hearts after I/R. Subsequently, both local and systemic inflammations were significantly reduced 24 hours after reperfusion.107 Intact exosomes restore bioenergetics, reduce oxidative stress, and activate pro-survival signaling, thereby enhancing cardiac function and geometry after myocardial I/R injury.107 Clearly, stem cell-derived exosomes may be a potential adjuvant to reperfusion therapy for myocardial infarction and heart failure.

The existing therapies for the ischemic heart disease have many limitations and efforts are underway for new treatments using the stem cell therapy to improve the clinical conditions by either replacing the damaged heart cells and/or improve cardiac performance. The cardiac tissue regeneration with stem cells, their exosomes or small vesicles and tissue engineering may be effective therapeutic options. Although the expectations have been high, the results from majority of clinical trials are negative. Due to very low engraftment and survival of stem cells injected into a cardiac muscle, there is convincing evidence that the release of paracrine factors from the stem cells contributes to myocardial cardioprotection and regeneration. It is likely that the future research will be focused on the biology of these endogenous signaling pathways, and will lead the way for different applications of exosomes and small vesicles in regenerative medicine. In the future there might be more successful approaches that would utilize stem cell technology with various bioengineering constructs having not only cardiomyocytes but other cardiac cells.

Regarding the regulatory agencies, one would need a significant efficacy/safety data and meaningful end points when compared with standard-of-care drugs that are used today for heart attacks and heart failures. So where are we today since most of the clinical trials did not achieve their primary efficacy end points? Because the cell-therapy studies for heart disease did not achieve this so far, more preclinical work might be necessary using current and other approaches in order to demonstrate compelling rationale for new clinical trials. Although the regenerative biology might not be very helpful to the cardiovascular patient in the near term, it is most likely that we will witness very impressive and exciting results over the next several decades.

This work was supported in part by grants P01GM066730 and T32 GM089586 from the National Institutes of Health, Bethesda, MD.

calcium ion

endothelial cells

endothelial nitric oxide synthase

endothelial nitric oxide synthase/protein kinase G

Extracellular signal-regulated protein kinases 1 and 2

human stem cells, induced pluripotent stem cells

human stem cells, induced pluripotent stem cells-derived cardiomyocytes

induced pluripotent stem cells

Nicotinamide adenine dinucleotide

Janus kinase and Signal Transducer and Activator of Transcription pathways

specific isoforms of mitogen-activated protein kinase and extracellular regulated kinase

Phosphatidylinositol 3-kinase, serine/threonine kinase also known as protein kinase B, and mammalian target of rapamycin pathway

protein kinase C

cGMP protein kinase G pathway

Disclosure

The authors declare that they have no disclosures.

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Maia Terashvili, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin, 53226, USA.

Zeljko J. Bosnjak, Departments of Medicine and Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin, 53226, USA.

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Mesenchymal stem cells derived exosomes: a new era in cardiac ...

The science around terminal inactivation and deletion of genetic codes of heredity in somatic cells was postulated by the Weismann barrier theory [1]. The somatic cell nuclear transfer (SCNT) demonstration asserted the fact that the genetic code in somatic cells is not discarded, and that reactivation of the same is a possibility through careful manipulations [2]. Developmental biology entered a new dimension of achievement when the discovery of embryonic stem cells (ESCs) and their pluripotency was exhibited, and further research identified that on fusion of somatic cells like fibroblasts, and T-lymphocytes with ESCs, reprogramming of the former through expression of genes associated with pluripotency becomes a possibility [3,4]. The findings around SCNT and ESC fusion identified the possibility of reversion in somatic cells indicating the presence of reprogramming factors that bear the potential to act as epigenetic memory erasing factors [5]. The earliest study around generation of pluripotent stem cells from fibroblasts was linked to introduction of four crucial transcription factors including octamer binding transcription factor 3/4 (Oct3/4), sex determining region Ybox 2 (SRY-Sox2), Krppel-like factor 4 (Klf4), and cellular-Myelocytomatosis (c-Myc) (OSKM) [6]. The allogenic trait of ESCs, risk of immune rejection in the recipient along with need for lifetime immunosuppression, and the ethicality around using the same, makes human induced pluripotent stem cells (iPSCs) an established candidate for regenerative therapies as they were found to not impact the host immune system [7]. The introduction of the iPSCs technology happened in the year 2006, and since then multiple observational studies have recounted its impact on cardiac diseases, ophthalmic conditions, as well as neurological disorders [8,9,10]. Figure 1 highlights the process of generating iPS cells.

Showing the process of progression and generating iPSC cells. Detailed description of creating iPSCs with reprogramming factors and differentiating them into a variety of cell types.

The nuclear reprogramming strategies, without compromising on safety and quality for therapeutic applications, include the integrative or nonintegrative transfer systems using viral or nonviral vectors. The first iPSCs were generated by integrating viral vectors, more popularly the retrovirus wherein the resultant iPSCs exhibited failure in complete expression of endogenous genes of pluripotency [11]. The more efficient viral vector has been documented to be the lentiviral vector (LV), which has recorded a reprogramming efficiency of between 0.11% [12,13,14]. To ensure increased safety for therapeutics, nonviral integrative systems have also been worked upon involving use of two plasmids; once encoding for c-Myc, and the other for the four reprogramming factors [15]. However, this system was also shown to have risk of integration, and low reprogramming efficiency. In case of nonintegrative nonviral systems for reprogramming, delivery of pluripotency marker genes has been done using self-replicating vectors, and cytoplasmic RNA. Though easy to work with, the reprogramming efficiency has been found to be lower than LV [16]. Today, research has identified possibility of successful reprogramming using microRNAs (miRNAs) which exhibit improved efficiency, wherein use of c-Myc has been replaced with miR-291-3p, miR-294, and miR-295 to generate homogenous colonies of human iPSCs [17]. The reprogramming methods have been highlighted in Table 1.

Reprogramming strategies for iPSCs in human species. Various programming strategies with ensuring safety and quality for therapeutic applications include the integrative or nonintegrative transfer systems using viral or nonviral.

There are many assays, including molecular and functional, to evaluate the developmental efficiency of iPSCs. These include alkaline phosphatase staining of pluripotency markers, DNA demethylation, retroviral silencing, and factor independence involving assessment of self-renewal in the absence of dox-inducible trans genes. The functional assays include teratoma formation, chimera development, tetraploid complementation, germline transmission, and in vitro differentiation [14]. Considering the low reprogramming efficiency in iPSCs, many studies have identified blocks in lineage conversion. Reprogramming pathway studies in fibroblasts have identified the repel factor to be involved in mesenchymal-to-epithelial transition (MET) and BMP receptor signaling [27,28]. Further studies on the refractory fibroblasts indicate negative iPSC generation in spite of prolonged culturing and presence of homogeneous factor expression indicating loss of somatic program, and activation of endogenous pluripotency genes to be the main roadblocks in formation of iPSCs [14]. The other limiting factor has been linked to expression levels of Nanog locus which are activated late in the reprogramming process and thus limit efficiency of conversion [29]. Gene silencing by DNA methylation, involving the pluripotency genes nanog and Oct4 which causes blockage in binding of transcription factors, has also been linked to causing interference in reprogramming [30]. Though the four most popular reprogramming factors have been Oct4, Sox2, Klf4, and c-Myc, human iPSCs have also been derived using expression of Oct4, Sox2, Nanog, and Lin28, indicating that pluripotent ground state becomes achievable through activation of different transcription factors [21]. The detailed derivation of iPSC along with the assay has been highlighted in Figure 2.

Schematic representation on derivation and assay for human iPSCs. Detailed schematic representation of derivation of iPSC with the various assays to evaluate the developmental efficiency.

The therapeutic potential of iPSC towards personalized cell therapy and disease modelling, has extended the functionality beyond laboratory tables as a research tool in murine and human models. Animal studies have identified promising potential of iPSC around treatment of genetic disorders, including sickle cell anemia; disease modelling of complex degenerative conditions like diabetes, Alzheimers disease, and the feasibility to be used in organ transplantation without risk of rejection and need of immunosuppression [14,31]. Few highlights on the therapeutic potential of iPSCs have been summarized in Table 2. The focus of the current review is to highlight and discuss the therapeutic roles of human iPSCs in different conditions and the future.

Few highlights of iPSC-disease models and the investigated therapy. The example of therapeutic potential of iPSC towards personalized cell therapy and disease modelling, has extended the functionality of the pluripotency beyond laboratory tables as a research tool in murine and human models.

Pluripotency and self-renewal are unique characteristics of iPSC that make them ideal for disease modelling and regenerative medicine. Their ability to indefinitely differentiate into cells of all the three germ layers makes them an important source for treating injuries as well as diseases. The availability of generating patient-specific iPSC with high efficiency and safety through protocols involving biochemical and epigenetic aspects expands the therapeutic potential of this tool. This can be assessed from the fact that a clinical trial involving iPSC-derived dopaminergic neurons have been initiated for Parkinsons disease after successful in vivo studies involving immunodeficient mice highlighted no risk of tumorigenicity [43]. Further, tissue resident macrophages, which are critical for immunity and derived from human-iPSCs, have been found to be immunologically different and better than the traditional monocyte-derived macrophages. Studies have shown human iPSC macrophages to restrict Mycobacterium tuberculosis growth in vitro by >75%, and were found to be capable of mounting antibacterial response when challenged with pathogens [44]. The greatest niche for iPSCs is the ability to generate the same from different donor categories including the diseased, and healthy making its application in the clinical setting at any stage a feasibility without the ethical issues around the ESCs.

The fundamental use of iPSC in regenerative medicine remains undisputed, but the tumorigenic potential of residual undifferentiated stem cells necessitates the need to devise strategies to remove the same from differentiated cells. Different study reports multiple treatment methodologies for eliminating undifferentiated iPSCs and one such recent publication identified undifferentiated hiPSCs to be sensitive to treatment involving medium supplemented with high concentration of L-alanine [45]. Another study assessed the efficacy of plasma-activated medium (PAM) in eliminating undifferentiated hiSPCs through inducing oxidative stress. This study found PAM to selectively eliminate undifferentiated hiPSCs cocultured with normal human dermal fibroblasts, which were the differentiated cells. Lower expression of oxidative-stress related genes in the undifferentiated hiPSCs were found to be the underlying cause for PAM-selective cell death [46]. A recent study report describes the use of salicylic diamines to remove residual undifferentiated cells from iPSC-derived cardiomyocytes. Salicylic diamines were found to exert their specific cytotoxic activity in the pluripotent stem cells by inhibiting the oxygen consumption rate. Teratoma formation was also found to be abolished in comparison to untreated cells [47].

Non-communicable diseases, including cardiovascular conditions, have emerged to be one of the leading causes for mortality in developed as well as developing nations. The trigger for myriad heart conditions exists both in genetics and the environment, which makes studying disease etiology in animal models complicated and inefficient. Animal model studies indicate up to 90% failure in new drug clinical trials, highlighting the limitation around prediction of safety and efficacy among humans. The iPSCs-based disease models have been studied for cardiac channelopathies including hereditary long QT syndrome (LQTS), dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC); the endothelial cell disease including familial pulmonary arterial hypertension (FPAH); the smooth muscle cell condition including Williams-Beuren syndrome (WBS), and Marfan syndrome (MFS) [8].

LQTS is an inherited fatal arrhythmia syndrome and around 17 genes have been associated with congenital LQTS, including the three main genes; KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3), together which account for ~75% of clinically definite cases. The current therapeutic intervention includes -blockers and a surgical procedure named left cardiac sympathetic denervation. Though genetic markers have been defined, the occurrence of variance of unknown significance (VUS) in 1 of 3 patients adds to the dilemma of inconclusive diagnosis. The need for better diagnostic platforms to assess outcome of genetic variants as well as different therapeutics led to the introduction of iPSCs. Many studies have worked to improve the differentiation efficiency, cellular maturation, and lineage specificity, develop new high-throughput assays for cellular phenotyping, and promote clinical implementation of patient-specific genetic models. A study by Wu J.C. et al. [48], utilized patient iPSC-derived cardiomyocytes (iPSC-CMs) and devised various strategies to reduce heterogeneity. These include derivation of chamber-specific cardiomyocytes, cultivation for extended period, 3-dimensional and mechanical conditioning, rapid electric stimulation, and hormonal stimulation; use of multicellular preparations to reduce intercellular variability; and development of high-throughput cellular phenotyping using optogenetic sensors including genetically coded voltage and calcium indicators. Further, this study also established the utility of iPSC-CMs to distinguish between pathogenic and benign variants to improve diagnosis and management of LQTS using CRISPR genome editing. This study, using iPSC-CMs, also identified factors causative for prolonged QT including upregulation of genes; DLG2, KCNE4, PTRF, and HTR2C and downregulation of CAMKV gene. Thus iPSC-based model platforms aid in developing a better understanding around intractable clinical problems associated with diseases like LQTS.

In case of DCM, characterized by ventricular chamber enlargement, and dilation as well as systolic dysfunction, human derived iPSCs have been used to investigate the excitation-contraction-coupling machinery, response to positive inotropic interventions, and study the proteome profile. This study utilized DCM patient specific-iPSC derived from skin fibroblasts and identified defects in assembly and maintenance of sarcomeric structure in the mutated iPSC-CM, as well as lower response to -adrenergic stimulation with isoproterenol, and increased [Ca2+] out and angiotensin-II. This indicates mutated CM from DCM patients to express blunted inotropic response [49]. In case of HCM which is the most common cause of sudden death among the young, iPSC models have been used to identify pathogenesis of the condition. Once such study involving iPSC-CM derived from patients in a maternally inherited HCM family positive for the mitochondrial 16s rRNA gene (MT-RNR2) mutation m.2336T > C identified mitochondrial dysfunction, and ultrastructure defects among the carriers. Further, reduction in levels of mitochondrial proteins, the ATP/ADP ratio, and mitochondrial potential was also found. These lead to increase in intracellular Ca2+ levels, that becomes causative for HCM-specific electrophysiological abnormalities [50]. Recent studies have also generated peripheral blood mononuclear cells-derived iPSC from HCM patient positive for the myosin binding protein C (MYBPC3) pathogenic mutation c.33693370 insC by the episomal method, which underwent successful differentiation to triblast cells with normal male karyotype, and expression of pluripotent markers indicating its usefulness as a tool to study HCM [51].

The iPSC models around FPAH have identified modification of BMPR2 signaling causing reduced endothelial cell adhesion, migration, survival, as well as angiogenesis. The autosomal dominant BMPR2 disease causing mutation has been found to be only 20% penetrant and the use of iPSC identified increased BIRC3 to be related to improved survival, indicating the potential to use protective modifiers of FPAH for developing treatment strategies in the future [52]. The iPSC model around WBS with haploinsufficiency found deficiency of elastin and the patient-derived smooth muscle cell to be immature and highly proliferative with defects in function and contractile properties. The rescue was done by upregulating elastin signaling and use of anti-proliferative drug rapamycin [53]. In case of MFS, disease pathogenesis investigation using iPSCs identified defects in fibrillin-1 accumulation, degradation of extracellular matrix, abnormal activation of transforming growth factor-, and cellular apoptosis [54].

The iPSC technology is also largely viewed to promote pre-clinical drug trials and screening over animal models to overcome differences in electrophysiological properties between human and animal cardiomyocytes. Studies have shown patient-derived iPSCs to exhibit higher sensitivity towards cardiotoxic drugs that could be the cause for change in action potential and arrhythmia [55]. Studies which have analyzed the beat characteristics of 3D engineered cardiac tissues have proven the occurrence of physiologically relevant changes in cardiac contraction in response to increasing concentrations of drugs like verapamil (multi-ion channel blocker) and metoprolol (-adrenergic antagonist) [56].

Thus, iPSC has been successfully used to model and understand pathogenesis of different cardiac diseases, providing insights on pathways around progression as well as for assessment of drug toxicity. These highlight the potential to use iPSC-based models for precision medicine in clinical use.

Theoretically iPSC has the potential to be programmed to form any cell in the human body, and coupled with improvements in reprogramming techniques, this technology has advanced our knowledge on disease pathology, developing precise therapeutics, as well as fuel advances in regenerative medicine [57]. In case of neurodegenerative conditions, and psychiatric disorders, the genetic predisposition and its relation to the disease pathophysiology is complex, and often there is alteration at structural as well as functional levels. In case of schizophrenia, which is aptly termed the disease of the synapses, studies have generated iPSC from family members positive for a frameshift mutation in schizophrenia 1 (DISC1) and used gene editing to generate isogenic iPS cell lines. This study found depletion of DISC1 protein among the mutation carriers, along with dysregulation of genes associated with synapses and psychiatric disorders in the forebrain. This mutation causes deficit of synaptic vesicles among the iPS-cell derived forebrain neurons. This identification of transcriptional dysregulation in human neurons, highlights a new facet involving synaptic dysregulation in mental disorders [58]. The technology of stem cell therapy has also been used to restore the functionality in many degenerative conditions including that of the retina that leads to loss of vision. Studies have evaluated the use iPSC to overcome challenges posed by use of stem cell therapy. The proposed strategy revolves around transplantation of photoreceptors with or without the retinal pigment epithelium cells for treating retinal degradation, with minimal risk using iPSC [59].

Degenerative disease generally progresses through multiple differentiation stages, and using iPSC models, these pathways of transition can be easily identified to assess cause as well as etiopathology better. Amyotrophic lateral sclerosis (ALS) involves loss of neurons from the spinal cord and motor cortex causing paralysis and death. The research around advancement of therapeutics, requires supply of human motor neurons positive for the causative genetic mutations that will also aid in understanding the root cause of motor neuron death. One study documented the production of iPS from ALS patient specific-skin fibroblasts from two sisters. Both were identified to be positive for the L144F (Leu144 Phe) mutation of the superoxide dismutase (SOD1) gene that is associated with a slowly progressing form of ALS. This study found successful reprogramming to be possible with only four factors; KLF4, SOX2, OCT4, and c-MYC. Further, the severe disability state of the patients used for harvesting in this case did not seem to block the transformation process or efficiency [60]. Fanconi anemia (FA) is an inherited bone marrow failure syndrome and is a chromosomal instability disorder needing transplantation of hematopoietic grafts from HLA-identical sibling donors. The reduced quality of the hematopoietic stem cells from the bone marrow of the affected limits the benefit of gene therapy trials. Studies have worked upon formation of genetically corrected FA-specific iPSCs through non-hematopoietic somatic cells reprogramming to generate large number of genetically-stable autologous hematopoietic stem cells for treating bone marrow failure in FA. The reprogramming was done on dermal fibroblasts involving two rounds of infection with mouse-stem-cell-virus-based retrovirus encoding amino-terminal flag-tagged version of the four transcription factors; OCT4, SOX2, KLF4, c-MYC. A batch of genetically corrected somatic cells using lentiviral vectors encoding FANCA or FANCD2 was also used for reprogramming to overcome the predisposition to apoptosis found in FA cells. The FANCA involved fibroblasts also underwent successful transformation to generate iPSCs. This study also found restoration of the FA pathway as a necessity to generate iPS from somatic cells of FA patients. The persistent FANCA expression in the FA-iPS cells indicated successful generation of genetically corrected FA-iPSCs with functional FA pathway, and disease-free status [61].

Parkinsons disease (PD) is a common chronic progressive disorder due to loss of nigrostriatal dopaminergic neurons. The pathophysiology of the disease is complex and research till date lacks complete understanding. Further, sporadic cases are not linked to any genetic variation. Development of patient-specific invitro iPSC models have been attempted to understand disease etiology better. Studies have worked upon generating iPSCs from sporadic cases of PD, which have been successfully reprogrammed to form dopaminergic neurons free of the reprogramming factors. This study utilized doxycycline-inducible lentiviral vectors that were excised with Cre-/lox-recombinase, resulting in generation of iPSC free of programming factors, and which retained all the pluripotent characteristics after removal of transgenes. This removal of promoter and transgene sequences from the vector reduced risk of oncogenic transformation and re-expression of the transduced transcription factors. This study highlighted the possibility of generating stable iPS-cell line in PD for better disease modelling [62]. Another study worked on improving the safety of human and non-human primate iPSC derived dopaminergic neurons for cell transplantation treatment in PD. This study found the protocol of NCAM(+)/CD29(low) sorting to result in enriching ventral midbrain dopaminergic neurons from the pluripotent stem cell-derived neural cell populations. Further, these neurons also exhibited increased expression of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, and NURR1 mRNA. These neurons were also found to bear the potential to restore motor function among the 6-hydroxydopamine lesioned rats, 16 weeks after transplantation. Further, the primate iPSC-derived neural cell was found to have survived without any immunosuppression after one year of autologous transplant, highlighting the proof-of-concept around feasibility and safety of iPSC-derived transplantation for PD [10].

Type 1 diabetes is an autoimmune condition involving destruction of the -cells of the pancreas wherein transplantation with -cells as islet tissues or the entire pancreas is suggested as an alternative over the traditional exogenous insulin supplementation. However, these come with risk of rejection, need of immunosuppression, apart from difficulty in the physiological control on blood glucose levels. To circumvent this block, generation of -cells or islet tissues from human pluripotent stem cells like iPSCs has been attempted. Many studies have generated pancreatic -like cells which secrete insulin in response to stimuli like potassium chloride [63]. However, co-excretion of glucagon, and somatostatin, apart from releasing unsuitable amounts of insulin; make these clinically inferior. iPSC-derived pancreatic endoderm cells have been shown to retain the potential to differentiate and are functionally comparable with adult -cells. Further, the shortage of donor islet has been overcome using iPSCs, as pancreatic cells generated from these have been evaluated in clinical trials as a new source for transplantation therapy. The differentiation of iPSCs through mimicking the natural in vivo process was facilitated using a combination of growth factors including Nodal-activin, Wnt, retinoic acid, hedgehog, epidermal and fibroblast growth factor, bone morphogenetic protein, and Notch to activate as well as inhibit the key signaling pathway. This study thus highlighted the possibility of generating patient-specific fully functional pancreatic tissue for transplantation over donor islet for diabetes treatment [64].

These studies highlight the development around iPSCs and transplantation technology for treatment of degenerative diseases as well as use them as disease models. The ability to generate patient-specific iPSC from skin biopsies, increases safety of autologous transplants without risk of immunorejection.

The treatment for blood disorders involves need for mature red blood cells/erythrocytes from the bone marrow or umbilical cord blood, for blood transfusion, and is limited due to incompatibility in blood group and Rh antigens, and risk of infections [65]. Erythropoiesis is a complex process for generation of mature erythrocytes from the precursor erythroblasts that are difficult to culture in vitro, as the entire process occurs in the bone marrow mediated by complex interaction between cellular and extracellular environment involving hormones, cytokines, and growth factors [66]. Further, the fully differentiated red blood cells (RBCs) are not proliferative, and setting up a system for erythropoiesis-like maturation in precursor cells is a challenge. Further, recruitment of donors, need for rare blood group types, as well as safety in sensitive population groups, add to the roadblock [67]. Studies have investigated human pluripotent stem cells, including iPSCs as an alternative source for unlimited supply of functional erythrocytes. Studies have discussed different methods devised for RBC production, including using PSCs by repeating the developmental haematopoiesis; reprogramming somatic cells through transcription factors including OCT4, SOX2, c-MYC, KLF4, NANOG, LIN28; and stimulating the maturation of hematopoietic stem cells isolated from peripheral or umbilical cord blood [67,68]. The advantage of using iPSCs is their ability to differentiate into any cell type, and can be maintained indefinitely, thus becoming a potential source for cell replacement therapies. The potential of iPSc becomes highlighted by the fact that the French National Registry of People with a Rare Blood Phenotype/Genotype claims a single iPSc clone from their database could meet 73% of the needs of sickle cell disease patients [69]. This highlights that a limited number or RBC clones have the potential to supply to the majority needs of alloimmunized patients with rare blood groups.

Studies have also worked on developing iPSC models for blood malignancies including myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), and myeloproliferative neoplasms (MPN). A study worked on generating iPSC clones from bone marrow and blood of patients by integrating mutational analysis with cell programming to generate different iPSC clones which represent different disease stage as well as spectrum of the diseases including predisposition, low- and high-risk conditions. Additionally, the researchers also utilized the CRISPR/Cas9 system to introduce as well as correct mutations in the iPSCs. This study found iPSC from AML patients upon differentiation exhibited the leukemic phenotype, and the derived hematopoietic stem cells contained two immunophenotypically distinct cell populations; an adherent and non-adherent fraction, wherein the adherent fraction cells continuously renewed and generated the non-adherent cells. The AML-iPSC thus generated was found to exhibit characteristics of the leukemia stem cell model thus becoming an efficient model for molecular analysis and studying key functional aspects to be utilized for developing better therapeutics [70]. In case of chronic myeloid leukemia (CML), the BCR-ABL gene fusion is the major disease driver, and treatment involves use of tyrosine kinase inhibitor (TKI), causing remission in the vast majority of the cases. Studies have shown the CML-iPSCs to not be affected by TKI even in presence of BCR-ABL expression, indicating absence of dependency in this state of differentiation. The CML-iPSCs factors essential for maintenance of BCR-ABL positive and iPSCs including phosphorylation of AKT, JNK, ERK1/2 remained unchanged while the expression of STAT5 and CRKL was decreased. Further, the hematopoietic cells derived from CML-iPSC regained TKI sensitivity thus facilitating understanding on the disease pathogenesis better [71,72]. In case of MDS, reprogramming to generate iPSCs has been done from patients with del7q mutation, which is the signature for the disease. The iPSCs with the mutation upon hematopoietic differentiation were found to generate low quantities of CD34+/CD45+ myeloid progenitor cells. Further, studying genetically engineered clones as well as the MDS-iPSC-del7q clone from the patient, the researchers functionally mapped MDS phenotype to regions 7q32.37q36.1, which is linked to loss of hematopoietic differentiation potential [73]. To highlight the efficiency of iPSC-technology in precision oncology, studies have also created isogenic iPSCs with del7q and mutation SRSF2 P95L, each of these connected to a specific phenotype and drug response [74].

Human iPSC preclinical models also exist for monogenic blood disorders including thalassemia, and hemoglobinopathies for gene and cell therapy. Pilot trial investigations have explored the safety and effectiveness of mobilizing CD34+ hematopoietic progenitor cells in beta-thalassemia major adults. Further, the CD34+ were transduced with globin lentiviral vector, wherein the vector-encoded beta-chain was found to be expressed at normal hemizygous protein output levels in NSG mice. This trial thus validated an effective protocol for beta-globin gene transfer among thalassemia major CD34+ hematopoietic progenitor cells [75]. The risk of insertional mutagenesis using hematopoietic stem cells can be overcome through iPSCs which can be cloned and the clones with vector integration in the safe harbor sites become possible. The genomic safe harbors (GSHs) ensure that the inserted new genetic material functions as predicted, and do not cause any alterations to the host genome [76]. Studies have shown the use of gene editing tools in case of beta-thalassemia to not be successful in expression of beta-globin in the corrected locus, because of the developmental immaturity of the iPSCs. In such cases, insertion of globin gene copy in the GSH site like AAVS1 has been recommended as an alternative approach [77]. Human iPSC models for gene therapy have also been developed and studied for primary immunodeficiency syndromes, including chronic granulomatous disease (CGD) caused by mutations in genes which code for the phagocyte NADPH oxidase that produces reactive oxygen species (ROS) that kill bacteria. Studies have shown genetically corrected CGD-iPSCs from macrophages and neutrophils using CRISPR/Cas9 system in the single intronic mutation of the CYBB gene to exhibit antimicrobial activity through generation of ROS and phagocytosis [78].

Thus, the potential of iPSCs to study etiology of complex diseases which manifest late in life, as well as to identify markers for precision therapeutics, is worth exploring in the arena of clinical biomedical research. Human iPSC-based models are a true success in our understanding of disease pathogenesis away from the animal models.

Organ donations are a key clinical need to treat end-stage organ failure conditions, and in often cases, patients are left to fight the acute shortage for the same. This apart, from identifying HLA-matched donors, handling risk of infections and rejection, as well as life-long immunosuppression, to a great extent damages quality of life for the affected as well as leads to loss of crucial time. Human iPSCs are being evaluated as a potential source for generating organs that can overcome roadblocks of shortage as well as risk of rejection. Studies have explored the possibility of generating a three-dimensional vascularized and functional liver organ from human iPSCs [79,80,81]. Generation of hepatocyte-like cells using iPSC technology has been reviewed to be fundamentally beneficial for treatment of severe liver disease, screening for drug toxicities, in liver transplantation, as well as to facilitate basic research [21]. Liver organogenesis involves delamination of specific hepatic cells from the foregut endodermal sheet to form a liver bud, which is then vascularized. One study prepared hepatic endoderm cells from human iPSCs through direct differentiation, wherein 80% of the treated cells were found to be positive for the cell fate determining hepatic marker; HNF4A. Further, to stimulate early organogenesis, the iPSCs were cocultured with stromal cells, human umbilical vein endothelial cells, and human mesenchymal stem cells, and after 48h of seeding, the human iPSCs were found to be self-organized into three-dimensional cell clusters visible macroscopically. This iPSC-derived liver bud, when further assessed by quantitative polymerase chain reaction (PCR) and microarray assay for expression analysis, highlighted the pattern to be similar to human fetal liver cell-derived liver buds. Hemodynamic stimulation to form organ was done by cranial window model, and the iPSC-derived tissue was found to perform liver-specific functions including protein synthesis and human-drug specific metabolism actions. This proof-of-concept study highlights the potential to use organ-bud transplantation for organ regeneration [82]. Figure 3 highlights the process of liver development and hepatic differentiation from hiPSCs.

Process of liver development and hepatic differentiation from hiPSCs. The process of isolated cells from patients can be cultured and reprogrammed into patient-specific hiPSCs and quick comparison from natural liver development.

Hepatocytes represent 80% of the liver mass and are the specialized epithelial cells crucial for maintaining homeostasis. The hepatic differentiation involves induction of endoderm differentiation by activin A, fibroblast growth factor 2 (FGF2), and bone morphogenetic protein 4 (BMP4), and such generated hepatocytes have been found to retain features of human liver including lipid and glycogen storage, urea synthesis, etc. Cholangiocytes in the inner space of the bile duct tree have also been generated from the common progenitor hepatoblast, through downregulation of signaling factors including epidermal growth factor (EGF), interleukin 6 (IL-6), Jagged 1, sodium taurocholate, and the generated cholangiocytes have been detected to express mature markers including SOX9 (SRY-Box Transcription Factor 9), OPN (Osteopontin), CK7 (Cytokeratin 7), CK19 (Cytokeratin 19), etc. The kupffer cells are the largest population of resident macrophages in the human body and also facilitate liver regeneration after an ischemic injury. Studies have demonstrated generation of iPSC-derived kupffer cells from macrophage precursors by adding a hepatic stimulus [83,84].

Another study evaluated lung regeneration by endogenous and exogenous stem cell mediated therapeutic approaches. Physiologically the tissue turnover rate in lung is slow and any insult to the regeneration process can lead to development of chronic obstructive pulmonary disease (COPD) as well as idiopathic pulmonary fibrosis. Bone marrow stem cells, embryonic stem cells, as well as iPSCs have shown excellent regenerative capacity to repair injured lung by generating whole lung in the lab using de-cellularized tissue scaffold and stem cells [85]. Lung organogenesis involves proximodistal patterning, branching morphogenesis, alveolarization, and cellular differentiation [86]. A study by Mou et al. [87], described generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis (CF) iPSCs. The definitive endoderm from mouse ESCs were converted to foregut endoderm and then into replicating lung endoderm+Nkx2.1 (earliest marker of lung endoderm), which further transformed to a multipotent embryonic lung progenitor and airway progenitor cells. This study further highlighted that precise timing of the BMP, WNT, FGF signaling pathways are crucial for induction of NKX2.1. This study also utilized the same strategy to develop disease-specific lung progenitor cells from CF-iPSCs to make a model platform to study lung diseases. Further, the disease-specific lung progenitors were also engrafted in immunodeficient mice. One study derived lung progenitor cells with ~80% efficiency from iPSCs which differentiated onto alveolar epithelium both in vitro and in vivo. This study used Activin/BMP-4/bFGF treatment to obtain definitive endoderm from iPSC, which was further exposed to a series of pathway inhibitors (BMP, TGF-, WNT), followed by longer exposure to FGF-19, KGF, BMP-4 and a small molecule CHIR99021 to mimic Wnt pathway to generate anterior foregut endoderm. The generated lung progenitors were further differentiated to many pulmonary progenitor cells including basal cells, goblet cells, ciliated cells, in vitro as well as in immunodeficient mice [88].

Studies have also utilized iPSC-derived organ models to study pathogenesis of the coronavirus disease-2019 (COVID-19). One study established a screening strategy to identify drugs that reduce angiotensin converting enzyme 2 (ACE2) using human ESCs-derived cardiac cells and lung organoids, as the infection occurs due to binding of the virus to ACE2 on the cell membrane. Target analysis revealed treatment with antiandrogenic drugs to reduce ACE2 expression, thus protecting the lung organoids from the SARS-CoV-2 infection. Clinical studies on COVID-19 identified patients with prostate disease, with elevated levels of circulating androgen to pose increased risk for high disease severity [89]. Another study utilized human lung stem-cell based alveolospheres to generate insights on SARS-CoV-2 mediated interferon response and pneumocyte dysfunction. This study described a chemically defined modular alveolosphere culture system for propagation and differentiation of the human alveolar type 2 (AT2) derived from primary lung tissue. The cultured cells were found to express ACE2 and transcriptome analysis of the infected alveolospheres were found to mirror features of the COVID-19 infected human lung, together with the interferon-mediated inflammatory response, loss of surfactant proteins, and apoptosis. Further, infected alveolospheres when treated with low dose interferons, a reduction in viral replication was noted. Thus, human stem-cell based models have also added insight to COVID-19 pathogenesis [90]. In case of use of iPSC three-dimensional model, a study by Huang et al. [91] found the derived AT2 to be susceptible to SARS-CoV-2 with decreased expression of surfactant proteins, and cell death, exhibiting delayed type I interferon response with multiplicities of infection of 5 and interferon-stimulated genes. Another study assessed inhibitor of SARS-CoV-2 infection using lung and colonic organoids from the gut. The derived iPSCs in three-dimensional, were positive for SARS-CoV-2 infection. In case of immune response, the tumor necrosis factor (TNF) and interleukin-17 (IL-17) signatures were noted after 24 h with multiplicities of infection of 0.1. This study also screened US Food and Drug Administration (USFDA) approved entry inhibitors including imatinib, mycophenolic acid, and quinacrine dihydrochloride; wherein treatment at physiologically relevant levels highlighted inhibition of SARS-CoV-2 infection both in iPSC-lung organoids and colonoids, indicating that iPSC models also prove to be a valuable source for safe drug screening [92].

Development of organ-specific progenitor cells which progress into the complete three-dimensional organ in a lab highlights the potential of iPSCs in regenerative medicine. Further, the impact of organ-system models to study infection pathology, highlights the wide clinical arena in which iPSC-technology can be used.

The iPSCs have been generated for modelling pathogenesis of many diseases, and one of the most notable additions to the same is cancer, including models for familial cancer syndromes. One such study reports on the successful establishment of Li-Fraumeni Syndrome (LFS) patient-derived iPSC to study role of p53 in development of osteosarcoma. LFS being a heterogenous cancer condition, osteosarcoma is one of the types wherein relevance of germline p53 mutations have been highly reported. The pre-existing murine LFS models have been insufficient in charting the entire tumor landscape and patient-derived iPSCs in this regard have demonstrated the feasibility to effectively study human cancer syndromes. Studies have found the LFS-derived mesenchymal stem cells to exhibit low expression of targets of p53 including p21 and MDM2; highlighting their ability to retain the defective p53 function from the parental fibroblasts. Further, p53 knockdown was found to cause upregulation of osteogenic markers in LFS osteoblasts, and the possibility to attain osteosarcoma-related phenotypes in LFS iPSC-derived osteoblasts was found. Further, gene expression analysis in LFS-derived osteoblasts was found to correlate with poor patient survival, and decreased time for recurrence. The impaired H19 restoration was also found to repress tumorigenic potential [36]. Another study involving modelling of osteosarcoma from LFS derived-iPSC identified the LFS osteoblasts to recapitulate oncogenic properties of osteosarcoma proving to be an excellent model to study disease pathogenesis [93]. In case of Noonan syndrome (NS) characterized by germline PTPN11 mutations, studies which have derived hiPSCs from hematopoietic cells and which harbor the PTPN11 mutations were found to successfully recapitulate features of NS. The iPSC-derived NS myeloid cells were found to exhibit increased STAT5 signaling and enhanced expression of micro-RNAs viz. miR-223 and miR-15a. Further, reducing miR-223 function was found to normalize myelogenesis, highlighting the role of micro-RNA dysregulation in early oncogenesis [94]. Human iPSC-derived hereditary cancer models have also aided in identifying BRCA1-deleted tumor niche to be the cause for disease progression [95].

The iPSC models around cancer aid in overcoming the hurdles posed by traditional cancer cell line systems, which may lose the characteristics of the original tumor with time, and further harnessing primary cancer cells at different stages of carcinogenesis is not feasible. The established iPSC reprogramming strategies can aid in differentiation of cancer cells to target cell lineages which can aid in studying each of the different stages in cancer progression [96]. The iPSCs developed from primary tumors, as well as cancer cell lines are invaluable tools to study genetic alterations early-on in familial cancer syndromes which is crucial in understand disease pathogenesis. Apart from cancer cell lines, patient-derived xenograft models have also been proven to be efficacious for understanding tumor heterogeneity, genetic alterations, and testing efficacy of cytotoxic drugs. However, the need for successful engraftment, technical challenges, and variable growth rates, are the key limitations. Even in case of animal models, high rate of mortality, and absence of metastasis are the limitations [97,98,99]. Advancements in iPSC models have also led researchers to be able to design autologous iPSC-based vaccine which presents a broad spectrum of tumor antigens to the immune system of the mice, and also found success in eliciting a prophylactic reaction against multiple cancer types. These studies highlight the great promise iPSC-based autologous vaccines present towards cancer prevention as well as therapy [100].

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Coordinated immune networks in leukemia bone marrow microenvironments distinguish response to cellular therapy  Science

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Coordinated immune networks in leukemia bone marrow microenvironments distinguish response to cellular therapy - Science

Continuing Education Activity

Hematopoietic stem cell transplant (HPSCT), sometimes referred to as bone marrow transplant, involves administering healthy hematopoietic stem cells to patients with dysfunctional or depleted bone marrow. There are several types of HPSCT in clinical use, and transplanted cells may be obtained from several sources. This procedure has several benefits and may be used to treat malignant and non-malignant conditions. It helps to augment bone marrow function. In addition, depending on the disease being treated, it may allow for the destruction of malignant tumor cells. It can also generate functional cells that replace dysfunctional ones in cases like immune deficiency syndromes, hemoglobinopathies, and other diseases. Survival rates after HPSCT are increasing, but morbidity due to complications of the procedure continues. This activity reviews the indications for HPSCT, the different options by which to obtain donor cells, including the advantages and disadvantages of each, and the acute and chronic complications of the procedure. Additionally, it highlights the role of the interprofessional team in managing patients who undergo HPSCT to improve patient outcomes and decrease procedure-associated morbidities.

Objectives:

Describe the malignant and non-malignant indications for hematopoietic stem cell transplants.

Contrast the advantages and disadvantages of different types of hematopoietic stem cells.

Outline the potential complications of hematopoietic stem cell transplants and apply strategies to ameliorate these risks.

Describe the need for a well-integrated, interprofessional team approach to improve care for patients undergoing hematopoietic stem cell transplants.

Hematopoietic stem cell transplant (HPSCT), sometimes referred to as bone marrow transplant, involves administering healthy hematopoietic stem cells to patients with dysfunctional or depleted bone marrow. This procedure has several benefits. It helps to augment bone marrow function. In addition, depending on the disease being treated, it may allow for the destruction of malignant tumor cells. It can also generate functional cells that replace dysfunctional ones in cases like immune deficiency syndromes, hemoglobinopathies, and other diseases.

History and Evolution

Hematopoietic stem cell transplantation (HPSCT) was first explored for use in humans in the 1950s. It was based on observational studies in mice models, which showed that infusion of healthy bone marrow components into a myelosuppressed bone marrow could induce recovery of its function in the recipient.[1]These animal-based studies soon found their clinical application in humans when the first successful bone marrow transplant was performedbetween monozygotic twins in New York in 1957 to treatacute leukemia.[2]The performing physician, E. Donnell Thomas, continued his research on the development of bone marrow transplantationand later received the Nobel Prize for Physiology and Medicine for his work. The first successfulallogeneicbone marrow transplant was reported in Minnesota in 1968 for a pediatric patient with severe combined immunodeficiency syndrome.[3]

Since then, allogeneic and autologous stem cell transplants have increased in the United States (US) and worldwide. The Center for International Blood and Marrow Transplant Research (CIBMTR) reported over 8000 allogeneic transplants performed in the US in 2016, with an evengreaternumber of autologous transplants; autologous transplants have steadily outpaced allogeneic transplants over time.[4][5]

Definitions

Major Histocompatibility Complex (MHC)

The human MHC genes on the short arm of chromosome 6 (6p) encode for human leukocyte antigens (HLA) and are highly polymorphic. These polymorphisms lead to significant differences in the resultant expressed human cell-surface proteins. They are divided into MHC class I and MHC class II.

Human Leukocyte Antigens (HLA)

The HLA proteins are expressed on the cellular surface and play an essential role in alloimmunity. HLA class I molecules, encoded by MHC class I, can be divided into HLA-A, HLA-B, and HLA-C. These proteins are expressed on all cell types and present peptides derived from the cytoplasm and recognized by CD8+ T cells. HLA class II molecules are classified as HLA- DP, HLA-DQ, and HLA-DR, are encoded by MHC class II, can be found on antigen-presenting cells (APCs), andare recognized by CD4+ T cells.

Syngeneic Bone Marrow Transplantation

The donor and the recipient are identical twins. The advantages of this type of transplant include no risk of graft versus host disease (GVHD) or graft failure. Unfortunately, however, only a very fewtransplant patients will have an identical twin available for transplantation.

Autologous Bone Marrow Transplantation

The bone marrow products are collected from the patient and are reinfused after purification methods. The advantage of this type of transplantis no risk of GVHD. The disadvantage is that the reinfused bone marrow products may contain abnormal cells that can cause relapse in the case of malignancy; hence, theoretically, this method cannot be used in all cases of abnormal bone marrow diseases.

Allogeneic Transplantation

The donor is an HLA-matched family member, an unrelated HLA-matched donor, or a mismatched family donor (haploidentical).

Engraftment

The process by which infused transplanted hematopoietic stem cells produce mature progeny in the peripheral circulation.

Preparative Regimen

This regimen comprises high-dose chemotherapy or total body irradiation (TBI) or both, which are administered to the recipient before stem cell infusion to eliminate the largest number of malignant cells and induce immunosuppression in the recipient so that engraftment can occur.

Malignant Disease

Multiple Myeloma

Studies have shown increased overall survival and progression-free survival in patients younger than 65 years when consolidation therapy with melphalan is initiated, followed by autologous stem cell transplantation and lenalidomide maintenance therapy.[6]The study showed a favorable outcome of high-dose melphalan plusHPSCT compared to consolidation therapy with melphalan, prednisone, and lenalidomide. It also showed better outcomes in patients who received maintenance therapy with lenalidomide.

Hodgkin and Non-Hodgkin Lymphoma

Studies have shown that in cases of recurrent Hodgkin and Non-Hodgkin lymphomas that do not respond to initial conventional chemotherapy, chemotherapy followed by autologous stem cell transplantation leads to better outcomes. A randomized controlled trial by Schmitz showed a better outcome at three years of high-dose chemotherapy with autologous stem cell transplant compared to aggressive conventional chemotherapy in relapsed chemosensitive Hodgkin lymphoma. However, the overall survival was not significantlydifferent between the two groups.[7]CIBMTR reports that his group of malignancies accounts for the second highest number of HPSCTs in the US, after multiple myeloma.

Acute Myeloid Leukemia (AML)

Allogeneic stem cell transplant has been shown to improve outcomes. It may prolong overall survival in patients with AML who fail primary induction therapy and do not achieve a complete response.[8]The study recommended that early HLA typing for patients with AML is beneficial if they fail induction therapy and are considered for HPSCT.

Acute Lymphocytic Leukemia (ALL)

Allogeneic stem cell transplant is indicated in refractory and resistant cases of ALL when induction therapy fails for a second time to induce remission. Some studies suggest an increased benefit of allogeneicHPSCT in patients with high-risk ALL, including patients with the Philadelphia chromosome and those with t(4;11).[9]

Myelodysplastic Syndrome (MDS)

Allogeneic stem cell transplant is considered curative in cases of disease progression and is only indicated in intermediate- or high-risk patients with MDS.

Chronic Myeloid Leukemia (CML) and Chronic Lymphocytic Leukemia (CLL)

Patients with CML and CLL received the fewest number of allogeneic transplants in 2020.HPSCT has high cure rates for CML, but because tyrosine kinase inhibitors pair high success rates with a low adverse risk profile, HPSCTis reserved for patients with refractory disease.

Myelofibrosis, Essential Thrombocytosis, and Polycythemia Vera

Allogeneic stem cell transplant has been shown to improve outcomes in patients with myelofibrosis and those diagnosed with myelofibrosis preceded by essential thrombocytosis or polycythemia vera.[10]

Solid Tumors

Autologous stem cell transplant is consideredthestandard of care in patients with testicular germ cell tumors that are refractory to chemotherapy; in this case, refractory is defined as the third recurrence with chemotherapy.[11]HPSCT has also been studied in medulloblastoma, metastatic breast cancer, and other solid tumors.

Non-Malignant Diseases

Aplastic Anemia

Systematic and retrospective studies have suggested an improved outcome with HPSCT in acquired aplastic anemia compared to conventional immunosuppressive therapy.[12]In a study of 1886 patients with acquired aplastic anemia, transplanted cells collected from the bone marrow produced superior outcomes compared to those collected from the peripheral blood.[13]Patients with aplastic anemia need a preparative regimen, as they still can develop immune rejection to the graft.

Severe Combined Immune Deficiency Syndrome (SCID)

Large retrospective studies have shown increased overall survival in infants with SCID when they received the transplant early after birth before the onset of infections.[14]

Thalassemia

Allogeneic stem cell transplant from a matched sibling donor is an option to treat certain types of thalassemia and has shown 15-year survival rates reaching near 80%. However, recent retrospective data showed similar overall survival compared to conventional treatments withmultiple blood transfusions.[15]

Sickle CellDisease

An allogeneic stem cell transplant is recommended to treat sickle cell disease.[16]

Other Non-malignant Diseases

HPSCT has been used to treat chronic granulomatous disease, leukocyte adhesion deficiency, Chediak-Higashi syndrome, Kostman syndrome, Fanconi anemia, Blackfan-Diamond anemia, and enzymatic disorders.Moreover, the role ofHPSCT is expanding in non-malignant autoimmune diseases, including systemic sclerosis and systemic lupus erythematosus, and has already shown promising results in cases like neuromyelitis optica.[17][18][19][20][21][22][23][24][25] It is also considered best practice for relapsing-remitting multiple sclerosis.[26][27]

There are no absolute contraindications for hematopoietic stem cell transplant.

Special equipment exists for collecting, preserving, and administering stem cell products.

An interprofessional team approach is amainstay of ensuring the high-quality collection and infusion of stem cell products.

Preparation includes:

Preparativeregimen:high-dose chemotherapy ortotal body irradiation (TBI) or both

Collection of hematopoietic stem cells

Instant infusion or cryopreservation followed by infusion

Mechanism of Action

The mechanism of action of HPSCT in leukemia is based on the effect of the graft and donor immunity against malignant cells in recipients. These findings were demonstrated in a study that involved over 2000 patients with different leukemias treated with HPSCT. The study showed the lowest relapse rates were in patients who received non-T-cell-depleted bone marrow cells and those who developed GVHD compared to patients who received T-cell-depleted stem cells, those who did not develop GVHD, and patients who received syngeneic grafts. These findings support the notion that donor cellular immunity is central to engraftment efficacy against tumor cells.[28]

The mechanism of action of HPSCT in autoimmune diseases is believed to be secondary to the increase in T-cell regulatory function, which promotes immune tolerance. However, more studies are needed to determine the exact physiology.

In hemoglobinopathies, the transplanted stem cells produce functional cells after engraftment that replace the diseased cells.

Administration

HLA Typing

HLA typing is essential to determine the most suitable donor for stem cell collection. In theory, matched, related donors are the best candidates, followed by matched unrelated donors, cord blood, and haploidentical donors. HLA typing is analyzed at either an intermediate-resolution level, which entails detecting a small number of matched alleles between the donor serum and the recipient, or at a high-resolution level to determine the specific number of polymorphic alleles at a higher level. Polymerase chain reaction and next-generation sequencing are used for HLA typing, and the results are reported as a score correlating with a match of two alleles for a specific HLA type. Different institutions use a different number of HLA subtypes for the eligibility of donors. However, studies that showed high-resolution matching for HLA-A, HLA-B, HLA-C, and HLA-DRB1 were associated with improved survival and outcomes.[29]The Blood and Marrow Transplant Clinical Trials Network (BM CTN) has proposed donor HLA assessment and matching recommendations.[30]

The process may vary depending on the source of the stem cell site collection, whether it is bone marrow, peripheral blood, or cord blood. Moreover, there is a slight difference based on whether it is autologous, allogeneic, or syngeneic HPSCT. For example, the procedure consists of the initial mobilization of stem cells, in which peripheral blood stem cells are collected, given the low number and the need for high levels of progeny cells. This is thenfollowed by a preparative regimen and, finally, infusion.

Mobilization and Collection

Mobilization and collection procedures involve using medication to increase the number of stem cells in the peripheral blood, given that there are insufficient stem cells in the peripheral blood. Medications include granulocyte colony-stimulating factors (G-CSF) or chemokine receptor 4 (CXCR4) blockers like plerixafor. G-CSF is believed to enhance neutrophils to release serine proteases, which break vascular adhesion molecules and promote the release of hematopoietic stem cells from the bone marrow. Plerixafor blocks the binding of stromal cell-derived factor-1-alpha (SDF-1) to CXCR, leading to stem cell mobilization to the peripheral blood.[31]CD34+ is considered the marker for progenitor hematopoietic stem cells in the peripheral blood, and usually, a dose of 2 to 10 x 10/kg CD34+ cells/kg is needed for proper engraftment. Chemotherapy can sometimes be used to mobilize hematopoietic stem cells; this process is termed chemoembolization.

The usual site of bone marrow collection is the anterior or posterior iliac crest. The aspiration procedure can be performed under local or general anesthesia. Common complications include pain and fever; serious iatrogenic complications occur in less than 1% of cases. Each aspiration contains 15 mL, and multiple aspirations are done. The goal is to collect 1 to 1.5 L of bone marrow product from the aspirations. The dose of nucleated cells from bone marrow should range between 2 to 4 x 10 cells/kg; overall survival and long-term engraftment are strongly influenced by cell dose in allogeneic HPSCT.[32]

Preparative Regimen

The preparative regimen consists of the administration of chemotherapy with or without total body irradiation for the eradication of malignant cells and induction of immune tolerance for the transfused cells to engraft properly. This process is not limited to patients with malignancies. It extends to cases like aplastic anemia and hemoglobinopathies, given that these patients have intact immune systems that could cause graft failure if there is no conditioning.

The administration of the preparative regimen should immediately precede the HPSCT. As a general rule, the effect of the regimen should produce bone marrow suppression within 1 to 3 weeks of administration. The preparative regimen is divided into myeloablative conditioning and reduced-intensity conditioning. Different combination regimens are used in the preparative period, depending on the disease being treated, existing comorbidities, previous radiation exposure, and the source of the harvested hematopoietic stem cells.

Reduced-intensity conditioning is preferred in patients who are older, have had prior radiotherapy, have comorbidities, and have a history of extensive chemotherapy before HPSCT.[33]The advantages of using reduced-intensity conditioning include less need for transfusion due to transient post-transplant pancytopenia, less chemotherapy-induced liver damage, and less radiation-induced lung damage.[34]However, the relapse rates after reduced-intensity conditioning are higher. Nevertheless, these regimens are better tolerated and have a better safety profile in specific patient populations.

Most chemotherapies used in preparative regimens consist of potent immunosuppressive agents like high doses of cyclophosphamide, alkylating agents like busulfan, nucleoside analogs like fludarabine, and many other agents like melphalan, anti-thymocyte globulin, rituximab, and gemcitabine. Totalbodyirradiation is performed using fractionated doses; there is less pulmonary toxicity than with a one-dose regimen.[35]

Reinfusion of either fresh or cryopreserved stem cells can occur in an ambulatory setting and takes up to two hours. Before the infusion begins, quality measures are performed to ensure the number of CD34+ cells is sufficient.

In the particular case of SCID, there is no need for a preparative regimen in patients receiving cells from HLA-matched siblings. This is because no abnormal cells need to be eliminated, and the immunosuppression caused by SCID can prevent graft rejection.

Advantages and Disadvantages of Different Hematopoietic Stem Cells

One advantage of peripheral blood stem cell transplant (PBSCT) is a more rapid engraftment rate than the bone marrow-derived stem cells; recovery in the former is two weeks and is delayed for five days more in the latter. Using a post-transplant immunosuppressive regimen to prevent GVHD can prolong the increase in bone marrow products.[36] Moreover, the rate of acute GVHD between PBSCT and bone marrow transplantation appears to be similar in HLA-identical matched related donors.[36]However, chronic GVHD is a more common occurrence after PBSCT, which could lead to more complications. Two-year overall survival rates seem to be similar regardless of stem cell origin.[37]Other studies comparing bone marrow-derived transplant andPSCT concluded that the psychological burden due to chronic GVHD and the 5-year ability to restore normal activities, including returning to work, was better in the bone marrow-derived transplant group.[38]

The advantages of cord blood transplant include the rapid collection and administration times, which facilitate treating urgent conditions, less frequent infections, lower rates of GVHD with the same rate of GVT, and less need for a stringent identical HLA. The disadvantages include delayed engraftment, a higher possibility of graft rejection, and higher rates of disease relapses. The cord blood transplant is most commonly used in patients without matched-related or unrelated donors. One major study demonstrated the utility of cord blood transplants in patients with thalassemia-major and sickle cell disease,indicating similar 6-year overall survival rates compared to the bone marrow-derived transplants.[39]

The most important factors affecting the success of cord blood transplant are the total nucleated cell dose and HLA matching; the recommended minimum dose of total nucleated cells for successful engraftment is 2 x 10^7 cells/kg. Theoretically, strict HLA matching is not required in the case of cord blood transplant as cord blood is devoid of mature T cells, but studies have shown better outcomes when matching recipients at HLA-A, HLA-B, HLA-C, and HLA-DRB1.[40]Given that a single cord blood unit might not contain the required amount of nucleated cells, a double cord transplant is used. However, only one cord blood transplant product will dominate within three months of infusion. Further, randomized controlled trials failed to show a significant difference in outcome, benefits, or risks between double cord blood and a single cord blood transplant.[41][42]

Haploidentical stem cell transplantation involves administering bone marrow products from a first-degree related haplotype-mismatched donor.[43]This helps underserved patients without broad access to resources as they have fewer chances of having a matched unrelated donor.[44]The advantages of this method include lower cost and rapid availability of hematopoietic cell products. However, the disadvantages include hyperacute GVHD, which increases mortality and graft rejection.[45]This has been overcome by the depletion of T cells responsible for the reaction mentioned above, but this also leads to delayed immune recovery and decreased graft versus tumor effect. Recently strategies including selective depletion of subsets of T cells, including alpha-beta, have shown improved outcomes compared to conventional ex vivo depletion of large T-cell populations.[46]

Complications after bone marrow transplant may be acute or chronic. Many factors can affect these adverse events, including age, baseline performance status, the source of stem cell transplant, and the type and intensity of the preparative regimen. Acute complications occur in the first 90 days, including myelosuppression with neutropenia, anemia, or thrombocytopenia; sinusoidal obstruction syndrome; mucositis; acute graft versus host disease; bacterial infections with gram-positive and gram-negative organisms; Herpesviridaeinfections; and fungal infection withCandidaand Aspergillus. Chronic complications include chronic GVHD, infection with encapsulated bacteria, and reactivation of the varicella-zoster virus.

Antimicrobial Prophylaxis

Levofloxacin is usually given orally or intravenously and initiated on the first day post-transplant. It is continued until the absolute neutrophil count is more than 1000 cells/microL or until the discontinuation of prednisonein cases of GVHD.[47]

Prophylaxis against Pneumocystis jirovecii (PCP)is warranted, given the immunosuppression following a hematopoietic stem cell transplant.[48]Trimethoprim-sulfamethoxazole (TMP-SMX) is usually used, and several dosing regimens have been proposed. TMP-SMX may be given twice weekly until the patient is off immunosuppression.[49]Antifungal infection prophylaxis with fluconazole is recommended for one month following the transplant as it has been shown to decrease the incidence of fungal infections. No difference was seen when fluconazole was compared to voriconazole.[50][51]However, voriconazole is used in patients with an elevated risk of developing severe antifungal infections.Anti-viral prophylaxis is achieved with acyclovir, continued for one month to prevent herpes-simplex virus and one year to prevent varicella-zoster virus.[52]Prophylaxis against cytomegalovirus is only recommended in patients who test positive by PCR, and the treatment of choice is ganciclovir.

One unique syndrome encountered with cord stem cell transplant is cord colitis which involves diarrhea in recipients of cord blood and is believed to be secondary to Bradyrhizobium enterica,which usually responds to a course of metronidazole or levofloxacin.[53]

Sinusoidal Obstruction Syndrome (SOS)

Sinusoidal obstruction syndrome (SOS), or veno-occlusive disease (VOD), results from chemotherapy during a preparative regimen and occurs within six weeks of HPSCT. This syndrome consists of tender hepatomegaly, jaundice due to hyperbilirubinemia, ascites, and weight gain due to fluid retention. The incidence is reported to be 13.6% in an analysis study assessing the existing literature on the incidence of the disease.[54]The pathophysiology consists of endothelial damage to the hepatic sinusoids leading to obstruction and necrosis of the centrilobular liver.[55]The destruction of the sinusoids leads to hepatic failure and hepatorenal syndrome, which areresponsible for the related mortality. The agents most commonly implicated in causing this syndrome are oral busulfan and cyclophosphamide. Using intravenous busulfan has been shown to decrease the occurrence of SOS.[56]

The diagnosis of SOS is clinical and is based on hyperbilirubinemia greater than 2 mg/dL in the presence of the aforementioned clinical findings. Treatment consists of ursodeoxycholic acid, which has been shown to significantly decrease the occurrence of SOS when given pre- and post-transplant.[57]Another medication, defibrotide, has shown efficacy in treating SOS when it occurs.[58][59]

Idiopathic Pneumonia Syndrome (IPS)

Idiopathic pneumonia syndrome usually occurs in the first 90 days post-transplant. The incidence is low and is related to the direct chemotoxicity of the preparative regimen. Treatment with steroids is standard, although no randomized controlled clinical trials have been done to support their efficacy. Recently, etanercept has been studied; adding soluble TNF-inhibitors to steroids has not shown added efficacy.[60]

Graft Rejection or Failure

A loss of bone marrow function after reconstitution following infusion of hematopoietic stem cells or no gain of function after infusion is termed graft rejection or failure. The incidence of failure is highest when there is a high HLA disparity; this disparity is highestin cases of cord blood and haploidentical donors and lowest with autologous and matched donor siblings. Factors responsible for graft failure include but are not limited to functional residual host immune response to the donor cells, a low number of infused cells, in vitro damage during collection and cryopreservation, inadequate preparative regimen, and infections.

Chimerism refers to the presence ofa cell population from a person in the blood of a different person. Evaluating for chimerism is an important step in ensuring engraftment and success of the transplantation. This evaluation is done by checking the expression of CD33, which indicates the presence ofgranulocytes, and CD3, which indicates the presence ofT cells, and confirming that most of thecells present are from the donor. The importance of effective chimerism has beendemonstrated in many studies that showed decreased relapse rates and increased survival in allogeneic transplantation.[61]

Graft Versus Host Disease (GVHD)

Graft versus host disease (GVHD) is a reaction between T cells from the donor in an allogeneic transplant and the recipient's HLA polymorphic epitopes, leading to a constellation of symptoms and manifestations. GVHD may be acute or chronic; each is sub-categorized into classic and late-onset, classic, and chronic overlap.[62]

Acute GVHD usually develops within three months. However, it can develop after three months and is then termed delayed acute GVHD. Prophylaxis is generally achieved with calcineurin inhibitors, methotrexate, and anti-thymocyte globulins. The severity of GVHD is estimatedusingthe Glucksberg scale, which classifies acute GVHD from grade I to VI. Treatment with either high-dose prednisone or methylprednisolone isindicated in higher-grade disease.[63]

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Hematopoietic Stem Cell Transplantation - StatPearls - NCBI ...

WARREN, N.J., Dec. 23, 2024 (GLOBE NEWSWIRE) -- Tevogen Bio (“Tevogen” or “Tevogen Bio Holdings Inc.”) (Nasdaq: TVGN), a clinical-stage specialty immunotherapy biotech developing off-the-shelf, genetically unmodified T cell therapeutics to treat infectious disease and cancers, announced today the company will host an AI panel during the 43rd Annual J.P. Morgan Healthcare Conference in San Francisco, California. The panel, titled "AI in Biopharma: Next Frontier of Medical Innovation," will explore the transformative potential of artificial intelligence in the biopharma industry. Panelists will include Dr. David Rhew, Global Chief Medical Officer & VP of Healthcare of Microsoft (Nasdaq: MSFT), Mittul Mehta, Chief Information Officer and Head of Tevogen.AI, and Dr. Sean Tunis, Principal of Rubix Health.

Excerpt from:
Tevogen Bio to Host Panel "AI In Biopharma: Next Frontier of Medical Innovation" During the 43rd Annual J.P. Morgan Healthcare Conference

Stem cell transplants are used to put blood stem cells back into the body after the bone marrow has been destroyed by disease, chemotherapy (chemo), or radiation. Depending on where the stem cells come from, the transplant procedure may go by different names:

All of these can also be calledhematopoietic stem cell transplants.

In a typical stem cell transplant for cancer, a person first gets very high doses of chemo, sometimes along with radiation therapy, to try to kill all the cancer cells. This treatment also kills the stem cells in the bone marrow. This is called myeloablation or myeloablative therapy.

Soon after treatment, blood stem cells are given (transplanted) to replace those that were destroyed. The replacement stem cells are given into a vein, much like ablood transfusion. The goal is that over time, the transplanted cells will settle in the bone marrow, where they will begin to grow and make healthy new blood cells. This process is called engraftment.

There are 2 main types of transplants. They are named based on who donates the stem cells.

In this type of transplant, the first step is to remove or harvest your own stem cells. Your stem cells are removed from either your bone marrow or your blood, and then frozen. (You can learn more about this process at Whats It Like to Donate Stem Cells?) After you get high doses of chemo and/or radiation as your myeloablative therapy, the stem cells are thawed and given back to you.

This kind of transplant is mainly used to treat certain leukemias, lymphomas, and multiple myeloma. Its sometimes used for other cancers, like testicular cancer and neuroblastoma, and certain cancers in children. Doctors can use autologous transplants for other diseases, too, like systemic sclerosis, multiple sclerosis (MS), Crohn's disease, and systemic lupus erythematosus (lupus).

An advantage of an autologous stem cell transplantis that youre getting your own cells back. When you get your own stem cells back, you dont have to worry about them (called the engrafted cells or the graft) being rejected by your body. You also dont have to worry about immune cells from the transplant attacking healthy cells in your body (known as graft-versus-host disease), which is a concern with allogeneic transplants.

An autologous transplant graft might still fail, which means the transplanted stem cells dont go into the bone marrow and make blood cells like they should.

Also, autologous transplants cant produce the graft-versus-cancer effect, in which the donor immune cells from the transplant help kill any cancer cells that remain.

Another possible disadvantage of an autologous transplant is that cancer cells might be collected along with the stem cells and then later be put back into your body.

To help prevent any remaining cancer cells from being transplanted along with stem cells, some centers treat the stem cells before theyre given back to the patient. This may be called purging. While this might work for some patients, there haven't been enough studies yet to know if this is really a benefit. A possible downside of purging is that some normal stem cells can be lost during this process. This may cause your body to take longer to start making normal blood cells, and you might have very low and unsafe levels of white blood cells or platelets for a longer time. This could increase the risk of infections or bleeding problems.

Another treatment to help kill cancer cells that might be in the returned stem cells involves giving anti-cancer drugs after the transplant. The stem cells are not treated. After transplant, the patient gets anti-cancer drugs to get rid of any cancer cells that may be in the body. This is called in vivo purging. For instance, lenalidomide (Revlimid) may be used in this way for multiple myeloma. The need to remove cancer cells from transplanted stem cells or transplant patients and the best way to do it continues to be researched.

Doing 2 autologous transplants in a row is known as a tandem transplant or a double autologous transplant. In this type of transplant, the patient gets 2 courses of high-dose chemo as myeloablative therapy, each followed by a transplant of their own stem cells. All of the stem cells needed are collected before the first high-dose chemo treatment, and half of them are used for each transplant. Usually, the 2 courses of chemo are given within 6 months. The second one is given after the patient recovers from the first one.

Tandem transplants have become the standard of care for certain cancers. High-risk types of the childhood cancer neuroblastoma and adult multiple myeloma are cancers where tandem transplants seem to show good results. But doctors dont always agree that these are really better than a single transplant for certain cancers. Because this treatment involves 2 transplants, the risk of serious outcomes is higher than for a single transplant.

Sometimes an autologous transplant followed by an allogeneic transplant might also be called a tandem transplant. (See Mini-transplants below.)

Allogeneic stem cell transplants use donor stem cells. In the most common type of allogeneic transplant, the stem cells come from a donor whose tissue type closely matches yours. (This is discussed in Matching patients and donors.) The best donor is a close family member, usually a brother or sister. If you dont have a good match in your family, a donor might be found in the general public through a national registry. This is sometimes called a MUD (matched unrelated donor) transplant. Transplants with a MUD are usually riskier than those with a relative who is a good match.

An allogeneic transplant works about the same way as an autologous transplant. Stem cells are collected from the donor and stored or frozen. After you get high doses of chemo and/or radiation as your myeloablative therapy, the donor's stem cells are thawed and given to you.

Allogeneic transplants are most often used to treat certain types of leukemia, lymphomas, multiple myeloma, myelodysplastic syndromes, and other bone marrow disorders such as aplastic anemia.

Blood taken from the placenta and umbilical cord after a baby is born can also be used for an allogeneic transplant. This small volume of cord blood has a high number of stem cells in it.

Cord blood transplants can have some advantages. For example, there are already a large number of donated units in cord blood banks, so finding a donor match might be easier. These units have already been donated, so they dont need to be collected once a match is found. A cord blood transplant is also less likely to be rejected by your body than is a transplant from an adult donor.

But cord blood transplants can have some downsides as well. There arent as many stem cells in a cord blood unit as there are in a typical transplant from an adult donor. Because of this, cord blood transplants are used more often for children, who have smaller body sizes. These transplants can be used for adults as well, although sometimes a person might need to get more than one cord blood unit to help ensure there are enough stem cells for the transplant.

Cord blood transplants can also take longer to begin making new blood cells, during which time a person is vulnerable to infections and other problems caused by having low blood cell counts. For a newer cord blood product, known as omidubicel (Omisirge), the cord blood cells are treated in a lab with a special chemical, which helps them get to the bone marrow and start making new blood cells quicker once theyre in the body.

A major benefit of allogeneic transplants is that the donor stem cells make their own immune cells, which could help kill any cancer cells that remain after high-dose treatment. This is called the graft-versus-cancer or graft-versus-tumor effect.

Other advantages are that the donor can often be asked to donate more stem cells or even white blood cells if needed (although this isn't true for a cord blood transplant), and stem cells from healthy donors are free of cancer cells.

As with any type of transplant, there is a risk that the transplant, or graft, might not take that is, the transplanted donor stem cells could die or be destroyed by the patients body before settling in the bone marrow.

Another risk is that the immune cells from the donor could attack healthy cells in the patients body. This is called graft-versus-host disease, and it can range from mild to life-threatening.

There is also a very small risk of certain infections from the donor cells, even though donors are tested before they donate.

Another risk is that some types of infections you had previously and which your immune system has had under control may resurface after an allogeneic transplant. This can happen when your immune system is weakened (suppressed) by medicines called immunosuppressive drugs. Such infections can cause serious problems and can even be life-threatening.

For some people, age or certain health conditions make it more risky to do myeloablative therapy that wipes out all of their bone marrow before a transplant. For those people, doctors can use a type of allogeneic transplant thats sometimes called a mini-transplant. Your doctor might refer to it as a non-myeloablative transplant or mention reduced-intensity conditioning (RIC). Patients getting a mini transplant typically get lower doses of chemo and/or radiation than if they were getting a standard myeloablative transplant. The goal in the mini-transplant is to kill some of the cancer cells (which will also kill some of the bone marrow), and suppress the immune system just enough to allow donor stem cells to settle in the bone marrow.

Unlike the standard allogeneic transplant, cells from both the donor and the patient exist together in the patients body for some time after a mini-transplant. But slowly, over the course of months, the donor cells take over the bone marrow and replace the patients own bone marrow cells. These new cells can then develop an immune response to the cancer and help kill off the patients cancer cells the graft-versus-cancer effect.

One advantage of a mini-transplant is that it uses lower doses of chemo and/or radiation. And because the stem cells arent all killed, blood cell counts dont drop as low while waiting for the new stem cells to start making normal blood cells. This makes it especially useful for older patients and those with other health problems. Rarely, it may be used in patients who have already had a transplant.

Mini-transplants treat some diseases better than others. They may not work well for patients with a lot of cancer in their body or people with fast-growing cancers. Also, although there might be fewer side effects from chemo and radiation than those from a standard allogeneic transplant, the risk of graft-versus-host disease is the same. Some studies have shown that for some cancers and some other blood conditions, both adults and children can have the same kinds of results with a mini-transplant as compared to a standard transplant.

This is a special kind of allogeneic transplant that can only be used when the patient has an identical sibling (twin or triplet) someone who has the exact same tissue type. An advantage of syngeneic stem cell transplant is that graft-versus-host disease will not be a problem. Also, there are no cancer cells in the transplanted stem cells, as there might be in an autologous transplant.

A disadvantage is that because the new immune system is so much like the recipients immune system, theres no graft-versus-cancer effect. Every effort must be made to destroy all the cancer cells before the transplant is done to help keep the cancer from coming back.

Improvements have been made in the use of family members as donors. This kind of transplant is called ahalf-match (haploidentical) transplant for people who dont have fully matching or identical family member. This can be another option to consider, along with cord blood transplant and matched unrelated donor (MUD) transplant.

If possible, it is very important that the donor and recipient are a close tissue match to avoid graft rejection. Graft rejection happens when the recipients immune system recognizes the donor cells as foreign and tries to destroy them as it would a bacteria or virus. Graft rejection can lead to graft failure, but its rare when the donor and recipient are well matched.

A more common problem is that when the donor stem cells make their own immune cells, the new cells may see the patients cells as foreign and attack their new home. This is called graft-versus-host disease. (See Stem Cell Transplant Side Effects for more on this). The new, grafted stem cells attack the body of the person who got the transplant. This is another reason its so important to find the closest match possible.

Many factors play a role in how the immune system knows the difference between self and non-self, but the most important for transplants is the human leukocyte antigen (HLA) system. Human leukocyte antigens are proteins found on the surface of most cells. They make up a persons tissue type, which is different from a persons blood type.

Each person has a number of pairs of HLA antigens. We inherit them from both of our parents and, in turn, pass them on to our children. Doctors try to match these antigens when finding a donor for a person getting a stem cell transplant.

How well the donors and recipients HLA tissue types match plays a large part in whether the transplant will work. A match is best when all 6 of the known major HLA antigens are the same a 6 out of 6 match. People with these matches have a lower chance of graft-versus-host disease, graft rejection, having a weak immune system, and getting serious infections. For bone marrow and peripheral blood stem cell transplants, sometimes a donor with a single mismatched antigen is used a 5 out of 6 match. For cord blood transplants a perfect HLA match doesnt seem to be as important, and even a sample with a couple of mismatched antigens may be OK.

Doctors keep learning more about better ways to match donors. Today, fewer tests may be needed for siblings, since their cells vary less than an unrelated donor. But to reduce the risks of mismatched types between unrelated donors, more than the basic 6 HLA antigens may be tested. For example, sometimes doctors to try and get a 10 out of 10 match. Certain transplant centers now require high-resolution matching, which looks more deeply into tissue types and allow more specific HLA matching.

There are thousands of different combinations of possible HLA tissue types. This can make it hard to find an exact match. HLA antigens are inherited from both parents. If possible, the search for a donor usually starts with the patients brothers and sisters (siblings), who have the same parents as the patient. The chance that any one sibling would be a perfect match (that is, that you both received the same set of HLA antigens from each of your parents) is 1 out of 4.

If a sibling is not a good match, the search could then move on to relatives who are less likely to be a good match parents, half siblings, and extended family, such as aunts, uncles, or cousins. (Spouses are no more likely to be good matches than other people who are not related.) If no relatives are found to be a close match, the transplant team will widen the search to the general public.

As unlikely as it seems, its possible to find a good match with a stranger. To help with this process, the team will use transplant registries, like those listed here. Registries serve as matchmakers between patients and volunteer donors. They can search for and access millions of possible donors and hundreds of thousands of cord blood units.

Be the Match (formerly the National Marrow Donor Program)Toll-free number: 1-800-MARROW-2 (1-800-627-7692)Website: http://www.bethematch.org

Blood & Marrow Transplant Information NetworkToll-free number: 1-888-597-7674Website: http://www.bmtinfonet.org

Depending on a persons tissue typing, several other international registries also are available. Sometimes the best matches are found in people with a similar racial or ethnic background. When compared to other ethnic groups, white people have a better chance of finding a perfect match for stem cell transplant among unrelated donors. This is because ethnic groups have differing HLA types, and in the past there was less diversity in donor registries, or fewer non-White donors. However, the chances of finding an unrelated donor match improve each year, as more volunteers become aware of registries and sign up for them.

Finding an unrelated donor can take months, though cord blood may be a little faster. A single match can require going through millions of records. Also, now that transplant centers are more often using high-resolution tests, matching is becoming more complex. Perfect 10 out of 10 matches at that level are much harder to find. But transplant teams are also getting better at figuring out what kinds of mismatches can be tolerated in which particular situations that is, which mismatched antigens are less likely to affect transplant success and survival.

Keep in mind that there are stages to this process there may be several matches that look promising but dont work out as hoped. The team and registry will keep looking for the best possible match for you. If your team finds an adult donor through a transplant registry, the registry will contact the donor to set up the final testing and donation. If your team finds matching cord blood, the registry will have the cord blood sent to your transplant center.

Originally posted here:
Types of Stem Cell and Bone Marrow Transplants

Heart disease, whether inherited or acquired, is the leading cause of mortality in both men and women worldwide, accounting for 17.3 million deaths per year.1 The urgent need to improve existing therapies has driven researchers to seek a better understanding of the diverse but inter-related mechanistic origins of heart development and failure, with the ultimate goals of identifying novel pharmacological treatments and/or cell-based engineering approaches to replace damaged heart tissue. Animal models are widely used as surrogates for studying human disease, both in order to recapitulate the complex clinical course of human heart failure and to generate in vitro tools for studying specific aspects of tissue dysfunction.2 Although useful insights have been gained, experimental findings from animal models have not always extrapolated to human disease presentation due to considerable species variation3. Here we describe prominent routes taken towards the goal of cardiac regeneration by focusing on key contributing papers published by Circulation Research in the 60 years since its establishment.

Multipotent adult stem cells have been the focus of most preclinical and clinical studies carried out to date in the field of cardiac regeneration. They represent an attractive source of stem cells since they are relatively abundant, accessible and autologous, and their mechanisms of action for any observed improvement in cardiac function can be potentially delineated. In 1998, Anversa et al. published a field-changing paper challenging the notion that the myocardium is a non-regenerating tissue, by describing the presence of multipotent cardiac stem cells (CSCs) in the adult myocardium that are positive for the hematopoietic progenitor marker c-kit.4 Methods for isolating functionally competent CSCs and mechanisms proving that their activation can reverse cardiac dysfunction were later published by the same group.5, 6 It was this pioneering work and the ability to adequately expand CSCs ex vivo that formed the basis for the first randomized clinical trial of CSC implant in ischemic heart disease patients (SCIPIO trial).7 Phase I of the trial demonstrated a sound safety profile and potential for efficacy in improving ventricular function. In 2004, Messina et al. were able to isolate and expand c-kit+ CSCs from adult murine hearts as self-adherent clusters of progenitor cells, termed cardiospheres.8 This isolation technique later became feasible for human hearts and was used to test the therapeutic efficacy of cardiosphere-derived cells (CDCs) in the CADUCEUS trial.9 The Phase I trial demonstrated a good safety profile and potential for reducing in scar size and regional function compared to controls. More recently, Dey et al. performed detailed characterization of multiple stem cell populations and concluded that c-kit+ CSCs represent the most primitive population of multipotent cardiac progenitors when compared to bone marrow-derived c-kit+ populations, and that CDCs are more closely related to bone marrow stem cells in terms of their gene and protein expression profiles.10 The exact mechanistic and functional outcome implications of such differences are not yet known, but may aid ongoing clinical trials in understanding the biology of these promising cell populations.

Bone marrow-derived mononuclear cells (MNCs) have also garnered considerable interest in regenerative cell therapy as they are easily accessible and autologous, and require minimal expansion. Significantly, evidence of MNC mobilization after myocardial infarction (MI) in mice have supported that bone marrow cells play a role in myocardial healing following injury.11, 12 Randomized human clinical studies of injected MNCs demonstrated a modest increase in left ventricular ejection fraction (LVEF) and a decrease in the New York Heart Association (NYHA) functional classification system.13 Ischemic cardiomyopathy patients receiving MNCs also demonstrated a significant reduction in natriuretic peptide levels.14 Notably, infusion of MNCs with higher colony-forming capacity was associated with lower mortality, raising awareness to the notion that cell viability and quality have a significant impact on therapeutic effect. Mechanistic investigations have suggested that beneficial effects of MNC therapy were a result of neovascularization and paracrine effects rather than cardiomyocyte differentiation.15

Studies of bone marrow-derived mesenchymal stem cells (MSCs) revealed yet another adult stem cell source thought to be suitable for cardiac regeneration. MSCs were reported to readily express phenotypic characteristics of CMs and, when introduced into infarcted animal hearts by intravenous injections, to localize at sites of myocardial injury, prevent tissue remodeling, and improve cardiac recovery.16, 17 Intracoronary infusion of allogeneic mesenchymal precursors (Stro-3+ subpopulation) was also shown to decrease infarct size, improve systolic function, and increase neovascularization in animal MI models.18 These observations led to a pilot human clinical study which confirmed the safety and tolerability of MSCs in humans, and subsequently to a Phase I/II randomized trial.19, 20 More recently, additional evidence has questioned the ability of MSCs to transdifferentiate into cardiomyocytes, instead attributing the mechanism of their therapeutic properties to paracrine effects, neovascularization, and activation of endogenous CSCs.19, 21

Another class of multipotent adult stem cells of particular interest in cardiac cell therapy are CD34+ angiogenic precursors. This interest stems from the relatively impaired angiogenesis seen in ischemic heart disease patients as well as from findings that patients with coronary artery disease have reduced number and migratory activity of angiogenic precursors.22 It has also been observed that CD34+ cell injection ameliorates cardiac recovery in human MI patients by improving perfusion and/or by paracrine effects rather than cardiomyocyte differentiation.23 In one of the largest cell therapy trials to date, Losordo et al. demonstrated that patients with refractory angina who received intramyocardial injections of CD34+ cells experienced significant improvements in angina frequency and exercise tolerance.24 In a subsequent publication, the group identified that CD34+ cells secrete exosomes that might account for some of the improved phenotypes.25 The benefit of CD34+ cells was also shown for non-ischemic cardiomyopathy, when intracoronary injections resulted in a small, but significant improvement in ventricular function and survival.26 More importantly, this study demonstrated that higher intramyocardial homing was associated with better cell therapy response, providing support to prior observations with MNCs that cell delivery method and quality play a significant role in their therapeutic efficacy.

Finally, adipose-derived stem cells (ADSCs) abundantly available from liposuction surgeries have been considered as potential sources of CMs. In 2004, Planart-Blenard et al. reported potential derivation of CMs from human ADSCs by treatment with transferrin, IL-3, IL-6, and VEGF, although at very low event rate (Figure 1).27 Ongoing trials are evaluating the efficacy of this cell population in regeneration of ischemic myocardium, and although complete results have yet to be published, preliminary data are encouraging (Trial identifier: NCT00426868).

Timeline of important discoveries contributing to the field of stem cell cardiac differentiation and characterization (purple and green boxes, above timeline), including the key Top 100 Circulation Research papers discussed in this review (red boxes, below timeline). ESC, embryonic stem cell; iPSC, induced pluripotency stem cell; CMs, cardiomyocytes.

Early attempts at inducing cardiac regeneration involved transplant of skeletal myoblasts or fetal CMs to infarcted canine or rat hearts. Unfortunately, these studies ultimately disappointed the field as myoblasts remained firmly committed to form mature skeletal muscle in the heart28, while extensive cell death coupled with limited proliferation after transplant prevented fetal cardiomyocytes from repairing injury.29 Transplantation of non-contractile committed cells such as fibroblasts and smooth muscle cells into infarcted rat hearts was then briefly thought to enhance heart function, possibly due to aforementioned paracrine effects.30 More recently, several studies have demonstrated in vitro31 and in vivo32 transdifferentiation of mouse fibroblasts into seemingly functional CMs by over-expressing combinations of the cardiac transcription factors Gata4, Mef2c, Tbx5, Hand2, and Nkx2.5. Mouse CMs generated by direct transdifferentiation are positive for CM-specific sarcomeric markers, exhibit electrophysiological and gene expression profiles similar to those of fetal CMs, although this was disputed by other investigators.33In vitro transdifferentiation towards CM-like cells was also reported for human fibroblasts, albeit by more time consuming and less efficient protocols that generated mostly partially reprogrammed CMs.34 Current efforts in this research area focus on optimizing transdifferentiation efficiency and CM maturation, further characterizing derived CMs, and validating that in vitro and in vivo transdifferentiation occur in the absence of experimental artifacts, which can include incomplete silencing of transgene expression from Cre-lox systems, cell fusion events, as well as the possibility of retrovirus transfecting not only dividing fibroblasts but also non-dividing cardiomyocytes in vivo. For this technology to be fully applied in the clinic, a greater understanding of issues that have plagued the field must be reached: (1) the potential consequences of depleting endogenous cardiac fibroblasts to replenish cardiomyocytes; (2) the ability to transfect bystander cells such as smooth muscle and endothelial cells with cardiac transcription factors; and (3) the challenge of triggering immune response against the host cells transfected with viral versus non-viral vectors.

The isolation by Evans and Kaufman of mouse embryonic stem cells (mESCs) in 198135 and the generation of human embryonic stem cells (hESCs) by Thomson in 199836 opened new horizons for in vitro generation of CMs. Many protocols have been developed over the years to maximize the yield and efficiency of pluripotent ESC differentiation to CMs.37 One of the most utilized methods has been the formation of 3D aggregates named embryoid bodies within which cardiac differentiation occurs. In 2002, Xu et al. were amongst the first to optimize cardiac differentiation protocols for hESCs by using DNA demethylating agent 5-azacytidine and enrichment with Percoll separation gradients to obtain up to 70% pure cardiomyocyte populations (Figure 1).38 Later on, rigorous protocol standardization and the use of key signaling factors such as BMP4 and Activin A enabled conversion of hESCs to CMs with over 90% efficiency.39 Consequently, the formation of 3D aggregates, a labor intensive process, has now been largely replaced by differentiation in monolayer cultures, which are more amenable to scale-up and automation.40

The discovery of induced pluripotent stem cell (iPSC) technology41, based partly on principles highlighted by early somatic cell nuclear transfer experiments42, has meant that mature somatic cells such as skin fibroblasts and peripheral blood mononuclear cells (PBMCs) can be reprogrammed with relative ease to acquire an ESC-like phenotype. iPSCs retain the same capacity for high efficiency cardiac differentiation as ESCs, with the added advantages of avoiding ethical debates related to use of human embryos and enabling autologous transplantation of CMs without the need for immunosuppression. These characteristics make iPSCs ideal cellular models to provide a renewable source of CMs for basic research, pharmacological testing, and cell therapy (Figure 2).43

iPSCs are ideal cellular models to provide a renewable source of cardiomyocytes for in vitro disease modeling, pharmacological testing, and therapeutic applications in regenerative medicine.

The use of pluripotent stem cell-derived cardiomyocytes (PSC-CMs), which include both hESC-CMs and iPSC-CMs, for downstream applications requires that their properties be physiologically analogous to human cardiomyocytes in vivo. Assays for CM characterization, such as assessment for cross striations, ultrastructure, and chronotropic drug response, were established decades ago for primary rodent myocytes and published in a highly cited Circulation Research paper by Simpson and Savion in 1982.44 In 1994, Maltsev et al. were able to apply the same assays for extensive characterization of mESC-CMs.45 In addition, rigorous experimental optimization enabled them to identify internal and external solutions for patch-clamp electrophysiological analysis to confirm that CM populations comprised of ventricular, atrial, and nodal sub-types, and exhibited most basic cardiac-specific ionic currents (L-type, ICa, INa, Ito, IK, IK1, IK, ATP, IK, Ach, and If). In 2003, He et al. were among the first to perform similar characterizations of hESC-CMs.46

In vitro derived PSC-CMs have been assessed as potential screening platforms for drug discovery and toxicology studies. Despite their immature fetal phenotype, extensive pharmacological validation confirms their potential utility in drug evaluation.47 Clinically relevant drugs (e.g., adrenergic receptor agonists, anti-arrhythmic agents) have been shown to exert chronotropic and inotropic effects on PSC-CMs. In addition, experimental drugs have been used for in vitro amelioration of diseased phenotypes in human iPSC models of cardiovascular diseases48 and prediction of cytotoxic drug-induced side-effects.49, 50 Accumulated evidence suggests that PSC-CMs can offer the pharmaceutical industry a valuable physiologically relevant tool for validation of novel drug candidates and identification of potential cardiotoxic effects in early drug development stages, thereby easing the huge associated economic and patient care burdens.51, 52

The most successful and widely acknowledged use of PSCs-CMs has so far been in disease modeling. The development of disease models by genome editing of mESCs, a technology that led to award of the Nobel Prize in 2007 for Sir Martin Evans, Mario Capecchi, and Oliver Smithies (Figure 1), has offered new tools for in vivo mechanistic investigation into cardiac illnesses. The discovery of induced pluripotency technologies, which likewise led to the Nobel Prize in 2012 for Sir John Gurdon and Shinya Yamanaka, allowed the generation of patient-specific iPSC-CMs for studying human disease models of familial hypertrophic cardiomyopathy53, familial dilated cardiomyopathy54, long QT syndrome55, Timothy syndrome56, arrhythmogenic right ventricular dysplasia57, and others44 (Figure 2). Beyond the potential ability of these models to reveal insights into pathological disease mechanisms, they also offer unique opportunities to explore promising new genetic therapies58 and to identify loci or pathways related to predisposition towards cardiac disorders, thus enabling refinement of phenotype-to-genotype correlations to improve risk stratification and disease management.

The use of PSC-CMs has also expanded to in vivo applications, with transplantation shown to improve cardiac function in rat and guinea pig models of acute myocardial infarct (MI).59, 60 Effective strategies to deplete potential tumorigenic cells61, 62, induce immunotolerance63, 64, and enhance cell survival65 are being sought. Novel tissue engineering approaches to create engineered heart tissues (EHTs) for aiding cell delivery, survival, alignment and functionality of transplanted PSC-CMs are being developed in parallel.66 Notably, these technologies were pioneered by Thomas Eschenhagens group, who published one of the very first EHT papers in Circulation Research in 2002.67

See the article here:
Cardiac stem cell biology: a glimpse of the past, present, and future - PMC

Abstract

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cellsthat can be obtained by reprogramming animal and human differentiated cells. In this review,issues related to the nature of iPSCs are discussed and different methods ofiPSC production are described. We particularly focused on methods of iPSC production withoutthe genetic modification of the cell genome and with means for increasing the iPSC productionefficiency. The possibility and issues related to the safety of iPSC use in cell replacementtherapy of human diseases and a study of new medicines are considered.

Keywords: induced pluripotent stem cells, directed stem cell differentiation, cell replacement therapy

Pluripotent stem cells are a unique model for studying a variety of processes that occur inthe early development of mammals and a promising tool in cell therapy of human diseases. Theunique nature of these cells lies in their capability, when cultured, for unlimitedselfrenewal and reproduction of all adult cell types in the course of theirdifferentiation [1]. Pluripotency is supported by acomplex system of signaling molecules and gene network that is specific for pluripotent cells.The pivotal position in the hierarchy of genes implicated in the maintenance of pluripotency isoccupied by Oct4, Sox2 , and Nanog genes encodingtranscription factors [2, 3]. The mutual effect of outer signaling molecules and inner factors leads tothe formation of a specific expression pattern, as well as to the epigenome statecharacteristic of stem cells. Both spontaneous and directed differentiations are associatedwith changes in the expression pattern and massive epigenetic transformations, leading totranscriptome and epigenome adjustment to a distinct cell type.

Until recently, embryonic stem cells (ESCs) were the only wellstudied source ofpluripotent stem cells. ESCs are obtained from either the inner cell mass or epiblast ofblastocysts [46]. A series of protocols has been developed for the preparation of variouscell derivatives from human ESCs. However, there are constraints for ESC usein cell replacement therapy. The first constraint is the immune incompatibility between thedonor cells and the recipient, which can result in the rejection of transplanted cells. Thesecond constraint is ethical, because the embryo dies during the isolation of ESCs. The firstproblem can be solved by the somatic cell nuclear transfer into the egg cell and then obtainingthe embryo and ESCs. The nuclear transfer leads to genome reprogramming, in which ovariancytoplasmic factors are implicated. This way of preparing pluripotent cells from certainindividuals was called therapeutic cloning. However, this method is technologyintensive,and the reprogramming yield is very low. Moreover, this approach encounters theabovementioned ethic problem that, in this case, is associated with the generation ofmany human ovarian cells [7].

In 2006, the preparation of pluripotent cells by the ectopic expression of four genes Oct4 , Sox2 , Klf4 , and cMyc in both embryonic and adult murine fibroblasts was first reported[8]. The pluripotent cells derived from somatic ones werecalled induced pluripotent stem cells (iPSCs). Using this set of factors(Oct4, Sox2, Klf4, and cMyc), iPSCs were prepared later from variousdifferentiated mouse [914] and human [1517] cell types. Human iPSCs were obtainedwith a somewhat altered gene set: Oct4 , Sox2 , Nanog , and Lin28 [18].Induced PSCs closely resemble ESCs in a broad spectrum of features. They possess similarmorphologies and growth manners and are equally sensitive to growth factors and signalingmolecules. Like ESCs, iPSCs can differentiate in vitro intoderivatives of all three primary germ layers (ectoderm, mesoderm, and endoderm) and formteratomas following their subcutaneous injection into immunodeficient mice. MurineiPSCs injected into blastocysts are normally included in the development toyield animals with a high degree of chimerism. Moreover, murine iPSCs, wheninjected into tetraploid blastocycts, can develop into a whole organism [19, 20]. Thus, an excellent method thatallows the preparation of pluripotent stem cells from various somatic cell types whilebypassing ethical problems has been uncovered by researchers.

In the first works on murine and human iPSC production, either retro or lentiviralvectors were used for the delivery of Oct4 , Sox2 , Klf4 , and cMyc genes into somatic cells. Theefficiency of transduction with retroviruses is high enough, although it is not the same fordifferent cell types. Retroviral integration into the host genome requires a comparatively highdivision rate, which is characteristic of the relatively narrow spectrum of cultured cells.Moreover, the transcription of retroviral construct under the control of a promoter localizedin 5LTR (long terminal repeat) is terminated when the somatic celltransform switches to the pluripotent state [21]. Thisfeature makes retroviruses attractive in iPSC production. Nevertheless, retroviruses possesssome properties that make iPSCs that are produced using them improper for celltherapy of human diseases. First, retroviral DNA is integrated into the host cell genome. Theintegration occurs randomly; i.e., there are no specific sequences or apparent logic forretroviral integration. The copy number of the exogenous retroviral DNA that is integrated intoa genome may vary to a great extent [15]. Retrovirusesbeing integrated into the cell genome can introduce promoter elements and polyadenylationsignals; they can also interpose coding sequences, thus affecting transcription. Second, sincethe transcription level of exogenous Oct4 , Sox2 , Klf4 , and cMyc in the retroviral constructdecreases with cell transition into the pluripotent state, this can result in a decrease in theefficiency of the stable iPSC line production, because the switch from the exogenous expressionof pluripotency genes to their endogenous expression may not occur. Third, some studies showthat the transcription of transgenes can resume in the cells derived fromiPSCs [22]. The high probability thatthe ectopic Oct4 , Sox2 , Klf4 , and cMyc gene expression will resume makes it impossible to applyiPSCs produced with the use of retroviruses in clinical trials; moreover,these iPSCs are hardly applicable even for fundamental studies onreprogramming and pluripotency principles. Lentiviruses used for iPSC production can also beintegrated into the genome and maintain their transcriptional activity in pluripotent cells.One way to avoid this situation is to use promoters controlled by exogenous substances added tothe culture medium, such as tetracycline and doxycycline, which allows the transgenetranscription to be regulated. iPSCs are already being produced using suchsystems [23].

Another serious problem is the gene set itself that is used for the induction of pluripotency[22]. The ectopic transcription of Oct4 , Sox2 , Klf4 , and cMyc can lead to neoplastic development from cells derived from iPSCs,because the expression of Oct4 , Sox2 , Klf4, and cMyc genes is associated with the development ofmultiple tumors known in oncogenetics [22, 24]. In particular, the overexpression of Oct4 causes murine epithelial cell dysplasia [25],the aberrant expression of Sox2 causes the development of serrated polypsand mucinous colon carcinomas [26], breast tumors arecharacterized by elevated expression of Klf4 [27] , and the improper expression of cMyc is observed in 70% of human cancers [28].Tumor development is oberved in ~50% of murine chimeras obtained through the injection ofretroviral iPSCs into blastocysts, which is very likely associated with thereactivation of exogenous cMyc [29, 30].

Several possible strategies exist for resolving the above-mentioned problems:

The search for a less carcinogenic gene set that is necessary and sufficient for reprogramming;

The minimization of the number of genes required for reprogramming and searching for the nongenetic factors facilitating it;

The search for systems allowing the elimination of the exogenous DNA from the host cell genome after the reprogramming;

The development of delivery protocols for nonintegrated genetic constructs;

The search for ways to reprogram somatic cells using recombinant proteins.

The ectopic expression of cMyc and Klf4 genes isthe most dangerous because of the high probability that malignant tumors will develop [22]. Hence the necessity to find other genes that couldsubstitute cMyc and Klf4 in iPSC production. Ithas been reported that these genes can be successfully substituted by Nanog and Lin28 for reprogramming human somatic cells [18;] . iPSCs were prepared from murine embryonic fibroblastsby the overexpression of Oct4 and Sox2 , as well as the Esrrb gene encoding the murine orphan nuclear receptor beta. It has alreadybeen shown that Esrrb , which acts as a transcription activator of Oct4 , Sox2 , and Nanog , is necessary for theselfrenewal and maintenance of the pluripotency of murine ESCs. Moreover, Esrrb can exert a positive control over Klf4 . Thus, the genes causingelevated carcinogenicity of both iPSCs and their derivatives can besuccessfully replaced with less dangerous ones [31].

The Most Effectively Reprogrammed Cell Lines . Murine and humaniPSCs can be obtained from fibroblasts using the factors Oct4, Sox2, and Klf4,but without cMyc . However, in this case, reprogramming deceleratesand an essential shortcoming of stable iPSC clones is observed [32, 33]. The reduction of a number ofnecessary factors without any decrease in efficiency is possible when iPSCsare produced from murine and human neural stem cells (NSCs) [12, 34, 35]. For instance, iPSCs were produced fromNSCs isolated from adult murine brain using two factors, Oct4 and Klf4, aswell as even Oct4 by itself [12, 34]. Later, human iPSCs were produced by the reprogramming offetal NSCs transduced with a retroviral vector only carrying Oct4 [35] . It is most likely that the irrelevanceof Sox2, Klf4, and cMyc is due to the high endogenous expression level of these genes inNSCs.

Successful reprogramming was also achieved in experiments withother cell lines, in particular, melanocytes of neuroectodermal genesis [36]. Both murine and human melanocytes are characterized by a considerableexpression level of the Sox2 gene, especially at early passages.iPSCs from murine and human melanocytes were produced without the use of Sox2or cMyc. However, the yield of iPSC clones produced from murine melanocytes was lower(0.03% without Sox2 and 0.02% without cMyc) in comparison with that achieved when allfour factors were applied to melanocytes (0.19%) and fibroblasts (0.056%). A decreasedefficiency without Sox2 or cMyc was observed in human melanocyte reprogramming (0.05%with all four factors and 0.01% without either Sox2 or cMyc ). All attempts to obtain stable iPSC clones in the absence of both Sox2 andcMyc were unsuccessful [36]. Thus, theminimization of the number of factors required for iPSC preparation can be achieved by choosingthe proper somatic cell type that most effectively undergoes reprogramming under the action offewer factors, for example, due to the endogenous expression of pluripotencygenes. However, if human iPSCs are necessary, these somatic cellsshould be easily accessible and wellcultured and their method of isolation should be asnoninvasive as possible.

One of these cell types can be adipose stem cells (ASCs). This is aheterogeneous group of multipotent cells which can be relatively easily isolated in largeamounts from adipose tissue following liposuction. Human iPSCs weresuccessfully produced from ASCs with a twofold reprogramming rate and20fold efficiency (0.2%), exceeding those of fibroblasts [37].

However, more accessible resources for the effective production of humaniPSCs are keratinocytes. When compared with fibroblasts, human iPSC productionfrom keratinocytes demonstrated a 100fold greater efficiency and a twofold higherreprogramming rate [38].

It has recently been found that the reprogramming of murine papillary dermal fibroblasts(PDFs) into iPSCs can be highly effective with theoverexpression of only two genes, Oct4 and Klf4 ,inserted into retroviral vectors [39;].PDFs are specialized cells of mesodermal genesis surrounding the stem cells ofhair follicles . One characteristic feature of these cells is the endogenous expression of Sox2 , Klf4 , and cMyc genes,as well as the geneencoding alkaline phosphatase, one of the murine and humanESC markers. PDFs can be easily separated from other celltypes by FACS (fluorescenceactivated cell sorting) using life staining with antibodiesagainst the surface antigens characteristic of one or another cell type. The PDF reprogrammingefficiency with the use of four factors (Oct4, Sox2, Klf4, and cMyc) retroviral vectorsis 1.38%, which is 1,000fold higher than the skin fibroblast reprogramming efficiency inthe same system. Reprogramming PDFs with two factors, Oct4 and Klf4 , yields 0.024%, which is comparable to the efficiency of skinfibroblast reprogramming using all four factors. The efficiency of PDF reprogramming iscomparable with that of NSCs, but PDF isolation is steady and far lessinvasive [39]. It seems likely that human PDF lines arealso usable, and this cell type may appear to be one of the most promising for human iPSCproduction in terms of pharmacological studies and cell replacement therapy. The use of suchcell types undergoing more effective reprogramming, together with methods providing thedelivery of pluripotency genes without the integration of foreign DNA into thehost genome and chemical compounds increasing the reprogramming efficiency and substitutingsome factors required for reprogramming, is particularly relevant.

Chemical Compounds Increasing Cell Reprogramming Efficiency. As was noted above,the minimization of the factors used for reprogramming decreases the efficiency of iPSCproduction. Nonetheless, several recent studies have shown that the use of genetic mechanisms,namely, the initiation of ectopic gene expression, can be substituted by chemical compounds,most of them operating at the epigenetic level. For instance, BIX01294 inhibitinghistone methyltransferase G9a allows murine fibroblast reprogramming using only two factors,Oct4 and Klf4, with a fivefold increased yield of iPSC clones in comparison with the controlexperiment without BIX01294 [40]. BIX01294taken in combination with another compound can increase the reprogramming efficiency even more.In particular, BIX01294 plus BayK8644 elevated the yield of iPCSs 15 times, andBIX01294 plus RG108 elevated it 30 times when only two reprogramming factors, Oct4 andKlf4, were used. RG108 is an inhibitor of DNA methyltransferases, and its role in reprogrammingis apparently in initiating the more rapid and effective demethylation of promoters ofpluripotent cellspecific genes, whereas BayK8644 is an antagonist of Ltypecalcium channels, and its role in reprogramming is not understood very well [40]. However, more considerable results were obtained inreprogramming murine NSCs. The use of BIX01294 allowed a 1.5foldincrease in iPSC production efficiency with two factors, Oct4 and Klf4, in comparison withreprogramming with all four factors. Moreover, BIX01294 can even substitute Oct4 in thereprogramming of NSCs, although the yield is very low [41]. Valproic (2propylvaleric) acid inhibiting histone deacetylases canalso substitute cMyc in reprogramming murine and human fibroblasts. Valproic acid (VPA)increases the reprogramming efficiency of murine fibroblasts 50 times, and human fibroblastsincreases it 1020 times when three factors are used [42, 43]. Other deacetylase inhibitors,such as TSA (trichostatin A) and SAHA (suberoylanilide hyroxamic acid), also increase thereprogramming efficiency. TSA increases the murine fibroblast reprogramming efficiency 15times, and SAHA doubles it when all four factors are used [42]. Besides epigenetic regulators, the substances inhibiting the proteincomponents of signaling pathways implicated in the differentiation of pluripotent cells arealso applicable in the substitution of reprogramming factors. In particular, inhibitors of MEKand GSK3 kinases (PD0325901 and CHIR99021, respectively) benefit the establishment of thecomplete and stable pluripotency of iPSCs produced from murineNSCs using two factors, Oct4 and Klf4 [41, 44].

It has recently been shown that antioxidants can considerably increase the efficiency ofsomatic cell reprogramming. Ascorbic acid (vitamin C) can essentially influence the efficiencyof iPSC production from various murine and human somatic cell types [45]. The transduction of murine embryonic fibroblasts (mEFs) with retrovirusescarrying the Oct4 , Sox2 , and Klf4 genes results in a significant increase in the production level of reactive oxygen species(ROS) compared with that of both control and Efs tranduced with Oct4 , Sox2 , cMyc , and Klf4 . Inturn, the increase in the ROS level causes accelerated aging and apoptosis of the cell, whichshould influence the efficiency of cell reprogramming. By testing several substances possessingantioxidant activity such as vitamin B1, sodium selenite, reduced glutathione, and ascorbicacid, the authors have found that combining these substances increases the yield ofGFPpositive cells in EF reprogramming (the Gfp genewas under the control of the Oct4 gene promoter). The use of individualsubstances has shown that only ascorbate possesses a pronounced capability to increase thelevel of GFPpositive cells, although other substances keep theirROSdecreasing ability. In all likelihood, this feature of ascorbates is not directlyassociated with its antioxidant activity [45]. The scoreof GFPpositive iPSC colonies expressing an alkaline phosphatase hasshown that the efficiency of iPSC production from mEFs with three factors (Oct4, Sox2, andKlf4) can reach 3.8% in the presence of ascorbate. When all four factors (Oct4, Sox2, Klf4, andcMyc) are used together with ascorbate, the efficiency of iPSC production may reach8.75%. A similar increase in the iPSC yield was also observed in the reprogramming of murinebreast fibroblasts; i.e., the effect of vitamin C is not limited by one cell type. Moreover,the effect of vitamin C on the reprogramming efficiency is more profound than that of thedeacetylase inhibitor valproic (2propylvaleric) acid. The mutual effect of ascorbate andvalproate is additive; i.e., these substances have different action mechanisms. Moreover,vitamin C facilitates the transition from preiPSCs to stablepluripotent cells. This feature is akin to the effects of PD0325901 and CHIR99021, which areinhibitors of MEK and GSK3 kinases, respectively. This effect of vitamin C expands to humancells as well [45]. Following the transduction of humanfibroblasts with retroviruses carrying Oct4 , Sox2 , Klf4 , and cMyc and treatment with ascorbate, theauthors prepared iPSCs with efficiencies reaching 6.2%. The reprogrammingefficiency of ASCs under the same conditions reached 7.06%. The mechanism ofthe effect that vitamin C has on the reprogramming efficiency is not known in detail.Nevertheless, the acceleration of cell proliferation was observed at the transitional stage ofreprogramming. The levels of the p53 and p21 proteins decreased in cells treated withascorbate, whereas the DNA repair machinery worked properly [45]. It is interesting that an essential decrease in the efficiency of iPSCproduction has been shown under the action of processes initiated by p53 and p21 [4650].

As was mentioned above, for murine and human iPSC production, both retro andlentiviruses were initially used as delivery vectors for the genes required for cellreprogramming. The main drawback of this method is the uncontrolled integration of viral DNAinto the host cells genome. Several research groups have introduced methods fordelivering pluripotency genes into the recipient cell which either do notintegrate allogenic DNA into the host genome or eliminate exogenous genetic constructs from thegenome.

CreloxP Mediated Recombination. To prepareiPSCs from patients with Parkinsons disease, lentiviruses were used,the proviruses of which can be removed from the genome by Cre recombinase. To do this, the loxP site was introduced into thelentiviral 3LTRregions containing separate reprogramming genesunder the control of the doxycyclineinducible promoter. During viral replication, loxP was duplicated in the 5LTR of the vector. As aresult, the provirus integrated into the genome was flanked with two loxP sites. The inserts were eliminated using the temporary transfection ofiPSCs with a vector expressing Cre recombinase[51].

In another study, murine iPSCs were produced using a plasmid carrying the Oct4 , Sox2 , Klf4I, and cMyc genes in the same reading frame in which individual cDNAs were separatedby sequences encoding 2 peptides, and practically the whole construct was flanked with loxP sites [52]. The use ofthis vector allowed a notable decrease in the number of exogenous DNA inserts in the hostcells genome and, hence, the simplification of their following excision [52]. It has been shown using lentiviruses carrying similarpolycistronic constructs that one copy of transgene providing a high expression level of theexogenous factors Oct4, Sox2, Klf4, and cMyc is sufficient for the reprogramming ofdifferentiated cells into the pluripotent state [53,54].

The drawback of the CreloxP system is the incomplete excisionof integrated sequences; at least the loxP site remains in thegenome, so the risk of insertion mutations remains.

Plasmid Vectors . The application of lentiviruses and plasmids carrying the loxP sites required for the elimination of transgene constructsmodifies, although insignificantly, the host cells genome. One way to avoid this is touse vector systems that generally do not provide for the integration of the whole vector orparts of it into the cells genome. One such system providing a temporary transfectionwith polycistronic plasmid vectors was used for iPSC production from mEFs [29]. A polycistronic plasmid carrying the Oct4 , Sox2 , and Klf4 gene cDNAs, as well as aplasmid expressing cMyc , was transfected into mEFs one, three, five,and seven days after their primary seeding. Fibroblasts were passaged on the ninth day, and theiPSC colonies were selected on the 25th day. Seven out of ten experiments succeeded inproducing GFPpositive colonies (the Gfp gene wasunder the control of the Nanog gene promoter). The iPSCsthat were obtained were similar in their features to murine ESCs and did not contain inserts ofthe used DNA constructs in their genomes. Therefore, it was shown that wholesome murineiPSCs that do not carry transgenes can be reproducibly produced, and that thetemporary overexpression of Oct4 , Sox2 , Klf4 , and cMyc is sufficient for reprogramming. The maindrawback of this method is its low yield. In ten experiments the yield varied from 1 to 29 iPSCcolonies per ten million fibroblasts, whereas up to 1,000 colonies per ten millions wereobtained in the same study using retroviral constructs [29].

Episomal Vectors . Human iPSCs were successfully produced fromskin fibroblasts using single transfection with polycistronic episomal constructs carryingvarious combinations of Oct4 , Sox2 , Nanog , Klf4 , cMyc , Lin28 , and SV40LT genes. These constructs were designed on the basis of theoriP/EBNA1 (EpsteinBarr nuclear antigen1) vector [55]. The oriP/EBNA1 vector contains the IRES2 linker sequence allowing theexpression of several individual cDNAs (encoding the genes required for successfulreprogramming in this case) into one polycistronic mRNA from which several proteins aretranslated. The oriP/EBNA1 vector is also characterized by lowcopy representation in thecells of primates and can be replicated once per cell cycle (hence, it is not rapidlyeliminated, the way common plasmids are). Under nonselective conditions, the plasmid iseliminated at a rate of about 5% per cell cycle [56]. Inthis work, the broad spectrum of the reprogramming factor combinations was tested, resulting inthe best reprogramming efficiency with cotransfection with three episomes containing thefollowing gene sets: Oct4 + Sox2 + Nanog + Klf4 , Oct4 + Sox2 + SV40LT + Klf4 , and cMyc + Lin28 . SV40LT ( SV40 large T gene )neutralizes the possible toxic effect of overexpression [57]. The authors have shown thatwholesome iPSCs possessing all features of pluripotent cells can be producedfollowing the temporary expression of a certain gene combination in human somatic cells withoutthe integration of episomal DNA into the genome. However, as in the case when plasmid vectorsare being used, this way of reprogramming is characterized by low efficiency. In separateexperiments the authors obtained from 3 to 6 stable iPSC colonies per 106transfected fibroblasts [55]. Despite the fact that skinfibroblasts are wellcultured and accessible, the search for other cell types which arerelatively better cultured and more effectively subject themselves to reprogramming throughthis method is very likely required. Another drawback of the given system is that this type ofepisome is unequally maintained in different cell types.

PiggyBacTransposition . One promising system used foriPSC production without any modification of the host genome is based on DNA transposons.Socalled PiggyBac transposons containing2linkered reprogramming genes localized between the 5 and3terminal repeats were used for iPSC production from fibroblasts. The integrationof the given constructs into the genome occurs due to mutual transfection with a plasmidencoding transposase. Following reprogramming due to the temporary expression of transposase,the elimination of inserts from the genome took place [58, 59]. One advantage of the PiggyBac system on CreloxP is that the exogenous DNA iscompletely removed [60].

However, despite the relatively high efficiency of exogenous DNA excision from the genome by PiggyBac transposition, the removal of a large number of transposoncopies is hardly achievable.

Nonintegrating Viral Vectors . Murine iPSCs were successfullyproduced from hepatocytes and fibroblasts using four adenoviral vectors nonintegrating into thegenome and carrying the Oct4 , Sox2 , Klf4 , and cMyc genes. An analysis of the obtainediPSCs has shown that they are similar to murine ESCs in their properties(teratoma formation, gene promoter DNA methylation, and the expression of pluripotent markers),but they do not carry insertions of viral DNA in their genomes [61]. Later, human fibroblastderived iPSCs wereproduced using this method [62].

The authors of this paper cited the postulate that the use of adenoviral vectors allows theproduction of iPSCs, which are suitable for use without the risk of viral oroncogenic activity. Its very low yield (0.00010.001%), the deceleration ofreprogramming, and the probability of tetraploid cell formation are the drawbacks of themethod. Not all cell types are equally sensitive to transduction with adenoviruses.

Another method of gene delivery based on viral vectors was recently employed for theproduction of human iPSCs. The sendaivirus (SeV)based vector wasused in this case [63]. SeV is a singlestrandedRNA virus which does not modify the genome of recipient cells; it seems to be a good vector forthe expression of reprogramming factors. Vectors containing either all pluripotencyfactors or three of them (without ) were used for reprogramming the human fibroblast. The construct based on SeV is eliminatedlater in the course of cell proliferation. It is possible to remove cells with the integratedprovirus via negative selection against the surface HN antigen exposed on the infected cells.The authors postulate that reprogramming technology based on SeV will enable the production ofclinically applicable human iPSCs [63].

Cell Transduction with Recombinant Proteins . Although the methods for iPSCproduction without gene modification of the cells genome (adenoviral vectors, plasmidgene transfer, etc.) are elaborated, the theoretical possibility for exogenous DNA integrationinto the host cells genome still exists. The mutagenic potential of the substances usedpresently for enhancing iPSC production efficiency has not been studied in detail. Fullychecking iPSC genomes for exogenous DNA inserts and other mutations is a difficult task, whichbecomes impossible to solve in bulk culturing of multiple lines. The use of protein factorsdelivered into a differentiated cell instead of exogenous DNA may solve this problem. Tworeports have been published to date in which murine and human iPSCs wereproduced using the recombinant Oct4, Sox2, Klf4, and cMyc proteins [64, 65] . T he methodused to deliver the protein into the cell is based on the ability of peptides enriched withbasic residues (such as arginine and lysine) to penetrate the cells membrane. MurineiPSCs were produced using the recombinant Oct4, Sox2, Klf4, and cMycproteins containing eleven Cterminal arginine residues and expressed in E. coli . The authors succeeded in producing murine iPSCs during four roundsof protein transduction into embryonic fibroblasts [65].However, iPSCs were only produced when the cells were additionally treatedwith 2propylvalerate (the deacetylase inhibitor). The same principle was used for theproduction of human iPSCs, but protein expression was carried out in humanHEK293 cells, and the proteins were expressed with a fragment of nine arginins at the proteinCend. Researchers have succeeded in producing human iPSCs after sixtransduction rounds without any additional treatment [64]. The efficiency of producing human iPSC in this way was 0.001%, which isone order lower than the reprogramming efficiency with retroviruses. Despite some drawbacks,this method is very promising for the production of patientspecificiPSCs.

The first lines of human pluripotent ESCs were produced in 1998 [6]. In line with the obvious fundamental importance of embryonic stem cellstudies with regard to the multiple processes taking place in early embryogenesis, much of theinterest of investigators is associated with the possibility of using ESCs and theirderivatives as models for the pathogenesis of human diseases, new drugs testing, and cellreplacement therapy. Substantial progress is being achieved in studies on directed humanESC differentiation and the possibility of using them to correct degenerativedisorders. Functional cell types, such as motor dopaminergic neurons, cardiomyocytes, andhematopoietic cell progenitors, can be produced as a result of ESCdifferentiation. These cell derivatives, judging from their biochemical and physiologicalproperties, are potentially applicable for the therapy of cardiovascular disorders, nervoussystem diseases, and human hematological disorders [66].Moreover, derivatives produced from ESCs have been successfully used for treating diseasesmodeled on animals. Therefore, bloodcell progenitors produced from ESCs weresuccessfully used for correcting immune deficiency in mice. Visual functions were restored inblind mice using photoreceptors produced from human ESCs, and the normal functioning of thenervous system was restored in rats modeling Parkinsons disease using the dopaminergicneurons produced from human ESCs [6770]. Despite obvious success, the fullscale applicationof ESCs in therapy and the modeling of disorders still carry difficulties, because of thenecessity to create ESC banks corresponding to all HLAhaplotypes, whichis practically unrealistic and hindered by technical and ethical problems.

Induced pluripotent stem cells can become an alternative for ESCs in the area of clinicalapplication of cell replacement therapy and screening for new pharmaceuticals.iPSCs closely resemble ESCs and, at the same time, can be produced in almostunlimited amounts from the differentiated cells of each patient. Despite the fact that thefirst iPSCs were produced relatively recently, work on directed iPSCdifferentiation and the production of patientspecific iPSCs isintensive, and progress in this field is obvious.

Dopamine and motor neurons were produced from human iPSCs by directeddifferentiation in vitro [71, 72]. These types of neurons are damaged in many inherited oracquired human diseases, such as spinal cord injury, Parkinsons disease, spinal muscularatrophy, and amyotrophic lateral sclerosis. Some investigators have succeeded in producingvarious retinal cells from murine and human iPSCs [7375]. HumaniPSCs have been shown to be spontaneously differentiated in vitro into the cells of retinal pigment epithelium [76]. Another group of investigators has demonstrated that treating human andmurine iPSCs with Wnt and Nodal antagonists in a suspended culture induces theappearance of markers of cell progenitors and pigment epithelium cells. Further treating thecells with retinoic acid and taurine activates the appearance of cells expressing photoreceptormarkers [75].

Several research groups have produced functional cardiomyocytes (CMs) in vitro from murine and human iPSCs [7781]. Cardiomyocytes producedfrom iPSC are very similar in characteristics (morphology, marker expression,electrophysiological features, and sensitivity to chemicals) to the CMs ofcardiac muscle and to CMs produced from differentiated ESCs. Moreover, murineiPSCs, when injected, can repair muscle and endothelial cardiac tissuesdamaged by cardiac infarction [77].

Hepatocytelike cell derivatives, dendritic cells, macrophages, insulinproducingcell clusters similar to the duodenal islets of Langerhans, and hematopoietic and endothelialcells are currently produced from murine and human iPSCs, in addition to thealreadylisted types of differentiated cells [8285].

In addition to directed differentiation in vitro , investigators apply mucheffort at producing patientspecific iPSCs. The availability ofpluripotent cells from individual patients makes it possible to study pathogenesis and carryout experiments on the therapy of inherited diseases, the development of which is associatedwith distinct cell types that are hard to obtain by biopsy: so the use ofiPSCs provides almost an unlimited resource for these investigations.Recently, the possibility of treating diseases using iPSCs was successfullydemonstrated, and the design of the experiment is presented in the figure. A mutant allele wassubstituted with a normal allele via homologous recombination in murine fibroblastsrepresenting a model of human sickle cell anemia. iPSCs were produced fromrepaired fibroblasts and then differentiated into hematopoietic cell precursors.The hematopoietic precursors were then injected into a mouse from which the skin fibroblastswere initially isolated (Fig. 1). As a result, the initialpathological phenotype was substantially corrected [86].A similar approach was applied to the fibroblasts and keratinocytes of a patient withFanconis anemia. The normal allele of the mutant gene producing anemia was introducedinto a somatic cell genome using a lentivirus, and then iPSCs were obtainedfrom these cells. iPSCs carrying the normal allele were differentiated intohematopoietic cells maintaining a normal phenotype [87].The use of lentiviruses is unambiguously impossible when producing cells to be introduced intothe human body due to their oncogenic potential. However, new relatively safe methods of genomemanipulation are currently being developed; for instance, the use of synthetic nucleasescontaining zinc finger domains allowing the effective correction of genetic defects invitro [88].

Design of an experiment on repairing the mutant phenotype in mice modeling sickle cell anemia development [2]. Fibroblasts isolatedfrom the tail of a mouse (1) carrying a mutant allele of the gene encoding the human hemoglobin -chain (hs) were used for iPSCproduction (2). The mutation was then repaired in iPSCs by means of homological recombination (3) followed by cell differentiationvia the embryoid body formation (4). The directed differentiation of the embryoid body cells led to hematopoietic precursor cells (5)that were subsequently introduced into a mouse exposed to ionizing radiation (6).

The induced pluripotent stem cells are an excellent model for pathogenetic studies at the celllevel and testing compounds possessing a possible therapeutic effect.

The induced pluripotent stem cells were produced from the fibroblasts of a patient with spinalmuscular atrophy (SMA) (SMAiPSCs). SMA is an autosomalrecessive disease caused by a mutation in the SMN1 ( survival motorneuron 1 ) gene, which is manifested as the selective nonviability of lower motor neurons. Patients with this disorder usually die at the age of about two years.Existing experimental models of this disorder based on the use of flatworms, drosophila, andmice are not satisfactory. The available fibroblast lines from patients withSMA cannot provide the necessary data on the pathogenesis of this disordereither. It was shown that motor neurons produced from SMAiPSCs canretain the features of SMA development, selective neuronal death, and the lackof SMN1 transcription. Moreover, the authors succeeded in elevating the SMNprotein level and aggregation (encoded by the SMN2 gene, whose expressioncan compensate for the shortage in the SMN1 protein) in response to the treatment of motorneurons and astrocytes produced from SMAiPSCs with valproate andtorbomycin [89;]. iPSCs and theirderivatives can serve as objects for pharmacological studies, as has been demonstrated oniPSCs from patients with familial dysautonomia (FDA) [90]. FDA is an inherited autosomal recessive disorder manifested as thedegeneration of sensor and autonomous neurons. This is due to a mutation causing thetissuespecific splicing of the IKBKAP gene, resulting in a decreasein the level of the fulllength IKAP protein. iPSCs were produced fromfibroblasts of patients with FDA. They possessed all features of pluripotent cells. Neuralderivatives produced from these cells had signs of FDA pathogenesis and low levels of thefulllength IKBKAP transcript. The authors studied the effect of threesubstances, kinetin, epigallocatechin gallate, and tocotrienol, on the parameters associatedwith FDA pathogenesis. Only kinetin has been shown to induce an increase in the level offulllength IKBKAP transcript. Prolonged treatment with kinetininduces an increase in the level of neuronal differentiation and expression of peripheralneuronal markers.

Currently, a broad spectrum of iPSCs is produced from patients with variousinherited pathologies and multifactorial disorders, such as Parkinsons disease, Downsyndrome, type 1 diabetes, Duchenne muscular dystrophy, talassemia, etc., whichare often lethal and can scarcely be treated with routine therapy [51, 87, 89, 9194]. The data on iPSCs produced by reprogramming somaticcells from patients with various pathologies are given in the Table 1.

Functional categories of M. tuberculosis genes with changed expression level during transition to the NC state

One can confidently state that both iPSCs themselves and their derivativesare potent instruments applicable in biomedicine, cell replacement therapy, pharmacology, andtoxicology. However, the safe application of iPSCbased technologies requires the use ofmethods of iPSCs production and their directed differentiation which minimizeboth the possibility of mutations in cell genomes under in vitro culturingand the probability of malignant transformation of the injected cells. The development ofmethods for human iPSC culturing without the use of animal cells (for instance, the feederlayer of murine fibroblasts) is necessary; they make a viralorigin pathogen transferfrom animals to humans impossible. There is a need for the maximum standardization ofconditions for cell culturing and differentiation.

This study was supported by the Russian Academy of Sciences Presidium ProgramMolecular and Cell Biology.

embryonic stem cells

induced pluripotent stem cells

neural stem cells

adipose stem cells

papillary dermal fibroblasts

cardiomyocytes

spinal muscular atrophy

iPCSs derived from fibroblasts of SMA patients

green fluorescent protein

long terminal repeat

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Induced Pluripotent Stem Cells: Problems and Advantages when Applying ...

Abstract

Extensive research has explored the functional correlation between stem cells and progenitor cells, particularly in blood. Hematopoietic stem cells (HSCs) can self-renew and regenerate tissues within the bone marrow, while stromal cells regulate tissue function. Recent studies have validated the role of mammalian stem cells within specific environments, providing initial empirical proof of this functional phenomenon. The interaction between bone and blood has always been vital to the function of the human body. It was initially proposed that during evolution, mammalian stem cells formed a complex relationship with the surrounding microenvironment, known as the niche. Researchers are currently debating the significance of molecular-level data to identify individual stromal cell types due to incomplete stromal cell mapping. Obtaining these data can help determine the specific activities of HSCs in bone marrow. This review summarizes key topics from previous studies on HSCs and their environment, discussing current and developing concepts related to HSCs and their niche in the bone marrow.

Keywords: hematopoietic stem cells, hematopoietic progenitor cells, bone marrow microenvironment, niche

Blood is a bodily fluid that delivers oxygen and nutrients to cells while collecting and transporting carbon dioxide and waste products produced by cellular metabolism [1]. Blood consists of plasma (a liquid component), red blood cells, white blood cells, and platelets. Hematopoiesis is the biological process through which blood and immune cells are produced [2] (Figure 1). Hematopoietic stem cells (HSCs) in the bone marrow are responsible for continuously replenishing these cells due to their limited lifespan [3]. HSCs occupy the highest position in the hierarchy of hematopoietic cells. The HSC niche in bone marrow is a specialized microenvironment that regulates the maintenance and activity of HSCs [4]. This niche governs self-renewal and differentiation of HSCs, ensuring the continual maintenance of hematopoiesis [5]. The bone marrow microenvironment was first introduced as a niche for HSCs in the 1970s [6]. The niche supplies the necessary components for the self-renewal and differentiation of HSCs. Additionally, the niche controls the states of rest and progression at various stages of the cell cycle in stem cells [6] (Figure 2). It also communicates crucial information to stem cells regarding the surrounding tissue, influences the development of stem cell offspring, and helps prevent genetic mutations [7]. Numerous studies have revealed the significance of HSCs and their niche, leading to a better understanding of their relationship [7,8,9,10,11].

Hematopoietic stem cell (HSC) regulation in steady-state and hematological malignancies. This image shows the features of HSC regulation between normal conditions and hematological malignancy. In normal hematopoiesis, HSCs are activated in response to signals from the bone marrow microenvironment. Upon activation, HSCs undergo proliferation to increase their numbers and develop into multipotent progenitors (MPPs). MMPs can evolve into more committed lymphoid/myeloid progenitors and their respective sub-progenitors (e.g., GMP, MEP, etc.). These progenitor cells undergo further differentiation and maturation to give rise to the diverse range of blood cell types found in circulation. Each cell in the hematopoietic process can be distinguished by differentiation markers. This tightly regulated process of activation, proliferation, and differentiation ensures the continuous replenishment of blood cells to maintain homeostasis. When the HSCs and the progenitors within the developing HSCs become damaged, they can transform into leukemic stem cells (LSCs). LSCs possess self-renewal capabilities and aberrant differentiation, giving rise to leukemic blasts that result in leukemia. CLP: Common lymphoid progenitor. CMP: Common myeloid progenitor. GMP: GranulocyteMacrophage progenitor. MEP: Megakaryocyteerythrocyte progenitor. Pro-B: Progenitor cell-B. Pro-T: Progenitor cell-T. Pro-NK: Progenitor cell-NK. Pro-DC: Dendritic progenitor cell. MncP: Monocyte progenitor. GrP: Granulocytic progenitor. EryP: Erythrocytic progenitor. MkP: Megakaryocyte progenitor. NK cells: Natural killer cells.

An image showing bone marrow microenvironment with their components. It shows two BM niches, two bone marrow niches, and the endosteal and vascular niches. The endosteal niche and vascular niche are two crucial microenvironments within the BM. The endosteal niche, located near the bone surface, provides a specialized environment for hematopoietic stem cells (HSCs) to reside and self-renew. The osteoblast is considered the most important cell in the endosteal niche; hence, it is also referred to as the osteoblastic niche. In contrast, the vascular niche, adjacent to blood vessels, supports HSCs by supplying nutrients and signaling molecules necessary for their proliferation and differentiation. It is composed of endothelial cells lining the blood vessels, as well as pericytes and smooth muscle cells surrounding them. Together, these niches play integral roles in regulating the maintenance and function of HSCs in the bone marrow. CAR cell: CXCL12-abundant reticular cell. OPN: Osteopontin. ANG1: Angiopoietin-1, SCF: Stem cell factor.

Due to global advancements in aging research and the increase in life expectancy over the past 150 years, studies on the physiological changes that occur in organisms as they age have made substantial progress [12,13]. Aging is characterized by a progressive decline in the function of many organs and tissues that, in some cases, can contribute to the development of cancer [14]. The hematopoietic system undergoes alterations with age, which affects the performance and number of HSCs and the composition of blood cells [15], increasing the likelihood of acquiring age-related blood illnesses such as anemia, a weakened immune response, and blood cancer. After a defined period, blood cells undergo differentiation and maturation and are eventually destroyed, preserving the equilibrium state. Hematological disorders are medical ailments characterized by an imbalance in homeostasis [10]. Hematopoietic tissue cancer (blood cancer) is a malignancy that originates in bone marrow [8] and is characterized by the excessive growth of abnormal blood cells [16]. These disorders are due to abnormalities in HSCs, the initiating cells in the hematopoietic system. Therefore, targeting only specific cells while minimizing damage to normal cells remains challenging [17,18]. Consequently, stem cell therapy is emerging as a promising alternative for treating hematological diseases, including those related to aging.

Stem cell therapy is highly regarded for its potential in treating not only blood-related diseases but also for regenerating damaged tissues and organs. Stem cells used in related research encompass various types, including adult stem cells such as HSCs and mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) created by reprogramming somatic cells back to a pluripotent state [19,20]. MSCs, being multipotent stromal cells, exhibit the capacity to differentiate into a variety of cell types, including bone, cartilage, and adipocytes [20,21,22,23]. Consequently, numerous research findings have suggested their therapeutic potential in diverse diseases such as cartilage regeneration [24,25] and neurological disease recovery [25,26,27,28]. ESCs present immense therapeutic promise, as they can differentiate into all cell types in the body [29,30]. However, research in this domain is constrained by ethical dilemmas surrounding the extraction of stem cells from embryos [19]. iPSCs are anticipated to circumvent these ethical issues while offering utility akin to ESCs. Nonetheless, challenges persist in the reprogramming process, and uncertainties exist regarding their stability [19]. Despite active research and reporting on the therapeutic potential of stem cell therapy, many facets of stem cell biology remain unexplored, including fundamental mechanisms governing stem cell behavior and their interactions with the host environment. Consequently, stem cell therapy has not yet attained widespread adoption as a standard treatment. This review focuses on HSCs and their microenvironment to enhance our understanding of stem cell therapy, especially hematopoietic stem cell therapy.

HSCs are a rare population of multipotent cells, responsible for replenishing all blood cell types throughout an individuals lifetime. They have the unique ability to self-renew and differentiate into several types of blood and immune cells. This process, which produces all types of blood cells, is called hematopoiesis (Figure 1) [9]. HSCs produce hematopoietic progenitor cells through differentiation, which differentiate further to produce blood and immune cells [1]. However, hematopoiesis is a highly regulated process and typically unidirectional; once HSCs differentiate into hematopoietic progenitor cells, they cannot regenerate into HSCs [1]. Additionally, HSCs are used in transplantation therapy after irradiation to treat patients with blood cancer [19]. Unlike solid cancers, which can be selectively targeted and treated, blood cancers present significant challenges for treatment with conventional chemotherapy and radiation. For this reason, HSC transplantation remains one of the most effective and promising approaches, with significant ongoing research focusing on its potential [10].

HSCs predominantly reside in a specialized microenvironment within the bone marrow, known as the endosteum [2,9]. In this niche, HSCs remain dormant under stable conditions. When blood cells decrease due to stressors, such as bleeding, illness, or radiation, HSCs activate and reorganize the hematopoietic system by proliferating and differentiating into new cell types [1]. The equilibrium between the quiescent state and the division of HSCs is crucial for maintaining normal hematopoiesis. If this equilibrium is not adequately regulated, HSCs may decrease in number or give rise to blood malignancies such as leukemia (Figure 1). Thus, the equilibrium between the dormant and active phases of HSCs is tightly controlled by both internal and external mechanisms.

Blood is an essential regenerating tissue that is susceptible to changes and deterioration with age [12,13,31]. Aging is accompanied by various clinically significant conditions that affect the hematopoietic system [14], including a decline in the adaptive immune system, an increased occurrence of specific autoimmune diseases, a higher prevalence of hematological malignancies, and an increased likelihood of age-related anemia [32]. An age-related decline in the functional capacity of HSCs has been widely recognized in studies conducted on mouse models [33]. When comparing young HSCs to old ones, the latter exhibit a preference for the myeloid lineage and have a reduced ability to regenerate when transplanted [33]. In addition, like many other tissues, the hematopoietic system is more likely to develop cancer with age, including a higher incidence of chronic and acute leukemia [14]. Given that myeloid leukemia is more common in older individuals and juvenile leukemia typically affects the lymphatic system, age-related alterations in HSCs may directly influence the development of disorders associated with blood cell formation [15]. Aged HSCs show increased expressions of genes implicated in the progression of myeloid leukemia, such as AML, PML, and ETO. Alternation of these gene expressions during normal hematopoiesis can result in impaired self-renewal capacity of HSC, heightened susceptibility to DNA damage, and aberrant differentiation potential. These alternations on HSCs are characteristic features of aged HSCs. Consequently, they are deemed suitable targets for investigating HSC aging and comprehending the molecular mechanisms underlying age-related hematopoietic dysfunction and leukemogenesis [32,34,35,36,37,38].

Multiple studies have documented the deterioration of HSCs in older mice, although the specific molecular processes responsible for this aging phenomenon remain unclear [14,15,31,32,33]. The aging of HSCs is limited by their diversity. The purity of HSCs isolated using flow cytometry has consistently been poor, indicating that the population becomes more heterogeneous as individuals age [15]. Ongoing research aims to identify specific subsets of HSCs that contribute to the aging phenotype [11]. This is achieved through the examination of age-dependent diverse pools of HSCs using single-cell bone marrow transplantation, flow cytometry, and single-cell transcriptome sequencing [15,32,39]. Specifically, HSC clones that undergo myeloid differentiation progressively occupy the HSC reservoir with age [39]. In this aspect, multiple research findings have been reported concerning the correlation between clonal hematopoiesis and aging [40]. Clonal hematopoiesis (CH) is a condition characterized by the expansion of specific HSC clones that acquire somatic mutations (e.g., DNMT3A, TET2, and ASXL) [41,42]. These mutations are thought to confer a selective advantage to HSCs, leading to the predominance of these clones in the blood system and allowing them to outcompete normal HSCs and expand clonally. While the specific signaling pathways involved in this process may vary depending on the gene and context, some common themes have emerged. For example, mutations in DNMT3A [43,44], TET2 [45,46,47], and ASXL1 [48] are known to affect epigenetic regulation, leading to alterations in gene expression patterns and cellular differentiation pathways [42,49]. Additionally, these mutations may impact other signaling pathways related to cell survival, proliferation, and self-renewal [48]. However, the exact signaling pathways or mechanisms through which these mutations lead to clonal expansion are still under investigation and continue to be an active area of research. This phenomenon becomes increasingly common with age and is associated with a higher risk of hematologic malignancies and cardiovascular diseases [41,50,51]. Research indicates that approximately 1020% of individuals over 70 years old exhibit clonal hematopoiesis, highlighting its prevalence in the elderly population.

CH not only alters the composition of the hematopoietic system but also impacts the bone marrow microenvironment, known as the niche, which is crucial for maintaining HSC function and homeostasis. Aging induces significant changes in the bone marrow niche, including a decline in the number and function of MSCs, osteoblasts, and endothelial cells [41,52]. These alterations, coupled with the production of elevated levels of inflammatory cytokines such as IL-6 and TNF- by mutant HSCs and the aging niche, create a pro-inflammatory and oxidative stress environment [47,53,54]. This environment promotes the expansion of CH, impairs normal HSC function, and decreases the secretion of essential factors for HSC maintenance, thus exacerbating the proliferation of clonal HSCs and diminishing the niches ability to support normal hematopoiesis. Although there have been numerous reports on the heterogeneity of HSCs associated with aging, our understanding of the effects of aging remains uncertain, and requires further investigation.

A recent study investigated the functional alterations that occur in aged HSCs within the mitochondrial metabolic milieu [12,13,14]. Specifically, the properties and roles of young and aged HSCs are influenced by the mitochondrial membrane potential within these cells [55]. Researchers reversed aging in old mice by manipulating the mitochondrial membrane potential of aged HSCs using the antioxidant Mito-Q [31]. Clinical utilization of Mito-Q is a possible preventative measure and treatment for age-related blood disorders.

HSCs typically reside in the bone marrow (BM), which is composed of various components, including bone, blood vessels, and other cells and substrates filling the spaces between them [2]. This BM microenvironment, known as a Niche, provides a structural framework and communication networks to HSCs [2,7].

This microenvironment can control the state of HSCs by direct or indirect interactions and safeguard them from sustaining their undifferentiated state [2,7,9]. It engages HSCs to control their growth and specialization through distinct signal transduction processes, resulting in regular hematopoiesis [7]. Recent advancements in single-cell analysis techniques have revolutionized our understanding of the BM niche, shedding light on its cellular composition, spatial organization, and dynamic interactions with HSCs. One of the key insights gleaned from single-cell analysis is the dynamic nature of the BM niche [56]. Studies have revealed the presence of specialized niches within the BM, each tailored to support specific stages of hematopoietic development [57,58]. Moreover, single-cell analysis has unveiled the plasticity of niche cells, demonstrating their ability to dynamically respond to extrinsic signals and adapt to changing physiological conditions [59,60]. Furthermore, single-cell analysis has provided insights into the spatial organization of the bone marrow niche, uncovering intricate spatial relationships between niche components and HSCs. Spatial transcriptomics techniques have revealed specialized niches localized within specific anatomical regions of the BM, highlighting the importance of spatial context in regulating hematopoietic function [58,61,62,63].

Depending on their spatial location, niches can be divided into an osteoblastic niche, which is the area near the endosteum, and a vascular niche, where blood vessels and surrounding matrix exist in the BM [58]. In addition, various immune cells derived from HSCs (including T/B lymphocytes, macrophages, natural killer cells, and dendritic cells) or the stromal cells contribute to configuring the BM microenvironment. These cells interact with HSCs, participating in the regulation of their state. Non-cellular substances can also serve as nutrients for HSCs, providing essential support for their growth and maintenance. These substances may include growth factors, cytokines (e.g., SCF, interleukins, CXCL12), and extracellular matrix components present in the BM microenvironment. By interacting with HSCs, these non-cellular factors play a crucial role in regulating hematopoiesis and maintaining stem cell homeostasis.

The vascular niche is composed of endothelial cells and perivascular stromal cells (such as pericytes and smooth muscle cells) that make up blood vessels [64,65,66]. They provide structural support and produce niche factors essential for HSC maintenance, proliferation, and differentiation. Additionally, the extracellular matrix surrounding these niche cells serves as a dynamic scaffold that facilitates cellular interactions and regulates the release and localization of signaling molecules [67,68].

Vasculogenesis can be categorized into two stages: the embryonic and adult stages [2,9]. During the embryonic stage, there is a significant level of contact between HSCs and endothelial cells [69]. Hematopoietic and endothelial cells are derived from hemangioblasts, multipotent progenitor cells, during the embryonic stage [70]. Endothelial cells expressing RUNX1 can produce HSCs in the aorta, gonad, mesonephros, and placenta [71]. Both endothelial and hematopoietic stem cells co-express CD31, CD34, CD133, FLK1, and TIE2 [72]. HSCs release angiopoietin-1 (ANG1), which stimulates the growth of new blood vessels during angiogenesis [73]. Additionally, endothelial cells provide a similar microenvironment for HSCs as well as neural stem cells. In the hippocampus, neural stem and endothelial cells that generate fibroblast growth factor (FGF), another angiogenesis-promoting substance, are close to each other [74].

However, the precise nature of the interaction between endothelial cells and bone marrow HSCs in the adult stage remains unclear. BM-derived endothelial progenitor cells participate in postnatal angiogenesis [75]. A conceptual framework for the vascular environments in bone marrow has been suggested, wherein the activation of MMP9 expressed in the osteoblast region results in the separation of the Kit ligand from the cell membrane of stromal cells in the BM. Subsequently, the soluble Kit ligand stimulates the initiation of the cell cycle and enhances the activity of HSCs [76]. Thus, HSC activity, proliferation, and differentiation occur in the vascular niche within the BM [69]. Vascular endothelial growth factor (VEGF) and ANG1 are angiogenic factors that play crucial roles in preserving HSCs [77]. VEGF controls the development of blood vessels and hematopoiesis and regulates hematopoietic stem cells through an internal autocrine loop [78]. HSCs remain inactive in osteoblastic niches, whereas both hematopoietic stem and progenitor cells undergo division in vascular habitats. Hematopoietic cell migration commences in stem cells located in the osteoblast niche where they then proliferate, differentiate, and ultimately mature [7]; cells migrate toward the vascular niche via this process.

To maintain hematopoietic homeostasis, the process of homing, wherein hematopoietic stem and progenitor cells (HSPCs) circulating through the blood return to the BM niche, is also essential [79,80,81]. In this process, HSPCs directly interact with the endothelium via cellcell adhesive interaction. Sinusoidal endothelial cells express adhesion molecules, including P-selectin (CD62P), E-selectin (CD62E), and vascular cell adhesion molecule-1 (VCAM-1 or CD106). Several receptors for these molecules are expressed in HSPCs, including P-selectin glycoprotein ligand-1 (CD162) and CD44, along with other less well-defined E-selectin receptors. Additionally, receptors for VCAM-1, such as integrins 41, 47, and 91, are also expressed.

The other components such as pericytes and smooth muscle cells also play an important role in regulating the behavior of HSCs [82,83]. Leptin-receptor-positive (LepR+) cells and CXCL12-abundant reticular (CAR) [82] cells are well-established cells that secrete growth factors essential for the maintenance of HSCs. They are located along the blood vessels of mainly the sinusoids, playing a crucial role in regulating vascular stability and function. CXCL12 and SCF from them are key factors for HSC proliferation [84]. This was confirmed through experiments deleting CXCL12 secreted by LepR+ cells and CAR cells. Deletion of CXCL12 in these cells results in the removal of all quiescent and serially transplantable HSCs from adult bone marrow. This occurs because signaling with CXCR4, receptors on HSCs, is reduced, demonstrating that CXCL12 from LepR+ cells and CAR cells play a central role in the signaling that maintains the pool of HSCs [85].

Conversely, Nestin-positive (Nes+) cells found exclusively around arterioles provide support, contrasting with perivascular cells around sinusoids [86]. Nes+ cells also secrete soluble factors like CXCL12 and SCF, which tend to drive quiescent HSCs into early hematopoietic stages and promote HSC activation, leading to differentiation [87].

Osteoblasts, layering the endosteal bone surface and providing an osteoblastic niche to HSCs, regulate hematopoiesis [7,88]. They provide a supportive environment for HSCs, regulating their self-renewal, differentiation, and quiescence. Osteoblasts produce niche factors and adhesion molecules that interact with HSCs, influencing the maintenance of HSCs in a dormant state and their activation in response to hematopoietic demand [89]. Osteoblasts have a critical role in the regulation of the physical location and proliferation of HSCs by expressing osteopontin (OPN). OPN specifically binds to beta1 integrin expressed on HSCs [90]. The other key factor expressed in osteoblasts is angiopoietin-1 (ANG1). Interaction of Tie2 and ANG1, the receptor of ANG1 expressed on HSCs, vital for maintaining HSCs in the quiescent state, preserves their long-term self-renewal potential and prevents exhaustion [39]. This signaling helps to retain HSCs in the bone marrow niche and prevents their premature differentiation or migration [91,92,93].

Through long-term in vivo labeling with 5-bromodeoxyuridine (BrdU), most HSCs divide [94]. However, some HSCs were found to be dormant, retained their labels, and remained dormant for several months. Therefore, bone marrow cells can be classified into resting and dividing HSCs. Resting HSCs are located close to osteoblasts [7]. Using Bmpr1a KO mice, Zhang et al. showed that N-cadherin+ spindle-shaped osteoblasts resemble HSCs with a slow cell cycle [94]. Their study revealed that osteoblast cells expressing N-cadherin in the bone marrow act as nests for HSCs, and that an increase in the number of N-cadherin+ cells is associated with an increase in HSCs. Additionally, Visnjic et al. showed that hematopoiesis is suppressed in osteoblast-deficient mice [95]. Thus, it was confirmed that defects in HSC osteoblasts inhibit hematopoiesis. The Notch signaling pathway, characterized by membrane-bound ligands, regulates cell fate determination across various systems, including the self-renewal of HSCs [96,97,98,99,100]. In the study by Calvi et al. [101], they found that PPR-stimulated osteoblasts express a high level of Notch ligand jagged 1 using the transgenic mouse of PTH/PTHrP receptors (PPRs). In response, the activation of the Notch1 intracellular domain (NICD) in Lin-Sca-1+c-Kit+ HSCs increased. Additionally, when HSCs were long-term co-cultured with a Notch cleavage inhibitor, the support for HSCs observed in transgenic stroma decreased to a similar level to their isotype control. Another study, using RAG-1-deficient mice essential for V(D)J recombination and lymphocyte development, showed that Notch1 activation leads to inhibition of HSC differentiation [98]. This confirms that interaction between osteoblasts and HSCs via the Notch pathway plays a crucial role in regulating HSC behavior within the bone marrow niche.

In addition to spatially distinct osteoblastic and vascular niches, stromal cells and immune cells play roles within the microenvironments of HSCs in bone marrow [62,63,102,103,104]. They can either directly interact with HSCs or regulate them indirectly by secreting soluble factors such as growth factors, cytokines chemokines, and other signaling molecules.

Macrophages in the bone marrow play a crucial role in the formation of HSCs [63,105,106,107,108,109,110,111]. CD169+ macrophages, associated with the clearance of blood-borne pathogens and regulation of immune responses, play a crucial role in maintaining the quiescent state of HSCs [105]. They interact with Nestin-positive (Nes+) cells to promote the transcription of CXCL12 and other factors (such as HSC maintenance and retention factors ANG, KITl, VCAM1) essential for HSC maintenance. Depletion of macrophages leads to the loss of these factors and subsequent egress of HSCs from the bone marrow [105,106]. A subset of macrophages called Osteomacs reside adjacent to osteoblasts and megakaryocytes along the bone lining, distinct from osteoclasts. These osteomas have been identified to play crucial regulatory roles in modulating osteoblast function. Their interaction with osteoblasts is essential for the low-level activation of nuclear factor B (NF-B) in osteoblasts, enabling them to maintain HSCs through appropriate chemokine signaling. Furthermore, the presence of megakaryocytes supports the function of osteomacs, and their synergistic interactions with osteoblasts contribute to the regulation of HSC repopulating potential, as evidenced by transplantation assays [107,108,109,110,111]. Although significant progress has been made in understanding the role of macrophages in HSC behavior [106], the specific signaling pathways and the diverse functions associated with macrophage heterogeneity are not yet fully understood. Therefore, ongoing additional studies are needed to fully elucidate the multifaceted roles of macrophages in hematopoiesis and their potential therapeutic applications.

Megakaryocytes also govern the viability of HSCs [112,113,114]. Megakaryocyte removal from the bone marrow leads to an increase in the number of HSCs. HSCs exhibited a compensatory increase in mice experiencing bleeding. However, this compensatory increase is restricted when blood cells are introduced into the bloodstream [113]. Megakaryocytes have been suggested to restrict the proliferation of HSCs in two ways. The first mechanism involves the production of CXCL4 by megakaryocytes, which inhibits HSC proliferation [112]. The second mechanism involves the action of TGF, which controls the inactive state of the HSCs [113]. Additionally, megakaryocytes influence myeloid-biased HSC activity and act as a physical barrier to HSC migration. Thrombopoietin (TPO) production by megakaryocytes further regulates hematopoietic activity. Depletion of megakaryocytes in mice resulted in decreased megakaryopoiesis, alongside lower numbers of HSCs and reduced HSC quiescence [115,116,117,118].

Chemokines, also known as chemo-attractant proteins, play crucial roles in regulating the movement of HSCs and facilitating their contact with stromal cells [119]. CXCL12, also known as SDF1, is a chemokine involved in cell homing. Deletion of SDF1 or its receptor CXCR4 leads to normal fetal heart hematopoiesis; however, there is a failure of bone marrow engraftment by hematopoietic cells [120,121]. Upregulation of CXCR4 in human hematopoietic progenitor cells results in enhanced engraftment in nude mice, whereas the use of CXCR4-neutralizing antibodies demonstrates an inhibitory effect on engraftment [122]. However, CXCR4 is not typically found in HSCs that are not actively dividing. This identifies the factors for successful HSC attachment and the molecules responsible for binding to osteoblasts. Osteoblasts express the adhesion molecules ALCAM and osteopontin, which may play a role in the interaction between HSCs and osteoblasts [123]. Furthermore, it is assumed that external factors such as BMPs, NOTCH ligands, and angiopoietins in bone marrow niches play a role in the interaction between HSCs and osteoblasts [94,101]. In some research, depletion of CXCL12 in osteoblasts resulted in the selective loss of B-lymphoid progenitors. Studies have shown that acute inflammation can inhibit osteoblastic bone formation, leading to T and B lymphopenia due to decreased production of interleukin-7 (IL-7). This suggests that osteoblasts may regulate common lymphoid progenitors by supplying IL-7 [124,125,126].

Myeloid lineage cells, including granulocytes and dendritic cells, also impact the HSC niche [127]. Granulocytes produce factors like G-CSF (granulocyte colony-stimulating factor), which promotes HSC mobilization from the bone marrow into the bloodstream. Dendritic cells contribute to HSC maintenance by modulating the expression of adhesion molecules and cytokines within the niche.

Due to the characteristics of HSCs, their self-renewal, multiple differentiation, and interactions with niche components, they can be used for the therapy of some blood-related diseases. Transplanting HSCs can restore patients HSC pools and also regenerate immune cell populations, which means that abnormal hematopoiesis has been replaced with normal hematopoiesis [128]. Hematopoietic stem cell transplantation (HSCT), also known as bone marrow transplantation, is utilized as a therapeutic approach for various blood-related diseases. HSCT offers a powerful therapeutic option by essentially resetting the hematopoietic and immune systems, allowing for the restoration of normal function and providing a potential cure for many serious conditions. It can be applied to patients as a therapeutic approach for various blood-related diseases, including malignant blood disorders such as lymphoma, multiple myeloma, and leukemia, as well as aplastic anemia and immunodeficiency disorders. It is especially considered in relapsed or refractory cases that do not respond to conventional chemotherapy or radiotherapy and in aggressive forms (e.g., diffuse large B-cell lymphoma, mantle cell lymphoma, and follicular lymphoma) [22,129,130,131].

Unlike solid organ transplantation, where the main goal is organ replacement, allogeneic hematopoietic cell transplantation for hematologic malignancies focuses on regulating the immune response against the underlying cancerous condition [128,132]. In leukemia, normal hematopoietic microenvironments are transformed into leukemic microenvironments by leukemic stem cells (LSCs). LSCs exhibit a high propensity for proliferation rather than differentiation into subset populations and possess strong resistance to drugs, resulting in poor prognosis and leukemia relapse [132,133,134,135,136]. For bone marrow transplantation, the most important thing is donor selection [137]. It is crucial to match the donors human leukocyte antigen (HLA) with the recipients as closely as possible to minimize the risk of graft rejection and graft-versus-host disease (GVHD). GVHD is a significant complication following HSCT, where donor immune cells attack the recipients tissues, leading to organ damage [138,139]. Immune checkpoint molecules such as TIGIT, PD-1, CTLA-4, and TIM-3 play pivotal roles in regulating immune responses in GVHD [140,141]. TIGIT and PD-1 inhibit T cell activation and effector functions [140,142,143,144,145,146], while CTLA-4 competes with CD28 for ligand binding, thereby inhibiting T cell activation [141,147]. TIM-3 regulates T cell exhaustion and tolerance [148,149]. Dysregulation of these markers can disrupt immune homeostasis, exacerbating GVHD pathology. Understanding the functions of immune checkpoint molecules is crucial for developing targeted therapies to mitigate GVHD severity post-HSCT. In a German study, after transplantation, the graft versus leukemia (GvL) effect in acute myeloid leukemia (AML) was found to significantly improve the 7-year relapse-free survival of patients with AML in first complete remission compared to conventional chemotherapy alone. This highlights its efficacy in disease control. However, transplantation at an advanced disease stage yields lower survival rates, emphasizing the importance of early consideration and referral for transplantation in eligible patients [150,151,152].

For successful transplantation, the recipients (patients) blood and immune system must initially be depleted by combinations of chemotherapy and radiotherapy [153]. Drugs used in conditioning therapy before bone marrow transplantation include cyclophosphamide, busulfan, melphalan, and fludarabine. These drugs induce apoptosis by interfering with DNA replication, transcription, and synthesis, thereby destroying the patients existing cells and suppressing the immune system. This helps prevent transplant rejection by adequately suppressing the immune system [154,155,156,157]. If this pre-HCT conditioning is performed well, donor HSCs can home to and engraft the recipients bone marrow, thereby reconstituting all the blood cell lineages. Immune recovery after HSCT occurs in phases, with innate immune cells and platelets generally recovering within weeks after HSCT; fully complete reconstitution of adaptive immunity may extend over months to even years (Figure 3) [158,159,160,161].

Dynamics of immune reconstitution and associated risks in recipients bone marrow following hematopoietic stem cell transplantation. In the first few weeks after transplantation, innate immune cells recover swiftly. Common infections during this phase include bacterial and Candida infections due to the early deficiency in adaptive immune cells. Meanwhile, adaptive immune function, including T cells and B cells, exhibits prolonged deficiencies and gradually recovers, taking over 2 years to fully restore. Viral infections and those caused by non-Candidal molds become more common during this phase. Various clinical factors, including conditioning regimens, donor sources, and post-transplant events such as graft-versus-host disease (GVHD) and immunosuppression, exert influence over the immune reconstitution process, thereby modulating the associated infectious risks.

During this process, various cells within the bone marrow serve as niche components for donor HSCs. The BM niche provides the microenvironment necessary for hematopoietic stem cell (HSC) maintenance, differentiation, and proliferation. Endothelial cells play a significant role in the regulation of various processes, including the quiescence, proliferation, and mobilization of HSCs. It is anticipated that ECs will aid in the hematopoietic recovery of donor HSCs following transplantation. Although ECs are often damaged during conditioning for HCT, when transplanted alongside HSCs, they have been shown to confer beneficial effects in terms of HSC engraftment, reconstitution, and survival post-irradiation [162,163,164]. MSCs, as a rare component of the bone marrow niche, play a crucial role in regulating HSC homeostasis through the production of key soluble factors. Different subsets of MSCs have distinct impacts on HSC behavior, supporting either quiescent or proliferative states. Despite surviving conditioning regimens, MSCs may accumulate damage, potentially affecting their functionality. In clinical contexts, MSCs have shown promise in enhancing HSC engraftment and treating complications like steroid-resistant aGvHD, although further research is needed to elucidate their precise mechanisms of action [165,166,167].

Hematopoietic stem cells (HSCs) possess the remarkable ability to generate all lower cells of the hematopoietic hierarchy and regulate the entire process of hematopoiesis through self-renewal and proliferation. The uninterrupted generation of new blood cells is indispensable for the survival of organisms, underscoring the critical importance of maintaining the normal function of HSCs throughout life. Normal hematopoiesis involves maintaining a good balance between activated HSCs that produce blood and quiescent HSCs that do not function. However, when HSCs are damaged due to various factors, such as aging, their function is compromised, leading to aberrant hematopoiesis and potentially giving rise to hematological diseases, including aplastic anemia, myelodysplastic syndromes, and leukemia.

Aging affects the overall functioning of an organism, and blood production is also strongly affected. Various research results have revealed that aging affects the function of HSCs, causing their parts to change abnormally. Functional and genomic analysis has been conducted through mouse experiments, and the phenotype in elderly people is similar. Aging eventually causes diseases such as immune disorders, lymphoma, and leukemia, and the prognosis is worse for elderly patients whose hematopoiesis and immune systems have already collapsed.

The niche of HSCs interacts with cells in various aspects to regulate their functions. Osteoblast cells in the bone-adjacent area of the bone marrow lumen play a crucial role in regulating the state of HSCs through various mechanisms. Osteoblasts express Ang1 and OPN, which bind to specific receptors expressed on HSCs, causing them to remain stationary in a specific area. This interaction helps maintain the quiescent state of HSCs and regulates their retention within the bone marrow niche.

Vascular tissue refers to vascular components including vascular endothelial cells, pericytes, and SMCs, as well as stromal cells, which are supporting cells around them. Endothelial cells (ECs) and pericytes are classified according to the location of blood vessels (sinusoids or arterioles), and they both regulate HSCs by secreting various chemokines, including CXCL12 and SCF. These soluble factors perform different functions depending on their site of secretion, either promoting the quiescence or activation of HSCs.

HSC transplantation is gaining attention as a treatment for diseases stemming from HSC damage, particularly leukemia. Just as HSCs interact with niche components to sustain ongoing hematopoiesis, hematopoiesis can be restored by transplanting HSCs from a healthy donor into patients with HSC or niche defects. However, due to the limited understanding of the niche in the context of bone marrow transplantation, ongoing research is crucial to address issues like GVHD.

Writingoriginal draft preparation, M.K.; conceptualization, B.S.K., S.Y., S.-O.O. and D.L.; funding acquisition, D.L. All authors have read and agreed to the published version of the manuscript.

The authors declare no conflicts of interest.

This research was supported by grants from the Korean Cell-Based Artificial Blood Project funded by the Korean government (The Ministry of Science and ICT; the Ministry of Trade, Industry, and Energy; the Ministry of Health & Welfare; the Ministry of Food and Drug Safety) [grant no. HX23C1692], and grants from the Basic Science Research Program through the National Research Foundation (NRF) of Korea, funded by the Ministry of Education and the Ministry of Health & Welfare [grant nos. 2022R1A5A2027161, RS-2023-00223764, and RS-2024-00333287].

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Originally posted here:
Hematopoietic Stem Cells and Their Niche in Bone Marrow

Curr Opin Hematol. Author manuscript; available in PMC 2022 Jan 1.

Published in final edited form as:

PMCID: PMC7769132

NIHMSID: NIHMS1651634

1.Division of Experimental Hematology and Cancer Biology, Cincinnati Childrens Medical center, Cincinnati, Ohio, 25228, USA

2.Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45229, USA

1.Division of Experimental Hematology and Cancer Biology, Cincinnati Childrens Medical center, Cincinnati, Ohio, 25228, USA

2.Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45229, USA

The bone marrow is the main site for hematopoiesis. It contains a unique microenvironment that provides niches that support self-renewal and differentiation of hematopoietic stem cells (HSC), multipotent progenitors (MPP), and lineage committed progenitors to produce the large number of blood cells required to sustain life. The bone marrow is notoriously difficult to image; because of this the anatomy of blood cell production- and how local signals spatially organize hematopoiesis-are not well defined. Here we review our current understanding of the spatial organization of the mouse bone marrow with a special focus in recent advances that are transforming our understanding of this tissue.

Imaging studies of HSC and their interaction with candidate niches have relied on ex vivo imaging of fixed tissue. Two recent manuscripts demonstrating live imaging of subsets of HSC in unperturbed bone marrow have revealed unexpected HSC behavior and open the door to examine HSC regulation, in situ, over time. We also discuss recent findings showing that the bone marrow contains distinct microenvironments, spatially organized, that regulate unique aspects of hematopoiesis.

Defining the spatial architecture of hematopoiesis in the bone marrow is indispensable to understand how this tissue ensures stepwise, balanced, differentiation to meet organism demand; for deciphering alterations to hematopoiesis during disease; and for designing organ systems for blood cell production ex vivo.

Keywords: Hematopoiesis, bone marrow organization and architecture, hematopoietic stem cell niches, hematopoietic progenitor niches, bone marrow microenvironment

Hematopoiesis takes place in the bone marrow (BM) where hematopoietic stem cells and multipotent progenitors (HSPC) self-renew and progressively differentiate into lineage-specific, unipotent, progenitors responsible for production of each major blood lineage. The bone marrow has been studied in detail using multiple approaches including scRNAseq, and in vivo lineage tracing studies [19]. These and other studies have dramatically changed our understanding of how the different stem and progenitor populations differentiate, and how they are regulated by the BM microenvironment- the collection of hematopoietic and stromal cells and structures that supports differentiation- during normal and stress hematopoiesis. Our understanding of the spatial organization of hematopoiesis in the bone marrow is less comprehensive. Spatial analyses of differentiating progenitors, their offspring, and the supporting microenvironment are challenging due to several factors (reviewed in [10]); a) the bone marrow is fully enclosed by opaque bone which makes direct observation difficult and requires extensive preparation steps in order to generate high quality samples for imaging analyses; b) the hematology field has used increasingly complex combinations of cell surface markers -requiring simultaneous detection upwards of 15 antibodies- to define each hematopoietic progenitor and mature cell in the bone marrow. In contrast fluorescent analyses are generally limited to much fewer (4-7) parameters preventing simultaneous identification of multiple cell types. Further, many antibodies used to define cells by flow cytometry fail to detect the same cells in imaging analyses, either because the signals are too dim or because sample preparation destroyed the epitopes recognized by that antibody; c) scRNAseq analyses of stromal cells in the bone marrow have revealed extraordinary complexity [79**]. However, there are no validated antibodies to detect many of these stromal populations and the field relies in Cre/fluorescent reporter mice that identify some stromal components but fail to completely resolve the different populations [11]; d) the bone marrow contains large numbers of mature cells but stem cells and progenitors are exceedingly rare. This makes identification of sufficient numbers of HSPC for adequately powered statistical analyses very challenging and time consuming; e) different groups have used different statistical approaches and methods to define proximity of cells to structures and a global consensus on which approaches to use has yet to emerge. Despite numerous challenges the field has made tremendous progress in defining the architecture of the BM and deciphering how local cues from the microenvironment regulate stem and progenitor cells. Here we summarize our current understanding of the spatial organization of the bone marrow, its impact on hematopoiesis, and discuss recent discoveries that are transforming the field.

The main structures that spatially organize the bone marrow are the bone, the vasculature, and a network of reticular stromal cells. The bone completely encloses the bone marrow, defines its boundaries, and projects trabeculae that penetrate into the BM parenchyma (). The bone marrow vasculature is composed of rare arterioles that enter through the bone and transform into transitional vessels that give rise to an extremely dense network of fenestrated sinusoids that occupy most of the BM space (). The vasculature is tightly associated with a network of perivascular reticular cells that spreads through the BM. Hematopoiesis takes place in the spaces between vessels, bone, and reticular cells (). Many other types of stromal (non-hematopoietic) cells are present in the bone marrow including sympathetic nerves, Schwann cells, adipocytes, osteoblasts, osteocytes, osteoblastic precursors, and diverse types of fibroblasts. These are reviewed elsewhere [1214]. These cells and structures in association with different types of hematopoietic cells-cooperate to provide distinct microenvironments that regulate and regionally organize-hematopoiesis in the bone marrow.

Schematic representation of the spatial organization of the mouse bone marrow under homeostasis. The endosteum, the vasculature and a network of reticular stromal cells define the volumes available for hematopoiesis. vWF+ HSC reside in a sinusoidal/megakaryocytic/reticular niche far from arterioles and the endosteum while vWF- reside in an arteriolar niche enriched in Ng2+ cells [38]. Note that this arteriolar niche also contains sinusoids and reticular cells. HSC in the central BM constantly traffic between reticular cells [27**]. Subsets of HSC -reserve HSC [33*] and MFG-HSC [26] localize to endosteal regions where they proliferate in response to stress, likely in areas undergoing simultaneous bone deposition by osteoblasts and bone resorption by osteoclasts [26]. GMP are distributed through the BM but form clusters in respond to stress [50]. Lymphoid progenitors have been mapped to the endosteum [18] but also to different types of reticular cells [42,60]. Erythropoiesis takes place in erythroblastic islands presumably adjacent to the same sinusoids that support erythroid progenitors [61,62,64*].

The best studied microenvironments in the bone marrow are the hematopoietic stem cell niches, which are responsible for ensuring that HSC are maintained through the life of the organism. The discovery of a two color strategy (LinCD48CD41CD150+) to detect HSC using confocal microscopy [15] led to an explosion of studies that used imaging to identify candidate HSC niche components that were later validated using complementary approaches [1522]. These analyses have been further refined by the development of mouse fluorescent reporter lines that identify populations highly enriched in HSC [2327**]. These studies showed that in the steady-state- HSC are always found as single cells and adjacent to perivascular cells and sinusoids. Most HSC exclusively localize to sinusoids but smaller fractions localize to areas that also contain arterioles and endosteal surfaces. Cells associated with each of these structures produce cytokines and growth factors that regulate HSC self-renewal and function (). The precise components of HSC niches and how they regulate HSC have been reviewed in detail elsewhere [13,28,29]. Here we will highlight recent insights from live imaging analyses of HSC.

Until recently live imaging of HSC in the bone marrow was restricted to experiments were HSC were prospectively isolated, transferred into recipient mice, and then imaged [30]. This has changed with the development of live imaging approaches of unperturbed HSC. Christodoulou et al., [26**] used Mds1GFP+, and Mds1GFP/+Flt3-Cre mice. In Mds1GFP+ mice the Mds1 promoter drives GFP expression in HSC and multipotent progenitors. However, in the Mds1GFP/+Flt3-Cre mice, Cre expression results in excision of the GFP cassette in all cells except a small (12%) subset of quiescent LT-HSC (MFG-HSC). Using live imaging of the calvarium they found that both the Mds1GFP+ HSPC and MFG-HSC were adjacent (less than 10m) to blood vessels. However, HSPC preferentially associated with transition zone vessels when compared to the MFG-HSC. In contrast the MFG-HSC were closer to the endosteum and sinusoids suggesting the existence of different microenvironments for HSC and downstream progenitors. Live imaging demonstrated that in the steady-state- the MFG-HSC were largely non-motile (moving less than 10m over a period of two hours) whereas the Mds1GFP+ HSPC migrated more and further. Treatment with chemotherapy and G-CSF -which dramatically induces HSC proliferation and mobilization into the circulation- led to the formation of clonal MFG-HSC clusters in endosteal regions undergoing both bone deposition and remodeling. This study demonstrates live imaging of a subset of minimally motile LT-HSC and suggests that a unique endosteal microenvironment supports MFG-HSC expansion after chemotherapy injury. Note that multiple studies have shown that less than 10% of LT-HSC localize near the endosteum and that most are associated with sinusoids in the central BM [12,17,21,23]. These suggest that the MFG-HSC represents a subset of HSC that specifically associates with the endosteum (). A subset of macrophages also localizes near the endosteal surface (osteomacs). These macrophages promote HSC retention in the bone marrow, and are suppressed after mobilizing doses of G-CSF [31,32]. It would be of great interest to the field to examine whether these osteomacs localize near the MFG-HSC as this will further support the existence of a discrete niche for amplifying and mobilizing HSC in response to stress.

Upadhaya et al., [27**] used Pdzk1ip1-CreER:tdTomato mice for live imaging of HSC. In these mice low dose tamoxifen expression results in TdTomato expression in 23% of LT-HSC. Live imaging of mouse calvarium or tibia showed that the labeled HSC are highly motile with ~90% of the labeled HSC moving more than 20m whereas ~10% of the labeled HSC showed minimal movement. Combining Pdzk1ip1-CreER:tdTomato with Fgd5ZsGreen or KitLGFP/+ reporter mice allowed visualization of endothelial cells or stem cell factor (SCF)-producing perivascular cells. These confirmed the perivascular location of HSC but also revealed that over short periods of time- HSC form multiple, close, transient contacts with various SCF-producing cells. Thus HSC might travel between different niches/microenvironments to receive different signals that regulate their behavior (). Surprisingly a drug treatment that inhibits the CXCR4 receptor and 41/91 integrins -and mobilizes HSC to the circulation- also blocked HSC movement in the BM. This indicates that HSC movement requires CXCL12-CXCR4 and/or integrin signaling.

Although additional analyses are needed to resolve the observed discrepancies in motility between the HSC examined, the Christodoulou et al., and Upadhaya et al., studies open the door to deciphering HSC regulation by different signals, in situ, with single cell resolution.

It is becoming increasingly clear that hematopoiesis in the bone marrow is spatially and regionally organized and that local cues produced by distinct microenvironments are responsible for regulating different HSPC. The best characterized example of this spatial heterogeneity is the data supporting the existence of distinct sinusoidal and arteriolar niches for HSC. As discussed in the previous sections most HSC localize reside exclusively in sinusoidal locations whereas smaller fractions also associate with arterioles and/or the endosteum [12,17,21,23,24,33*,34,35]. Note that the precise fractions of HSC associated which each structure and whether these associations are specific-remains a source of controversy. Each group has used different methods to identify HSC and different criteria to define proximity to each type of structure. These further highlight a need for a common criteria in the field for defining cell proximity to niche components. Also note that due to the abundance of sinusoids- almost all hematopoietic cells locate within 30m of a sinusoid [36]. Therefore an arteriolar niche or endosteal niche is going to also contain sinusoids [12,26**,36]. Kunisaki et al., showed that 30% of LinCD48CD150+ HSC localized near arterioles ensheathed by Ng2+ periarteriolar cells and that Ng2+ cell ablation caused loss of HSC quiescence and function [17]; another 30% of HSC specifically map within 5m of megakaryocytes and loss of megakaryocytes or megakaryocyte-derived CXCL4 or TGF resulted in HSC proliferation in sinusoidal locations without affecting HSC in arteriolar locations [21,22]. Pinho et al., found that von Willebrand factor (vWF) positive HSC, which are biased towards megakaryocyte fates [37] selectively localized near megakaryocytes (60% of vWF+ HSC are within 5m of a megakaryocyte) whereas vWF- HSC localized near arterioles. Megakaryocyte ablation specifically expanded vWF+ HSC [38]. Itkin et al., discovered that HSC could be fractionated based on intracellular ROS (reactive oxygen species) levels and that HSC with lower levels of ROS where enriched near arterioles whereas HSC with higher levels of ROS located near sinusoids [39]. Further data supporting a distinct arteriolar HSC niche was provided by Kusumbe et al., which showed that constitutive Notch signaling in the vasculature increased the number of arterioles in the BM followed by accumulation of HSC suggesting that arteriole number controls HSC abundance. Together these studies support the existence of a megakaryocyte/sinusoidal niche that maintains HSC biased towards megakaryocyte fates and an arteriolar niche that maintains more quiescent HSC ().

There is also evidence supporting the existence of a distinct endosteal HSC niche. After adoptive transfer into recipient mice the donor HSC are selectively enriched near endosteal cells [30,40,41]. In agreement Zhao et al., discovered that CD48CD49b HSC are resistant to chemotherapy and proposed that they represent a reserve HSC (rHSC) population. Sixteen percent of these rHSC localize -and amplify after chemotherapy near the endosteum, adjacent to N-cadherin+ stromal cells that support them [33*]. Live imaging analyses also demonstrated that after chemotherapy- a subset of HSC selectively proliferate in endosteal regions undergoing bone remodeling and deposition [26**]. Together these studies support the concept that the endosteum might provide a niche for regenerating HSC ().

The localization of progenitors downstream of HSC is less characterized. Multipotent progenitors are immediately below HSC in the hematopoietic hierarchy and are major contributors to blood cell production in the steady-state [13]. Live animal imaging of transplanted HSC -or a population enriched in MPP- into non-irradiated recipients showed that the MPP located further away from the endosteum [30]. In agreement live animal imaging of Mds1GFP+ HSPC also enriched in MPP- and MFG-HSC [26**] showed different spatial distributions for these two populations and increased HSPC localization near transitional vessels. These studies suggest that HSC and MPP might occupy different niches. In contrast, Cordeiro-Gomes et al., found similar spatial organization for HSC and MPP and in rare occasions- observed colocalization of HSC and MPP suggesting that they occupy the same niche [42]. Note that each of these studies used different markers/reporter mice to define HSC/HSPC/MPP as well as different imaging approaches (live imaging of transplanted cells/live imaging of subsets of HSC and HSPC in the calvarium/fixed femur whole mounts) and additional studies are needed to resolve the question on whether HSC and MPP (of which there are multiple subsets [43]) occupy the same or distinct niches.

Several components of the bone marrow microenvironment including endothelial cells [44,45], perivascular cells [4648], osteocytes [49], megakaryocytes [50], and even neutrophils [51] produce signals that support and regulate myeloid cell production in the steady-state and after stress (reviewed in [52]). However, the specific sites for myelopoiesis in the bone marrow, or whether myeloid progenitors and HSC share the same niche, remain unknown. This is mainly due to lack of approaches to image myeloid progenitors. Herault et al., were able to image a population of classically defined granulocyte monocyte progenitors (GMP) as Lin-Sca1-CD150-c-kit+FcR+ cells [50]. Note that subsequent studies have shown that these phenotypically defined GMP are heterogeneous and contain bipotent and unipotent monocyte and granulocyte progenitors [4,53]. Herault et al., showed that these heterogeneous GMP were almost always found as single cells, distributed through the bone marrow. Insults that trigger emergency myeloid cell production induced formation of tightly packed GMP clusters. This cluster formation required signals provided from the microenvironment [50] suggesting that specific regions of the bone marrow support emergency myeloid progenitor expansion in response to stress.

Similarly, lymphopoiesis is dependent on signals produced by perivascular stromal cells [42], endothelial cells [42], and osteoblastic lineage cells, which include osteoblastic progenitors, osteoblasts, and osteocytes [18,20,41,5459]. While these studies support the concept of an endosteal niche for lymphopoiesis the spatial localization of lymphoid progenitors is not clear. Ding et al., found that 30% of LinIL7Ra+ cells which are enriched in lymphoid progenitor- were in contact with the endosteum [18]. In contrast, Tokoyoda et al., found that B220+flk2+ pre-pro-B cells and B220+c-kit+ pro-B cells were scattered through the central bone marrow. Additionally, 65% of Pre-pro-B cells and 0% pro-B cells were in contact with CXCL12 producing reticular cells whereas 11% of Pre-pro-B cells and 89% of pro-B cells contacted IL7-producing reticular cells. These suggest a perivascular location for lymphopoiesis and that CXCL12 and IL7 producing stromal cells provide niches for different stages of lymphocyte maturation [60]. Surprisingly, Cordeiro-Gomes et al., found that IL7-producing cells are a subset of CXCL12-producing reticular cells and that Ly6D+ common lymphoid progenitors localize to this subset. Since HSC also associate with IL7+CXCL12+ reticular stromal cells [42] this also suggested that common lymphoid progenitors and HSC occupy the same niche. Additional studies are necessary to reconcile these findings.

Erythropoiesis is also regionally organized; classical studies demonstrated that a subset of macrophages that localize near sinusoids provides a niche that supports islands of erythroblasts maturation [61,62] (for a recent review see [63]). Recently, Comazzetto et al., were able to image Lin-Sca1-c-kit+CD105+ erythroid progenitors. These selectively localize to reticular stromal cells in perisinusoidal locations. These stromal cells maintained these adjacent progenitors via SCF secretion [64**]. These indicate that sinusoids are the site of erythropoiesis.

The bone marrow is highly organized and contains specialized regions that provide distinct microenvironments that selectively regulate unique types of hematopoietic cells (). The field has made tremendous progress in defining the spatial architecture of hematopoiesis. The development of approaches to image hematopoiesis in vivo will further transform the field by allowing visualization of cell decisions in real time. However, several challenges remain including: a) the lack of approaches to simultaneously image many types of hematopoietic progenitors and precursors. These prevent examination of stepwise differentiation in situ to determine how local signals impact progenitor function; b) scRNAseq analyses have identified several new types of stromal cells with unknown functions in hematopoiesis and shown that known populations e.g endothelial cells and perivascular cells- are highly heterogeneous with different subsets producing unique combinations of cytokines and growth factors [79**]. Visualization of these novel populations and subsets will likely lead to the identification of unique niches for hematopoietic progenitors and precursors. The development of novel techniques allowing imaging of cytokines in the BM [65*] will be invaluable for these approaches; c) the bone marrow extracellular matrix is increasingly being recognized as a key regulator of hematopoiesis [66] that is spatially organized [67]. How differentiating hematopoietic cells interact with the extracellular matrix remains poorly understood; d) most studies in the field have focused in dissecting how each type of cell in the microenvironment interacts with- and regulates- one type of HSPC. However, multiple stromal cell types cooperate to regulate each type of HSPC and different stromal cells regulate different stages of HSPC maturation [58,60,68]; how the bone marrow ensures that each HSPC localizes to the right microenvironment as they mature remains unknown. Answers to these questions will define how the spatial architecture of the bone marrow regulates hematopoiesis during homeostasis and disease and allow the development of culture systems containing all the niche structures to necessary to produce large amounts of blood ex vivo.

Key points:

The spatial organization of the bone marrow ensures that distinct microenvironments regulate different types of stem cells and progenitors.

The best studies microenvironments are HSC niches and mounting evidence supports the existence of distinct sinusoidal and arteriolar HSC niches.

It is becoming increasingly clear that the bone marrow contains distinct niches for progenitors downstream of HSC.

Despite tremendous progress the spatial organization of hematopoiesis remains poorly understood and new approaches are needed.

New live imaging studies of native HSC open the door to examine HSC regulation, in situ.

The author apologizes to colleagues whose work was not cited because of space constraints.

Financial support and sponsorship

This work was partially supported by the National Heart Lung and Blood Institute (R01HL136529 to D.L.).

Conflicts of interest

The author has no conflicts of interest

View original post here:
Structural organization of the bone marrow and its role in ...

World J Stem Cells. 2022 Jan 26; 14(1): 140.

Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Oral Pathology Department, Faculty of Dentistry/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Human Anatomy and Embryology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Histology and Cell Biology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Medical Biochemistry Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Medical Biochemistry Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Forensic Medicine and Clinical toxicology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt

Histology and Cell Biology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt. ge.ude.demxela@annahem.awdar

Radwa A Mehanna, Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Alexandria 21500, Egypt;

Supported by Science and Technology Development Fund, No. 28932; and Cardiovascular Research, Education, Prevention Foundation, CVREP - Dr. Wael Al Mahmeed Grant.

Corresponding author: Radwa A Mehanna, MD, PhD, Academic Research, Professor, Executive President, Medical Physiology Department/Center of Excellence for Research in Regenerative Medicine and Applications, Faculty of Medicine, Alexandria University, Al Khartoum Square, Azareeta, Alexandria 21500, Egypt. ge.ude.demxela@annahem.awdar

Received 2021 Feb 26; Revised 2021 Jul 2; Accepted 2022 Jan 6.

Regenerative medicine is the field concerned with the repair and restoration of the integrity of damaged human tissues as well as whole organs. Since the inception of the field several decades ago, regenerative medicine therapies, namely stem cells, have received significant attention in preclinical studies and clinical trials. Apart from their known potential for differentiation into the various body cells, stem cells enhance the organ's intrinsic regenerative capacity by altering its environment, whether by exogenous injection or introducing their products that modulate endogenous stem cell function and fate for the sake of regeneration. Recently, research in cardiology has highlighted the evidence for the existence of cardiac stem and progenitor cells (CSCs/CPCs). The global burden of cardiovascular diseases morbidity and mortality has demanded an in-depth understanding of the biology of CSCs/CPCs aiming at improving the outcome for an innovative therapeutic strategy. This review will discuss the nature of each of the CSCs/CPCs, their environment, their interplay with other cells, and their metabolism. In addition, important issues are tackled concerning the potency of CSCs/CPCs in relation to their secretome for mediating the ability to influence other cells. Moreover, the review will throw the light on the clinical trials and the preclinical studies using CSCs/CPCs and combined therapy for cardiac regeneration. Finally, the novel role of nanotechnology in cardiac regeneration will be explored.

Keywords: Cardiac stem and progenitor cells, Cardiac stem cells secretome, Cardiac stem cells niche and metabolism, Nanotechnology, Clinical trials, Combined therapy

Core Tip: With the growing evidence for the existence of regenerating cardiac stem and progenitor cells, studies to evaluate their therapeutic potential have received increasing attention. Although pre-clinical research and clinical trials have demonstrated promising results, yet the latter were often inconsistent in many aspects thus imposing the need for deeper exploration of the molecular biology and relevant pathways regulating cardiogenesis and cardiac muscle repair. This review gives an insight into cardiac stem and progenitor cells regarding their embryological origin, populations, niche, secretome, and metabolism. It overviews the current preclinical research, including medical nanotechnology, and the clinical trials generally applied for cardiac regeneration.

Cardiovascular diseases are the leading cause of death globally, as stated by the latest report 2019 for the World Health Organization, with 17.9 million deaths per year, accounting for 31% of all deaths worldwide.

The heart is one of the least proliferative organs in the human body, and its minimal regenerative capacity has been dogma for decades. Such dogma has been led by the belief that the heart cannot regenerate from ischemic damage. The absence of primary tumors in the heart has further supported the notion of low proliferation. In an alleged post-mitotic organ, it has been debatable whether cardiac cells repair through activation of resident cardiac stem cells (CSCs) and cardiac progenitor cells (CPCs) or by the proliferation of pre-existing cardiomyocytes (CMs). In 2009, Bergmann et al[1] were the first to refute that notion and have reported that the heart can in fact self-renew. Based on the results obtained from their carbon-14-labelled DNA study to track CMs, Bergmann et al[1] stated that about 50% of CMs renew over the lifespan of an adult. Hsieh et al[2] provided further evidence for the origin of newly generated CMs from progenitor cells in an alpha myosin heavy chain (MHC) transgenic model. They estimated that approximately 15% of CMs can regenerate in adult hearts following ischemic damage. With progression of research, lineage tracing of regenerated cardiac tissue confirmed that the newly regenerated CMs develop from a non-CM and possibly from stem cells (SCs)[2].

Further studies have revealed various CSC/CPC candidates that are morphologically and functionally distinct from each other yet act in a complementary fashion and contribute to the regeneration process. This complex cell aggregation is known as the CSC niche that has been a challenge to characterize and locate anatomically[3].

SC applications have been under intensive research interest since the early 20th century. Many types have been isolated, starting from the embryonic, amniotic, and cord blood mesenchymal stem cells (MSCs) and passing through the adult SCs till the induced pluripotent SCs (iPSCs). Adult MSCs are undifferentiated cells with the same potentials as progenitor cells regarding the ability to differentiate into all three germ layer cells[4]. Exogenous MSCs from various sources, including bone marrow, adipose tissue, umbilical cord, placenta, and amniotic fluid[5], have shown promising results in the treatment of cardiovascular diseases. However, the outcome of CSC therapy has shown superior results in experimental studies but to a lesser extent in human clinical trials[6]. The applications of SC therapy for cardiovascular regeneration still hold a plethora of queries to be answered as well as commandment of the molecular and signaling features for CSCs in order to standardize this therapy. Among the aspects that need optimization are the types of SCs and supporting cells to be used, the number of cells, the route of injection, the frequency, and best timing for transplantation. Standardization requires an advanced understanding of the full biological features of CSCs.

SC therapy in cardiac regeneration has dual beneficiary actions. Primarily, the transplanted exogenous SCs would directly differentiate into CMs. Concomitantly, SCs activate the endogenous progenitors through their rich secretome of extracellular vesicles, immunomodulatory and growth factors, protein, and nucleic acid families[7]. These paracrine factors act to activate resident SCs and enhance vascularization to potentiate cardiac repair.

This review aims to provide insight into CSCs/CPCs regarding their embryological origin, populations, niche, metabolism, secretome, and therapeutic potentials. Also discussed is the interplay of nanotechnology with SCs in several aspects, including differentiation, tracking, imaging, and assisted therapy, showing the prospects and limitations of nanoparticle (NP)-based cardiac therapy. Finally, preclinical trials and ongoing, completed, and future clinical trials using CSCs and combined therapy are shown to delineate the potential applications in treating cardiac disease.

The heart is formed of a wide range of cell types originating from the mesodermal precursor cells. They include CMs and endocardial cells forming the inner layer, while epicardial-derived cells (EPDCs) and smooth muscle cells (SMCs) are found on the external layer. Differentiation of the mesodermal cells is initiated by the T-box transcriptional factors Brachyury (Bry) and Eomes. Bry+ cells differentiate into insulin gene enhancer protein islet-1 (ISL1) and T-box transcription factor 5 (TBX5) expressing cells, while Eomes induce expression of mesoderm posterior 1 (MESP1). MESP1+ cells are identified before the first heart field (FHF) and the second heart field (SHF) separations, so MESP1 serves as an indicator of early CPCs for both heart fields[8]. Chemokine receptor type 4 (CXCR4), fetal liver kinase 1 (FLK-1), and platelet derived growth factor receptor A are other surface markers that coincide with MESP1 and are used in combination to isolate CPCs[9,10].

In addition, a novel cell surface marker known as G protein-coupled receptor lysophosphatidic acid receptor 4 is specific to CPCs and determines its functional significance. Interestingly, its transient expression peaks in cardiac progenitors after 3 to 7 d of human (h)PSCs differentiation toward cardiac lineage, then it declines. In vivo, lysophosphatidic acid receptor 4 shows high expression in the initial stages of embryonic heart development and decreases throughout development[11].

The FHF cells are the firstly differentiated myocardial cells that are derived from cells in the anterior lateral plate mesoderm; they give rise to the left ventricle, partially some of the right ventricle population, sinoatrial node, atrioventricular node, and both atria[12]. Meanwhile, the SHF cells originate from the pharyngeal mesoderm to the posterior side of the heart and further divide into anterior and posterior SHF. They contribute to the right ventricle, atria, and the cardiac outflow tract (OFT) formation. Addition of the SHF-derived CMs to the ventricles depend on myocyte enhancer factor 2C (MEF2C). It has been found that MEF2C null mice die at 9.5-d post conception with severe heart defects due to failure of heart looping[13]. In OFT formation, two waves of SHF progenitors and their derivatives have been identified, making a differential contribution to the aorta and pulmonary artery. The early wave of cells is favorably directed to the aorta, while the second wave of cells contributes to the pulmonary artery. Phosphoinositide-dependent kinase-1 critically regulates the second wave of cells, and its deletion results in pulmonary stenosis[14]. The epicardium of the heart is formed of a transient proepicardial organ. Proepicardium is formed from homeobox protein NKx2.5 (NKx2.5) and ISL1+ cells. After epicardial formation, subepicardial mesenchymal space is formed by epithelial to mesenchymal cell transformation of the epicardial cells[15] (Figure ).

Embryonic cardiac progenitors, Brachyury-positive mesoderm precursors and Pax3+ neural crest cells. Brachyury (Bry+) mesoderm precursors give rise to the mesoderm posterior 1+ primordial precursors, which are the origin of the first heart field, second heart field, and proepicardial progenitors, each population of which is responsible for the development of different parts in the heart. Pax3+ neural crest cells are responsible for the development of vascular smooth muscle, outflow tract, valves and the conductive system. Progenitors are tagged with their specific markers. Created with BioRender.com. CPC: Cardiac progenitor cell; LT: Left; RT: Right; FHF: First heart field; SHF: Second heart field; OFT: Outflow tract.

The differentiation in the posterior SHF is regulated by Hoxb1 gene. Stimulation of Hoxb1 in embryonic stem cells (ESCs) halts cardiac differentiation, while Hoxb1-deficiency shows premature cardiac differentiation in embryos. Moreover, an atrioventricular septal defect develops as a result of ectopic differentiation in the posterior SHF of embryos deficient in Hoxb1 and its paralog Hoxa1[16].

Multiple signaling pathways have essential roles in cardiogenesis with a sequential arrangement. The transforming growth factor- (TGF-) superfamily, retinoic acid, Hedgehog, Notch, Wnt, and fibroblast growth factors (FGFs) pathways comprise the chief signaling pathways involved in cardiac development. These pathways, along with transcription factors and epigenetic regulators, regulate cardiac progenitors specification, proliferation, and differentiation into the different cardiac cell lineages[17].

The TGF- superfamily members consist of over 30 structurally associated polypeptide growth factors including nodal and bone morphogenetic proteins (BMP)[18].

Nodal signaling is vital for the formation of sinoatrial node. Nodal inhibition during the cardiac mesoderm differentiation stage downregulates PITX2c, a transcription factor recognized to inhibit the formation of the sinoatrial in the left atrium during cardiac development[19]. Moreover, nodal signaling is dispensable for initiation of heart looping; however, it regulates asymmetries that result in a helical shape at the heart tube poles[20].

BMP signaling, as a member of TGF-, has an important role in the different stages of heart development including the OFT formation, endocardium, and lastly the epicardium. The cardiac neural crest cells have a crucial role in normal cardiovascular development. They give rise to the vascular smooth muscle of the pharyngeal arch arteries, OFT septation, valvulogenesis, and development of the cardiac conduction system[21] (Figure ). The role of BMP in OFT septation mainly depends on their gradient signaling, which arranges neural crest cell aggregation along the OFT; this Dullard-mediated tuning of BMP signaling ensures the fine timed zipper-like closure of the OFT by the neural crest cells[22]. Furthermore, the BMP signaling promotes the development of endocardial cells (ECs) from hPSC-derived cardiovascular progenitors[23]. It is also integrated with Notch signaling for influencing the proepicardium formation, where overexpression of Notch intracellular receptor in the endothelium enhances BMP expression and increases the number of phospho-Smad1/5+ cells for enhancing the formation of the proepicardium[24].

Retinoic acid signaling plays a role in heart development. It is a key factor for efficient lateral mesoderm differentiation into atrial-like cells in a confined time frame. The structural, electrophysiological, and metabolic maturation of CMs are significantly influenced by retinoic acid[25]. However, it is reported that retinoic acid receptor agonists transiently enhance the proliferation of human CPCs at the expense of terminal cardiac differentiation[26].

The downregulation of the retinoic acid responsive gene, ripply transcriptional repressor 3 (RIPPLY3), within the SHF progenitors by histone deacetylase 1 is required during OFT formation[27].

Hedgehog signaling has a role in OFT morphogenesis. Lipoprotein-related protein 2 (LRP2) is a member of the LDL receptor gene family, a class of multifunctional endocytic receptors that play crucial roles in embryonic development. LRP2 is expressed in the anterior SHF cardiac progenitor niche, which leads to the elongation of the OFT during separation into aorta and pulmonary trunk. Loss of LRP2 in mutant mice results in depleting a pool of sonic hedgehog-dependent progenitor cells in the anterior SHF as they migrate into the OFT myocardium due to premature differentiation into CMs. This depletion results in aberrant shortening of the OFT[28].

Four Notch receptors (Notch1Notch4) and five structurally similar Notch ligands [Delta-like (DLL) 1, DLL3, DLL4, Jagged1, and Jagged2] have been detected in mammals[29]. Activation of Notch signaling enhances CM differentiation from human PSCs. However, the CMs derived from Notch-induced cardiac mesoderm are developmentally immature[30]. In vivo, the Notch pathway plays a significant role in CPC biology. An arterial-specific Notch ligand known as DLL4 is expressed by SHF progenitors at critical time-points in SHF biology. The DLL4-mediated Notch signaling is a crucial requirement for maintaining an adequate SHF progenitor pool, in a way that DLL4 knockout results in decreased proliferation and increased apoptosis. Reduced SHF progenitor pool leads to an underdeveloped OFT and right ventricle[31].

The Wnt signaling pathway has an essential role in many developmental stages of embryogenesis. The Wnt family consists of 19 distinct Wnt proteins and other 10 types of Frizzled receptors. On the basis of their primary functions, the Wnt and Frizzled receptors are divided into two major classes, which are the canonical and non-canonical Wnt pathways[32]. Accumulating evidence suggests a role for the dynamic balance between canonical and non-canonical Wnt signaling in cardiac formation and differentiation. Wnt/-catenin signaling is required for proper mesoderm formation and proliferation of CMs but needs to be low for terminal differentiation and cardiac specification. In contrast, for cardiac specification in murine and human ESCs, non-canonical -catenin independent Wnt signaling is essential, while the non-canonical Wnt signaling is necessary for terminal differentiation later in development[33].

The activation of non-canonical Wnt is non-catenin-independent, and the downstream proteins involve several kinases, including protein kinase C, calcium/ calmodulin-dependent kinase, and Jun N terminal kinase (JNK). Wnt11 enhances angiogenesis and improves cardiac function through non-canonical Wnt-protein kinase C-Jun N terminal kinase dependent pathways in myocardial infarction (MI)[34]. In hypoxia, Wnt11 expression preserves the integrity of mitochondrial membrane and facilitates the release of insulin growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF), thus protecting CMs against hypoxia[35]. Canonical dependent Wnt signaling, Wnt 3 Ligand, favors the pacemaker lineage, while its suppression promotes the chamber CM lineage[36].

The regenerative capacity of most organs is contingent on the adult SC populations that exist in their niches and are activated by injury. Adult SC populations vary greatly in their molecular marker expression profile and hence in their possible role in regenerative medicine. The transcriptome is a representation of the gene read-outs, the cellular state, and is imperative for studying all genetic disease and biological processes. The genome-wide profiling using novel sequencing technology has made transcriptome research accessible.

Receptor tyrosine kinase (RTK) c-KIT (also referred to as SC factor receptor or CD117)-expressing CPCs are mainly located in the atria and the ventricular apex, comprising most of the ventricular and atrial myocardium[37]. c-KIT+ cells also express the cardiac transcription factors NKx2.5, GATA binding protein 4 (GATA4), and MEF2C but are negative for the hematopoietic markers CD45, CD3, CD34, CD19, CD16, CD20, CD14, and CD56[38,39]. SC factor ligand attaches to the c-KIT receptor and activates the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and p38 mitogen-activated protein kinase (MAPK) signaling pathways[40]. Both PI3K/AKT and MAPK pathways control various CPCs functions like self-renewal, proliferation, migration, and survival[41]. During embryonic development and the early post-natal time, c-KIT+ CPCs contribute to the generation of new CMs. Such capacity declines in the adult heart with only a few new CMs originating from CPCs[42]. In a rat MI model, the c-KIT+ CPCs have migrated through the collagen type I and type III matrices into the infarcted area. The transplanted CPCs have shown overexpressed matrix metalloproteinases (MMPs; MMP2, MMP9, and MMP14) that degrade extracellular matrix (ECM), concluding that c-KIT+ CPCs hold an invasive capacity[43]. Transplanted CPCs (c-KIT+ CPCs and cardiospheres) also show an endogenous proliferative potential in vivo and additionally activate endogenous CPCs[44].

Stem cell antigen 1 (SCA-1) expressing CPC population exists predominantly in the atrium, intra-atrial septum, and atrium-ventricular boundary and dispersed inside the epicardial layer of adult hearts[45]. SCA-1 is a cell surface protein of the lymphocyte antigen-6 (Ly6) gene family, which has roles in cell survival, proliferation, and differentiation[46]. A population of SCA-1+ cells from murine adult myocardium hold a telomerase activity comparable to that of a neonatal heart. This SCA-1+ population is different from hematopoietic SCs as they lack CD45, CD34, c-KIT, LIM domain only 2, GATA2, VEGF receptor 1, and T-cell acute lymphoblastic leukemia 1/SC leukemia proteins. SCA-1+ cells are also distinct from endothelial progenitor cells and express cardiac lineage transcriptional factors such as GATA4, MEF2C, and translation elongation factor 1 yet lack transcripts for cardiomyocytic structural genes such as BMP1r1 and -, -MHC[47,48]. Although this population exhibits the endothelial marker CD31, it is suggested to be due to the contaminating endothelial CD31+/SCA-1+ cells. In vitro studies have revealed that 5-azacytidine (5-aza), a demethylating agent, pushed SCA-1+ cells to differentiate into CMs[48,49]. Further studies have isolated SCA-1+ cells that lack CD31 and CD45 markers, referring to them as lineage negative (Lin). The SCA-1+/Lin cells display a mesenchymal cell-surface profile (CD34, CD29+, CD90+, CD105+, and CD44+) and are able to differentiate, to a certain extent, into CMs and endothelial and smooth muscle-like cells[50,51].

Human SCA-1+-like cells also express early cardiac transcription factors (GATA4, MEF2C, insulin gene enhancer protein ISL-1, and Nkx-2.5) and can differentiate into contractile CMs[52]. Although a human ortholog of the SCA-1 protein has not been yet identified, an anti-mouse SCA-1 antibody is used to isolate SCA-1+-like cells from the adult human heart.

MESP1 expressing cells mainly contribute to the mesoderm and to the myocardium of the heart tube during development[53]. Transient expression of MESP1 seems to accelerate and enhance the appearance of cardiac progenitor. However, homologous disruption of the MESP1 gene has resulted in aberrant cardiac morphogenesis. MESP1 interacts with the promoter area of main cardiac transcription factors, including heart and neural crest derivatives expressed 2, Nkx2-5, myocardin, and GATA4[54]. These factors induce fibroblasts to express a full battery of cardiac genes, form sarcomeres, develop CM-like electrical activity, and in a few cases elicit beating activity[55]. Several studies have shown that the addition of MESP1 could enhance the efficacy of direct reprogramming of fibroblasts into CMs[56,57]. The transdifferentiation of fibroblasts to CMs via MESP1 suggests that MESP1 chiefly modulates the gene regulatory network for cardiogenesis[52].

Kinase insert domain receptor (KDR), also known as Flk-1, is one of the earliest discovered cardiogenic progenitor cell markers acting during the early stages of cardiac development in human[58]. Nelson et al[59] have reported that Flk-1 has a distinctive transcriptome that has been evident at day 6, immediately after gastrulation but prior to the expression of the cardiac transcription factors. KDR+ population lack the pluripotent octamer-binding transcription factor 4, sex determining region Y-Box transcription factor (SOX) 2, and endoderm SOX17 markers. On the other hand, KDR+ CPCs have shown a noteworthy upregulation in SOX7, a vasculogenic transcription factor, overlapping with the emergence of primordial cardiac transcription factors GATA4, myocardin, and NKx2.5. Moreover, KDR subpopulations that overexpress SOX7 are associated with a vascular phenotype rather than a cardiogenic phenotype. These outcomes offer insights for refining the therapeutic regenerative interventions.

The FHF cells express hyperpolarization activated cyclic nucleotide gated potassium channel 4 and TBX5, while SHF progenitors express TBX1, FGF 8, FGF10, and sine oculis homeobox2 (Figure ). Cells from the SHF exhibit high proliferative and migratory capacities and are mostly responsible for the elongation and winding of the heart tube. Moreover, SHF cells differentiate to CMs, SMCs, fibroblasts, and endothelial cells (ECs) along their journey in the heart tube to form the right ventricle, right ventricular OFT, and most of the atria[60,61]. However, FHF cells hold less proliferative and migratory potentials and differentiate predominantly to CMs that form the left ventricle and small parts of the atria[62]. The cells of the cardiac crescent, theoretically the progeny of FHF CPCs, are terminally differentiated cells expressing the markers of CMs, such as actin alpha cardiac muscle 1 and myosin light chain 7[63,64], hence they are unlikely to be multipotent progenitors. Therefore, it is difficult to identify FHF before Nkx2.5 and TBX5 expressions. Conversely, multipotent SHF CPCs were validated with a clonal tracing experiment and identified by ISL1 expression[65]. However, ISL1 expression is not specific for SHF and has been proposed to represent only the developmental stages[66]. Tampakakis et al[67] generated ESCs by using hyperpolarization activated cyclic nucleotide gated potassium channel 4-green fluorescent protein and TBX1-Cre; Rosa-red fluorescent protein reporters of the FHF and the SHF respectively, and also by using live immunostaining of the cell membrane CXCR4, a SHF marker and the reporters. The ESC-derived progenitor cells have shown functional properties and transcriptome similar to their in vivo equivalents. Thus, chamber-specific cardiac cells have been generated for modelling of heart diseases in vitro.

The EPDCs are important as a signaling source for heart development, cardiac regeneration, and post-MI heart repair. Throughout the development of the heart in mice, EPDCs aid in the formation of various cardiac cell types and secrete paracrine factors for myocardial maturation[68]. In the adult heart, EPDCs are normally dormant and become stimulated following myocardial injury. Transcriptional analysis of the EPDCs derived from human (h)iPSCs cells have revealed several markers of EPDCs including Wilms tumor protein 1, endoglin, thymus cell antigen 1, and aldehyde dehydrogenase 1 family member A2[69] (Figure ). Following MI in mice, EPDCs undergo an epithelial-to-mesenchymal transition, with overexpression of Wilms tumor protein 1, and differentiate mainly into SMCs/fibroblasts[70,71]. EPDC-secreted paracrine factors include VEGF-A, FGF2, and PDGF-C, which support the growth of blood vessels, protect the myocardium, and recover cardiac functions in an acute MI-mouse model[70].

Side population (SP) cells have been detected in the heart and other various tissues and hold enhanced stem and progenitor cell activity[72]. SP cells, when stained in vitro, hold the ability to flush out the DNA Hoechst dye from their nuclei[73]. Gene expression profiling of SP cells after MI has revealed a downregulation of Wnt-related signals coupled with increased SP cell proliferation. This has been validated in vitro by treatment of isolated SP cells with canonical Wnt agonists or recombinant Wnt, where the proliferation of SP cells has been repressed with partial arresting the G1 cell cycle phase[74]. Consistent with this observation, delivery of secreted Frizzled-related proteins (SFRP; the Wnt antagonizer) improves post-MI remodeling[75,76].

SP cells can be identified by surface marker adenosine triphosphate (ATP) binding cassette subfamily G member 2 (ABCG2), also referred to as the breast cancer resistance protein1[77]. ABCG2+ cells have been also observed in the adult heart and can differentiate in vitro into CMs[78]. When SP cells have been injected into the injured hearts of rats, they have been recruited to the injured regions, where they differentiate into CMs, ECs, and SMCs, suggesting that they may be endogenous SP cells[79]. However, ABCG2CreER based genetic lineage tracing has demonstrated that ABCG2+ cells could only differentiate into the multiple cardiac cell lineages during the embryonic stages but not in adulthood[80,81]. The combination of ABCG2+ cells with pre-existing CMs is more likely to stimulate CM proliferation rather than differentiation into CMs directly[82]. Therefore, genetic fate mapping investigations have disproved the SP cells property of the adult endogenous ABCG2+ SP and their in vivo renewing myogenic ability[83].

Cardiospheres contain a combination of stromal, mesenchymal, and progenitor cells that are isolated from cultures of human heart biopsy[39,84]. They represent a niche-like environment, with cardiac-committed cells in the center and supporting cells in the periphery of the spherical cluster[85]. The cardiosphere-derived cells (CDCs) were originally isolated from mouse heart explants and human ventricular biopsies based on their ability to form three-dimensional (3D) spheroids in suspension cultures[86]. CDCs have grabbed much attention due to their proliferation and differentiation abilities by inherent stimulation of cardio-specific differentiation factors [GATA4, MEF2C, Nkx2.5, heart and neural crest derivatives expressed 2, and cardiac troponin T (TNNT2)] using a clustered regularly interspaced short palindromic repeat/dead Cas9 (CRISPR/dCas9) assisted transcriptional enhancement system[87,88]. Sano et al[89] have postulated that the CRISPR/dCas9 system may provide a proficient method of modifying TNNT2 gene activation in SCs. Consequently, CRISPR/dCas9 can improve the therapeutic outcomes of patients with ischemic heart disease by enhancing the transplanted CDCs differentiation capacity within the ischemic myocardium. Heart tissue is usually obtained by endomyocardial biopsy or during open cardiac surgery and grown in explants to form CDCs. CDCs have shown a superior myogenic differentiation potential, angiogenesis, and paracrine factor secretion as compared to other cell types. In heart failure animal models, the injected CDCs potentially differentiated into CMs and vascular cells. Additionally, CDCs have diminished unfavorable remodeling and infarct size, and hence improve cardiac function[90]. Accordingly, cardiospheres and CDCs may be some of the most promising sources of CPCs for cardiac repair.

The niche in the heart integrates several heterogeneous cell types, including CSCs, progenitors, fibroblasts, SMCs, CMs, capillaries, and supporting telocytes (TCs)[91], together with the junctions and cementing ECM that hold the niche together. Such architectural arrangement is essential for protection against external damaging stimuli and for preserving the stemness of the CSCs (Figure ). Without the niche microenvironment, CSCs lose their stemness and initiate differentiation eventually, leading to the exhaustion of the CSC pool. Similarly, in vitro studies require feeder layers and cytokines supplements in the culture media to ensure that SCs remain in their undifferentiated state[37].

Invivo arrangement of the central cardiac stem cells and the surrounding cells that comprise the niche (right side) and the in vitro derived cardio spheres (left side). The key delineates the types of cells identified in the niche and cardio spheres. Created with BioRender. CSC: Cardiac stem cell.

In vitro studies have recapitulated the niche theory using cardiospheres, which are 20150 m spheres (Figure ) of cells generated from the explant outgrowth of heart tissues[92,93]. Cardiospheres consist of CSCs in the core and cells committed to the cardiac lineage such as myofibroblasts, while vascular SMCs and ECs form the outer layer of the spheres. The 3D structure of cardiospheres protects the interiorly located CSCs from oxidative stress as well as maintain their stemness and function[84].

Accurate anatomical identification of CSCs in vivo remains a challenge due to the lack of basal-apical anatomical orientation as seen in epithelial organs such as the intestines[94]. Moreover, the heart does not comprise a specific compartment, where cells form a well-defined lining as seen in the bone marrow osteoblasts[95]. The adult heart epicardial lining anatomically contains several classes of niches, which are not limited to the sub epicardium[96] but dispersed throughout the myocardium, more in the atria and apex away from hemodynamic stress[97]. Some niches have been described in the atrio-ventricular junction of adult mouse and rat hearts[98] and interestingly in the human hearts[99]. The young mouse heart has been studied morphometrically to identify the location of CSCs niche and has been defined as a randomly positioned ellipsoid structure consisting of cellular and extracellular components. Within the niches, undifferentiated CSCs are usually assembled together with early committed cells that express c-KIT on surface, Nkx2.5 in the nucleus, and the contractile protein -sarcomeric actin in the cytoplasmic[97].

CSCs niche consists of clusters of c-kit+, MDR1+, and Sca-1+ cells[98] but lack the expression of the transcription factors and cytoplasmic or membrane proteins of cardiac cells[99,100]. Cardiac c-kit+/CD45- cells comprise about 1% of the CSC niche[97], are self-renewing clonogenic, and possess a cardiac multilineage differentiation potential comprise[101].

Within the niche, gap junctions (connexins) and (cadherins) connect SCs to their supporting cells, myocytes/fibroblasts. Conversely, ECs and SMCs do not act as supporting cells. Hence, the communication between CSCs with CMs and fibroblasts has been investigated by using in vitro assays[102]. The transmission of dyes via gap junctions between CSCs and CMs or fibroblasts was demonstrated previously and verified the functional coupling of these three cell populations[97]. In addition, micro ribonucleic acid (miRNA-499) translocates from CMs to CSCs comprising to the initiation of lineage specification and formation of myocytes[103].

Identification of SC niches is contingent upon the fulfillment of explicit criteria, including the recognition and determination of the affixing of SCs to their supporting cells as well as assuring the existence of an ancestor-progeny association[104]. Chemical and physical signals modulate the behavior of SCs within the niche. Amongst these signals are cytokines, cell surface adhesion molecules, shear forces, oxygen tension, innervation, and ions that serve as major determinants of SCs function[97]. Cell-to-cell signaling mediates the fate of SCs within the niches to promote self-renewal and favors their migration and differentiation. The fine-tuned crosstalk between SCs and their supporting cells regulates the state of the niche regarding quiescence or activity[105].

CSC niches, similar to the bone marrow, characteristically live in low oxygen tension, which favors a quiescent primitive state for SCs[106]. The longstanding perpetuation of the CSC niche requires a hypoxic environment, while physiological normoxia could be required for active cardiomyogenesis[107]. Hypoxic c-KIT+ CSCs within niches have been found throughout the myocardium, especially at the atria and apex. Throughout all ages, bundles of CSCs with low oxygen content coexist with normoxic CSCs niches. Hypoxic CSCs, especially in the atria, are quiescent cells undergoing cell cycle arrest and cannot divide. Normoxic CSCs are pushed into intense proliferation and differentiation with continuous telomere erosion, resulting finally in dysfunctional aged CMs[108]. Additionally, Nkx2.5 and GATA4 expressions are only restricted to the normoxic CSC niche. A balance between the hypoxic and normoxic niche is essential for the preservation of the CSC compartment and for the maintenance of myocardial homeostasis during the organ lifespan. Some factors such as aging cause an imbalance by expanding the hypoxic quiescent CSCs so that less pools of cycling CSCs maintain cell turnover[100]. Hypoxic cardiac niches are abundant in the epicardium and subepicardium in an adult mouse heart, which also fosters a metabolically distinctive population of glycolytic progenitor cells[109].

The pool of CSCs seems to be heterogeneous, incorporating quiescent and actively proliferating cells, migratory and adherent cells, uncommitted and early committed cells, with young and senescent cells. Additional surface epitopes remain to be disclosed to classify pools of CSCs holding specific properties. Surface Notch1 expression distinguishes multipotent CSCs that are poised for lineage commitment, while c-Met and ephrin type-A receptor 2 receptors reveal cells with particular migratory potential out of the niche area. A specific compartment of CSCs, expressing IGF-1 receptor, can be stimulated to regenerate damaged myocardium, while those expressing IGF-2 receptor hold higher probability for senescence and apoptosis. Although this arrangement of cells seems to equip properly the CSC with homeostasis regulation, it does not effectively protect against aging or ischemic injury of the heart[100].

Circulatory angiogenic cells (CACs) are endothelial progenitor cells involved in vasculogenesis, angiogenesis, and stimulating myocardial repair, mainly through paracrine action. Latham et al[110] demonstrated that conditioned medium from CACCSC co-cultures exhibited greatly mobilized CACs, with induction of tubule formation in human umbilical vein endothelial cells, mainly through the upregulation of the angiogenic factors angiogenin, stromal cell-derived factor 1 (SDF-1), and VEGF. Moreover, administration of CACs and CSCs in infarcted hearts of non-obese/severe combined immunodeficient mice restored substantially the left ventricular ejection fraction (LVEF), with reduction of scar formation as revealed by echocardiography. Successful yet modest SMCs, ECs, and CM differentiation has been also reported.

Pericytes (also called Rouget cells, mural cells, or perivascular mesenchymal precursor cells) are mesodermal cells that border the endothelial lining. They are highly proliferative cells and express neural/glial antigen 2, SOX-2, PDGFR-, CD34, and several mesenchymal markers such as CD105, CD90, and CD44. It was previously reported that the transplantation of saphenous vein-derived pericytes (SVPs) into an ischemic limb of an immunodeficient mice restored the local circulatory network via angiogenesis[111]. Moreover, treatment with SVP reduced fibrotic scar, CM death, and vascular permeability in a mouse model of MI via miRNA-132 facilitated angiogenesis[112]. Avolio et al[113] were the first to describe the relationship between SVP and the endogenous CSCs. Combined CSC and SVP transplantation in the infarcted myocardium of severe combined immunodeficient/Beige-immunodeficient mice showed similar results to treatment with CSCs or SVP cells per se, regarding scar size and ventricular function, indicating that SVPs alone are as potent as CSCs.

TCs represent a recently described cell population in the stromal spaces located in many organs, including the heart. They are broadly dispersed throughout the heart and comprise a network in the three cardiac layers, heart valves, and in CSC niches. TCs have been documented also in primary culture from heart tissues[114,115]. The ratio of cardiac TCs (0.5%-1%) exceeds that of CSCs. Although they still represent a minute portion of human cardiac interstitial cells, their extremely long and extensive telopodes allow them to occupy more surface area, forming a 3D platform probably that extends to support other cells[116]. The telopodes act as tracks for the sliding of precursor cells towards mature CMs and their integration into heart architecture[91]. TCs form a tandem with CSCs/CPCs in niches, where they communicate through direct physical contact by atypical junctions or indirect paracrine signaling[115].

TC-CSC co-culturing have suggested that TCs and CSCs act synergistically to control the level of secreted proteins, as shown by the increased levels of monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein1 and 2 (MIP-1 and MIP-2), and interleukin (IL)-13. Whereas, the level of IL-2 decreased compared to the monoculture of CSCs or TCs. IL-6 found in TC culture is behind the upregulation of these chemokines. Chemokines elucidated the role of TCs in directing the formation of CMs. Within the context, MIP-1 and MCP-1 play roles in the formation of SMCs in the airway. Additionally, MCP-1 is also involved in mouse skeletal muscle regeneration by recruiting macrophages. The enhancement of MCP-1 secretion serves as an activator of another cell population, primarily macrophages, which are generally involved in such processes[117].

IL-6 also activates downstream signaling pathways and contributes to cardioprotection and vessel formation in the heart through activation of gp130/signal transducer and activator of transcription 3. The Gp130/signal transducer and activator of transcription 3 is essential for the commitment of cardiac SCA-1+ cells into endothelial lineage[118].

Furthermore, IL-6 targets VEGF and hepatocyte growth factor (HGF) genes. VEGF has a mitogenic effect on CMs[119]. It is known to mobilize bone marrow-derived mesenchymal stem cells (BM-MSCs) into the peripheral blood in MI patients[120]. HGF and its receptor (c-Met) are also involved in cardiogenesis, as it is expressed early during cardiac development[121]. The level of HGF mRNA is normally low in the heart, but it is upregulated for at least 14 d after ischemic insult in rats, enhancing CMs survival under ischemic conditions[122,123]. Moreover, it has the potential to generate an adhesive micro-environment for SCs, as demonstrated in a study of transplantation of HGF transfected BM-MSCs in the infarcted myocardium[124]. HGF is also a powerful angiogenic agent, conducting its mitogenic and morphogenic effects through the expression of its specific receptor in various types of cells, including myocytes. Moreover, HGF exerts antifibrotic and antiapoptotic effects on the myocardium[125,126].

Transcriptomic analysis also has disclosed that TCs express pro-angiogenic miRNAs including let-7e, miRNA-21, miRNA-27b, miRNA-126, miRNA-130, miRNA-143, miRNA-503, and miRNA-100[127]. The TCs and CSCs interact in vitro forming atypical junctions, such as puncta adherentia and stromal synapses. The puncta adherentia consists of cadherincatenin clusters. It controls the symmetry of division by facilitating the proper positioning of centrosomes. Therefore, an increased number of CSCs has been reported to be encountered in the presence of cardiac TCs[128,129].

The paracrine potential of CSCs/CPCs has been recently under focus. CSC-derived cytokines and growth factors include epidermal growth factor (EGF), HGF, IGF-1, IGF-2, IL-6, IL-1, and TGF-1[130,131]. Exosomes appear to harbor relevant reparative signals, which mechanistically underlie the beneficial effects of CSCs transplantation[132].

Structurally, exosomes are lipid bilayer nano-sized organelles, 20-150 nm in diameter, secreted from all cell types, and function as intercellular communicators. Exosomes are highly heterogenic in content, and this stems from the unique packaging process that occurs inside progenitor and SCs. Exosomes carry lipids, proteins, and nucleic acids, with an abundance of miRNAs that hold profound post-transcriptional gene regulatory effects[133].

Amongst the distinctive protein content of cardiac exosomes are the chaperone proteins heat shock protein (HSP) 70 and HSP60. The HSP70 and HSP60, which under normal conditions assist in protein folding processes and deter misfolding and protein aggregation under pathological states induced by stress, also play major roles in apoptosis[134]. Circulating exosomes from healthy individuals have been found to activate cardioprotective pathways in CMs via HSP70 through extracellular signal-regulated kinase and HSP27 phosphorylation[135].

The exosome protein cargo of CPCs is distinct from BM-MSCs, fibroblasts, and other sources as it contains ample amounts of the pregnancy-associated plasma protein-A (PAPP-A). PAPP-A is present on the surface of human exosomes and interacts with IGF binding proteins (IGFBPs) to release IGF-1[136]. The cardioprotective role of CPCs-exosomes has been proven experimentally in in vitro ischemia/reperfusion and MI models and on CMs apoptosis to surpass that of BM-MSC-exosomes owing to their rich content of PAPP-A[137].

Like all exosomes, mouse CPCs-derived exosomes are positive for the surface markers CD63, CD81, and CD9, TSG-101, and Alix, however, they express a high-level of GATA4-responsive-miRNA-451. MiRNA-451 has been shown to inhibit CM apoptosis in an acute mouse myocardial ischemia-reperfusion model through inhibition of the caspases 3/7. The expression of miRNA-21 in the mouse CPCs-exosomes additionally justifies their CM protection against oxidative stress and antiapoptotic effects via inhibition of programmed cell death protein 4 (PDCD4)[138]. Human CPCs-exosomes are enriched with miRNA-210, miRNA-132, and miRNA-146a-3p, which account for the diminished CM apoptosis, enhanced angiogenesis, and improved LVEF[139]. MiRNA-146a-5p is the most highly upregulated miRNA in human CPCs-exosomes and targets genes involved in inflammatory and cell death pathways[137].

The CDCs contain CD34+ stromal cells of cardiac origin and are multipotent and clonogenic but not self-renewing[140]. CDCs secrete exosomes that induce cardiomyogenesis and angiogenesis, regulate the immune response, downgrade fibrosis, and improve the overall cardiac function[141,142]. Moreover, CDCs homogeneously express CD105 but not CD45 or other hematopoietic markers. They also exhibit a high expression of miRNA-126[143]. Circulating miRNA-126 may participate in cardiac repair during acute MI and has been demonstrated to be downregulated in heart damage[144].

While exosomes are constitutively secreted, changes in the surrounding microenvironment, such as hypoxia, can induce modifications in CPCs- and CM- derived extracellular vesicles. Hypoxic CMs secrete large extracellular vesicles containing long noncoding RNA neat 1 (LNCRNA NEAT1), which is transcriptionally regulated under basal conditions by p53, while during hypoxia it is regulated by the hypoxia inducible factor 2A. An uptake of the hypoxic CM-derived extracellular vesicles by fibroblasts can prompt the expression of profibrotic genes[145]. Oxidative stress may also induce the release of cardiac CPCs exosomes, which in turn inhibit apoptosis when taken up by H9C2 (rat cardiomyoblast cell line)[132]. Furthermore, oxidative stress stimulates secretion of miRNA-21 rich exosomes, which could inhibit H9C2 apoptosis by targeting PDCD4 and hence can be accounted as a new method to treat ischemia-reperfusion[138].

Intercellular communication via exosomes occurs as part of various biological processes, including immune modulation, vasculogenesis, transport of genetic materials, and pathological conditions such as inflammation, apoptosis, and fibrosis, which can lead to cardiovascular disease when altered[146]. Hence, isolation and analysis of cardiac exosomes contents, mainly miRNA and proteins, could offer diagnostic information for several cardiovascular diseases[147] (Figure ).

Schematic diagram elucidating the diverse exosomal contents that serve as biomarkers for several cardiovascular diseases. Created with BioRender.com. HSP: Heat shock protein; lncRNA: Long non-coding RNA; miR: MicroRNA.

Functionally, exosomes mediate several intra-cardiac inter-cellular communications such as:

CPC-CM crosstalk through factors, such as miRNA-146a and PAPP-A, which activate extracellular signal-regulated kinases 1/2 pathway and inhibit apoptosis[139].

CPC-macrophage (M1) crosstalk via miRNA-181b and Y-RNA fragment transforms M1 to M2 macrophages with attenuated proinflammatory cytokines and increased IL-10[148,149] (Figure ).

Possible cardiac reparative effects of cardiac stem cell/cardiosphere-derived cell-derived exosomes in myocardial ischemia and ischemia/reperfusion injury. Created with BioRender.com. CSC: Cardiac stem cell; IL: Interleukin; IR: Ischemia/reperfusion; miRNA: MicroRNA; PI3K: Phosphoinositide 3-kinase; SDF-1: Stromal cell-derived factor 1; VEGF: Vascular endothelial growth factor.

CPC-fibroblast interaction via exosomes primes the fibroblasts and increases expression of VEGF and SDF-1. Experimental injection of fibroblasts primed with CPCs-exosomes into the myocardium of a MI model proved to reduce infarct size and improve cardiac function. In addition, cardiosphere-isolated exosomes have been used to prime inert fibroblasts, leading to an intensification of their angiogenic, cardiomyogenic, antifibrotic, and collective regenerative effects[150] (Figure ).

CPC-self regulatory mechanisms: Exosomes derived from CPCs may play critical roles in maintaining the self-renewal state of CPCs themselves and balance their differentiation, i.e. preserve their stemness[151] (Figure ). The CPC-derived exosomes activate the endogenous CPCs by transferring signal molecules directly within their niche[152].

CPC-derived exosomes release various RNA species in the extracellular space, modulating endogenous SC plasticity and tissue regeneration through their cytoprotective, immunomodulatory, pro-angiogenic, and anti-apoptotic actions[153].

Fibroblasts and pericytes interact after transdifferentiating to myofibroblasts and deposit ECM causing cardiac fibrosis. These fibrotic changes are usually induced by cardiac damage and lead to scar formation. Exosomes serve as messengers for cell-to-cell communication during cardiac fibrosis[154]. Molecular mechanisms of cardiac fibrosis are primarily related to TGF- pathways, IL-11 signaling pathway, nuclear factor- pathway, and Wnt pathways[155]. Accordingly, the bioactive substances targeted at these pathways could hypothetically be applied in the treatment of cardiac fibrosis. Wnt3a, being highly expressed in exosomes, could activate the Wnt/-catenin pathway in cardiac fibroblasts by restricting GSK3 activation[156]. Moreover, tumor necrosis factor contained in exosomes can be transferred between cardiac myocytes. In general activation/inhibition of the exosomes conveying remodeling substance secretion or uptake can control the myocardial remodeling and repair following MI[154,157].

The highlighted complex cell-to-cell communication from endogenous or exogenous CSCs provides an optimal microenvironment for resident CPC proliferation and differentiation (Figure ), rendering the environment receptive to transplanted CPCs. This adaptation is promoted through activation of pro-survival kinases, leading to the induction of a glycolytic switch in recipient CPCs[158].

Data from experimental models suggest that the exosomal component of the CPC secretome can fully recapitulate the effects of cellular therapy on ischemic and non-ischemic heart models[140]. In an ischemia-reperfusion injury rat model, Ciullo and partners[159] have shown that the systemic injection of exosomes (genetically manipulated to overexpress CXCR4ExoCXCR4) improve cardiac function. Additionally, expression of hypoxia-inducible factor 1 (HIF-1) in the infarcted myocardium is upregulated through the stimulation of SDF-1. The latter is one of the CXC chemokine family overexpressed in heart post-MI that readily attaches to the CXCR4 receptor and acts as a potent chemoattractant for CXCR4 expressing circulating progenitor cells. The ExoCXCR4 are more bioactive in the infarcted zone than naturally occurring exosomes injected via tail-vein, confirming their superior homing and cardioprotective properties in the damaged heart.

Gallet et al[160] postulated the safety and efficiency of CDC-derived exosomes in acute and chronic myocardial injury animal models. Within the context of experimental research to validate the paracrine hypothesis for CDCsderived exosomes, it has been proven that human CDC-exosomes can recapitulate CDC therapy and boost cardiac function post-MI in pig models. Intramyocardial injection of human CDC-exosomes has resulted in higher exosome retention and efficacy as compared to intracoronary injection, with great reduction of scar size and increased ejection fraction. This indicates that the route of administration is imperative for full functional capacity of the exosomes. Subsequently, the researchers have devised a randomized preclinical study by means of a NOGA-guided intramyocardial exosome injection. Decreased collagen content in the infarct and border zone and increased neovascularization and Ki67+ CMs are indicative of the reparative functions of CDC-exosomes. Notably, human CDC-exosomes have shown a lack of an immune reaction, as seen by the lack of inflammatory reactions or CM necrosis in pig models. These observations strongly support the view that CDC-exosomes are ready to be tested in clinical trials.

Similar promising outcomes were observed in a Duchenne muscular dystrophy model (mdx), in which intramyocardial injection of CDC-exosomes efficiently recapitulated the effects of CDC injection on cardiac function, leading to recovery of movement. Administration of CPC-derived exosomes has resulted in transient restoration of partial expression of full-length dystrophin in mdx mice[161]. Further studies assessed the therapeutic potential of CPC-exosomes in a doxorubicin cardiotoxicity model and non-ischemic heart disease[162]. In addition, two concluded phase I clinical trials in patients with heart failure and revealed the capacity of CDCs to enhance cardiac function by reducing ventricular remodeling and scar formation. Despite receiving a single injection at the beginning of the study, the improvement in cardiac function was noted after the 1-year follow-up. This finding consequently leads to the proposition that transplanted CDCs mainly have imposed their actions at the site of injury by secreting paracrine factors including exosomes. In other words, CDC-exosomes achieved a biphasic beneficiary regenerative effect involving acute cardio protection coupled with long-term stimulation of endogenous cardiac repair[163].

While the fetal heart obtains most of its ATP supply via glycolysis[164], the adult heart relies mainly on fatty acid oxidation to fulfill the contracting myocardium high energy demand[164,165]. The loss of the regenerative phenotype is related to the oxidative metabolism of glucose and fatty acids[166,167] and is mediated by various physiological changes including increased workload and the demand for growth, which cannot be solely met by glycolysis[168,169], as well as postnatal increase in both circulating levels of free fatty acids and blood oxygen levels[164,165]. Studies have shown the involvement of the HIF-1 signaling pathway[170], peroxisome proliferator-activated receptor (PPAR)[171], and peroxisome proliferator-activated receptor coactivator-1 (PGC-1) in the switch toward oxidative metabolism[172], which is accompanied by dramatic increase in the number of mitochondria in CMs[173].

Notably, similar metabolic reprogramming occurs during differentiation from cardiac SCs to CMs[167]. Studies reported that after differentiation into CMs, there is an increase in the mitochondrial number and activity[174], increased oxidative metabolism[175], and increased respiratory capacity resulting in an increased adenosine diphosphate:ATP ratio[173] after differentiation into CMs.

The fact of the various metabolic changes that accompany the transition from glycolysis to fatty acids oxidation affect cardiac cell maturation[164,167] has mandated the consideration of substrate composition in cardiac differentiation protocols[167].

A study by Malandraki-Miller et al[176] investigated the effect of fatty acid supplementation, which mimics the metabolic switch from glucose to fatty acid oxidation, on adult cardiac progenitors. The study used radiolabeled substrate consumption for metabolic flux to investigate the role of the PPAR/PGC-1 axis during metabolic maturation. Oleic acid stimulated the PPAR pathway, enhanced the maturation of the cardiac progenitor, and increased the expression of MHC and connexin after differentiation. Moreover, total glycolytic metabolism, mitochondrial membrane potential, the expression of glucose, and fatty acid transporter increased. The recorded results contributed greatly in highlighting the role of fatty acids and PPAR in CPC differentiation.

Another study by Correia et al[177] has linked substrate utilization and functional maturation of CMs via studying the effect of the metabolic shift from glucose to galactose and fatty acid-containing medium in the maturation of hPSCs-derived CMs (hPSCs-CMs). The shift accelerated hPSC-CM maturation into adult-like CMs with higher oxidative metabolism, mature transcriptional signatures, higher myofibril density, improved calcium influx, and enhanced contractility. Galactose improved total oxidative capacity with reduction of fatty acid oxidation, thereby protecting the cells from lipotoxicity.

In CDCs, oxidative metabolism and cell differentiation reciprocally affect each other. In vitro cultures for CDCs revealed a PPAR agonist that triggers fatty acid oxidation. Metabolic changes have been characterized as the CDC differentiated towards a cardiac phenotype. Addition of a PPAR agonist at the onset of differentiation has induced a switch towards oxidative metabolism, as shown by changes in gene expression with decreasing glycolytic flux and increasing oxidation of glucose and palmitate. Undifferentiated CDCs have generated high levels of ATP from glycolysis and from oxidation of acetoacetate. Upon differentiation, oxidative metabolism of glucose and fatty acids is upregulated with decreased oxidation of acetoacetate, a metabolic phenotype similar to that of the adult heart[178].

Taken together, the metabolic hallmarks of differentiated CMs vary from their undifferentiated SCs. Energy substrate metabolism during cardiac development and differentiation shows gradual decrease in the contribution of glycolysis to ATP synthesis with simultaneous increase in fatty aciddependent mitochondrial respiration[179].

Common methods for the investigation of substrate metabolism include the measurement of metabolic fluxes using radio-labeled substrates, such as D-U-14C-glucose[180,181] as well as measurement of mitochondrial oxygen consumption rate and extracellular acidification rate using the XF Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, United States)[182,183].

Recently, a detailed protocol for metabolic characterization of hiPSCs-CMs has been developed. The hiPSCs are obtained from adult somatic cells via novel cell reprogramming approaches, followed by differentiation to CMs. The novel in vitro cardiac cellular model provided new insights into studying cardiac disease mechanisms and therapeutic potentials. The characterization protocol measures small metabolites and combines gas- and liquid-chromatography-mass spectrometry metabolic profiling, lactate/pyruvate, and glucose uptake assays as important tools[184]. Integration between the implemented assays has provided complementary metabolic characteristics besides the already established electrophysiological and imaging techniques, such as monitoring ion channel activities[185], measurement of action potentials, changes in Ca+2 fluxes[186], and mitochondria viability and apoptosis[187].

An alternative pathway for glucose metabolism in CMs involves the entry of glucose-6-phosphate (G6P) in the pentose phosphate pathway, with resultant generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH)[188]. Reduced NADPH helps to regenerate reduced glutathione and thus acts protectively against reactive oxygen species induced cell injury.

The cardioprotective role of the pentose/G6P/NADPH/glutathione pathway has been emphasized by Jain et al[189] who demonstrated that G6P dehydrogenase (G6PD) lacking mice have more severe heart damage induced by the myocardial ischemia reperfusion injury in Langendorff-perfused hearts as compared with wild-type mice.

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Cardiac stem cells: Current knowledge and future prospects