Specific location in the body containing stem cells

Stem-cell niche refers to a microenvironment, within the specific anatomic location where stem cells are found, which interacts with stem cells to regulate cell fate.[1] The word 'niche' can be in reference to the in vivo or in vitro stem-cell microenvironment. During embryonic development, various niche factors act on embryonic stem cells to alter gene expression, and induce their proliferation or differentiation for the development of the fetus. Within the human body, stem-cell niches maintain adult stem cells in a quiescent state, but after tissue injury, the surrounding micro-environment actively signals to stem cells to promote either self-renewal or differentiation to form new tissues. Several factors are important to regulate stem-cell characteristics within the niche: cellcell interactions between stem cells, as well as interactions between stem cells and neighbouring differentiated cells, interactions between stem cells and adhesion molecules, extracellular matrix components, the oxygen tension, growth factors, cytokines, and the physicochemical nature of the environment including the pH, ionic strength (e.g. Ca2+ concentration) and metabolites, like ATP, are also important.[2] The stem cells and niche may induce each other during development and reciprocally signal to maintain each other during adulthood.

Scientists are studying the various components of the niche and trying to replicate the in vivo niche conditions in vitro.[2] This is because for regenerative therapies, cell proliferation and differentiation must be controlled in flasks or plates, so that sufficient quantity of the proper cell type are produced prior to being introduced back into the patient for therapy.

Human embryonic stem cells are often grown in fibroblastic growth factor-2 containing, fetal bovine serum supplemented media. They are grown on a feeder layer of cells, which is believed to be supportive in maintaining the pluripotent characteristics of embryonic stem cells. However, even these conditions may not truly mimic in vivo niche conditions.

Adult stem cells remain in an undifferentiated state throughout adult life. However, when they are cultured in vitro, they often undergo an 'aging' process in which their morphology is changed and their proliferative capacity is decreased. It is believed that correct culturing conditions of adult stem cells needs to be improved so that adult stem cells can maintain their stemness over time.[citation needed]

A Nature Insight review defines niche as follows:

"Stem-cell populations are established in 'niches' specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. It constitutes a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets. The interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-cell therapeutics ... The simple location of stem cells is not sufficient to define a niche. The niche must have both anatomic and functional dimensions."[3]

Though the concept of stem cell niche was prevailing in vertebrates, the first characterization of stem cell niche in vivo was worked out in Drosophila germinal development.

By continuous intravital imaging in mice, researchers were able to explore the structure of the stem cell niche and to obtain the fate of individual stem cells (SCs) and their progeny over time in vivo. In particular in intestinal crypt,[4] two distinct groups of SCs have been identified: the "border stem cells" located in the upper part of the niche at the interface with transit amplifying cells (TAs), and "central stem cells" located at the crypt base. The proliferative potential of the two groups was unequal and correlated with the cells' location (central or border). It was also shown that the two SC compartments acted in accord to maintain a constant cell population and a steady cellular turnover. A similar dependence of self-renewal potential on proximity to the niche border was reported in the context of hair follicle, in an in vivo live-imaging study.[5]

This bi-compartmental structure of stem cell niche has been mathematically modeled to obtain the optimal architecture that leads to the maximum delay in double-hit mutant production.[6] They found that the bi-compartmental SC architecture minimizes the rate of two-hit mutant production compared to the single SC compartment model. Moreover, the minimum probability of double-hit mutant generation corresponds to purely symmetric division of SCs with a large proliferation rate of border stem cells along with a small, but non-zero, proliferation rate of central stem cells.[citation needed]

Stem cell niches harboring continuously dividing cells, such as those located at the base of the intestinal gland, are maintained at small population size. This presents a challenge to the maintenance of multicellular tissues, as small populations of asexually dividing individuals will accumulate deleterious mutations through genetic drift and succumb to mutational meltdown.[7] Mathematical modeling of the intestinal gland reveals that the small population size within the stem cell niche minimizes the probability of carcinogenesis occurring anywhere, at the expense of gradually accumulated deleterious mutations throughout organismal lifetimea process that contributes to tissue degradation and aging.[8] Therefore, the population size of the stem cell niche represents an evolutionary trade-off between the probability of cancer formation and the rate of aging.

Germline stem cells (GSCs) are found in organisms that continuously produce sperm and eggs until they are sterile. These specialized stem cells reside in the GSC niche, the initial site for gamete production, which is composed of the GSCs, somatic stem cells, and other somatic cells. In particular, the GSC niche is well studied in the genetic model organism Drosophila melanogaster and has provided an extensive understanding of the molecular basis of stem cell regulation.[citation needed]

In Drosophila melanogaster, the GSC niche resides in the anterior-most region of each ovariole, known as the germarium. The GSC niche consists of necessary somatic cells-terminal filament cells, cap cells, escort cells, and other stem cells which function to maintain the GSCs.[9] The GSC niche holds on average 23 GSCs, which are directly attached to somatic cap cells and Escort stem cells, which send maintenance signals directly to the GSCs.[10] GSCs are easily identified through histological staining against vasa protein (to identify germ cells) and 1B1 protein (to outline cell structures and a germline specific fusome structure). Their physical attachment to the cap cells is necessary for their maintenance and activity.[10] A GSC will divide asymmetrically to produce one daughter cystoblast, which then undergoes 4 rounds of incomplete mitosis as it progresses down the ovariole (through the process of oogenesis) eventually emerging as a mature egg chamber; the fusome found in the GSCs functions in cyst formation and may regulate asymmetrical cell divisions of the GSCs.[11] Because of the abundant genetic tools available for use in Drosophila melanogaster and the ease of detecting GSCs through histological stainings, researchers have uncovered several molecular pathways controlling GSC maintenance and activity.[12] [13]

The Bone Morphogenetic Protein (BMP) ligands Decapentaplegic (Dpp) and Glass-bottom-boat (Gbb) ligand are directly signalled to the GSCs, and are essential for GSC maintenance and self-renewal.[14] BMP signalling in the niche functions to directly repress expression of Bag-of-marbles (Bam) in GSCs, which is up-regulated in developing cystoblast cells.[15] Loss of function of dpp in the niche results in de-repression of Bam in GSCs, resulting in rapid differentiation of the GSCs.[10] Along with BMP signalling, cap cells also signal other molecules to GSCs: Yb and Piwi. Both of these molecules are required non-autonomously to the GSCs for proliferation-piwi is also required autonomously in the GSCs for proliferation.[16] In the germarium, BMP signaling has a short-range effect, therefore the physical attachment of GSCs to cap cells is important for maintenance and activity.[citation needed]

The GSCs are physically attached to the cap cells by Drosophila E-cadherin (DE-cadherin) adherens junctions and if this physical attachment is lost GSCs will differentiate and lose their identity as a stem cell.[10] The gene encoding DE-cadherin, shotgun (shg), and a gene encoding Beta-catenin ortholog, armadillo, control this physical attachment.[17] A GTPase molecule, rab11, is involved in cell trafficking of DE-cadherins. Knocking out rab11 in GSCs results in detachment of GSCs from the cap cells and premature differentiation of GSCs.[18] Additionally, zero population growth (zpg), encoding a germline-specific gap junction is required for germ cell differentiation.[19]

Both diet and insulin-like signaling directly control GSC proliferation in Drosophila melanogaster. Increasing levels of Drosophila insulin-like peptide (DILP) through diet results in increased GSC proliferation.[20] Up-regulation of DILPs in aged GSCs and their niche results in increased maintenance and proliferation.[21] It has also been shown that DILPs regulate cap cell quantities and regulate the physical attachment of GSCs to cap cells.[21]

There are two possible mechanisms for stem cell renewal, symmetrical GSC division or de-differentiation of cystoblasts. Normally, GSCs will divide asymmetrically to produce one daughter cystoblast, but it has been proposed that symmetrical division could result in the two daughter cells remaining GSCs.[22][23] If GSCs are ablated to create an empty niche and the cap cells are still present and sending maintenance signals, differentiated cystoblasts can be recruited to the niche and de-differentiate into functional GSCs.[24]

As the Drosophila female ages, the stem cell niche undergoes age-dependent loss of GSC presence and activity. These losses are thought to be caused in part by degradation of the important signaling factors from the niche that maintains GSCs and their activity. Progressive decline in GSC activity contributes to the observed reduction in fecundity of Drosophila melanogaster at old age; this decline in GSC activity can be partially attributed to a reduction of signaling pathway activity in the GSC niche.[25][26] It has been found that there is a reduction in Dpp and Gbb signaling through aging. In addition to a reduction in niche signaling pathway activity, GSCs age cell-autonomously. In addition to studying the decline of signals coming from the niche, GSCs age intrinsically; there is age-dependent reduction of adhesion of GSCs to the cap cells and there is accumulation of Reactive Oxygen species (ROS) resulting in cellular damage which contributes to GSC aging. There is an observed reduction in the number of cap cells and the physical attachment of GSCs to cap cells through aging. Shg is expressed at significantly lower levels in an old GSC niche in comparison to a young one.[26]

Males of Drosophila melanogaster each have two testes long, tubular, coiled structures and at the anterior most tip of each lies the GSC niche. The testis GSC niche is built around a population of non-mitotic hub cells (a.k.a. niche cells), to which two populations of stem cells adhere: the GSCs and the somatic stem cells (SSCs, a.k.a. somatic cyst stem cells/cyst stem cells). Each GSC is enclosed by a pair of SSCs, though each stem cell type is still in contact with the hub cells. In this way, the stem cell niche consists of these three cell types, as not only do the hub cells regulate GSC and SSC behaviour, but the stem cells also regulate the activity of each other. The Drosophila testis GSC niche has proven a valuable model system for examining a wide range of cellular processes and signalling pathways.[27]

The process of spermatogenesis begins when the GSCs divide asymmetrically, producing a GSC that maintains hub contact, and a gonialblast that exits the niche. The SSCs divide with their GSC partner, and their non-mitotic progeny, the somatic cyst cells (SCCs, a.k.a. cyst cells) will enclose the gonialblast. The gonialblast then undergoes four rounds of synchronous, transit-amplifying divisions with incomplete cytokinesis to produce a sixteen-cell spermatogonial cyst. This spermatogonial cyst then differentiates and grows into a spermatocyte, which will eventually undergo meiosis and produce sperm.[27]

The two main molecular signalling pathways regulating stem cell behaviour in the testis GSC niche are the Jak-STAT and BMP signalling pathways. Jak-STAT signalling originates in the hub cells, where the ligand Upd is secreted to the GSCs and SSCs.[28][29] This leads to activation of the Drosophila STAT, Stat92E, a transcription factor which effects GSC adhesion to the hub cells,[30] and SSC self-renewal via Zfh-1.[31] Jak-STAT signalling also influences the activation of BMP signalling, via the ligands Dpp and Gbb. These ligands are secreted into the GSCs from the SSCs and hub cells, activate BMP signalling, and suppress the expression of Bam, a differentiation factor.[32] Outside of the niche, gonialblasts no longer receive BMP ligands, and are free to begin their differentiation program. Other important signalling pathways include the MAPK and Hedgehog, which regulate germline enclosure [33] and somatic cell self-renewal,[34] respectively.

The murine GSC niche in males, also called spermatogonial stem cell (SSC) niche, is located in the basal region of seminiferous tubules in the testes. The seminiferous epithelium is composed of sertoli cells that are in contact with the basement membrane of the tubules, which separates the sertoli cells from the interstitial tissue below. This interstitial tissue comprises Leydig cells, macrophages, mesenchymal cells, capillary networks, and nerves.[35]

During development, primordial germ cells migrate into the seminiferous tubules and downward towards the basement membrane whilst remaining attached to the sertoli cells where they will subsequently differentiate into SSCs, also referred to as Asingle spermatogonia.[35][36] These SSCs can either self-renew or commit to differentiating into spermatozoa upon the proliferation of Asingle into Apaired spermatogonia. The 2 cells of Apaired spermatogonia remain attached by intercellular bridges and subsequently divide into Aaligned spermatogonia, which is made up of 416 connected cells. Aaligned spermatogonia then undergo meiosis I to form spermatocytes and meiosis II to form spermatids which will mature into spermatozoa.[37][38] This differentiation occurs along the longitudinal axis of sertoli cells, from the basement membrane to the apical lumen of the seminiferous tubules. However, sertoli cells form tight junctions that separate SSCs and spermatogonia in contact with the basement membrane from the spermatocytes and spermatids to create a basal and an adluminal compartment, whereby differentiating spermatocytes must traverse the tight junctions.[35][39] These tight junctions form the blood testis barrier (BTB) and have been suggested to play a role in isolating differentiated cells in the adluminal compartment from secreted factors by the interstitial tissue and vasculature neighboring the basal compartment.[35]

The basement membrane of the seminiferous tubule is a modified form of extracellular matrix composed of fibronectin, collagens, and laminin.[35] 1- integrin is expressed on the surface of SSCs and is involved in their adhesion to the laminin component of the basement membrane although other adhesion molecules are likely also implicated in the attachment of SSCs to the basement membrane.[40] E cadherin expression on SSCs in mice, unlike in Drosophila, have been shown to be dispensable as the transplantation of cultured SSCs lacking E-cadherin are able to colonize host seminiferous tubules and undergo spermatogenesis.[41] In addition the blood testis barrier provides architectural support and is composed of tight junction components such as occludins, claudins and zonula occludens (ZOs) which show dynamic expression during spermatogenesis.[42] For example, claudin 11 has been shown to be a necessary component of these tight junctions as mice lacking this gene have a defective blood testis barrier and do not produce mature spermatozoa.[40]

GDNF (Glial cell-derived neurotrophic factor) is known to stimulate self-renewal of SSCs and is secreted by the sertoli cells under the influence of gonadotropin FSH. GDNF is a related member of the TGF superfamily of growth factors and when overexpressed in mice, an increase in undifferentiated spermatogonia was observed which led to the formation of germ tumours.[35][40] In corroboration for its role as a renewal factor, heterozygous knockout male mice for GDNF show decreased spermatogenesis that eventually leads to infertility.[40] In addition the supplementation of GDNF has been shown to extend the expansion of mouse SSCs in culture. However, the GDNF receptor c-RET and co-receptor GFRa1 are not solely expressed on the SSCs but also on Apaired and Aaligned, therefore showing that GDNF is a renewal factor for Asingle to Aaligned in general rather than being specific to the Asingle SSC population. FGF2 (Fibroblast growth factor 2), secreted by sertoli cells, has also been shown to influence the renewal of SSCs and undifferentiated spermatogonia in a similar manner to GDNF.[35]

Although sertoli cells appear to play a major role in renewal, it expresses receptors for testosterone that is secreted by Leydig cells whereas germ cells do not contain this receptor- thus alluding to an important role of Leydig cells upstream in mediating renewal. Leydig cells also produce CSF 1 (Colony stimulating factor 1) for which SSCs strongly express the receptor CSF1R.[37] When CSF 1 was added in culture with GDNF and FGF2 no further increase in proliferation was observed, however, the longer the germ cells remained in culture with CSF-1 the greater the SSC density observed when these germ cells were transplanted into host seminiferous tubules. This showed CSF 1 to be a specific renewal factor that tilts the SSCs towards renewal over differentiation, rather than affecting proliferation of SSCs and spermatogonia. GDNF, FGF 2 and CSF 1 have also been shown to influence self-renewal of stem cells in other mammalian tissues.[35]

Plzf (Promyelocytic leukaemia zinc finger) has also been implicated in regulating SSC self-renewal and is expressed by Asingle, Apaired and Aaligned spermatogonia. Plzf directly inhibits the transcription of a receptor, c-kit, in these early spermatogonia. However, its absence in late spermatogonia permits c-kit expression, which is subsequently activated by its ligand SCF (stem cell factor) secreted by sertoli cells, resulting in further differentiation. Also, the addition of BMP4 and Activin-A have shown to reduce self-renewal of SSCs in culture and increase stem cell differentiation, with BMP4 shown to increase the expression of c-kit.[37]

Prolonged spermatogenesis relies on the maintenance of SSCs, however, this maintenance declines with age and leads to infertility. Mice between 12 and 14 months of age show decreased testis weight, reduced spermatogenesis and SSC content. Although stem cells are regarded as having the potential to infinitely replicate in vitro, factors provided by the niche are crucial in vivo. Indeed, serial transplantation of SSCs from male mice of different ages into young mice 3 months of age, whose endogenous spermatogenesis had been ablated, was used to estimate stem cell content given that each stem cell would generate a colony of spermatogenesis.[35][43] The results of this experiment showed that transplanted SSCs could be maintained far longer than their replicative lifespan for their age. In addition, a study also showed that SSCs from young fertile mice could not be maintained nor undergo spermatogenesis when transplanted into testes of old, infertile mice. Together, these results points towards a deterioration of the SSC niche itself with aging rather than the loss of intrinsic factors in the SSC.[43]

Vertebrate hematopoietic stem cells niche in the bone marrow is formed by cells subendosteal osteoblasts, sinusoidal endothelial cells and bone marrow stromal (also sometimes called reticular) cells which includes a mix of fibroblastoid, monocytic and adipocytic cells (which comprise marrow adipose tissue).[1]

The hair follicle stem cell niche is one of the more closely studied niches thanks to its relative accessibility and role in important diseases such as melanoma. The bulge area at the junction of arrector pili muscle to the hair follicle sheath has been shown to host the skin stem cells which can contribute to all epithelial skin layers. There cells are maintained by signaling in concert with niche cells signals include paracrine (e.g. sonic hedgehog), autocrine and juxtacrine signals.[44] The bulge region of the hair follicle relies on these signals to maintain the stemness of the cells. Fate mapping or cell lineage tracing has shown that Keratin 15 positive stem cells' progeny participate in all epithelial lineages.[45] The follicle undergoes cyclic regeneration in which these stem cells migrate to various regions and differentiate into the appropriate epithelial cell type. Some important signals in the hair follicle stem cell niche produced by the mesenchymal dermal papilla or the bulge include BMP, TGF- and Fibroblast growth factor (FGF) ligands and Wnt inhibitors.[46] While, Wnt signaling pathways and -catenin are important for stem cell maintenance,[47] over-expression of -catenin in hair follicles induces improper hair growth. Therefore, these signals such as Wnt inhibitors produced by surrounding cells are important to maintain and facilitate the stem cell niche.[48]

Intestinal organoids have been used to study intestinal stem cell niches. An intestinal organoid culture can be used to indirectly assess the effect of the manipulation on the stem cells through assessing the organoid's survival and growth. Research using intestinal organoids have demonstrated that the survival of intestinal stem cells is improved by the presence of neurons and fibroblasts,[49] and through the administration of IL-22.[50]

Cardiovascular stem cell niches can be found within the right ventricular free wall, atria and outflow tracks of the heart. They are composed of Isl1+/Flk1+ cardiac progenitor cells (CPCs) that are localized into discrete clusters within a ColIV and laminin extracellular matrix (ECM). ColI and fibronectin are predominantly found outside the CPC clusters within the myocardium. Immunohistochemical staining has been used to demonstrate that differentiating CPCs, which migrate away from the progenitor clusters and into the ColI and fibronectin ECM surrounding the niche, down-regulate Isl1 while up-regulating mature cardiac markers such as troponin C.[51] There is a current controversy over the role of Isl1+ cells in the cardiovascular system. While major publications have identified these cells as CPC's and have found a very large number in the murine and human heart, recent publications have found very few Isl1+ cells in the murine fetal heart and attribute their localization to the sinoatrial node,[52] which is known as an area that contributes to heart pacemaking. The role of these cells and their niche are under intense research and debate.[citation needed]

Neural stem cell niches are divided in two: the Subependymal zone (SEZ) and the Subgranular zone (SGZ).

The SEZ is a thin area beneath the ependymal cell layer that contains three types of neural stem cells: infrequently dividing neural stem cells (NSCs), rapidly dividing transit amplifying precursors (TaPs) and neuroblasts (NBs). The SEZ extracellular matrix (ECM) has significant differences in composition compared to surrounding tissues. Recently, it was described that progenitor cells, NSCs, TaPs and NBs were attached to ECM structures called Fractones.[53] These structures are rich in laminin, collagen and heparan sulfate proteoglycans.[54] Other ECM molecules, such as tenascin-C, MMPs and different proteoglycans are also implicated in the neural stem cell niche.[55]

Cancer tissue is morphologically heterogenous, not only due to the variety of cell types present, endothelial, fibroblast and various immune cells, but cancer cells themselves are not a homogenous population either.[citation needed]

In accordance with the hierarchy model of tumours, the cancer stem cells (CSC) are maintained by biochemical and physical contextual signals emanating from the microenvironment, called the cancer stem cell niche.[56] The CSC niche is very similar to normal stem cells niche (embryonic stem cell (ESC), Adult Stem Cell ASC) in function (maintaining of self-renewal, undifferentiated state and ability to differentiate) and in signalling pathways (Activin/Noda, Akt/PTEN, JAK/STAT, PI3-K, TGF-, Wnt and BMP).[57] It is hypothesized that CSCs arise form aberrant signalling of the microenvironment and participates not only in providing survival signals to CSCs but also in metastasis by induction of epithelial-mesenchymal transition (EMT).[citation needed]

Hypoxic condition in stem cell niches (ESC, ASC or CSC) is necessary for maintaining stem cells in an undifferentiated state and also for minimizing DNA damage via oxidation. The maintaining of the hypoxic state is under control of Hypoxia-Inducible transcription Factors (HIFs).[58] HIFs contribute to tumour progression, cell survival and metastasis by regulation of target genes as VEGF, GLUT-1, ADAM-1, Oct4 and Notch.[57]

Hypoxia plays an important role in the regulation of cancer stem cell niches and EMT through the promotion of HIFs.[59] These HIFs help maintain cancer stem cell niches by regulating important stemness genes such as Oct4, Nanog, SOX2, Klf4, and cMyc.[60][61] HIFs also regulate important tumor suppressor genes such as p53 and genes that promote metastasis.[62][63] Although HIFs increase the survival of cells by decreasing the effects of oxidative stress, they have also been shown to decrease factors such as RAD51 and H2AX that maintain genomic stability.[64] In the hypoxic condition there is an increase of intracellular Reactive Oxygen Species (ROS) which also promote CSCs survival via stress response.[65][66] ROS stabilizes HIF-1 which promotes the Met proto-oncogene, which drives metastasis or motogenic escape in melanoma cells.[67] All of these factors contribute to a cancer stem cell phenotype which is why it is often referred to as a hypoxic stem cell niche. Hypoxic environments are often found in tumors where the cells are dividing faster that angiogenesis can occur. It is important to study hypoxia as an aspect of cancer because hypoxic environments have been shown to be resistant to radiation therapy.[68] Radiation has been shown to increase the amounts of HIF-1.[69] EMT induction by hypoxia though interactions between HIF-1 and ROS is crucial for metastasis in cancers such as melanoma. It has been found that many genes associated with melanoma are regulated by hypoxia such as MXI1, FN1, and NME1.[70]

Epithelialmesenchymal transition is a morphogenetic process, normally occurs in embryogenesis that is "hijacked" by cancer stem cells by detaching from their primary place and migrating to another one. The dissemination is followed by reverse transition so-called Epithelial-Mesenchymal Transition (EMT). This process is regulated by CSCs microenvironment via the same signalling pathways as in embryogenesis using the growth factors (TGF-, PDGF, EGF), cytokine IL-8 and extracellular matrix components. These growth factors' interactions through intracellular signal transducers like -catenin has been shown to induce metastatic potential.[71][72] A characteristic of EMT is loss of the epithelial markers (E-cadherin, cytokeratins, claudin, occluding, desmoglein, desmocolin) and gain of mesenchymal markers (N-cadherin, vimentin, fibronectin).[73]

There is also certain degree of similarity in homing-mobilization of normal stem cells and metastasis-invasion of cancer stem cells. There is an important role of Matrix MetalloProteinases (MMP), the principal extracellular matrix degrading enzymes, thus for example matrix metalloproteinase-2 and 9 are induced to expression and secretion by stromal cells during metastasis of colon cancer via direct contact or paracrine regulation. The next sharing molecule is Stromal cell-Derived Factor-1 (SDF-1).[73][74]

The EMT and cancer progression can be triggered also by chronic inflammation. The main roles have molecules (IL-6, IL-8, TNF-, NFB, TGF-, HIF-1) which can regulate both processes through regulation of downstream signalling that overlapping between EMT and inflammation.[57] The downstream pathways involving in regulation of CSCs are Wnt, SHH, Notch, TGF-, RTKs-EGF, FGF, IGF, HGF.

NFB regulates the EMT, migration and invasion of CSCs through Slug, Snail and Twist. The activation of NFB leads to increase not only in production of IL-6, TNF- and SDF-1 but also in delivery of growth factors.

The source of the cytokine production are lymphocytes (TNF-), Mesenchymal Stem Cells (SDF-1, IL-6, IL8).

Interleukin 6 mediates activation of STAT3. The high level of STAT3 was described in isolated CSCs from liver, bone, cervical and brain cancer. The inhibition of STAT3 results in dramatic reduction in their formation. Generally IL-6 contributes a survival advantage to local stem cells and thus facilitates tumorigenesis.[57]

SDF-1 secreted from Mesenchymal Stem Cells (MSCs) has important role in homing and maintenance of Hematopoietic Stem Cell (HSC) in bone marrow niche but also in homing and dissemination of CSC.[74]

Hypoxia is a main stimulant for angiogenesis, with HIF-1 being the primary mediator. Angiogenesis induced by hypoxic conditions is called an "Angiogenic switch". HIF-1 promotes expression of several angiogenic factors: Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), Placenta-Like Growth Factor (PLGF), Platelet-Derived Growth Factor (PDGF) and Epidermal Growth Factor. But there is evidence that the expression of angiogenic agens by cancer cells can also be HIF-1 independent. It seems that there is an important role of Ras protein, and that intracellular levels of calcium regulate the expression of angiogenic genes in response to hypoxia.[73]

The angiogenic switch downregulates angiogenesis suppressor proteins, such as thrombospondin, angiostatin, endostatin and tumstatin. Angiogenesis is necessary for the primary tumour growth.[citation needed]

During injury, support cells are able to activate a program for repair, recapitulating aspects of development in the area of damage. These areas become permissive for stem cell renewal, migration and differentiation. For instance in the CNS, injury is able to activate a developmental program in astrocytes that allow them to express molecules that support stem cells such as chemokines i.e. SDF-1[75] and morphogens such as sonic hedgehog.[76]

It is evident that biophysio-chemical characteristics of ECM such as composition, shape, topography, stiffness, and mechanical strength can control the stem cell behavior. These ECM factors are equally important when stem cells are grown in vitro. Given a choice between niche cell-stem cell interaction and ECM-stem cell interaction, mimicking ECM is preferred as that can be precisely controlled by scaffold fabrication techniques, processing parameters or post-fabrication modifications. In order to mimic, it is essential to understand natural properties of ECM and their role in stem cell fate processes. Various studies involving different types of scaffolds that regulate stem cells fate by mimicking these ECM properties have been done.[2])

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Clinical translation of stem cell therapy for spinal cord injury still premature: results from a single-arm meta-analysis based on 62 clinical trials...

Introduction

Traumatic brain injury is one of the main causes of deaths, disabilities, and hospitalization in the world. In the USA, around 30% of all injury-related deaths are due to traumatic brain injury.1 Globally, traumatic brain injury affects the lives of about 10 million people each year.2 It happened as the brain tissue is damaged by an external force, the result of direct impact, rapid acceleration or deceleration, a piercing object, and blast waves from an explosion.3 Visual impairment, cognitive dysfunction, hearing loss, and mental health disorders are among the most common complications affecting traumatic brain injury patients and their families. The pathophysiology of traumatic brain injury is not clear since the structure of the brain is complex with many cell types such as neurons, astrocytes, oligodendrocytes, microglia, and multiple subtypes of these cells. Traumatic brain injury occurs in two phases. These are primary (acute) and secondary (late) brain injuries. The primary injury is the initial blow to the head; in this phase, brain tissue and cells such as neurons, glial cells, endothelial cells, and the bloodbrain barrier are damaged by mechanical injury. The secondary injury occurs after primary injury and in these late phases, several toxins are released from the injured cells leading to the formation of cytotoxic cascades, which increase the initial brain damage.4 The primary brain injury causes the dysfunction of the bloodbrain barrier and initiates local inflammation and secondary neuronal injury. In addition, severe and long-term inflammation causes severe neurodegenerative and inflammatory diseases. Repairing of tissue damage needs the inhibition of secondary injury and rapid regeneration of injured tissue.5 Depending on the nature of the injury, neurons and neuroglial cells may be damaged; excessive bleeding may happen, axons may be destroyed and a contusion may occur.6 Moreover, the pathogenesis of traumatic brain injury involves bloodbrain barrier damage, neural inflammation, and diffuse neuronal degeneration.7 Unlike other organs, it has long been thought that mature brain tissue cannot be able to repair itself after injury.8 However, the current research indicated that multipotent neural stem/progenitor cells are residing in some areas of the brain throughout the lifespan of an animal, implying the mature brains ability to produce new neurons and neuroglial cells.9 In the previous decades, several studies have shown that the mature neurons in the hippocampal dentate gyrus of the brain play significant roles in hippocampal-induced learning and memory activities,9 while new olfactory interneurons produced from the subventricular zone are essential for the appropriate functioning of the olfactory bulb network and some specific olfactory behaviors.10 After traumatic brain injuries, clinical evidence indicated that endogenous neural progenitor cells might play an important role in regenerative medicine to treat brain injury because an increased neurogenic regeneration ability has been reported in different types of brain injury models of animal and human studies.11 Nowadays, there is a new therapeutic approach for traumatic brain injury that involves the use of stem cells for neural regeneration and restoration. Exogenous stem cell transplantation has been found to accelerate immature neuronal development and increase endogenous cellular proliferation in the damaged brain region.12 A better understanding of the endogenous neural stem cells regenerative ability as well as the effect of exogenous neural stem cells on proliferation and differentiation may help researchers better understand how to increase functional recovery and brain tissue repair following injury. Therefore, in this study, we discussed the therapeutic effects of stem cells in the repair of traumatic brain injury.

Traumatic brain injury causes severe stress on the brain, making it extremely hard to keep appropriate cognitive abilities. Even though many organs in the body, for example, the skin, can regenerate following injury, the brain tissue may not easily repair. In the adult brain, endogenous neural stem cells are primarily localized to the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus.13 In the subventricular zone, neural stem/progenitor cells generate neuronal and oligodendroglial progenies.14 Most of the new neurons produced from the subventricular zone migrate via the rostral migratory stream, eventually becoming olfactory interneurons in the olfactory bulb.15 A few subventricular zone-derived new neurons travel into cortical areas for an unknown cause but may be related to tissue repair or renewal mechanisms.16 Similarly, newly produced dentate gyrus cells travel laterally into the dentate granule cell layer and become fully mature in a few weeks through a process known as adult hippocampus neurogenesis.17 However, it is still unknown whether these neural stem cells in the subventricular zone and dentate gyrus regions can replace the lost neurons following injury.

So far, several studies have assessed the degree of neurogenesis in these two areas and have demonstrated that significant numbers of new cells are continuously generated.9,18 For example, the rat dentate gyrus generates about 9000 new cells each day or 270,000 cells every month.18 A current clinical finding indicated that the whole granular cell population in the deep layer and half of the superficial layer of the olfactory bulb were replaced by newly produced mature neurons for a year.19 A similar study also revealed that adult-produced neurons account for around 10% of the overall number of dentate granule cells in the hippocampus and they are uniformly distributed along the anterior-posterior axis of the dentate gyrus.19 After the finding of continuous adult neurogenesis during the lifetime in the adult animal brain, the functional roles and the significance of this adult neurogenesis, mainly hippocampal neurogenesis concerning learning and memory processes, have been widely explored. Previous studies showed factors that increase hippocampal neurogenesis such as exposure to enriched environments, physical activity, or growth factor therapy may improve cognitive abilities.2022

The newly formed granular cells in the mature dentate gyrus can become functional neurons in the normal hippocampus by demonstrating passive membrane characteristics, generating action potentials, and receiving functional synaptic inputs, as seen in the adult dentate gyrus neurons.23 For instance, mouse strains hereditarily having poor levels of neurogenesis carry out low learning activities than those with a higher level of baseline neurogenesis.2325 A variety of physical and chemical signals influence the proliferation and maturational destiny of cells in the subventricular zone and dentate gyrus. For instance, biochemical variables including serotonin, glucocorticoids, ovarian hormones, and growth factors strongly regulate the proliferative response, implying that cell proliferation in these areas has a significant physiological role.26,27 Besides, physical factors such as exercise and stress produce changes in cell proliferation implying a significant role in network adaptation.28,29 For example, physical exercise might cognitively and physically enhance the production of cells and neurogenesis within the subventricular zone and dentate gyrus, but stress inhibits this type of cellular activity. Furthermore, the physiologic role of these new cells depends on the number of cells being produced, survival rate, differentiation ability, and integration of cells into existing neuronal circuity.24,30

The subventricular zone and hippocampus contain neural stem cells that respond to a variety of stimuli. Different kinds of experimental traumatic brain injury models such as fluid percussive injury,31,32 controlled cortical impact injury,33,34 closed-head weight drop injury,35 and acceleration-impact injury36 have shown increased neural stem cells activation. All of these experimental studies have shown the most prevalent and notable endogenous cell response after traumatic brain injury is an elevated cell proliferation within neurogenic areas of the dentate gyrus and subventricular zone. It is well accepted that enhanced production of new neurons following the traumatic brain injury was detected predominantly in the hippocampus in the more seriously injured animals in many experimental studies.37 More studies have discovered that injury-enhanced new granule neurons send out axonal projections into the targeted CA3 region implying their integration into the existing hippocampal circuitry,37,38 and this injury-induced endogenous neurogenic stem cells response is directly associated with the inherent cognitive functional recovery after traumatic brain injury of rodents.39,40

In the human brain, the extent and physiology of the adult neural generation are not well understood. A study on human brain samples taken from the autopsy revealed neural stem cells with proliferative ability have been observed within the subventricular zone and the hippocampus.41,42 Conversely, a more recent study has shown that neurogenesis in the subventricular zone and movement of new neurons from the subventricular zone to the olfactory bulbs and neocortex are restricted and only seen in the early childhood period.43,44 Therefore, credible evidence of traumatic brain injury-initiated neurogenesis in the human brain is inadequate because of the difficulties of collecting human brain samples and technical challenges to birth-dating neural stem cells.

After traumatic brain injury, injury-initiated neural cell loss is permanent. Given the restricted amount of endogenous neurogenic stem cells, neural transplantation supplementing exogenous stem cells to the damaged brain tissue is a potential treatment for post-traumatic brain injury regeneration.45 Especially, the transplanted cells will not only be able to replace the damaged neural cells but also give neurotrophic support in hopes of reestablishing and stabilizing the damaged brain tissue.45 Clinical evidence revealed intervention with stem cell secretome may significantly improve neural inflammation after traumatic brain injury and other neurological deficits in humans.46 Besides, the combined effects of bioscaffold and exosomes can aid in the transportation of stem cells to damaged areas as well as enhance their survival and facilitate successful treatment.47 Despite the rapid progression of brain infarction, the decreased proliferation of neural stem cells, and the delayed initiation of neurological recovery were observed in the aged rat model compared with a young rat after stroke, the restorative capability of the brain by stem cell therapy is still present in the aged rat.48 Compared to stem cell monotherapies which are still uniformly failed in clinical practice, combination therapy with hypothermia has potential therapeutic effects on the physiology of the aged brain and may be required for effective protection of the brain following stroke.49 After several years of biomaterials study for regeneration of peripheral nerve, a new 3D printing strategy is developing as a good substitution for nerve autograft over large gap injuries. The applications of 3D printing technologies can help in improving long-distance peripheral nerve regeneration since it is a leading device to give one path for better nerve guidance.50 Up to now, various categories of stem cell therapy have been tested for post-traumatic brain injury. These include embryonic stem cells, adult-derived neural stem cells, mesenchymal stem cells, and induced pluripotent stem cells.

Embryonic stem cells obtained from fetal or embryonic brain tissues are highly considered for neural transplantation because of their ability of plasticity and have the capacity to self-repair and differentiation into all germinal layers. They can differentiate, migrate, and innervate as transplanted into a receiver brain tissue.51 In previous clinical brain injury studies, neural stem cells derived from the embryonic human brain could survive for a long time, migrating to the contralateral cortex and differentiating into mature neural cells and microglia following transplantation into the damaged brain tissue.52 Implanted neurogenic stem cells obtained from human fetal stem cells may differentiate into adult neurons and release growth factors increasing the cognitive functional recovery of the damaged brain.53 Interestingly, the long-term survival rate of transplanted neural stem cells obtained from mice embryonic brains was seen for up to 1 year with a high degree of migration in the damaged brain and maturation into neurons or neuroglial cells along with enhanced motor and spatial learning functions of the brain tissue.5456 In addition, embryonic stem cells expressing growth factors or early differentiated into neurotransmitter expressing adult neurons after in vitro manipulation have revealed improved transplant survival and neuronal differentiation following grafted into the damaged brain, and the receivers have better recovery in motor and cognitive activities.5759 Even though embryonic stem cells have a high rate of survival and plasticity in neuronal transplantation, the ethical concerns, risk of transplant rejection, and the likelihood of teratoma development restrict their therapeutic use for traumatic brain injury.45

Neural stem cells are multipotent cells that can differentiate into neural cells but have a limited ability to differentiate into other tissue types.60 Neurogenic stem cells are located in the subventricular zones of the lateral ventricle, the hippocampal dentate gyrus, and other areas of the brain like the cerebral cortex, amygdala, hypothalamus, and substantia nigra. They could be isolated, developed in culture media, and produce many neural lineages that can be used in the treatment of neurological disorders as an important element of cellular-replacement therapy.61 Adult neural stem cells were transplanted into damaged parts of the brain in a traumatic brain injury rat model. These cells survived the transplantation process and moved to a damaged site when expressing markers for adult microglia and oligodendrocytes.62 Interestingly, one most recent study indicated that Korean red ginseng extract-mediated astrocytic heme oxygenase-1 induction contributes to the proliferation and differentiation of adult neural stem cells by upregulating astrocyteneuronal system cooperation.63 Another study revealed that following neural stem cell transplantation to the hippocampal region, injured rats had developed better cognitive function.64 The administration of combined therapies such as human neural stem/progenitor cells and curcumin-loaded noisome nanoparticles significantly improve brain edema, gliosis, and inflammatory responses in the traumatic brain injury rat model.65 Furthermore, in traumatic brain injury rat models, as neural stem cells were injected intravenously, they resulted in a decreased neurologic impairment and less edema because of the anti-inflammatory and anti-apoptotic features of neural stem cells.60,66 The ideal transplantation timeframe is 714 days,60 beyond which the glial scar forms, restricting perfusion and graft survival.67 The ability to transport cells to the desired location is a key obstacle with neural stem cell transplantation. Neural stem cells can be administered intrathecally, intravenously, and intra-arterial infusion. Conversely, a nanofiber scaffold implantation was proposed by Walker et al as a new strategy to be implemented to give the support essential for cell proliferation, which provides direction to future research.68

Mesenchymal stem cells are multipotent stromal that can differentiate into mesenchymal and non-mesenchymal tissue, such as neural tissue.69 They are obtained from different types of tissues.70 The accessibility, availability, and differentiation ability of these cells have drawn the attention of researchers performing studies in regenerative medicine. A previous study revealed the differentiation capacity of mesenchymal stem cells into neuronal cells. This study found that when rat and human mesenchymal stem cells are exposed to various experimental culture conditions, they can differentiate into neural and neuroglial cells.69 Besides, mesenchymal stem cells have also been demonstrated to enhance the proliferation and differentiation of native neural stem cells; the mechanism of which may be directly associated with chemokines produced by mesenchymal stem cells or indirectly through stimulation of adjacent astrocytes.70 In addition to their capacity to differentiate, mesenchymal stem cells selectively move to damaged tissues in traumatic brain injury rat models, where they develop into neurons and astrocytes and enhance motor function.71 The possible mechanism of action through which this occurs is linked to chemokines, growth factors,72 and adhesion factors, like the vascular cell adhesion molecule (VCAM-1), which permits mesenchymal stem cells to adhere to the endothelium of damaged organ.73 Mesenchymal stem cell transplantation has become a potential and safe treatment of choice for traumatic brain injuries because of its anti-inflammatory capability by regulating leukocyte and inflammatory factors such as IL-6, CRP, and TNF-a.74,75 Treatment with mesenchymal stem cell-derived extracellular vesicles greatly increased neurogenesis and neuroplasticity in a pig model of hemorrhagic stroke and traumatic brain damage.76 Currently, stem cell therapy using mesenchymal stromal cells has been widely investigated in preclinical models and clinical trials for the treatment of several neurological illnesses, including traumatic brain injury. Mesenchymal stem cells investigated for the treatment of traumatic brain injury in these clinical trials include bone marrow-derived stem cells, amnion-derived multipotent progenitor cells, adipose-derived stem cells, umbilical cord-derived stem cells, and peripheral blood-derived stem cells.7779 Those undifferentiated mesenchymal-derived cells have a heterogeneous cell population that includes stem and progenitor cells. They can be stimulated to differentiate into a neuronal cell phenotype in vitro. In the damaged brain tissue, these cells can generate a large number of growth factors, cytokines, and extracellular matrix substances that have neurotrophic or neuroprotective effects.80,81

From all mesenchymal stem cells, the effect of bone marrow-derived mesenchymal stem cells on traumatic brain injury has been fully investigated. According to previous studies, mesenchymal stem cells injected directly into the injured brain, or through intravenous or intra-arterial injections during the acute, sub-acute, or chronic phase following traumatic brain injury, have been shown to significantly reduce neurological abnormalities in motor and cognitive abilities.7779,82 The therapeutic effect of mesenchymal stem cells is mostly because of the bioactive molecules they produced to facilitate the endogenous plasticity and remodeling of the recipient brain tissue instead of direct neural repair as direct neuronal differentiation and long-term viability were rarely seen.80 A more recent study found that the injection of cell-free exosomes obtained from human bone marrow-derived mesenchymal stromal cells can increase the functional recovery of damaged animals after traumatic brain injury.83 Another study used a traumatic rodent model to evaluate the anti-inflammatory and immunoregulatory properties of mesenchymal stem cells. When compared to the control group, neurological function was improved in the treatment groups from 3 to 28 days. Mesenchymal stem cell therapy significantly decreased the amount of microglia or macrophages, neutrophils, CD3 lymphocytes, apoptotic cells in the damaged cortex, and proinflammatory cytokines.81 The main challenge of using mesenchymal stem cells for traumatic brain injury treatment is the long-term possibility of brain malignancy development because of the mesenchymal stromal cells ability to antitumor response suppression.84

In a recent study, seven traumatic brain injury patients were given a mesenchymal stem cells transplant during a cranial operation and then administered a second dose intravenously. At the end of the 6-month follow-up period, patients exhibited better neurological function with no signs of toxicity.85

Recent studies revealed that the administration of exosomes-derived human umbilical cord mesenchymal stem improves sensorimotor function and spatial learning activities in rat models following brain injuries. Furthermore, the applications of these cells extensively decreased proinflammatory cytokine expression via inhibiting the NF-B signaling pathway, reduced neuronal apoptosis, reduced inflammation, and increased neural regeneration ability in the injured cortex of rats following the injuries.86 Human umbilical cord-derived mesenchymal stem cells have better anti-inflammatory activity that may prevent and decrease secondary brain injury caused by the immediate discharge of inflammatory factors following traumatic brain injury.87 In traumatic brain injury rat models, the transplantation of umbilical cord-derived mesenchymal stem cells triggers the trans-differentiation of T-helper 17 into T regulatory, which in turn repairs neurological deficits and improves learning and memory function.88

To see the therapeutic effects of transplanted induced pluripotent stem cells compared to that of embryonic stem cells, Wang et al demonstrated animal models of ischemia and three different treatment options, which consist of pluripotent stem cells, embryonic stem cells, and phosphate-buffered saline for the control. The rodents were given an injection into the left lateral ventricle of the brain. Embryonic stem cell treatment group rodents showed a significant improvement in glucose metabolism within two-week period. However, 1 month following treatment, neuroimaging tests were done and it was revealed that both pluripotent stem cell and embryonic stem cell treatment groups had improved neurologic scores as compared to the control group, suggesting that the treatment groups showed better recovery of their cognitive function. Further investigation indicated that the implanted cells survived and traveled to the area of injury. Finally, the investigator of this study concluded that induced pluripotent stem cells may be a better option than embryonic stem cells.57 Different studies showed that induced pluripotent stem cells improved motor and cognitive function in the host mouse brain tissue, and these cells migrate the injured brain areas from the injection site.89,90 Until now, there are limited studies on induced pluripotent stem cell therapy for brain injuries. This is because of the difficulty of obtaining induced pluripotent stem cells, high therapy costs, and technique limitations.

In preclinical and clinical trials, advanced progress has been made in stem cell-based therapy for traumatic brain injury patients. Various studies reported the therapeutic effect of stem cells for regenerating damaged brain tissue. However, because of the complexity and variability of brain injuries, post-traumatic brain injury neuronal regeneration and repair remain a long-term goal. There are numerous unresolved challenges for successful stem cell treatment. For endogenous restoration via mature neural regeneration, methods guiding the movement of new neuronal cells to the area of damaged tissue and maintaining long-term survival are very important. In stem cell therapy, the inherent features of transplanted cells and the local host micro-environment influences the fate of grafted cells, an appropriate cell source, and a host environment, which are required for effective transplantation. Therefore, these problems should be solved in preclinical traumatic brain injury trials before stem cell-based treatments could be used in the clinic. The therapeutic application of neural stem cell treatment, whether via manipulation of endogenous or implantation of exogenous neural stem cells, is a method that has been shown in multiple studies to have substantial potential to increase brain function recovery in persons suffering from traumatic brain injury-related disability. However, further studies need to be done on the therapeutic application of stem cells for traumatic brain injury due to our poor understanding of possible consequences, unknown ethical issues, routes of administration, and the use of mixed treatment.

All authors declared no conflicts of interest for this study.

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Repair of Traumatic Brain Injury | SCCAA - Dove Medical Press

Stem Cells. Author manuscript; available in PMC 2020 Jul 1.

Published in final edited form as:

PMCID: PMC6658105

NIHMSID: NIHMS1024291

1NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

1NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

1NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

2Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA

3Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD, USA

1NeuroRepair Department, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

2Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA

3Cellular Imaging Section and Vascular Biology Program, Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD, USA

Author contributions:

Barbara Lukomska: Conception and design, financial support, collection and/or assembly of data, final approval of manuscript

Miroslaw Janowski: Conception and design, financial support, collection and/or assembly of data, manuscript writing, final approval of manuscript

It was shown as long as half a century ago that bone marrow is a source of not only hematopoietic stem cells, but also stem cells of mesenchymal tissues. Then the term of mesenchymal stem cells (MSCs) has been coined in early 1990s and over a decade later the criteria for defining MSCs have been released by International Society for Cellular Therapy. The easy derivation from a variety of fetal and adult tissues and not demanding cell culture conditions made MSCs an attractive research object. It was followed by the avalanche of reports from preclinical studies on potentially therapeutic properties of MSCs such as immunomodulation, trophic support and capability for a spontaneous differentiation into connective tissue cells, and differentiation into majority of cell types upon specific inductive conditions. While ontogenesis, niche and heterogeneity of MSCs are still under investigation, there is a rapid boost of attempts in clinical applications of MSCs, especially for a flood of civilization-driven conditions in so quickly aging societies in not only developed countries, but also very populous developing world. The fields of regenerative medicine and oncology are particularly extensively addressed by MSC applications, in part due to paucity of traditional therapeutic options for these highly demanding and costly conditions. There are currently almost 1000 clinical trials from entire world registered at clinicaltrials.gov and it seems that we are starting to witness the snowball effect with MSCs becoming a powerful global industry, however spectacular effects of MSCs in clinic still need to be shown.

Keywords: Mesenchymal stem cells, clinical, differentiation, immunomodulation, paracrine activity, history

Friedenstein was one of the pioneers of the theory that bone marrow is a reservoir of stem cells of mesenchymal tissues in adult organisms. It was based on his observation at the turn of the 1960s and 1970s., that ectopic transplantation of bone marrow into the kidney capsule, results not only the proliferation of bone marrow cells, but also the formation of bone [1] (). This indicated the existence in the bone marrow of a second, in addition to hematopoietic cells, stem cell population giving rise to bone precursors. Due to the ability of these cells to create osteoblasts, Friedenstein gave them the name of osteogenic stem cells. Friedenstein was also the first to isolate from bone marrow adherent fibroblast-like cells with the ability to grow rapidly in vitro in the form of clonogenic colonies (CFU-F; colony forming unit-fibroblast). These cells derived from CFU-F colonies were characterized by the ability to differentiate in vitro not only to osteocytes, but also to chondrocytes and adipocytes. After transplantation of CFU-F colonies into the recipient, they were capable of co-formation of the bone marrow micro-environment [2,3]. The term mesenchymal stem cells has been proposed by Caplan in 1991 because of their ability to differentiate into more than one type of cells that form connective tissue in many organs [4]. This name has become very popular and is currently the most commonly used, even though it raised doubts about the degree of their stemness [5]. Today, there are many substitutes in the literature for the abbreviation of MSCs, including Multipotent Stromal Cells, Marrow Stromal Cells, Mesodermal Stem Cells, Mesenchymal Stromal Cells and many more. In its latest work, Caplan recommends renaming these cells to Medicinal Signaling Cells due to the emphasis on the mechanism of their therapeutic effects after transplantation, which is believed to be based mainly on the secretion of factors facilitating regenerative processes [6].

The roots of research on bone marrow-derived stem cells of connective tissue, which has been then named: mesenchymal stem cells

Due to the growing controversy regarding the nomenclature, the degree of stemness and the characteristics of the cells discovered by Friedenstein, the International Society for Cellular Therapy (ISCT) in 2006 published its position specifying the criteria defining the population of MSCs, which was accepted by the global scientific community. These guidelines recommend the use of the name multipotent mesenchymal stromal cells, however, the name mesenchymal stem cells still remains the most-used. The condition for the identification of MSCs is the growth of cells in vitro as a population adhering to the substrate, as well as in the case of cells of human origin, a phenotype characterized by the presence of CD73, CD90, CD105 surface antigens and the lack of expression of proteins such as: CD45, CD34, CD14, CD11b, CD79a or CD19 or class II histocompatibility complex antigens (HLA II, human leukocyte antigens class II). Moreover, these cells must have the ability to differentiate towards osteoblasts, adipocytes and chondroblasts [7,8]. In addition to the markers mentioned in the ISCT guidelines, the following antigens turned out to be useful in isolating the human MSCs from the bone marrow: STRO-1 (antigen of the bone marrow stromal-1 antigen, cell surface antigen expressed by stromal elements in human bone marrow-1), VCAM / CD106 (vascular cell adhesion molecule 1) and MCAM / CD146 (melanoma cell adhesion molecule), which characterizes cells growing in vitro in a adherent form, with a high degree of clonogenicity and multidirectional differentiation ability [911].

The common mesenchymal core in both versions of MSC abbreviation comes from the term mesenchyme, which is synonymous with mesenchymal tissue or embryonic connective tissue. It is used to refer to a group of cells present only in the developing embryo derived mainly from the third germ layer - mesoderm. During the development these cells migrate and diffuse throughout the body of the embryo. They give rise to cells that build connective tissue in adult organisms, such as bones, cartilage, tendons, ligaments, muscles and bone marrow. The view about the differentiation of MSCs during embryonic development from mesenchymal cells is widely spread [4]. This is due, inter alia, to the observed convergence in the expression of markers such as: vimentin, laminin 1, fibronectin and osteopontin, which are typical for mesoderm cells during embryonic development, as well as characteristic for in vitro adherent bone marrow stroma cells [12]. However, the true origin of MSCs is unknown. In the literature, we can find also reports indicating that they are ontogenetically associated with a group of cells derived from ectoderm, which originate from Sox1 + cells (SRY - sex determining region Y) that appear during the development of embryonic neuroectoderm and neural crest. These cells inhabit newborn bone marrow and meet the criteria corresponding to their designation as MSCs. However, with the development of animals, the population of these cells disappears and is replaced by cells with a different, unidentified origin [13]. It has also been shown that in the bone marrow of the developing mouse embryo, at least two MSCs populations with distinct expression of the nestin protein and the intensity of cell divisions can be distinguished. The former one originates from mesoderm that does not express nestin, and is characterized by intense proliferation and is involved in the process of creating the embryo skeleton. The latter one is derived from the cells of the neural crest, which expresses nestin and is non-dividing and remains passive during bone formation while in the adult organism contributes to a niche of hematopoietic cells [14]. It seems, therefore, that the ontogenesis of MSCs is associated with cells belonging to different germ layers and their original source determines the role and functions that they play in the adult body.

In 1978, the concept of a niche was defined as a place in the body that is settled by stem cells and whose environment allows them to be maintained in an undifferentiated state [15]. MSCs were first obtained from the bone marrow stroma where they constitute an element of stromal cells, participating in the production of signals modulating the maturation of hematopoietic cells. However, the precise location of the niche for MSCs has not been known so far. In the context of research results indicating that MSCs can be isolated from many mesoderm-derived tissues during embryonic development, a common element was sought for all sources from which MSCs can be isolated and a theory was proposed about the existence of their niche within the blood vessels that are present in all structures from which these cells were isolated.

Crisan and colleagues have shown that cells inhabiting the perivascular space of blood vessels, isolated from human tissues such as skeletal muscle, pancreas, adipose tissue and placenta, with the phenotype CD146 +, NG2 + (neuroglycan-2), PDGF-R + (-type platelet-derived growth factor receptor), ALP + expressing endothelial, hematopoietic and muscle cell markers described as pericytes were precursors for cells that after in vitro expansion meet the criteria for determining them as MSCs [16]. Analogously to the described by Friedenstein MSCs, CD146 + cells colonizing the perivascular space of sinusoidal sinus vessels, are responsible for the production of signals allowing the reconstruction of the bone marrow microenvironment after transplantation to heterotopic location [11]. Whats more, tracing the fate of pericytes in the process of rebuilding a damaged tooth in rodents has shown that they are transforming into odontoblasts, which arise from MSCs found in the pulp. However, the same studies showed that in the process of reconstruction of incisors in mice, a different population of odontoblasts, which is not formed from pericytes, but from MSCs of different origin migrating to the area of damage, prevailed quantitatively [17]. The second cell population associated with blood vessels, proposed as a counterpart of MSCs in the body is advent building cells with the CD34+ CD31- CD146- phenotype, which after isolation and in vitro culture meet the criteria defining the population as MSCs. However, these cells also have the ability to differentiate into pericytes [18,19]. Although pericytes and MSCs have a very similar gene expression profile as well as an analogical capacity for differentiation, it has been shown that the functionality of these cells varies. In vitro studies of endothelial cell interactions in co-culture with MSCs or pericytes have shown that only pericytes are able to form highly branched, dense, cylindrical structures with large diameter, typical for well-organized blood vessels, while isolated from the bone marrow MSCs do not have such abilities. Currently, it is believed that there is a link between pericytes and MSCs, but their mutual relations are not well defined. There are speculations that MSCs are an intermediate form of pericytes or their subpopulation, but there is still no conclusive evidence confirming this hypothesis [20,21].

While the cells fulfilling criteria for MSCs can be harvested from various tissues at all developmental stages (fetal, young, adult and aged) using their plastic adherence property, there are profound differences between obtained MSC populations [22,23]. Bone marrow was historically the first source from which MSCs were obtained, however, over time, there have been reports of the possibility of isolation from other sources of cells with similar properties. Mesenchymal cells are obtained from both tissues and secretions of the adult body, such as adipose tissue, peripheral blood, dental pulp, yellow ligament, menstrual blood, endometrium, milk from mothers, as well as fetal tissues: amniotic fluid, membranes, chorionic villi, placenta, umbilical cord, Wharton jelly, and umbilical cord blood [2437]. MSCs of fetal origin as compared to cells isolated from tissues of adult organisms are characterized by a faster rate of proliferation as well as a greater number of in vitro passages until senescence [38]. However, MSCs derived from bone marrow and adipose tissue are able to create a larger number of CFU-F colonies, which indirectly indicates a higher degree of their stemness. The comparison of gene expression typical for pluripotent cells shows that only in cells isolated from the bone marrow we can observe the expression of the SOX2 gene, the activation of which is associated with the self-renewal process of stem cells as well as with neurogenesis during embryonic development [39]. Discrepancies in the ability of MSCs obtained from various sources to differentiate have also been described. The lack of differentiation of MSCs derived from umbilical cord blood towards adipocytes as well as the greater tendency of MSCs from bone marrow and adipose tissue to differentiate towards osteoblasts were observed [39,40].

In addition to the diverseness observed between MSCs from different sources, there are also differences associated with obtaining them from individual donors. Among the cells isolated from the bone marrow from donors of different ages and sexes, up to 12-fold differences in the rate of their proliferation and osteogenesis were found, combined with a 40-fold difference in the level of bone remodeling marker activity - ALP (alkaline phosphatase). At the same time, no correlations were found resulting from differences in the sex or age of donors [41]. However, the results of studies by other authors indicate that the properties of MSCs isolated from the bone marrow are strongly associated with the age of the donor. Cells collected from older donors are characterized by an increased percentage of apoptotic cells and slower rate of proliferation, which is associated with an increased population doubling time. There is also a weakened ability of MSCs from older donors to differentiate towards osteoblasts [42]. Heo in his work shows the different ability of MSCs to osteogenesis combining it with different levels of DLX5 gene expression (transcription factor with the homeodomain 5 motif) in individual donors, however independent of the type of tissue from which the cells were isolated [39].

The next stage in which we can observe diversity among the MSCs population is in vitro culture. The morphology of cultured cells that originate from the same isolation allows for differentiation into three sub-populations. There are observed spindle-shaped proliferating cells resembling fibroblasts (type I); large, flat cells with a clearly marked cytoskeleton structure, containing a number of granules (type II) and small, round cells with high self-renewal capacity [43,44]. The original hypothesis assumed that all cells that make up the MSCs population are multipotent, and each colony of CFU is capable of differentiating into adipocytes, chondrocytes and osteoblasts, as confirmed by appropriate studies [45]. However, in the literature we can find reports that cell lines derived from a common colony of CFU-F differ in their properties, characterized by uni-, di- or multipotence [46]. Some of the authors showed the division of clonogenic MSCs colonies into as much as eight groups distinct in their potential for differentiation. At the same time, it is suggested that there is a hierarchy within which cells subordinate to each other are increasingly directed towards osteo- chondro- or adipocytes and gradually lose their multipotential properties to di- and unipotential ones. This transformation may also be associated with a decrease in the rate of cell proliferation and the level of CD146 protein expression (CD; cluster of differentiation) - proposed as a marker of multipotency [47].

One of the main advantages of MSCs are their immunomodulatory properties. MSCs grown in vitro have the ability to interact and regulate the function of the majority of effector cells involved in the processes of primary and acquired immune response () [48]. They exert their immunomodulatory effects by inhibiting the complement-mediated effects of peripheral blood mononuclear cell proliferation [49,50], blocking apoptosis of native and activated neutrophils, as well as reducing the number of neutrophils binding to vascular endothelial cells, limiting the mobilization of these cells to the area of damage [51,52]. In addition, cytokines synthesized by activated MSCs stimulate neutrophil chemotaxis and secretion of pro-inflammatory chemokines involved in recruitment and stimulation of phagocytic macrophage properties [53]. Moreover MSCs limit mast cell degranulation, secretion of pro-inflammatory cytokines by these cells as well as their migration towards the chemotactic factors [54]. Native MSCs have the ability to block the proliferation of de novo-induced NK cells, but they are only able to partially inhibit the proliferation of already activated cells [55]. They also contribute to the reduction of cytotoxic activity of NK cells [56]. Moreover MSCs can block the differentiation of CD34 + cells isolated from the bone marrow or blood monocytes into mature dendritic cells both by direct contact as well as by secreted paracrine factors [57,58]. They inhibit the transformation of immature dendritic cells into mature forms and limit the mobilization of dendritic cells to the tissues [59]. Under their influence, M1 (pro-inflammatory) macrophages are transformed into M2 type cells with an anti-inflammatory phenotype, and the IL-10 (IL, interleukin) secreted by them inhibits T-cell proliferation [60,61]. In vitro studies have demonstrated a direct immunomodulatory effect of MSCs on lymphocytes. During the co-culture of MSCs with lymphocytes, suppression of activated CD4 + and CD8 + T cells and B lymphocytes was observed [62]. In addition, MSCs reduce the level of pro-inflammatory cytokines synthesized by T-lymphocytes, such as TNF- (tumor necrosis factor ) and IFN- (interferon ) [63], and increase synthesis of anti-inflammatory cytokines, e.g. IL-4. In the presence of MSCs, the inhibition of the differentiation of naive CD4 + T lymphocytes to Th17 + lymphocytes (Th; T helper cells) was observed, while the percentage of T cells differentiating towards CD4 + CD25 + regulatory T cells was found to increase [64,65]. Glennie et al. described this condition as anergy of activated T cells in the presence of MSCs [62]. MSCs also have the ability to limit the synthesis of immunoglobulins like IgM, IgG and IgA (Ig; immunoglobulin) classes secreted by activated B cells, thereby blocking the differentiation of these cells to plasma cells. They also reduce the expression of chemokines and their receptors on the surface of B lymphocytes, which probably have a negative effect on their ability to migrate [66].

The schematic representation of immunomodulatory capabilities of MSCs

Mesenchymal stem cells secrete a wide range of paracrine factors, collectively referred to as the secretome, which support regenerative processes in damaged tissues. They comprise the components of the extracellular matrix, proteins involved in the adhesion process, enzymes as well as their activators and inhibitors, growth factors and binding proteins, cytokines and chemokines, and probably many more [67]. These factors can have distinct impact on the processes they regulate (). MSCs secrete factors promoting angiogenesis, such as: vascular endothelial growth factor (VEGF) but they may also inhibit this process, through expression of monokine induced by interferon and tissue inhibitors of metalloproteinases 1 and 2 [68,69]. An important role is also played by chemokines secreted by MSCs in the process of blocking or stimulating cell chemotaxis, such as: CCL5 (RANTES, regulated by activation, expression and secretion by normal T lymphocytes), CXCL12 (SDF-1, stromal cell-derived factor 1) or CCL8 (MCP-2; monocyte chemoattractant protein 2). An essential group of factors from the point of view of regeneration processes are growth factors with an anti-apoptotic effect, including: HGF (hepatocyte growth factor), IGF-1 (insulin-like growth factor 1), VEGF, CINC-3 (cytokine induced by a chemoattractant for neutrophil chemoattractant), TIMP-1 (tissue inhibitor of metalloproteinases 1), TIMP-2 (tissue inhibitor of metalloproteinases 2), osteopontin, growth hormone, FGF-BP (bFGF binding protein), and BDNF (brain-derived growth factor; -derived neurotrophic factor) and stimulating proliferation as: TGF- (transforming growth factor ), HGF, EGF (epidermal growth factor), NGF (nerve growth factor; nerve growth factor), bFGF (basic fibroblast growth factor), IGFBP-1, IGFBP-2 (IGFBP; insulin-like growth factor 1 binding protein, IGF-Protein-1 protein) and M-CSF (stimulant factor t molar macrophage colony; macrophage colony-stimulating factor) [68,70,71]. Growth factors secreted by MSCs have also ability to reduce fibrosis of tissues during regeneration. These include KGF (keratinocyte growth factor), HGF, VEGF, and Ang-1 (angiopoietin-1), SDF1, IGF-1, EGF, HGF, NGF, TGF- [71,72]. There are reports about the antibacterial properties and interaction of the MSC secretome with cancer cells. Data on the impact of MSCs on neoplasia are not conclusive, however, it is assumed that both the tumor type and the origin of MSCs are of great importance for the final effect [73]. It was shown that factors enclosed within the MSCs secretome are able to reduce the proliferation, viability and migration of certain types of cancer cells (such as non-small-cell lung carcinoma) [74]. Others have shown that factors released by MSCs may increase motility, invasiveness and the ability to form metastases (including, for example, breast cancer cells) [75]. In response to bacteria, levels of cytokines such as IL- 6, IL-8, CCL5, PGE2, TNF-, IL-1, IL-10, VEGF and SDF-1 secreted by MSCs are subject to change [76]. MSCs contain also substances with antibacterial, anti-parasitic and antiviral activity [77].

The mechanisms mediating MSC-dependent trophic support

Another broad and dynamically developing field in recent years which is related to paracrine MSCs activity is their ability to secrete extracellular vesicles (EVs), which include exosomes, microvesicles and apoptotic bodies. Their composition largely coincides with the components contained in the cells from which they originate. Physiologically they play an important role in the regulation of biological functions, homeostasis and the immune response of the body. It is also postulated that the biological activity of microvesicles is comparable to that of MSCs [78]. Experiments conducted using supernatant derived from in vitro culture of MSCs showed that the factors contained in their secretome are responsible for a large part of the effects exerted by MSCs during the regeneration of the damaged area including the protection of other cells against apoptosis, induction of their proliferation, prevention of excessive fibrosis of tissues, stimulation of the angiogenesis process and immunomodulatory effects, as well as the induction of endogenous stem cells differentiation [65,68,69,7982].

As mentioned above, the ability to differentiate into three types of cells such as: osteocytes, chondrocytes and adipocytes is one of the criterion for MSCs [8]. This phenomenon can be traced in vitro by placing MSCs in a medium containing specific supplements, for the adipogenesis process they are mainly dexamethasone, indomethacin, insulin and isobutylmethylxanthin [83], for chondrogenesis cell culture in DMEM medium (Dulbecco / Vogt Modified Eagles Minimal Essential Medium) supplemented with insulin, transferrin, selenium, linoleic acid, selenium acid, pyruvate, ascorbic phosphate, dexamethasone and TGF- III [84], which may additionally be aided by the addition of IGF-1 and BMP-2 (BMP; bone morphogenetic proteins) [85]. In turn the osteogenesis is induced by the presence of ascorbic acid, -glycerophosphate and dexamethasone [86]. Differentiation of MSCs in the appropriate cell type is assessed by identifying the production of respectively: fat droplets (adipogenesis), proteoglycans and type II collagen synthesis (chondrogenesis) or mineralization of calcium deposits and the increase of alkaline phosphatase expression (osteogenesis). However, many literature reports indicate that by the treatment with appropriate factors MSCs might be also a source of other cell types. Caplan and Dennis in their work from 2006 present a process that they call mesengenesis, in which MSCs give also rise to myoblasts, bone marrow stromal cells, fibroblasts, cells co-creating connective tissue of the body as well as ligaments and tendons [87]. Addition of 5-azacytidine to MSCs allows to obtain muscle cells, including cardiomyocytes and myoblasts having the ability to create multinucleated miotubes and expressing markers such as: -myosin heavy chain, -actin cardiac form and desmin [88]. In addition, in vitro studies have made it possible to obtain from MSCs at least two types of cells derived from the endoderm through their transdifferentiation into hepatocytes and -cells of pancreatic islets. The liver cells are obtained from MSCs in two stages by culturing them in modified Dulbeccos medium supplemented with EGF, bFGF and nicotinamide, and in the next stage with the addition of oncostatin M, dexamethasone, insulin, transferrin and selenium. The resulting cells show the presence of markers typical for hepatocytes such as albumin, -fetoprotein and hepatocyte nuclear factor 4 (HNF-4) [89]. By the treatment with a mixture of growth factors secreted by regenerating cells of the pancreas as well as by the use of acitin A, sodium butyrate, taurine and nicotinamide the pancreatic islets of -cells capable of producing insulin were obtained from MSCs [90,91]. It has also been shown that stimulation with appropriate factors may result in the differentiation of MSCs into cells derived ontogenetically from ectoderm, such as neurons. The use of BME stimulation in vitro (-mercaptoethanol) followed by NGF leads to the differentiation of MSCs into cholinergic nerve cells expressing their typical proteins such as NF-68 neurofilaments (68 kDa Neurofilament protein with 68 kDa molecular mass), NF-200 (neurofilament protein with a molecular weight 200kDa, 200kDa neurofilament protein), NF-160 (neurofilament protein molecular weight 160kDa, 160kDa neurofilament protein), choline acetyltransferase and synapsin I [92]. Other factors mentioned as compounds inducing the transformation of MSCs into nerve cells are insulin, retinoic acid, bFGF, EGF, valproic acid, BME and hydrocortisol [93]. In addition, GNDF (glial cell-derived neurotrophic factor), BDNF (brain-derived neurotrophic factor), retinoic acid, 5-azacytidine, isobutylmethylxanthine and indomethacin stimulate the transformation of MSCs into mature neurons that express markers of nervous systems cells such as: nestin, -III tubulin, microtubule associated protein - MAP2 (microtubule associated protein 2) and neuron-specific enolase (ENO2; enolase 2) [94]. These studies show that under strictly controlled conditions prevailing during in vitro culture, in the presence of chemicals and growth factors, MSCs are able to turn into cells derived from all three embryonic germ layers ().

The differentiation potential of MSCs

It has been more than half a century since the curiosity has been revealed that not only hematopoietic cells, but also those capable of forming connective tissue reside in the bone marrow. Subsequent studies have begun to reveal the increasingly fascinating properties of these cells, which go far beyond forming connective tissue. This, combined with their easy derivation from various tissues, made them an attractive research object. Immunomodulatory properties, aiding repair of various tissues as well as differentiation potential to practically any types of cells stunned a whole host of scientists and established MSCs as a driving force of regenerative medicine and began also to play an increasingly important role in oncology [95]. We are currently observing a flood of clinical trials with the use of MSCs, and their number doubles every few years and currently reaches almost 1000 registered items on the clinicaltrials.gov website.

MSCs compose a negligible fraction of cells derived from in vivo tissues and there is no effective method to capture them directly. Therefore, MSCs need to be subjected to the process of in vitro expansion, which in clinical context is called biomanufacturing and biobanking and both terms are frequently used interchangeably to describe the process from procurement of cell source to deliver cells to the patients bed. The processing of MSCs must be performed according to current Good Manufacturing Practice (cGMP) as any other therapeutic agent and is subjected to extensive regulatory effort. Food and Drug Administration (FDA) is the main authority responsible for acceptance of medical products including those containing living cells such as MSCs in the USA. FDA has issued a perspective on MSC-based product characterization [96] and up-dated it in FDA Grand Round delivered by Steven Bauer, PhD, Chief of Cell and Tissue Therapies Branch at FDA on March 08, 2018. Both sources are an excellent overview of regulatory challenges related to the biobanking of MSCs. In general, any new product must obtain investigational new drug status (INDs) to be used in clinical trial before filing application for marketing, and there were 66 INDs submitted to FDA between 2006 and 2012. Based on that FDA engaged into regulatory research project called MSC consortium to characterize MSC based-products with an output of 16 research papers. The main organ responsible for the regulation of medical market in all Member States is European Medicines Agency (EMA) consisting of seven smaller committees. The MSCs-containing products should be classified as Advanced Therapy Medical Product (ATMP) and in detail considered as Somatic Cell Therapy Medicinal Product (CTMP) [97]. Its release on medical market has to be first accredited by Committee for Advanced Therapies (CAT) which creates the general opinion and evaluates the quality, safety and efficiency of the product. After CAT assessment the final acceptance should be then approved by Committee for the Medicinal Products for Human Use (CHMP). This type of legalization is called Centralized Marketing Authorization and it allows to use ATMP products in all European Union countries. Currently, there is a variety of protocols used for biomanufacturing and biobanking of MSCs, and once the successful stories become strong, the landscape of MSC production will probably solidify with predicted reduction of MSC production approaches due to economic and regulatory pressures.

Summing up, it seems that the MSCs are becoming a powerful global industry, ready to respond to the unmet needs of modern medicine struggling with the proper care and quality of life of rapidly aging societies, which is already affecting not only developed countries, but also very populous developing countries. In conclusion, we are beginning to observe the effect of the snowball in which ever new discoveries related to MSC are increasingly stimulating clinical applications of the MSC, which is beginning to contribute to the transformation of medical care.

Significance Statement

The research on bone marrow-derived stem cells of connective tissue is evolving and continuously expanding with a recent boost of interest in clinical applications reflected by an avalanche of nearly 1000 registered clinical trials. While, the current name: mesenchymal stem cells (MSCs) have been coined as late as early 90-ies, it is important to commemorate of the fiftieth anniversary of research on them and provide a big picture from roots of first paper in 1968, through identification of their various potential therapeutic activities such as immunomodulation, trophic support and capability for differentiation and taking role in cell replacement strategies.

This work was funded by NCR&D grant EXPLORE ME within the STRATEGMED I program and by NIH R01 NS091100-01A1.

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Mesenchymal stem cells: from roots to boost - PMC

While historically lacking in foreign investments, Japans biotech scene is thriving with global investors showing increasing interest. Here are five of the hottest Japanese private companies innovating in the healthcare space.

Japan boasts one of the highest life expectancies in the world, and, faced with a rapidly aging population, is witnessing a growing burden of chronic conditions including cardiovascular disease and type 2 diabetes. For this reason, the Japanese healthcare authorities are encouraging research into the treatment and prevention of these diseases, in addition to promoting the potential of regenerative medicine.

In addition to having a roster of healthcare giants including Takeda, Astellas Pharma and Eisai, Japan is also an Asian hotspot for biotech companies. Upcoming startups have historically been limited in foreign funding and reliant on local venture capital players such as Nippon Venture Capital, Shinsei Capital Partners, and the University of Tokyo Edge Capital Partners.

In 2021, however, the amount of foreign investment flowing into the Japanese biotech space rose to $98 million, almost triple the haul of previous years. The most prominent global backers included Newton Biocapital, F-Prime Capital, and SoftBank Group. This trend arose as the COVID-19 pandemic triggered a wave of investor enthusiasm in biotechnology around the world.

With the help of local experts, weve listed five of the hottest private biotech companies in Japan. These firms, shown in alphabetical order, have raised large funding rounds in the last two years and are developing innovative treatments for a range of conditions including cancer, cardiovascular disease and inflammatory disorders.

Source: Shutterstock

Founded: 2017

Headquarters: Fujisawa

Chordia Therapeutics derives its name from the English term chord referring to a collection of musical notes normally played in harmony. In a similar way, the company aims to work in harmony with stakeholders and collaborators to develop first-in-class small molecule treatments for cancer.

Chordias lead program is a drug that disrupts the processing of RNA in tumor cells. In a healthy cell, RNA molecules are typically transcribed from a DNA template and spliced together to guide the production of new proteins. Some cancer cells accumulate mutations in the RNA splicing machinery and become vulnerable to Chordias drugs that interfere with this process.

Chordia raised $31 million (4 billion yen) in a Series C round in May 2022. The aim of the round was to push the companys lead drug through phase I testing and fund the preclinical development of the rest of its pipeline.

This month, the company announced interim results from the phase I trial of its lead candidate, with four of the recruited patients so far showing signs of responding to the treatment.

Founded: 2015

Headquarters: Tokyo

Heart failure occurs when the heart muscle is irreparably damaged and is unable to pump blood. While this deadly condition can be treated with a heart transplant, there is a general shortage of donors available, making a pressing need for alternatives.

In June 2021, the stem cell therapy developer Heartseed raised $36.5 million (4 billion yen) in a Series C round. The mission is to provide a regenerative route to saving the heart via stem cell therapy.

In the lab, Heartseed reprograms skin cells from the patient into a type of stem cell called induced pluripotent stem cells and grows these stem cells into heart muscle cells. The company then injects the muscle cells as a small cluster, or seed, into heart tissue to repair the muscle.

The proceedings from its Series C round will allow Heartseed to take its lead candidate into clinical development, including a phase I/II trial scheduled for later this year. Last year, Heartseed also licensed its treatment to Novo Nordisk in Denmark to co-develop the treatment outside of Japan.

Founded: 2018

Headquarters: Tokyo

LUCA Science hit the headlines in the last week for raising an impressive $30.3 million (3.86 billion yen) in a Series B round. The company is developing an unusual approach for treating a wide range of diseases: delivering a therapy based on mitochondria, the energy production plants in human cells.

One example where the technology could work well is in strokes and heart attacks, where blood flow is blocked to critical tissue in the brain and heart respectively. The reperfusion of blood to these tissues after the blockage can kill the tissue by damaging its mitochondria. Delivering healthy mitochondria could keep the tissue working properly and protect it from harm.

LUCA Science plans to use its recent Series B winnings to accelerate the preclinical development of its mitochondrial therapies and establish its manufacturing process. In May 2022, the firm also inked a collaboration deal with compatriot pharmaceutical company Kyowa Kirin Co., Ltd. to co-develop a mitochondrial therapy for rare genetic diseases.

Founded: 2016

Headquarters: Boston, U.S., and Tokyo

Modulus Discovery is a preclinical-stage drug discovery specialist. The company focuses on developing small molecule treatments for conditions such as cancer, inflammatory disorders and rare genetic conditions.

The firm uses a mixture of strategies to speed up the drug discovery process. These include simulating target proteins using a supercomputer; structural protein biology; forming collaborations such as with the peptide drug expert PeptiDream; and tapping into global networks for biological expertise. Modulus most advanced drug program is in late-stage preclinical testing for the treatment of chronic inflammatory diseases.

In March 2022, Modulus bagged $20.4 million (2.34 billion yen) in a Series C round. The cash is earmarked to advance the companys R&D programs by expanding its infrastructure, collaborations and headcount.

Founded: 2015

Headquarters: Tokyo

The name Noile-Immune is derived from blending together the phrases no illness and no immunity, no life. This company is developing CAR-T cell therapies for the treatment of cancer, which traditionally consist of extracting the patients immune T cells, engineering them in the lab to hunt down cancer cells, and reinfusing them into the patient.

Unlike approved CAR-T cell therapies, which are limited to treating forms of blood cancer, Noile-Immune aims its therapies at treating solid tumors. The company does this by engineering immune T cells to produce proteins that cause immune cells to migrate into the tumor site.

Noile-Immune is testing its lead candidate in a phase I in patients with solid tumors. The firm is also co-developing therapies with partners including Takeda and the European cell therapy specialists Adaptimmune and Autolus. Additionally, Noile-Immune has an allogeneic version of its cell therapy in the pipeline where immune T cells are sourced from healthy donors rather than the patient.

To finance the clinical development of its lead candidate, Noile-Immune raised $21.8 million (2.38 billion yen) in a Series C round in early 2021. The company hit a setback in January 2022 when a collaboration deal fell through with the U.S. player Legend Biotech. Nonetheless, other external companies remain interested in Noile-Immunes offering, including Japan-based Daiichi Sankyo Company Ltd., which opted to assess Noile-Immunes technology in late 2021.

Cover image via Elena Resko.

Thanks to feedback from Shiohara Azusa, VC Investor at The University of Tokyo Edge Capital, and Hironoshin Nomura, Chief Financial Officer, Sosei Group Corporation.

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Japan's five hottest biotech companies in healthcare - Labiotech.eu

Stem Cell Investig. 2019; 6: 19.

1Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;

2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;

3Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

1Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran;

2Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran;

3Hematology and Oncology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

Contributions: (I) Conception and design: E Fathi, R Farahzadi; (II) Administrative support: E Fathi, R Farahzadi; (III) Provision of study materials or patients: None; (IV) Collection and assembly of data: R Farahzadi, N Rajabzadeh; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

#These authors contributed equally to this work.

Received 2018 Nov 11; Accepted 2019 Mar 17.

Recent developments in the stem cell biology provided new hopes in treatment of diseases and disorders that yet cannot be treated. Stem cells have the potential to differentiate into various cell types in the body during age. These provide new cells for the body as it grows, and replace specialized cells that are damaged. Since mesenchymal stem cells (MSCs) can be easily harvested from the adipose tissue and can also be cultured and expanded in vitro they have become a good target for tissue regeneration. These cells have been widespread used for cell transplantation in animals and also for clinical trials in humans. The purpose of this review is to provide a summary of our current knowledge regarding the important and types of isolated stem cells from different sources of animal models such as horse, pig, goat, dog, rabbit, cat, rat, mice etc. In this regard, due to the widespread use and lot of attention of MSCs, in this review, we will elaborate on use of MSCs in veterinary medicine as well as in regenerative medicine. Based on the studies in this field, MSCs found wide application in treatment of diseases, such as heart failure, wound healing, tooth regeneration etc.

Keywords: Mesenchymal stem cells (MSCs), animal model, cell-based therapy, regenerative medicine

Stem cells are one of the main cells of the human body that have ability to grow more than 200 types of body cells (1). Stem cells, as non-specialized cells, can be transformed into highly specialized cells in the body (2). In the other words, Stem cells are undifferentiated cells with self-renewal potential, differentiation into several types of cells and excessive proliferation (3). In the past, it was believed that stem cells can only differentiate into mature cells of the same organ. Today, there are many evidences to show that stem cells can differentiate into the other types of cell as well as ectoderm, mesoderm and endoderm. The numbers of stem cells are different in the tissues such as bone marrow, liver, heart, kidney, and etc. (3,4). Over the past 20 years, much attention has been paid to stem cell biology. Therefore, there was a profound increase in the understanding of its characteristics and the therapeutic potential for its application (5). Today, the utilization of these cells in experimental research and cell therapy represents in such disorders including hematological, skin regeneration and heart disease in both human and veterinary medicine (6).The history of stem cells dates back to the 1960s, when Friedenstein and colleagues isolated, cultured and differentiated to osteogenic cell lineage of bone marrow-derived cells from guinea pigs (7). This project created a new perspective on stem cell research. In the following, other researchers discovered that the bone marrow contains fibroblast-like cells with congenic potential in vitro, which were capable of forming colonies (CFU-F) (8). For over 60 years, transplantation of hematopoietic stem cells (HSCs) has been the major curative therapy for several genetic and hematological disorders (9). Almost in 1963, Till and McCulloch described a single progenitor cell type in the bone marrow which expand clonally and give rise to all lineages of hematopoietic cells. This research represented the first characterization of the HSCs (10). Also, the identification of mouse embryonic stem cells (ESCs) in 1981 revolutionized the study of developmental biology, and mice are now used extensively as one of the best option to study stem cell biology in mammals (11). Nevertheless, their application a model, have limitations in the regenerative medicine. But this model, relatively inexpensive and can be easily manipulated genetically (12). Failure to obtain a satisfactory result in the selection of many mouse models, to recapitulate particular human disease phenotypes, has forced researchers to investigate other animal species to be more probably predictive of humans (13). For this purpose, to study the genetic diseases, the pig has been currently determined as one the best option of a large animal model (14).

Stem cells, based on their differentiation ability, are classified into different cell types, including totipotent, pluripotent, multipotent, or unipotent. Also, another classification of these cells are based on the evolutionary stages, including embryonic, fetal, infant or umbilical cord blood and adult stem cells (15). shows an overview of stem cells classifications based on differentiation potency.

An overview of the stem cell classification. Totipotency: after fertilization, embryonic stem cells (ESCs) maintain the ability to form all three germ layers as well as extra-embryonic tissues or placental cells and are termed as totipotent. Pluripotency: these more specialized cells of the blastocyst stage maintain the ability to self-renew and differentiate into the three germ layers and down many lineages but do not form extra-embryonic tissues or placental cells. Multipotency: adult or somatic stem cells are undifferentiated cells found in postnatal tissues. These specialized cells are considered to be multipotent; with very limited ability to self-renew and are committed to lineage species.

Toti-potent cells have the potential for development to any type of cell found in the organism. In the other hand, the capacity of these cells to develop into the three primary germ cell layers of the embryo and into extra-embryonic tissues such as the placenta is remarkable (15).

The pluripotent stem cells are kind of stem cells with the potential for development to approximately all cell types. These cells contain ESCs and cells that are isolated from the mesoderm, endoderm and ectoderm germ layers that are organized in the beginning period of ESC differentiation (15).

The multipotent stem cells have less proliferative potential than the previous two groups and have ability to produce a variety of cells which limited to a germinal layer [such as mesenchymal stem cells (MSCs)] or just a specific cell line (such as HSCs). Adult stem cells are also often in this group. In the word, these cells have the ability to differentiate into a closely related family of cells (15).

Despite the increasing interest in totipotent and pluripotent stem cells, unipotent stem cells have not received the most attention in research. A unipotent stem cell is a cell that can create cells with only one lineage differentiation. Muscle stem cells are one of the example of this type of cell (15). The word uni is derivative from the Latin word unus meaning one. In adult tissues in comparison with other types of stem cells, these cells have the lowest differentiation potential. The unipotent stem cells could create one cell type, in the other word, these cells do not have the self-renewal property. Furthermore, despite their limited differentiation potential, these cells are still candidates for treatment of various diseases (16).

ESCs are self-renewing cells that derived from the inner cell mass of a blastocyst and give rise to all cells during human development. It is mentioned that these cells, including human embryonic cells, could be used as suitable, promising source for cell transplantation and regenerative medicine because of their unique ability to give rise to all somatic cell lineages (17). In the other words, ESCs, pluripotent cells that can differentiate to form the specialized of the various cell types of the body (18). Also, ESCs capture the imagination because they are immortal and have an almost unlimited developmental potential. Due to the ethical limitation on embryo sampling and culture, these cells are used less in research (19).

HSCs are multipotent cells that give rise to blood cells through the process of hematopoiesis (20). These cells reside in the bone marrow and replenish all adult hematopoietic lineages throughout the lifetime of the human and animal (21). Also, these cells can replenish missing or damaged components of the hematopoietic and immunologic system and can withstand freezing for many years (22).The mammalian hematopoietic system containing more than ten different mature cell types that HSCs are one of the most important members of this. The ability to self-renew and multi-potency is another specific feature of these cells (23).

Adult stem cells, as undifferentiated cells, are found in numerous tissues of the body after embryonic development. These cells multiple by cell division to regenerate damaged tissues (24). Recent studies have been shown that adult stem cells may have the ability to differentiate into cell types from various germ layers. For example, bone marrow stem cells which is derived from mesoderm, can differentiate into cell lineage derived mesoderm and endoderm such as into lung, liver, GI tract, skin, etc. (25). Another example of adult stem cells is neural stem cells (NSCs), which is derived from ectoderm and can be differentiate into another lineage such as mesoderm and endoderm (26). Therapeutic potential of adult stem cells in cell therapy and regenerative medicine has been proven (27).

For the first time in the late 1990s, CSCs were identified by John Dick in acute myeloid diseases. CSCs are cancerous cells that found within tumors or hematological cancers. Also, these cells have the characteristics of normal stem cells and can also give rise to all cell types found in a particular cancer sample (28). There is an increasing evidence supporting the CSCs hypothesis. Normal stem cells in an adult living creature are responsible for the repair and regeneration of damaged as well as aged tissues (29). Many investigations have reported that the capability of a tumor to propagate and proliferate relies on a small cellular subpopulation characterized by stem-like properties, named CSCs (30).

Embryonic connective tissue contains so-called mesenchymes, from which with very close interactions of endoderm and ectoderm all other connective and hematopoietic tissues originate, Whereas, MSCs do not differentiate into hematopoietic cell (31). In 1924, Alexander A. Maxi mow used comprehensive histological detection to identify a singular type of precursor cell within mesenchyme that develops into various types of blood cells (32). In general, MSCs are type of cells with potential of multi-lineage differentiation and self-renewal, which exist in many different kinds of tissues and organs such as adipose tissue, bone marrow, skin, peripheral blood, fallopian tube, cord blood, liver and lung et al. (4,5). Today, stem cells are used for different applications. In addition to using these cells in human therapy such as cell transplantation, cell engraftment etc. The use of stem cells in veterinary medicine has also been considered. The purpose of this review is to provide a summary of our current knowledge regarding the important and types of isolated stem cells from different sources of animal models such as horse, pig, goat, dog, rabbit, cat, rat, mice etc. In this regard, due to the widespread use and lot of attention of MSCs, in this review, we will elaborate on use of MSCs in veterinary medicine.

The isolation method, maintenance and culture condition of MSCs differs from the different tissues, these methods as well as characterization of MSCs described as (36). MSCs could be isolated from the various tissues such as adipose tissue, bone marrow, umbilical cord, amniotic fluid etc. (37).

Diagram for adipose tissue-derived mesenchymal stem cell isolation (3).

Diagram for bone marrow-derived MSCs isolation (33). MSC, mesenchymal stem cell.

Diagram for umbilical cord-derived MSCs isolation (34). MSC, mesenchymal stem cell.

Diagram for isolation of amniotic fluid stem cells (AFSCs) (35).

Diagram for MSCs characterization (35). MSC, mesenchymal stem cell.

The diversity of stem cell or MSCs sources and a wide aspect of potential applications of these cells cause to challenge for selecting an appropriate cell type for cell therapy (38). Various diseases in animals have been treated by cell-based therapy. However, there are immunity concerns regarding cell therapy using stem cells. Improving animal models and selecting suitable methods for engraftment and transplantation could help address these subjects, facilitating eventual use of stem cells in the clinic. Therefore, for this purpose, in this section of this review, we provide an overview of the current as well as previous studies for future development of animal models to facilitate the utilization of stem cells in regenerative medicine (14). Significant progress has been made in stem cells-based regenerative medicine, which enables researchers to treat those diseases which cannot be cured by conventional medicines. The unlimited self-renewal and multi-lineage differentiation potential to other types of cells causes stem cells to be frontier in regenerative medicine (24). More researches in regenerative medicine have been focused on human cells including embryonic as well as adult stem cells or maybe somatic cells. Today there are versions of embryo-derived stem cells that have been reprogrammed from adult cells under the title of pluripotent cells (39). Stem cell therapy has been developed in the last decade. Nevertheless, obstacles including unwanted side effects due to the migration of transplanted cells as well as poor cell survival have remained unresolved. In order to overcome these problems, cell therapy has been introduced using biocompatible and biodegradable biomaterials to reduce cell loss and long-term in vitro retention of stem cells.

Currently in clinical trials, these biomaterials are widely used in drug and cell-delivery systems, regenerative medicine and tissue engineering in which to prevent the long-term survival of foreign substances in the body the release of cells are controlled (40).

Today, the incidence and prevalence of heart failure in human societies is a major and increasing problem that unfortunately has a poor prognosis. For decades, MSCs have been used for cardiovascular regenerative therapy as one of the potential therapeutic agents (41). Dhein et al. [2006] found that autologous bone marrow-derived mesenchymal stem cells (BMSCs) transplantation improves cardiac function in non-ischemic cardiomyopathy in a rabbit model. In one study, Davies et al. [2010] reported that transplantation of cord blood stem cells in ovine model of heart failure, enhanced the function of heart through improvement of right ventricular mass, both systolic and diastolic right heart function (42). In another study, Nagaya et al. [2005] found that MSCs dilated cardiomyopathy (DCM), possibly by inducing angiogenesis and preventing cardial fibrosis. MSCs have a tremendous beneficial effect in cell transplantation including in differentiating cardiomyocytes, vascular endothelial cells, and providing anti-apoptotic as well angiogenic mediators (43). Roura et al. [2015] shown that umbilical cord blood mesenchymal stem cells (UCBMSCs) are envisioned as attractive therapeutic candidates against human disorders progressing with vascular deficit (44). Ammar et al., [2015] compared BMSCs with adipose tissue-derived MSCs (ADSCs). It was demonstrated that both BMSCs and ADSCs were equally effective in mitigating doxorubicin-induced cardiac dysfunction through decreasing collagen deposition and promoting angiogenesis (45).

There are many advantages of small animal models usage in cardiovascular research compared with large animal models. Small model of animals has a short life span, which allow the researchers to follow the natural history of the disease at an accelerated pace. Some advantages and disadvantages are listed in (46).

Despite of the small animal model, large animal models are suitable models for studies of human diseases. Some advantages and disadvantages of using large animal models in a study protocol planning was elaborated in (47).

Chronic wound is one of the most common problem and causes significant distress to patients (48). Among the types of tissues that stem cells derived it, dental tissuederived MSCs provide good sources of cytokines and growth factors that promote wound healing. The results of previous studies showed that stem cells derived deciduous teeth of the horse might be a novel approach for wound care and might be applied in clinical treatment of non-healing wounds (49). However, the treatment with stem cells derived deciduous teeth needs more research to understand the underlying mechanisms of effective growth factors which contribute to the wound healing processes (50). This preliminary investigation suggests that deciduous teeth-derived stem cells have the potential to promote wound healing in rabbit excisional wound models (49). In the another study, Lin et al. [2013] worked on the mouse animal model and showed that ADSCs present a potentially viable matrix for full-thickness defect wound healing (51).

Many studies have been done on dental reconstruction with MSCs. In one study, Khorsand et al. [2013] reported that dental pulp-derived stem cells (DPSCs) could promote periodontal regeneration in canine model. Also, it was shown that canine DPSCs were successfully isolated and had the rapid proliferation and multi-lineage differentiation capacity (52). Other application of dental-derived stem cells is shown in .

Diagram for application of dental stem cell in dentistry/regenerative medicine (53).

As noted above, stem cells have different therapeutic applications and self-renewal capability. These cells can also differentiate into the different cell types. There is now a great hope that stem cells can be used to treat diseases such as Alzheimer, Parkinson and other serious diseases. In stem cell-based therapy, ESCs are essentially targeted to differentiate into functional neural cells. Today, a specific category of stem cells called induced pluripotent stem (iPS) cells are being used and tested to generate functional dopamine neurons for treating Parkinson's disease of a rat animal model. In addition, NSC as well as MSCs are being used in neurodegenerative disorder therapies for Alzheimers disease, Parkinsons disease, and stroke (54). Previous studies have shown that BMSCs could reduce brain amyloid deposition and accelerate the activation of microglia in an acutely induced Alzheimers disease in mouse animal model. Lee et al. [2009] reported that BMSCs can increase the number of activated microglia, which effective therapeutic vehicle to reduce A deposits in AD patients (55). In confirmation of previous study, Liu et al. [2015] showed that transplantation of BMSCs in brain of mouse model of Alzheimers disease cause to decrease in amyloid beta deposition, increase in brain-derived neurotrophic factor (BDNF) levels and improvements in social recognition (56). In addition of BMSCs, NSCs have been proposed as tools for treating neurodegeneration disease because of their capability to create an appropriate cell types which transplanted. kerud et al. [2001] demonstrated that NSCs efficiently express high level of glial cell line-derived neurotrophic factor (GDNF) in vivo, suggesting a use of these cells in the treatment of neurodegenerative disorders, including Parkinsons disease (57). In the following, Venkataramana et al. [2010] transplanted BMSCs into the sub lateral ventricular zones of seven Parkinsons disease patients and reported encouraging results (58).

The human body is fortified with specialized cells named MSCs, which has the ability to self-renew and differentiate into various cell types including, adipocyte, osteocyte, chondrocyte, neurons etc. In addition to mentioned properties, these cells can be easily isolated, safely transplanted to injured sites and have the immune regulatory properties. Numerous in vitro and in vivo studies in animal models have successfully demonstrated the potential of MSCs for various diseases; however, the clinical outcomes are not very encouraging. Based on the studies in the field of stem cells, MSCs find wide application in treatment of diseases, such as heart failure, wound healing, tooth regeneration and etc. In addition, these cells are particularly important in the treatment of the sub-branch neurodegenerative diseases like Alzheimer and Parkinson.

The authors wish to thank staff of the Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Funding: The project described was supported by Grant Number IR.TBZMED.REC.1396.1218 from the Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflicts of Interest: The authors have no conflicts of interest to declare.

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Stem cell-based regenerative medicine - PMC

Inflammation represents one of the leading drivers of disease. Biotech company 180 Life Sciences is developing novel, anti-TNF therapies for treating distinct inflammatory diseases.

DocWire News spoke to James Woody, CEO of 180 Life Sciences, to learn more about the company, its mission, its treatment assets, and current clinical trials its involved in.

*Interview recorded in March 2022.

DocWire News:Can you give us some background on yourself, and the company, 180 Life Sciences?

James Woody:So by background, Im a pediatric immunologist, and in my prior life, I was Chief Scientific Officer of a company called Centocor, which was one of the very early biotech companies. And we were the first ones ever to make a anti-TNF antibody and to test it in patients, and we were able to show that it was remarkably effective in patients with rheumatoid arthritis, Crohns disease and psoriasis and ulcerative colitis. And that actually began the pretty much the whole antibody based biologics industry. We were the first ones to do this with a humanized antibody.

I went on from there to run a pharmaceutical company called Syntex, former Syntex that was after Roche bought it and did that for eight years, we invented a lot of small molecules. And then I went on to start a company in oncology, cancer stem cells. And from there I went over to the dark side and joined a venture capital group and helped start companies for about 10 years and some of them are really successful. Some of them are okay and some crashed and burned, but thats the nature of the business. And then more recently I helped start a couple companies on my own. And then I was approached by the founders of 180 LS to help them out and also to be CEO of their company, so thats how I came to be CEO of 180 Life Sciences.

180 Life Sciences is repurposing anti-TNF for unmet needs. What is anti-TNF?

So in your body, you have lots of protein circulating around in your blood. These tell the body cells what to do, and some of them are called cytokines and cytokines are the ones that kind of tell your immune system what to do. And theres quite a lot of these. And theres some of them that are very good. Theres some of them that are bad actors and one of them is called tumor necrosis factor. It was named that totally by accident because it seemed to eliminate tumors in mice, but thats never been able to be shown in humans, but the name has stuck with it. So tumor necrosis factor is the thing that causes some types of inflammation, if theres an overproduction. For example, in rheumatoid arthritis, its the tumor necrosis factor that drives the destruction of the joints of your fingers and knees and shoulders and everything, so its a destructive cytokine. And what we did is we made a specialized antibody against TNF that binds it up and blocks it and prevents it from causing the inflammation. And that was the basis of infliximab or Remicade that we discovered from Centocor.

What is Dupuytrens disease, how is it characterized?

Dupuytrens Contracture is kind of a chronic disease, but it affects quite a lot of people, maybe 16 or 20 million in the US, same in Europe. It starts out as a small nodule in your palm. And over time, maybe a couple of years, some faster, some slower, it begins to form cords underneath the palm of your hand, it pulls your fingers together and contracts them. Sometimes this is inherited in families and sometimes it just occurs. So what happens is that this nodule starts, and as I said, over time, the fingers become contracted. So theres no therapies for the early stage when the nodules just form, but thats the basis of what were doing, Ill talk about that in a minute.

Later on, after the fingers are already contracted and you have the disability, you cant button your clothes, you cant type with that hand. You cant do many of the things that you like to do with your hand. Theres several therapies that they try. One of them is injecting a collagenase thats partially effective, but they all, about half of those recur. You can try to disrupt these cords with a needle called needle aponeurectomy or alternatively, what happens is you end up going to surgery and they cut these cords out. Ironically, my wife had this and went through a whole year of steroid injections into her hand, finally had to have the surgery. So Im familiar with the process. But thats what happens, and I think people, as soon as the nodule forms, people these days, because they have Dr. Google, can immediately know whats going to happen in the long run, so the information out there is quite impressive.

180 Life Sciences recently completed a Phase 2 study for Dupuytrens. Tell us about the study protocol, the drug used and other updates on the study.

Our colleague in England, Dr. Jagdeep Nanchahal, was able to look at Dupuytrens Contracture and especially the nodules, and through a series of very elegant experiments, he was able to show that the nodule was driven by the TNF, the bad actor. And in this case, the inflammation caused the fibrosis that were talking about, that leads to the finger contracture. And so he was able to work out that if you inject anti-TNF into this nodule, you can impact the course of the disease.

And so he did a very large trial of about 150 patients in the UK and was able to inject anti-TNF into the nodules of their hands. And in that trial, which took over a year, there were three or four injections, but we were able to show that both the primary and secondary endpoints of the trial were met and the endpoints had to do with the size of the nodule, whether it was growing, whether it was shrinking, whether it was harder or whether it was softer or whether the fingers were contracting, all of that, but we met the primary endpoints and the full publication with all the details will be out, hopefully in the next couple of months.

You have another trial planned for Frozen Shoulder. What is Frozen Shoulder, and how will the trial aim to address it?

Yes, Frozen Shoulder is another kind of inflammatory condition where fibrosis forms in the shoulder. And it initially starts out as being extremely painful. And that goes on for several months and then eventually the pain subsides, but the shoulder becomes totally immobile. And eventually you have to have surgery to remove the fibrotic tissues. Interestingly enough, this occurs more common in patients with diabetes, but about half of those patients also have Dupuytrens. And so we think that the fibrosis in the Dupuytrens and the fibrosis in the shoulder is the same mechanism. And so Dr. Nanchahal will be injecting anti-TNF into the shoulder very early, as soon as the pain is evident, then hell try to inject anti-TNF and maybe relieve the pain and also the formation of the fibrosis, so that one can avoid the surgery, which is actually quite expensive. And also, theres quite a long course of physical therapy after the surgery, so its something youd like to avoid. And so were trying to treat patients both with Dupuytrens and Frozen Shoulder before the disability develops.

A third program, which is soon to be clinical, is anti-TNF for post-operative cognition delirium or POCD. Tell me about POCD, and the preliminary research that led the team to pursue this indication?

We know that now that theyre doing fairly aggressive surgery in older patients, either hip replacements or emergency hip corrections or CABG procedure, coronary artery bypass graft, or cardiac surgery, that a fair percentage of these people after the surgery, just have a foggy brain. And the fog goes on for some time and we call it postoperative cognitive dementia, as the technical term. And in some patients, maybe 15 or 20%, it doesnt go away. And they end up in nursing homes and they actually dont live very long after that. And so our colleagues in the UK, Dr. Nanchahal and Dr. Feldmann and his colleagues, have shown that during the surgery, any kind of aggressive surgery, that TNF is released from the tissue damage, and the TNF goes to the brain and opens it up and lets inflammatory cells get into the area of the brain thats where your cognitive areas are, and so that leads to the dementia.

And in the past, theyve thought this all had to do with the anesthesia, but we think its the TNF thats actually causing this dementia going forward. And so were actually going to do a trial in patients that are having their hip repaired that are older, and were going to administer one dose of anti-TNF just before the surgery starts with a view towards preventing the dementia going forward. So this will be a long trial, but if it works, itll be something that everybody who goes into major surgery would want to have. So its another exciting opportunity for 180 LS and our investigators.

180 Life Sciences recently announced licensing of a compound called HMGB1. Tell us more about HMGB1 and the companys plans for it.

The company is also working on other areas of fibrosis, not just Dupuytrens Contracture and Frozen Shoulder, but other areas like liver fibrosis, which occurs with NASH. And we are working on ways to prevent that as well, much like were working on Dupuytrens and Frozen Shoulder. The fibrosis in the liver is really hard to reverse, and there are no real agents that do that, but theres a lot of people trying different things. Now what the HMGB-1 does, it doesnt change the fibrosis, but once the fibrosis is stopped, it could help the liver cells to regenerate. So this is kind of a regenerative medicine. It makes the tissues regenerate, whether its heart or whether its liver or whether its lung or whatever. And so its going to be used after the fibrosis is stopped. And so thats kind of what were interested in. And were just getting that program off the ground and making the initial compounds to do our testing.

Any closing thoughts?

Well, Id like to talk about our team. The company was founded by Dr. Mark Feldmann, who was the one, he was the original person who figured out that TNF was causing the joint destruction and arthritis, and with he and I and others, that actually did the very first trials ever. And this was done in patients with wheelchairs, and they actually got up out of their wheelchairs and walked around. It was a phenomenal moment. We had no idea it would work that well. And some of them actually did a pirouette down some stairs. We have videos of this. So its kind of like The Awakening movie where they gave them the L-DOPA and they all woke up. Well, in this case, they got up out of their wheelchairs and theres no patients in wheelchairs with rheumatoid arthritis in the whole world because of that drug, and the ones that followed on.

The current Humira from AbbVie is the preferred one. But the whole idea and concept, we started back then. Other founders, Dr. Larry Steinman, he and Mark put 180 LS together. And he developed Tysabri, the very first drug to help MS patients. And it was another phenomenal discovery that he made. And hes also working on MS and other areas. But so we have the leaders in inflammation as the people who actually founded the company. So its a pleasure to work with them. Ive been acquainted with them off and on for the past, maybe 25 years, so working with them again is a real pleasure.

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James Woody, CEO of 180 Life Sciences: Developing New Therapies to Treat Inflammatory Diseases - DocWire News

Introduction

Given the multi-lineage differentiation abilities of mesenchymal stem cells (MSCs) isolated from different tissues and organs, MSCs have been widely used in various medical fields, particularly regenerative medicine.13 The representative sources of MSCs are bone marrow, adipose, periodontal, muscle, and umbilical cord blood.410 Interestingly, slight differences have been reported in the characteristics of MSCs depending on the different sources, including their population in source tissues, immunosuppressive activities, proliferation, and resistance to cellular aging.11 Bone marrow-derived MSCs (BM-MSCs) are the most intensively studied and show clinically promising results for cartilage and bone regeneration.11 However, the isolation procedures for BM-MSCs are complicated because bone marrow contains a relatively small fraction of MSCs (0.0010.01% of the cells in bone marrow).12 Furthermore, bone marrow aspiration to harvest MSCs in human bones is a painful procedure and the slower proliferation rate of BM-MSCs is a clinical limitation.13 In comparison with BM-MSCs, adipose-derived MSCs (AD-MSCs) are relatively easy to collect and can produce up to 500 times the cell population of BM-MSCs.14 AD-MSCs showed a greater ability to regenerate damaged cartilage and bone tissues with increased immunosuppressive ability.14,15 Umbilical cord blood-derived MSCs (UC-MSCs) proliferate faster than BM-MSCs and are resistant to significant cellular aging.11

MSCs have been investigated and gained worldwide attention as potential therapeutic candidates for incurable diseases such as arthritis, spinal cord injury, and cardiac disease.3,1623 In particular, the inherent tropism of MSCs to inflammatory sites has been thoroughly studied.24 This inherent tropism, also known as homing ability, originates from the recognition of various chemokine sources in inflamed tissues, where profiled chemokines are continuously secreted and the MSCs migrate to the chemokines in a concentration-dependent manner.24 Rheumatoid arthritis (RA) is a representative inflammatory disease that primarily causes inflammation in the joints, and this long-term autoimmune disorder causes worsening pain and stiffness following rest. RA affects approximately 24.5 million people as of 2015, but only symptomatic treatments such as pain medications, steroids, and nonsteroidal anti-inflammatory drugs (NSAIDs), or slow-acting drugs that inhibit the rapid progression of RA, such as disease-modifying antirheumatic drugs (DMARDs) are currently available. However, RA drugs have adverse side effects, including hepatitis, osteoporosis, skeletal fracture, steroid-induced arthroplasty, Cushings syndrome, gastrointestinal (GI) intolerance, and bleeding.2527 Thus, MSCs are rapidly emerging as the next generation of arthritis treatment because they not only recognize and migrate toward chemokines secreted in the inflamed joints but also regulate inflammatory progress and repair damaged cells.28

However, MSCs are associated with many challenges that need to be overcome before they can be used in clinical settings.2931 One of the main challenges is the selective accumulation of systemically administered MSCs in the lungs and liver when they are administered intravenously, leading to insufficient concentrations of MSCs in the target tissues.32,33 In addition, most of the administered MSCs are typically initially captured by macrophages in the lungs, liver, and spleen.3234 Importantly, the viability and migration ability of MSCs injected in vivo differed from results previously reported as favorable therapeutic effects and migration efficiency in vitro.35

To improve the delivery of MSCs, researchers have focused on chemokines, which are responsible for MSCs ability to move.36 The chemokine receptors are the key proteins on MSCs that recognize chemokines, and genetic engineering of MSCs to overexpress the chemokine receptor can improve the homing ability, thus enhancing their therapeutic efficacy.37 Genetic engineering is a convenient tool for modifying native or non-native genes, and several technologies for genetic engineering exist, including genome editing, gene knockdown, and replacement with various vectors.38,39 However, safety issues that prevent clinical use persist, for example, genome integration, off-target effects, and induction of immune response.40 In this regard, MSC mimicking nanoencapsulations can be an alternative strategy for maintaining the homing ability of MSCs and overcoming the current safety issues.4143 Nanoencapsulation involves entrapping the core nanoparticles of solids or liquids within nanometer-sized capsules of secondary materials.44

MSC mimicking nanoencapsulation uses the MSC membrane fraction as the capsule and targeting molecules, that is chemokine receptors, with several types of nanoparticles, as the core.45,46 MSC mimicking nanoencapsulation consists of MSC membrane-coated nanoparticles, MSC-derived artificial ectosomes, and MSC membrane-fused liposomes. Nano drug delivery is an emerging field that has attracted significant interest due to its unique characteristics and paved the way for several unique applications that might solve many problems in medicine. In particular, the nanoscale size of nanoparticles (NPs) enhances cellular uptake and can optimize intracellular pathways due to their intrinsic physicochemical properties, and can therefore increase drug delivery to target tissues.47,48 However, the inherent targeting ability resulting from the physicochemical properties of NPs is not enough to target specific tissues or damaged tissues, and additional studies on additional ligands that can bind to surface receptors on target cells or tissues have been performed to improve the targeting ability of NPs.49 Likewise, nanoencapsulation with cell membranes with targeting molecules and encapsulation of the core NPs with cell membranes confer the targeting ability of the source cell to the NPs.50,51 Thus, MSC mimicking nanoencapsulation can mimic the superior targeting ability of MSCs and confer the advantages of each core NP. In addition, MSC mimicking nanoencapsulations have improved circulation time and camouflaging from phagocytes.52

This review discusses the mechanism of MSC migration to inflammatory sites, addresses the potential strategy for improving the tropism of MSCs using genetic engineering, and discusses the promising therapeutic agent, MSC mimicking nanoencapsulations.

The MSC migration mechanism can be exploited for diverse clinical applications.53 The MSC migration mechanism can be divided into five stages: rolling by selectin, activation of MSCs by chemokines, stopping cell rolling by integrin, transcellular migration, and migration to the damaged site (Figure 1).54,55 Chemokines are secreted naturally by various cells such as tumor cells, stromal cells, and inflammatory cells, maintaining high chemokine concentrations in target cells at the target tissue and inducing signal cascades.5658 Likewise, MSCs express a variety of chemokine receptors, allowing them to migrate and be used as new targeting vectors.5961 MSC migration accelerates depending on the concentration of chemokines, which are the most important factors in the stem cell homing mechanism.62,63 Chemokines consist of various cytokine subfamilies that are closely associated with the migration of immune cells. Chemokines are divided into four classes based on the locations of the two cysteine (C) residues: CC-chemokines, CXC-chemokine, C-chemokine, and CX3 Chemokine.64,65 Each chemokine binds to various MSC receptors and the binding induces a chemokine signaling cascade (Table 1).56,66

Table 1 Chemokine and Chemokine Receptors for Different Chemokine Families

Figure 1 Representation of stem cell homing mechanism.

The mechanisms underlying MSC and leukocyte migration are similar in terms of their migratory dynamics.55 P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) are major proteins involved in leukocyte migration that interact with P-selectin and E-selectin present in vascular endothelial cells. However, these promoters are not present in MSCs (Figure 2).53,67

Figure 2 Differences in adhesion protein molecules between leukocytes and mesenchymal stem cells during rolling stages and rolling arrest stage of MSC. (A) The rolling stage of leukocytes starts with adhesion to endothelium with ESL-1 and PSGL-1 on leukocytes. (B) The rolling stage of MSC starts with the adhesion to endothelium with Galectin-1 and CD24 on MSC, and the rolling arrest stage was caused by chemokines that were encountered in the rolling stage and VLA-4 with a high affinity for VACM present in endothelial cells.

Abbreviations: ESL-1, E-selectin ligand-1; PSGL-1, P-selectin glycoprotein ligand-1 VLA-4, very late antigen-4; VCAM, vascular cell adhesion molecule-1.

The initial rolling is facilitated by selectins expressed on the surface of endothelial cells. Various glycoproteins on the surface of MSCs can bind to the selectins and continue the rolling process.68 However, the mechanism of binding of the glycoprotein on MSCs to the selectins is still unclear.69,70 P-selectins and E-selectins, major cell-cell adhesion molecules expressed by endothelial cells, adhere to migrated cells adjacent to endothelial cells and can trigger the rolling process.71 For leukocyte migration, P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1) expressed on the membranes of leukocytes interact with P-selectins and E-selectins on the endothelial cells, initiating the process.72,73 As already mentioned, MSCs express neither PSGL-1 nor ESL-1. Instead, they express galectin-1 and CD24 on their surfaces, and these bind to E-selectin or P-selectin (Figure 2).7476

In the migratory activation step, MSC receptors are activated in response to inflammatory cytokines, including CXCL12, CXCL8, CXCL4, CCL2, and CCL7.77 The corresponding activation of chemokine receptors of MSCs in response to inflammatory cytokines results in an accumulation of MSCs.58,78 For example, inflamed tissues release inflammatory cytokines,79 and specifically, fibroblasts release CXCL12, which further induces the accumulation of MSCs through ligandreceptor interaction after exposure to hypoxia and cytokine-rich environments in the rat model of inflammation.7982 Previous studies have reported that overexpressing CXCR4, which is a receptor to recognize CXCL12, in MSCs improves the homing ability of MSCs toward inflamed sites.83,84 In short, cytokines are significantly involved in the homing mechanism of MSCs.53

The rolling arrest stage is facilitated by integrin 41 (VLA-4) on MSC.85 VLA-4 is expressed by MSCs which are first activated by CXCL-12 and TNF- chemokines, and activated VLA-4 binds to VCAM-1 expressed on endothelial cells to stop the rotational movement (Figure 2).86,87

Karp et al categorized the migration of MSCs as either systemic homing or non-systemic homing. Systemic homing refers to the process of migration through blood vessels and then across the vascular endothelium near the inflamed site.67,88 The process of migration after passing through the vessels or local injection is called non-systemic homing. In non-systemic migration, stem cells migrate through a chemokine concentration gradient (Figure 3).89 MSCs secrete matrix metalloproteinases (MMPs) during migration. The mechanism underlying MSC migration is currently undefined but MSC migration can be advanced by remodeling the matrix through the secretion of various enzymes.9093 The migration of MSCs to the damaged area is induced by chemokines released from the injured site, such as IL-8, TNF-, insulin-like growth factor (IGF-1), and platelet-derived growth factors (PDGF).9496 MSCs migrate toward the damaged area following a chemokine concentration gradient.87

Figure 3 Differences between systemic and non-systemic homing mechanisms. Both systemic and non-systemic homing to the extracellular matrix and stem cells to their destination, MSCs secrete MMPs and remodel the extracellular matrix.

Abbreviation: MMP, matrix metalloproteinase.

RA is a chronic inflammatory autoimmune disease characterized by distinct painful stiff joints and movement disorders.97 RA affects approximately 1% of the worlds population.98 RA is primarily induced by macrophages, which are involved in the innate immune response and are also involved in adaptive immune responses, together with B cells and T cells.99 Inflammatory diseases are caused by high levels of inflammatory cytokines and a hypoxic low-pH environment in the joints.100,101 Fibroblast-like synoviocytes (FLSs) and accumulated macrophages and neutrophils in the synovium of inflamed joints also express various chemokines.102,103 Chemokines from inflammatory reactions can induce migration of white blood cells and stem cells, which are involved in angiogenesis around joints.101,104,105 More than 50 chemokines are present in the rheumatoid synovial membrane (Table 2). Of the chemokines in the synovium, CXCL12, MIP1-a, CXCL8, and PDGF are the main ones that attract MSCs.106 In the RA environment, CXCL12, a ligand for CXCR4 on MSCs, had 10.71 times higher levels of chemokines than in the normal synovial cell environment. MIP-1a, a chemokine that gathers inflammatory cells, is a ligand for CCR1, which is normally expressed on MSC.107,108 CXCL8 is a ligand for CXCR1 and CXCR2 on MSCs and induces the migration of neutrophils and macrophages, leading to ROS in synovial cells.59 PDGF is a regulatory peptide that is upregulated in the synovial tissue of RA patients.109 PDGF induces greater MSC migration than CXCL12.110 Importantly, stem cells not only have the homing ability to inflamed joints but also have potential as cell therapy with the anti-apoptotic, anti-catabolic, and anti-fibrotic effect of MSC.111 In preclinical trials, MSC treatment has been extensively investigated in collagen-induced arthritis (CIA), a common autoimmune animal model used to study RA. In the RA model, MSCs downregulated inflammatory cytokines such as IFN-, TNF-, IL-4, IL-12, and IL1, and antibodies against collagen, while anti-inflammatory cytokines, such as tumor necrosis factor-inducible gene 6 protein (TSG-6), prostaglandin E2 (PGE2), transforming growth factor-beta (TGF-), IL-10, and IL-6, were upregulated.112116

Table 2 Rheumatoid Arthritis (RA) Chemokines Present in the Pathological Environment and Chemokine Receptors Present in Mesenchymal Stem Cells

Genetic engineering can improve the therapeutic potential of MSCs, including long-term survival, angiogenesis, differentiation into specific lineages, anti- and pro-inflammatory activity, and migratory properties (Figure 4).117,118 Although MSCs already have an intrinsic homing ability, the targeting ability of MSCs and their derivatives, such as membrane vesicles, which are utilized to produce MSC mimicking nanoencapsulation, can be enhanced.118 The therapeutic potential of MSCs can be magnified by reprogramming MSCs via upregulation or downregulation of their native genes, resulting in controlled production of the target protein, or by introducing foreign genes that enable MSCs to express native or non-native products, for example, non-native soluble tumor necrosis factor (TNF) receptor 2 can inhibit TNF-alpha signaling in RA therapies.28

Figure 4 Genetic engineering of mesenchymal stem cells to enhance therapeutic efficacy.

Abbreviations: Sfrp2, secreted frizzled-related protein 2; IGF1, insulin-like growth factor 1; IL-2, interleukin-2; IL-12, interleukin-12; IFN-, interferon-beta; CX3CL1, C-X3-C motif chemokine ligand 1; VEGF, vascular endothelial growth factor; HGF, human growth factor; FGF, fibroblast growth factor; IL-10, interleukin-10; IL-4, interleukin-4; IL18BP, interleukin-18-binding protein; IFN-, interferon-alpha; SDF1, stromal cell-derived factor 1; CXCR4, C-X-C motif chemokine receptor 4; CCR1, C-C motif chemokine receptor 1; BMP2, bone morphogenetic protein 2; mHCN2, mouse hyperpolarization-activated cyclic nucleotide-gated.

MSCs can be genetically engineered using different techniques, including by introducing particular genes into the nucleus of MSCs or editing the genome of MSCs (Figure 5).119 Foreign genes can be transferred into MSCs using liposomes (chemical method), electroporation (physical method), or viral delivery (biological method). Cationic liposomes, also known as lipoplexes, can stably compact negatively charged nucleic acids, leading to the formation of nanomeric vesicular structure.120 Cationic liposomes are commonly produced with a combination of a cationic lipid such as DOTAP, DOTMA, DOGS, DOSPA, and neutral lipids, such as DOPE and cholesterol.121 These liposomes are stable enough to protect their bound nucleic acids from degradation and are competent to enter cells via endocytosis.120 Electroporation briefly creates holes in the cell membrane using an electric field of 1020 kV/cm, and the holes are then rapidly closed by the cells membrane repair mechanism.122 Even though the electric shock induces irreversible cell damage and non-specific transport into the cytoplasm leads to cell death, electroporation ensures successful gene delivery regardless of the target cell or organism. Viral vectors, which are derived from adenovirus, adeno-associated virus (AAV), or lentivirus (LV), have been used to introduce specific genes into MSCs. Recombinant lentiviral vectors are the most widely used systems due to their high tropism to dividing and non-dividing cells, transduction efficiency, and stable expression of transgenes in MSCs, but the random genome integration of transgenes can be an obstacle in clinical applications.123 Adenovirus and AAV systems are appropriate alternative strategies because currently available strains do not have broad genome integration and a strong immune response, unlike LV, thus increasing success and safety in clinical trials.124 As a representative, the Oxford-AstraZeneca COVID-19 vaccine, which has been authorized in 71 countries as a vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which spread globally and led to the current pandemic, transfers the spike protein gene using an adenovirus-based viral vector.125 Furthermore, there are two AAV-based gene therapies: Luxturna for rare inherited retinal dystrophy and Zolgensma for spinal muscular atrophy.126

Figure 5 Genetic engineering techniques used in the production of bioengineered mesenchymal stem cells.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 were recently used for genome editing and modification because of their simpler design and higher efficiency for genome editing, however, there are safety issues such as off-target effects that induce mutations at sites other than the intended target site.127 The foreign gene is then commonly transferred into non-integrating forms such as plasmid DNA and messenger RNA (mRNA).128

The gene expression machinery can also be manipulated at the cytoplasmic level through RNA interference (RNAi) technology, inhibition of gene expression, or translation using neutralizing targeted mRNA molecules with sequence-specific small RNA molecules such as small interfering RNA (siRNA) or microRNA (miRNA).129 These small RNAs can form enzyme complexes that degrade mRNA molecules and thus decrease their activity by inhibiting translation. Moreover, the pre-transcriptional silencing mechanism of RNAi can induce DNA methylation at genomic positions complementary to siRNA or miRNA with enzyme complexes.

CXC chemokine receptor 4 (CXCR4) is one of the most potent chemokine receptors that is genetically engineered to enhance the migratory properties of MSCs.130 CXCR4 is a chemokine receptor specific for stromal-derived factor-1 (SDF-1), also known as CXC motif chemokine 12 (CXCL12), which is produced by damaged tissues, such as the area of inflammatory bone destruction.131 Several studies on engineering MSCs to increase the expression of the CXCR4 gene have reported a higher density of the CXCR4 receptor on their outer cell membrane and effectively increased the migration of MSCs toward SDF-1.83,132,133 CXC chemokine receptor 7 (CXCR7) also had a high affinity for SDF-1, thus the SDF-1/CXCR7 signaling axis was used to engineer the MSCs.134 CXCR7-overexpressing MSCs in a cerebral ischemia-reperfusion rat hippocampus model promoted migration based on an SDF-1 gradient, cooperating with the SDF-1/CXCR4 signaling axis (Figure 6).37

Figure 6 Engineered mesenchymal stem cells with enhanced migratory abilities.

Abbreviations: CXCR4, C-X-C motif chemokine receptor 4; CXCR7, C-X-C motif chemokine receptor 7; SDF1, stromal cell-derived factor 1; CXCR1, C-X-C motif chemokine receptor 1; IL-8, interleukin-8; Aqp1, aquaporin 1; FAK, focal adhesion kinase.

CXC chemokine receptor 1 (CXCR1) enhances MSC migratory properties.59 CXCR1 is a receptor for IL-8, which is the primary cytokine involved in the recruitment of neutrophils to the site of damage or infection.135 In particular, the IL-8/CXCR1 axis is a key factor for the migration of MSCs toward human glioma cell lines, such as U-87 MG, LN18, U138, and U251, and CXCR1-overexpressing MSCs showed a superior capacity to migrate toward glioma cells and tumors in mice bearing intracranial human gliomas.136

The migratory properties of MSCs were also controlled via aquaporin-1 (Aqp1), which is a water channel molecule that transports water across the cell membrane and regulates endothelial cell migration.137 Aqp1-overexpressing MSCs showed enhanced migration to fracture gap of a rat fracture model with upregulated focal adhesion kinase (FAK) and -catenin, which are important regulators of cell migration.138

Nur77, also known as nerve growth factor IB or NR4A1, and nuclear receptor-related 1 (Nurr1), can play a role in improving the migratory capabilities of MSCs.139,140 The migrating MSCs expressed higher levels of Nur77 and Nurr1 than the non-migrating MSCs, and overexpression of these two nuclear receptors functioning as transcription factors enhanced the migration of MSCs toward SDF-1. The migration of cells is closely related to the cell cycle, and normally, cells in the late S or G2/M phase do not migrate.141 The overexpression of Nur77 and Nurr1 increased the proportion of MSCs in the G0/G1-phase similar to the results of migrating MSCs had more cells in the G1-phase.

MSC mimicking nanoencapsulations are nanoparticles combined with MSC membrane vesicles and these NPs have the greatest advantages as drug delivery systems due to the sustained homing ability of MSCs as well as the advantages of NPs. Particles sized 10150 nm have great advantages in drug delivery systems because they can pass more freely through the cell membrane by the interaction with biomolecules, such as clathrin and caveolin, to facilitate uptake across the cell membrane compared with micron-sized materials.142,143 Various materials have been used to formulate NPs, including silica, polymers, metals, and lipids.144,145 NPs have an inherent ability, called passive targeting, to accumulate at specific sites based on their physicochemical properties such as size, surface charge, surface hydrophilicity, and geometry.146148 However, physicochemical properties are not enough to target specific tissues or damaged tissues, and thus active targeting is a clinically approved strategy involving the addition of ligands that can bind to surface receptors on target cells or tissues.149,150 MSC mimicking nanoencapsulation uses natural or genetically engineered MSC membranes to coat synthetic NPs, producing artificial ectosomes and fusing them with liposomes to increase their targeting ability (Figure 7).151 Especially, MSCs have been studied for targeting inflammation and regenerative drugs, and the mechanism and efficacy of migration toward inflamed tissues have been actively investigated.152 MSC mimicking nanoencapsulation can mimic the well-known migration ability of MSCs and can be equally utilized without safety issues from the direct application of using MSCs. Furthermore, cell membrane encapsulations have a wide range of functions, including prolonged blood circulation time and increased active targeting efficacy from the source cells.153,154 MSC mimicking encapsulations enter recipient cells using multiple pathways.155 MSC mimicking encapsulations can fuse directly with the plasma membrane and can also be taken up through phagocytosis, micropinocytosis, and endocytosis mediated by caveolin or clathrin.156 MSC mimicking encapsulations can be internalized in a highly cell type-specific manner that depends on the recognition of membrane surface molecules by the cell or tissue.157 For example, endothelial colony-forming cell (ECFC)-derived exosomes were shown CXCR4/SDF-1 interaction and enhanced delivery toward the ischemic kidney, and Tspan8-alpha4 complex on lymph node stroma derived extracellular vesicles induced selective uptake by endothelial cells or pancreatic cells with CD54, serving as a major ligand.158,159 Therefore, different source cells may contain protein signals that serve as ligands for other cells, and these receptorligand interactions maximized targeted delivery of NPs.160 This natural mechanism inspired the application of MSC membranes to confer active targeting to NPs.

Figure 7 Mesenchymal stem cell mimicking nanoencapsulation.

Cell membrane-coated NPs (CMCNPs) are biomimetic strategies developed to mimic the properties of cell membranes derived from natural cells such as erythrocytes, white blood cells, cancer cells, stem cells, platelets, or bacterial cells with an NP core.161 Core NPs made of polymer, silica, and metal have been evaluated in attempts to overcome the limitations of conventional drug delivery systems but there are also issues of toxicity and reduced biocompatibility associated with the surface properties of NPs.162,163 Therefore, only a small number of NPs have been approved for medical application by the FDA.164 Coating with cell membrane can enhance the biocompatibility of NPs by improving immune evasion, enhancing circulation time, reducing RES clearance, preventing serum protein adsorption by mimicking cell glycocalyx, which are chemical determinants of self at the surfaces of cells.151,165 Furthermore, the migratory properties of MSCs can also be transferred to NPs by coating them with the cell membrane.45 Coating NPs with MSC membranes not only enhances biocompatibility but also maximizes the therapeutic effect of NPs by mimicking the targeting ability of MSCs.166 Cell membrane-coated NPs are prepared in three steps: extraction of cell membrane vesicles from the source cells, synthesis of the core NPs, and fusion of the membrane vesicles and core NPs to produce cell membrane-coated NPs (Figure 8).167 Cell membrane vesicles, including extracellular vesicles (EVs), can be harvested through cell lysis, mechanical disruption, and centrifugation to isolate, purify the cell membrane vesicles, and remove intracellular components.168 All the processes must be conducted under cold conditions, with protease inhibitors to minimize the denaturation of integral membrane proteins. Cell lysis, which is classically performed using mechanical lysis, including homogenization, sonication, or extrusion followed by differential velocity centrifugation, is necessary to remove intracellular components. Cytochalasin B (CB), a drug that affects cytoskeletonmembrane interactions, induces secretion of membrane vesicles from source cells and has been used to extract the cell membrane.169 The membrane functions of the source cells are preserved in CB-induced vesicles, forming biologically active surface receptors and ion pumps.170 Furthermore, CB-induced vesicles can encapsulate drugs and NPs successfully, and the vesicles can be harvested by centrifugation without a purification step to remove nuclei and cytoplasm.171 Clinically translatable membrane vesicles require scalable production of high volumes of homogeneous vesicles within a short period. Although mechanical methods (eg, shear stress, ultrasonication, or extrusion) are utilized, CB-induced vesicles have shown potential for generating membrane encapsulation for nano-vectors.168 The advantages of CB-induced vesicles versus other methods are compared in Table 3.

Table 3 Comparison of Membrane Vesicle Production Methods

Figure 8 MSC membrane-coated nanoparticles.

Abbreviations: EVs, extracellular vesicles; NPs, nanoparticles.

After extracting cell membrane vesicles, synthesized core NPs are coated with cell membranes, including surface proteins.172 Polymer NPs and inorganic NPs are adopted as materials for the core NPs of CMCNPs, and generally, polylactic-co-glycolic acid (PLGA), polylactic acid (PLA), chitosan, and gelatin are used. PLGA has been approved by FDA is the most common polymer of NPs.173 Biodegradable polymer NPs have gained considerable attention in nanomedicine due to their biocompatibility, nontoxic properties, and the ability to modify their surface as a drug carrier.174 Inorganic NPs are composed of gold, iron, copper, and silicon, which have hydrophilic, biocompatible, and highly stable properties compared with organic materials.175 Furthermore, some photosensitive inorganic NPs have the potential for use in photothermal therapy (PTT) and photodynamic therapy (PDT).176 The fusion of cell membrane vesicles and core NPs is primarily achieved via extrusion or sonication.165 Cell membrane coating of NPs using mechanical extrusion is based on a different-sized porous membrane where core NPs and vesicles are forced to generate vesicle-particle fusion.177 Ultrasonic waves are applied to induce the fusion of vesicles and NPs. However, ultrasonic frequencies need to be optimized to improve fusion efficiency and minimize drug loss and protein degradation.178

CMCNPs have extensively employed to target and treat cancer using the membranes obtained from red blood cell (RBC), platelet and cancer cell.165 In addition, membrane from MSC also utilized to target tumor and ischemia with various types of core NPs, such as MSC membrane coated PLGA NPs targeting liver tumors, MSC membrane coated gelatin nanogels targeting HeLa cell, MSC membrane coated silica NPs targeting HeLa cell, MSC membrane coated PLGA NPs targeting hindlimb ischemia, and MSC membrane coated iron oxide NPs for targeting the ischemic brain.179183 However, there are few studies on CMCNPs using stem cells for the treatment of arthritis. Increased targeting ability to arthritis was introduced using MSC-derived EVs and NPs.184,185 MSC membrane-coated NPs are proming strategy for clearing raised concerns from direct use of MSC (with or without NPs) in terms of toxicity, reduced biocompatibility, and poor targeting ability of NPs for the treatment of arthritis.

Exosomes are natural NPs that range in size from 40 nm to 120 nm and are derived from the multivesicular body (MVB), which is an endosome defined by intraluminal vesicles (ILVs) that bud inward into the endosomal lumen, fuse with the cell surface, and are then released as exosomes.186 Because of their ability to express receptors on their surfaces, MSC-derived exosomes are also considered potential candidates for targeting.187 Exosomes are commonly referred to as intracellular communication molecules that transfer various compounds through physiological mechanisms such as immune response, neural communication, and antigen presentation in diseases such as cancer, cardiovascular disease, diabetes, and inflammation.188

However, there are several limitations to the application of exosomes as targeted therapeutic carriers. First, the limited reproducibility of exosomes is a major challenge. In this field, the standardized techniques for isolation and purification of exosomes are lacking, and conventional methods containing multi-step ultracentrifugation often lead to contamination of other types of EVs. Furthermore, exosomes extracted from cell cultures can vary and display inconsistent properties even when the same type of donor cells were used.189 Second, precise characterization studies of exosomes are needed. Unknown properties of exosomes can hinder therapeutic efficiencies, for example, when using exosomes as cancer therapeutics, the use of cancer cell-derived exosomes should be avoided because cancer cell-derived exosomes may contain oncogenic factors that may contribute to cancer progression.190 Finally, cost-effective methods for the large-scale production of exosomes are needed for clinical application. The yield of exosomes is much lower than EVs. Depending on the exosome secretion capacity of donor cells, the yield of exosomes is restricted, and large-scale cell culture technology for the production of exosomes is high difficulty and costly and isolation of exosomes is the time-consuming and low-efficient method.156

Ectosome is an EV generated by outward budding from the plasma membrane followed by pinching off and release to the extracellular parts. Recently, artificially produced ectosome utilized as an alternative to exosomes in targeted therapeutics due to stable productivity regardless of cell type compared with conventional exosome. Artificial ectosomes, containing modified cargo and targeting molecules have recently been introduced for specific purposes (Figure 9).191,192 Artificial ectosomes are typically prepared by breaking bigger cells or cell membrane fractions into smaller ectosomes, similar size to natural exosomes, containing modified cargo such as RNA molecules, which control specific genes, and chemical drugs such as anticancer drugs.193 Naturally secreted exosomes in conditioned media from modified source cells can be harvested by differential ultracentrifugation, density gradients, precipitation, filtration, and size exclusion chromatography for exosome separation.194 Even though there are several commercial kits for isolating exosomes simply and easily, challenges in compliant scalable production on a large scale, including purity, homogeneity, and reproducibility, have made it difficult to use naturally secreted exosomes in clinical settings.195 Therefore, artificially produced ectosomes are appropriate for use in clinical applications, with novel production methods that can meet clinical production criteria. Production of artificially produced ectosomes begins by breaking the cell membrane fraction of cultured cells and then using them to produce cell membrane vesicles to form ectosomes. As mentioned above, cell membrane vesicles are extracted from source cells in several ways, and cell membrane vesicles are extracted through polycarbonate membrane filters to reduce the mean size to a size similar to that of natural exosomes.196 Furthermore, specific microfluidic devices mounted on microblades (fabricated in silicon nitride) enable direct slicing of living cells as they flow through the hydrophilic microchannels of the device.197 The sliced cell fraction reassembles and forms ectosomes. There are several strategies for loading exogenous therapeutic cargos such as drugs, DNA, RNA, lipids, metabolites, and proteins, into exosomes or artificial ectosomes in vitro: electroporation, incubation for passive loading of cargo or active loading with membrane permeabilizer, freeze and thaw cycles, sonication, and extrusion.198 In addition, protein or RNA molecules can be loaded by co-expressing them in source cells via bio-engineering, and proteins designed to interact with the protein inside the cell membrane can be loaded actively into exosomes or artificial ectosomes.157 Targeting molecules at the surface of exosomes or artificial ectosomes can also be engineered in a manner similar to the genetic engineering of MSCs.

Figure 9 Mesenchymal stem cell-derived exosomes and artificial ectosomes. (A) Wound healing effect of MSC-derived exosomes and artificial ectosomes,231 (B) treatment of organ injuries by MSC-derived exosomes and artificial ectosomes,42,232234 (C) anti-cancer activity of MSC-derived exosomes and artificial ectosomes.200,202,235

Most of the exosomes derived from MSCs for drug delivery have employed miRNAs or siRNAs, inhibiting translation of specific mRNA, with anticancer activity, for example, miR-146b, miR-122, and miR-379, which are used for cancer targeting by membrane surface molecules on MSC-derived exosomes.199201 Drugs such as doxorubicin, paclitaxel, and curcumin were also loaded into MSC-derived exosomes to target cancer.202204 However, artificial ectosomes derived from MSCs as arthritis therapeutics remains largely unexplored area, while EVs, mixtures of natural ectosomes and exosomes, derived from MSCs have studied in the treatment of arthritis.184 Artificial ectosomes with intrinsic tropism from MSCs plus additional targeting ability with engineering increase the chances of ectosomes reaching target tissues with ligandreceptor interactions before being taken up by macrophages.205 Eventually, this will decrease off-target binding and side effects, leading to lower therapeutic dosages while maintaining therapeutic efficacy.206,207

Liposomes are spherical vesicles that are artificially synthesized through the hydration of dry phospholipids.208 The clinically available liposome is a lipid bilayer surrounding a hollow core with a diameter of 50150 nm. Therapeutic molecules, such as anticancer drugs (doxorubicin and daunorubicin citrate) or nucleic acids, can be loaded into this hollow core for delivery.209 Due to their amphipathic nature, liposomes can load both hydrophilic (polar) molecules in an aqueous interior and hydrophobic (nonpolar) molecules in the lipid membrane. They are well-established biomedical applications and are the most common nanostructures used in advanced drug delivery.210 Furthermore, liposomes have several advantages, including versatile structure, biocompatibility, low toxicity, non-immunogenicity, biodegradability, and synergy with drugs: targeted drug delivery, reduction of the toxic effect of drugs, protection against drug degradation, and enhanced circulation half-life.211 Moreover, surfaces can be modified by either coating them with a functionalized polymer or PEG chains to improve targeted delivery and increase their circulation time in biological systems.212 Liposomes have been investigated for use in a wide variety of therapeutic applications, including cancer diagnostics and therapy, vaccines, brain-targeted drug delivery, and anti-microbial therapy. A new approach was recently proposed for providing targeting features to liposomes by fusing them with cell membrane vesicles, generating molecules called membrane-fused liposomes (Figure 10).213 Cell membrane vesicles retain the surface membrane molecules from source cells, which are responsible for efficient tissue targeting and cellular uptake by target cells.214 However, the immunogenicity of cell membrane vesicles leads to their rapid clearance by macrophages in the body and their low drug loading efficiencies present challenges for their use as drug delivery systems.156 However, membrane-fused liposomes have advantages of stability, long half-life in circulation, and low immunogenicity due to the liposome, and the targeting feature of cell membrane vesicles is completely transferred to the liposome.215 Furthermore, the encapsulation efficiencies of doxorubicin were similar when liposomes and membrane-fused liposomes were used, indicating that the relatively high drug encapsulation capacity of liposomes was maintained during the fusion process.216 Combining membrane-fused liposomes with macrophage-derived membrane vesicles showed differential targeting and cytotoxicity against normal and cancerous cells.217 Although only a few studies have been conducted, these results corroborate that membrane-fused liposomes are a potentially promising future drug delivery system with increased targeting ability. MSCs show intrinsic tropism toward arthritis, and further engineering and modification to enhance their targeting ability make them attractive candidates for the development of drug delivery systems. Fusing MSC exosomes with liposomes, taking advantage of both membrane vesicles and liposomes, is a promising technique for future drug delivery systems.

Figure 10 Mesenchymal stem cell membrane-fused liposomes.

MSCs have great potential as targeted therapies due to their greater ability to home to targeted pathophysiological sites. The intrinsic ability to home to wounds or to the tumor microenvironment secreting inflammatory mediators make MSCs and their derivatives targeting strategies for cancer and inflammatory disease.218,219 Contrary to the well-known homing mechanisms of various blood cells, it is still not clear how homing occurs in MSCs. So far, the mechanism of MSC tethering, which connects long, thin cell membrane cylinders called tethers to the adherent area for migration, has not been clarified. Recent studies have shown that galectin-1, VCAM-1, and ICAM are associated with MSC tethering,53,220 but more research is needed to accurately elucidate the tethering mechanism of MSCs. MSC chemotaxis is well defined and there is strong evidence relating it to the homing ability of MSCs.53 Chemotaxis involves recognizing chemokines through chemokine receptors on MSCs and migrating to chemokines in a gradient-dependent manner.221 RA, a representative inflammatory disease, is associated with well-profiled chemokines such as CXCR1, CXCR4, and CXCR7, which are recognized by chemokine receptors on MSCs. In addition, damaged joints in RA continuously secrete cytokines until they are treated, giving MSCs an advantage as future therapeutic agents for RA.222 However, there are several obstacles to utilizing MSCs as RA therapeutics. In clinical settings, the functional capability of MSCs is significantly affected by the health status of the donor patient.223 MSC yield is significantly reduced in patients undergoing steroid-based treatment and the quality of MSCs is dependent on the donors age and environment.35 In addition, when MSCs are used clinically, cryopreservation and defrosting are necessary, but these procedures shorten the life span of MSCs.224 Therefore, NPs mimicking MSCs are an alternative strategy for overcoming the limitations of MSCs. Additionally, further engineering and modification of MSCs can enhance the therapeutic effect by changing the targeting molecules and loaded drugs. In particular, upregulation of receptors associated with chemotaxis through genetic engineering can confer the additional ability of MSCs to home to specific sites, while the increase in engraftment maximizes the therapeutic effect of MSCs.36,225

Furthermore, there are several methods that can be used to exploit the targeting ability of MSCs as drug delivery systems. MSCs mimicking nanoencapsulation, which consists of MSC membrane-coated NPs, MSC-derived artificial ectosomes, and MSC membrane-fused liposomes, can mimic the targeting ability of MSCs while retaining the advantages of NPs. MSC-membrane-coated NPs are synthesized using inorganic or polymer NPs and membranes from MSCs to coat inner nanosized structures. Because they mimic the biological characteristics of MSC membranes, MSC-membrane-coated NPs can not only escape from immune surveillance but also effectively improve targeting ability, with combined functions of the unique properties of core NPs and MSC membranes.226 Exosomes are also an appropriate candidate for use in MSC membranes, utilizing these targeting abilities. However, natural exosomes lack reproducibility and stable productivity, thus artificial ectosomes with targeting ability produced via synthetic routes can increase the local concentration of ectosomes at the targeted site, thereby reducing toxicity and side effects and maximizing therapeutic efficacy.156 MSC membrane-fused liposomes, a novel system, can also transfer the targeting molecules on the surface of MSCs to liposomes; thus, the advantages of liposomes are retained, but with targeting ability. With advancements in nanotechnology of drug delivery systems, the research in cell-mimicking nanoencapsulation will be very useful. Efficient drug delivery systems fundamentally improve the quality of life of patients with a low dose of medication, low side effects, and subsequent treatment of diseases.227 However, research on cell-mimicking nanoencapsulation is at an early stage, and several problems need to be addressed. To predict the nanotoxicity of artificially synthesized MSC mimicking nanoencapsulations, interactions between lipids and drugs, drug release mechanisms near the targeted site, in vivo compatibility, and immunological physiological studies must be conducted before clinical application.

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2019M3A9H1103690), by the Gachon University Gil Medical Center (FRD2021-03), and by the Gachon University research fund of 2020 (GGU-202008430004).

The authors report no conflicts of interest in this work.

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Stem Cell Mimicking Nanoencapsulation for Targeting Arthrit | IJN - Dove Medical Press

Int J Cell Biol. 2016; 2016: 6940283.

Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Bhopal, Madhya Pradesh 462066, India

Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Bhopal, Madhya Pradesh 462066, India

Academic Editor: Paul J. Higgins

Received 2016 Mar 13; Accepted 2016 Jun 5.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Regenerative medicine, the most recent and emerging branch of medical science, deals with functional restoration of tissues or organs for the patient suffering from severe injuries or chronic disease. The spectacular progress in the field of stem cell research has laid the foundation for cell based therapies of disease which cannot be cured by conventional medicines. The indefinite self-renewal and potential to differentiate into other types of cells represent stem cells as frontiers of regenerative medicine. The transdifferentiating potential of stem cells varies with source and according to that regenerative applications also change. Advancements in gene editing and tissue engineering technology have endorsed the ex vivo remodelling of stem cells grown into 3D organoids and tissue structures for personalized applications. This review outlines the most recent advancement in transplantation and tissue engineering technologies of ESCs, TSPSCs, MSCs, UCSCs, BMSCs, and iPSCs in regenerative medicine. Additionally, this review also discusses stem cells regenerative application in wildlife conservation.

Regenerative medicine, the most recent and emerging branch of medical science, deals with functional restoration of specific tissue and/or organ of the patients suffering with severe injuries or chronic disease conditions, in the state where bodies own regenerative responses do not suffice [1]. In the present scenario donated tissues and organs cannot meet the transplantation demands of aged and diseased populations that have driven the thrust for search for the alternatives. Stem cells are endorsed with indefinite cell division potential, can transdifferentiate into other types of cells, and have emerged as frontline regenerative medicine source in recent time, for reparation of tissues and organs anomalies occurring due to congenital defects, disease, and age associated effects [1]. Stem cells pave foundation for all tissue and organ system of the body and mediates diverse role in disease progression, development, and tissue repair processes in host. On the basis of transdifferentiation potential, stem cells are of four types, that is, (1) unipotent, (2) multipotent, (3) pluripotent, and (4) totipotent [2]. Zygote, the only totipotent stem cell in human body, can give rise to whole organism through the process of transdifferentiation, while cells from inner cells mass (ICM) of embryo are pluripotent in their nature and can differentiate into cells representing three germ layers but do not differentiate into cells of extraembryonic tissue [2]. Stemness and transdifferentiation potential of the embryonic, extraembryonic, fetal, or adult stem cells depend on functional status of pluripotency factors like OCT4, cMYC, KLF44, NANOG, SOX2, and so forth [35]. Ectopic expression or functional restoration of endogenous pluripotency factors epigenetically transforms terminally differentiated cells into ESCs-like cells [3], known as induced pluripotent stem cells (iPSCs) [3, 4]. On the basis of regenerative applications, stem cells can be categorized as embryonic stem cells (ESCs), tissue specific progenitor stem cells (TSPSCs), mesenchymal stem cells (MSCs), umbilical cord stem cells (UCSCs), bone marrow stem cells (BMSCs), and iPSCs (; ). The transplantation of stem cells can be autologous, allogenic, and syngeneic for induction of tissue regeneration and immunolysis of pathogen or malignant cells. For avoiding the consequences of host-versus-graft rejections, tissue typing of human leucocyte antigens (HLA) for tissue and organ transplant as well as use of immune suppressant is recommended [6]. Stem cells express major histocompatibility complex (MHC) receptor in low and secret chemokine that recruitment of endothelial and immune cells is enabling tissue tolerance at graft site [6]. The current stem cell regenerative medicine approaches are founded onto tissue engineering technologies that combine the principles of cell transplantation, material science, and microengineering for development of organoid; those can be used for physiological restoration of damaged tissue and organs. The tissue engineering technology generates nascent tissue on biodegradable 3D-scaffolds [7, 8]. The ideal scaffolds support cell adhesion and ingrowths, mimic mechanics of target tissue, support angiogenesis and neovascularisation for appropriate tissue perfusion, and, being nonimmunogenic to host, do not require systemic immune suppressant [9]. Stem cells number in tissue transplant impacts upon regenerative outcome [10]; in that case prior ex vivo expansion of transplantable stem cells is required [11]. For successful regenerative outcomes, transplanted stem cells must survive, proliferate, and differentiate in site specific manner and integrate into host circulatory system [12]. This review provides framework of most recent (; Figures ) advancement in transplantation and tissue engineering technologies of ESCs, TSPSCs, MSCs, UCSCs, BMSCs, and iPSCs in regenerative medicine. Additionally, this review also discusses stem cells as the tool of regenerative applications in wildlife conservation.

Promises of stem cells in regenerative medicine: the six classes of stem cells, that is, embryonic stem cells (ESCs), tissue specific progenitor stem cells (TSPSCs), mesenchymal stem cells (MSCs), umbilical cord stem cells (UCSCs), bone marrow stem cells (BMSCs), and induced pluripotent stem cells (iPSCs), have many promises in regenerative medicine and disease therapeutics.

ESCs in regenerative medicine: ESCs, sourced from ICM of gastrula, have tremendous promises in regenerative medicine. These cells can differentiate into more than 200 types of cells representing three germ layers. With defined culture conditions, ESCs can be transformed into hepatocytes, retinal ganglion cells, chondrocytes, pancreatic progenitor cells, cone cells, cardiomyocytes, pacemaker cells, eggs, and sperms which can be used in regeneration of tissue and treatment of disease in tissue specific manner.

TSPSCs in regenerative medicine: tissue specific stem and progenitor cells have potential to differentiate into other cells of the tissue. Characteristically inner ear stem cells can be transformed into auditory hair cells, skin progenitors into vascular smooth muscle cells, mesoangioblasts into tibialis anterior muscles, and dental pulp stem cells into serotonin cells. The 3D-culture of TSPSCs in complex biomaterial gives rise to tissue organoids, such as pancreatic organoid from pancreatic progenitor, intestinal tissue organoids from intestinal progenitor cells, and fallopian tube organoids from fallopian tube epithelial cells. Transplantation of TSPSCs regenerates targets tissue such as regeneration of tibialis muscles from mesoangioblasts, cardiac tissue from AdSCs, and corneal tissue from limbal stem cells. Cell growth and transformation factors secreted by TSPSCs can change cells fate to become other types of cell, such that SSCs coculture with skin, prostate, and intestine mesenchyme transforms these cells from MSCs into epithelial cells fate.

MSCs in regenerative medicine: mesenchymal stem cells are CD73+, CD90+, CD105+, CD34, CD45, CD11b, CD14, CD19, and CD79a cells, also known as stromal cells. These bodily MSCs represented here do not account for MSCs of bone marrow and umbilical cord. Upon transplantation and transdifferentiation these bodily MSCs regenerate into cartilage, bones, and muscles tissue. Heart scar formed after heart attack and liver cirrhosis can be treated from MSCs. ECM coating provides the niche environment for MSCs to regenerate into hair follicle, stimulating hair growth.

UCSCs in regenerative medicine: umbilical cord, the readily available source of stem cells, has emerged as futuristic source for personalized stem cell therapy. Transplantation of UCSCs to Krabbe's disease patients regenerates myelin tissue and recovers neuroblastoma patients through restoring tissue homeostasis. The UCSCs organoids are readily available tissue source for treatment of neurodegenerative disease. Peritoneal fibrosis caused by long term dialysis, tendon tissue degeneration, and defective hyaline cartilage can be regenerated by UCSCs. Intravenous injection of UCSCs enables treatment of diabetes, spinal myelitis, systemic lupus erythematosus, Hodgkin's lymphoma, and congenital neuropathies. Cord blood stem cells banking avails long lasting source of stem cells for personalized therapy and regenerative medicine.

BMSCs in regenerative medicine: bone marrow, the soft sponge bone tissue that consisted of stromal, hematopoietic, and mesenchymal and progenitor stem cells, is responsible for blood formation. Even halo-HLA matched BMSCs can cure from disease and regenerate tissue. BMSCs can regenerate craniofacial tissue, brain tissue, diaphragm tissue, and liver tissue and restore erectile function and transdifferentiation monocytes. These multipotent stem cells can cure host from cancer and infection of HIV and HCV.

iPSCs in regenerative medicine: using the edge of iPSCs technology, skin fibroblasts and other adult tissues derived, terminally differentiated cells can be transformed into ESCs-like cells. It is possible that adult cells can be transformed into cells of distinct lineages bypassing the phase of pluripotency. The tissue specific defined culture can transform skin cells to become trophoblast, heart valve cells, photoreceptor cells, immune cells, melanocytes, and so forth. ECM complexation with iPSCs enables generation of tissue organoids for lung, kidney, brain, and other organs of the body. Similar to ESCs, iPSCs also can be transformed into cells representing three germ layers such as pacemaker cells and serotonin cells.

Stem cells in wildlife conservation: tissue biopsies obtained from dead and live wild animals can be either cryopreserved or transdifferentiated to other types of cells, through culture in defined culture medium or in vivo maturation. Stem cells and adult tissue derived iPSCs have great potential of regenerative medicine and disease therapeutics. Gonadal tissue procured from dead wild animals can be matured, ex vivo and in vivo for generation of sperm and egg, which can be used for assistive reproductive technology oriented captive breeding of wild animals or even for resurrection of wildlife.

Application of stem cells in regenerative medicine: stem cells (ESCs, TSPSCs, MSCs, UCSCs, BMSCs, and iPSCs) have diverse applications in tissue regeneration and disease therapeutics.

For the first time in 1998, Thomson isolated human ESCs (hESCs) [13]. ESCs are pluripotent in their nature and can give rise to more than 200 types of cells and promises for the treatment of any kinds of disease [13]. The pluripotency fate of ESCs is governed by functional dynamics of transcription factors OCT4, SOX2, NANOG, and so forth, which are termed as pluripotency factors. The two alleles of the OCT4 are held apart in pluripotency state in ESCs; phase through homologues pairing during embryogenesis and transdifferentiation processes [14] has been considered as critical regulatory switch for lineage commitment of ESCs. The diverse lineage commitment potential represents ESCs as ideal model for regenerative therapeutics of disease and tissue anomalies. This section of review on ESCs discusses transplantation and transdifferentiation of ESCs into retinal ganglion, hepatocytes, cardiomyocytes, pancreatic progenitors, chondrocytes, cones, egg sperm, and pacemaker cells (; ). Infection, cancer treatment, and accidents can cause spinal cord injuries (SCIs). The transplantation of hESCs to paraplegic or quadriplegic SCI patients improves body control, balance, sensation, and limbal movements [15], where transplanted stem cells do homing to injury sites. By birth, humans have fixed numbers of cone cells; degeneration of retinal pigment epithelium (RPE) of macula in central retina causes age-related macular degeneration (ARMD). The genomic incorporation of COCO gene (expressed during embryogenesis) in the developing embryo leads lineage commitment of ESCs into cone cells, through suppression of TGF, BMP, and Wnt signalling pathways. Transplantation of these cone cells to eye recovers individual from ARMD phenomenon, where transplanted cone cells migrate and form sheet-like structure in host retina [16]. However, establishment of missing neuronal connection of retinal ganglion cells (RGCs), cones, and PRE is the most challenging aspect of ARMD therapeutics. Recently, Donald Z Jacks group at John Hopkins University School of Medicine has generated RGCs from CRISPER-Cas9-m-Cherry reporter ESCs [17]. During ESCs transdifferentiation process, CRIPER-Cas9 directs the knock-in of m-Cherry reporter into 3UTR of BRN3B gene, which is specifically expressed in RGCs and can be used for purification of generated RGCs from other cells [17]. Furthermore, incorporation of forskolin in transdifferentiation regime boosts generation of RGCs. Coaxing of these RGCs into biomaterial scaffolds directs axonal differentiation of RGCs. Further modification in RGCs generation regime and composition of biomaterial scaffolds might enable restoration of vision for ARMD and glaucoma patients [17]. Globally, especially in India, cardiovascular problems are a more common cause of human death, where biomedical therapeutics require immediate restoration of heart functions for the very survival of the patient. Regeneration of cardiac tissue can be achieved by transplantation of cardiomyocytes, ESCs-derived cardiovascular progenitors, and bone marrow derived mononuclear cells (BMDMNCs); however healing by cardiomyocytes and progenitor cells is superior to BMDMNCs but mature cardiomyocytes have higher tissue healing potential, suppress heart arrhythmias, couple electromagnetically into hearts functions, and provide mechanical and electrical repair without any associated tumorigenic effects [18, 19]. Like CM differentiation, ESCs derived liver stem cells can be transformed into Cytp450-hepatocytes, mediating chemical modification and catabolism of toxic xenobiotic drugs [20]. Even today, availability and variability of functional hepatocytes are a major a challenge for testing drug toxicity [20]. Stimulation of ESCs and ex vivo VitK12 and lithocholic acid (a by-product of intestinal flora regulating drug metabolism during infancy) activates pregnane X receptor (PXR), CYP3A4, and CYP2C9, which leads to differentiation of ESCs into hepatocytes; those are functionally similar to primary hepatocytes, for their ability to produce albumin and apolipoprotein B100 [20]. These hepatocytes are excellent source for the endpoint screening of drugs for accurate prediction of clinical outcomes [20]. Generation of hepatic cells from ESCs can be achieved in multiple ways, as serum-free differentiation [21], chemical approaches [20, 22], and genetic transformation [23, 24]. These ESCs-derived hepatocytes are long lasting source for treatment of liver injuries and high throughput screening of drugs [20, 23, 24]. Transplantation of the inert biomaterial encapsulated hESCs-derived pancreatic progenitors (CD24+, CD49+, and CD133+) differentiates into -cells, minimizing high fat diet induced glycemic and obesity effects in mice [25] (). Addition of antidiabetic drugs into transdifferentiation regime can boost ESCs conservation into -cells [25], which theoretically can cure T2DM permanently [25]. ESCs can be differentiated directly into insulin secreting -cells (marked with GLUT2, INS1, GCK, and PDX1) which can be achieved through PDX1 mediated epigenetic reprogramming [26]. Globally, osteoarthritis affects millions of people and occurs when cartilage at joints wears away, causing stiffness of the joints. The available therapeutics for arthritis relieve symptoms but do not initiate reverse generation of cartilage. For young individuals and athletes replacement of joints is not feasible like old populations; in that case transplantation of stem cells represents an alternative for healing cartilage injuries [27]. Chondrocytes, the cartilage forming cells derived from hESC, embedded in fibrin gel effectively heal defective cartilage within 12 weeks, when transplanted to focal cartilage defects of knee joints in mice without any negative effect [27]. Transplanted chondrocytes form cell aggregates, positive for SOX9 and collagen II, and defined chondrocytes are active for more than 12wks at transplantation site, advocating clinical suitability of chondrocytes for treatment of cartilage lesions [27]. The integrity of ESCs to integrate and differentiate into electrophysiologically active cells provides a means for natural regulation of heart rhythm as biological pacemaker. Coaxing of ESCs into inert biomaterial as well as propagation in defined culture conditions leads to transdifferentiation of ESCs to become sinoatrial node (SAN) pacemaker cells (PCs) [28]. Genomic incorporation TBox3 into ESCs ex vivo leads to generation of PCs-like cells; those express activated leukocyte cells adhesion molecules (ALCAM) and exhibit similarity to PCs for gene expression and immune functions [28]. Transplantation of PCs can restore pacemaker functions of the ailing heart [28]. In summary, ESCs can be transdifferentiated into any kinds of cells representing three germ layers of the body, being most promising source of regenerative medicine for tissue regeneration and disease therapy (). Ethical concerns limit the applications of ESCs, where set guidelines need to be followed; in that case TSPSCs, MSCs, UCSCs, BMSCs, and iPSCs can be explored as alternatives.

TSPSCs maintain tissue homeostasis through continuous cell division, but, unlike ESCs, TSPSCs retain stem cells plasticity and differentiation in tissue specific manner, giving rise to few types of cells (). The number of TSPSCs population to total cells population is too low; in that case their harvesting as well as in vitro manipulation is really a tricky task [29], to explore them for therapeutic scale. Human body has foundation from various types of TSPSCs; discussing the therapeutic application for all types is not feasible. This section of review discusses therapeutic application of pancreatic progenitor cells (PPCs), dental pulp stem cells (DPSCs), inner ear stem cells (IESCs), intestinal progenitor cells (IPCs), limbal progenitor stem cells (LPSCs), epithelial progenitor stem cells (EPSCs), mesoangioblasts (MABs), spermatogonial stem cells (SSCs), the skin derived precursors (SKPs), and adipose derived stem cells (AdSCs) (; ). During embryogenesis PPCs give rise to insulin-producing -cells. The differentiation of PPCs to become -cells is negatively regulated by insulin [30]. PPCs require active FGF and Notch signalling; growing more rapidly in community than in single cell populations advocates the functional importance of niche effect in self-renewal and transdifferentiation processes. In 3D-scaffold culture system, mice embryo derived PPCs grow into hollow organoid spheres; those finally differentiate into insulin-producing -cell clusters [29]. The DSPSCs, responsible for maintenance of teeth health status, can be sourced from apical papilla, deciduous teeth, dental follicle, and periodontal ligaments, have emerged as regenerative medicine candidate, and might be explored for treatment of various kinds of disease including restoration neurogenic functions in teeth [31, 32]. Expansion of DSPSCs in chemically defined neuronal culture medium transforms them into a mixed population of cholinergic, GABAergic, and glutaminergic neurons; those are known to respond towards acetylcholine, GABA, and glutamine stimulations in vivo. These transformed neuronal cells express nestin, glial fibrillary acidic protein (GFAP), III-tubulin, and voltage gated L-type Ca2+ channels [32]. However, absence of Na+ and K+ channels does not support spontaneous action potential generation, necessary for response generation against environmental stimulus. All together, these primordial neuronal stem cells have possible therapeutic potential for treatment of neurodental problems [32]. Sometimes, brain tumor chemotherapy can cause neurodegeneration mediated cognitive impairment, a condition known as chemobrain [33]. The intrahippocampal transplantation of human derived neuronal stem cells to cyclophosphamide behavioural decremented mice restores cognitive functions in a month time. Here the transplanted stem cells differentiate into neuronal and astroglial lineage, reduce neuroinflammation, and restore microglial functions [33]. Furthermore, transplantation of stem cells, followed by chemotherapy, directs pyramidal and granule-cell neurons of the gyrus and CA1 subfields of hippocampus which leads to reduction in spine and dendritic cell density in the brain. These findings suggest that transplantation of stem cells to cranium restores cognitive functions of the chemobrain [33]. The hair cells of the auditory system produced during development are not postmitotic; loss of hair cells cannot be replaced by inner ear stem cells, due to active state of the Notch signalling [34]. Stimulation of inner ear progenitors with -secretase inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY411575","term_id":"1257853995","term_text":"LY411575"}}LY411575) abrogates Notch signalling through activation of transcription factor atonal homologue 1 (Atoh1) and directs transdifferentiation of progenitors into cochlear hair cells [34]. Transplantation of in vitro generated hair cells restores acoustic functions in mice, which can be the potential regenerative medicine candidates for the treatment of deafness [34]. Generation of the hair cells also can be achieved through overexpression of -catenin and Atoh1 in Lrg5+ cells in vivo [35]. Similar to ear progenitors, intestine of the digestive tract also has its own tissue specific progenitor stem cells, mediating regeneration of the intestinal tissue [34, 36]. Dysregulation of the common stem cells signalling pathways, Notch/BMP/TGF-/Wnt, in the intestinal tissue leads to disease. Information on these signalling pathways [37] is critically important in designing therapeutics. Coaxing of the intestinal tissue specific progenitors with immune cells (macrophages), connective tissue cells (myofibroblasts), and probiotic bacteria into 3D-scaffolds of inert biomaterial, crafting biological environment, is suitable for differentiation of progenitors to occupy the crypt-villi structures into these scaffolds [36]. Omental implementation of these crypt-villi structures to dogs enhances intestinal mucosa through regeneration of goblet cells containing intestinal tissue [36]. These intestinal scaffolds are close approach for generation of implantable intestinal tissue, divested by infection, trauma, cancer, necrotizing enterocolitis (NEC), and so forth [36]. In vitro culture conditions cause differentiation of intestinal stem cells to become other types of cells, whereas incorporation of valproic acid and CHIR-99021 in culture conditions avoids differentiation of intestinal stem cells, enabling generation of indefinite pool of stem cells to be used for regenerative applications [38]. The limbal stem cells of the basal limbal epithelium, marked with ABCB5, are essential for regeneration and maintenance of corneal tissue [39]. Functional status of ABCB5 is critical for survival and functional integrity of limbal stem cells, protecting them from apoptotic cell death [39]. Limbal stem cells deficiency leads to replacement of corneal epithelium with visually dead conjunctival tissue, which can be contributed by burns, inflammation, and genetic factors [40]. Transplanted human cornea stem cells to mice regrown into fully functional human cornea, possibly supported by blood eye barrier phenomena, can be used for treatment of eye diseases, where regeneration of corneal tissue is critically required for vision restoration [39]. Muscle degenerative disease like duchenne muscular dystrophy (DMD) can cause extensive thrashing of muscle tissue, where tissue engineering technology can be deployed for functional restoration of tissue through regeneration [41]. Encapsulation of mouse or human derived MABs (engineered to express placental derived growth factor (PDGF)) into polyethylene glycol (PEG) fibrinogen hydrogel and their transplantation beneath the skin at ablated tibialis anterior form artificial muscles, which are functionally similar to those of normal tibialis anterior muscles [41]. The PDGF attracts various cell types of vasculogenic and neurogenic potential to the site of transplantation, supporting transdifferentiation of mesoangioblasts to become muscle fibrils [41]. The therapeutic application of MABs in skeletal muscle regeneration and other therapeutic outcomes has been reviewed by others [42]. One of the most important tissue specific stem cells, the male germline stem cells or spermatogonial stem cells (SSCs), produces spermatogenic lineage through mesenchymal and epithets cells [43] which itself creates niche effect on other cells. In vivo transplantation of SSCs with prostate, skin, and uterine mesenchyme leads to differentiation of these cells to become epithelia of the tissue of origin [43]. These newly formed tissues exhibit all physical and physiological characteristics of prostate and skin and the physical characteristics of prostate, skin, and uterus, express tissue specific markers, and suggest that factors secreted from SSCs lead to lineage conservation which defines the importance of niche effect in regenerative medicine [43]. According to an estimate, more than 100 million people are suffering from the condition of diabetic retinopathy, a progressive dropout of vascularisation in retina that leads to loss of vision [44]. The intravitreal injection of adipose derived stem cells (AdSCs) to the eye restores microvascular capillary bed in mice. The AdSCs from healthy donor produce higher amounts of vasoprotective factors compared to glycemic mice, enabling superior vascularisation [44]. However use of AdSCs for disease therapeutics needs further standardization for cell counts in dose of transplant and monitoring of therapeutic outcomes at population scale [44]. Apart from AdSCs, other kinds of stem cells also have therapeutic potential in regenerative medicine for treatment of eye defects, which has been reviewed by others [45]. Fallopian tubes, connecting ovaries to uterus, are the sites where fertilization of the egg takes place. Infection in fallopian tubes can lead to inflammation, tissue scarring, and closure of the fallopian tube which often leads to infertility and ectopic pregnancies. Fallopian is also the site where onset of ovarian cancer takes place. The studies on origin and etiology of ovarian cancer are restricted due to lack of technical advancement for culture of epithelial cells. The in vitro 3D organoid culture of clinically obtained fallopian tube epithelial cells retains their tissue specificity, keeps cells alive, which differentiate into typical ciliated and secretory cells of fallopian tube, and advocates that ectopic examination of fallopian tube in organoid culture settings might be the ideal approach for screening of cancer [46]. The sustained growth and differentiation of fallopian TSPSCs into fallopian tube organoid depend both on the active state of the Wnt and on paracrine Notch signalling [46]. Similar to fallopian tube stem cells, subcutaneous visceral tissue specific cardiac adipose (CA) derived stem cells (AdSCs) have the potential of differentiation into cardiovascular tissue [47]. Systemic infusion of CA-AdSCs into ischemic myocardium of mice regenerates heart tissue and improves cardiac function through differentiation to endothelial cells, vascular smooth cells, and cardiomyocytes and vascular smooth cells. The differentiation and heart regeneration potential of CA-AdSCs are higher than AdSCs [48], representing CA-AdSCs as potent regenerative medicine candidates for myocardial ischemic therapy [47]. The skin derived precursors (SKPs), the progenitors of dermal papilla/hair/hair sheath, give rise to multiple tissues of mesodermal and/or ectodermal origin such as neurons, Schwann cells, adipocytes, chondrocytes, and vascular smooth muscle cells (VSMCs). VSMCs mediate wound healing and angiogenesis process can be derived from human foreskin progenitor SKPs, suggesting that SKPs derived VSMCs are potential regenerative medicine candidates for wound healing and vasculature injuries treatments [49]. In summary, TSPSCs are potentiated with tissue regeneration, where advancement in organoid culture (; ) technologies defines the importance of niche effect in tissue regeneration and therapeutic outcomes of ex vivo expanded stem cells.

MSCs, the multilineage stem cells, differentiate only to tissue of mesodermal origin, which includes tendons, bone, cartilage, ligaments, muscles, and neurons [50]. MSCs are the cells which express combination of markers: CD73+, CD90+, CD105+, CD11b, CD14, CD19, CD34, CD45, CD79a, and HLA-DR, reviewed elsewhere [50]. The application of MSCs in regenerative medicine can be generalized from ongoing clinical trials, phasing through different state of completions, reviewed elsewhere [90]. This section of review outlines the most recent representative applications of MSCs (; ). The anatomical and physiological characteristics of both donor and receiver have equal impact on therapeutic outcomes. The bone marrow derived MSCs (BMDMSCs) from baboon are morphologically and phenotypically similar to those of bladder stem cells and can be used in regeneration of bladder tissue. The BMDMSCs (CD105+, CD73+, CD34, and CD45), expressing GFP reporter, coaxed with small intestinal submucosa (SIS) scaffolds, augment healing of degenerated bladder tissue within 10wks of the transplantation [51]. The combinatorial CD characterized MACs are functionally active at transplantation site, which suggests that CD characterization of donor MSCs yields superior regenerative outcomes [51]. MSCs also have potential to regenerate liver tissue and treat liver cirrhosis, reviewed elsewhere [91]. The regenerative medicinal application of MSCs utilizes cells in two formats as direct transplantation or first transdifferentiation and then transplantation; ex vivo transdifferentiation of MSCs deploys retroviral delivery system that can cause oncogenic effect on cells. Nonviral, NanoScript technology, comprising utility of transcription factors (TFs) functionalized gold nanoparticles, can target specific regulatory site in the genome effectively and direct differentiation of MSCs into another cell fate, depending on regime of TFs. For example, myogenic regulatory factor containing NanoScript-MRF differentiates the adipose tissue derived MSCs into muscle cells [92]. The multipotency characteristics represent MSCs as promising candidate for obtaining stable tissue constructs through coaxed 3D organoid culture; however heterogeneous distribution of MSCs slows down cell proliferation, rendering therapeutic applications of MSCs. Adopting two-step culture system for MSCs can yield homogeneous distribution of MSCs in biomaterial scaffolds. For example, fetal-MSCs coaxed in biomaterial when cultured first in rotating bioreactor followed with static culture lead to homogeneous distribution of MSCs in ECM components [7]. Occurrence of dental carries, periodontal disease, and tooth injury can impact individual's health, where bioengineering of teeth can be the alternative option. Coaxing of epithelial-MSCs with dental stem cells into synthetic polymer gives rise to mature teeth unit, which consisted of mature teeth and oral tissue, offering multiple regenerative therapeutics, reviewed elsewhere [52]. Like the tooth decay, both human and animals are prone to orthopedic injuries, affecting bones, joint, tendon, muscles, cartilage, and so forth. Although natural healing potential of bone is sufficient to heal the common injuries, severe trauma and tumor-recession can abrogate germinal potential of bone-forming stem cells. In vitro chondrogenic, osteogenic, and adipogenic potential of MSCs advocates therapeutic applications of MSCs in orthopedic injuries [53]. Seeding of MSCs, coaxed into biomaterial scaffolds, at defective bone tissue, regenerates defective bone tissues, within fourwks of transplantation; by the end of 32wks newly formed tissues integrate into old bone [54]. Osteoblasts, the bone-forming cells, have lesser actin cytoskeleton compared to adipocytes and MSCs. Treatment of MSCs with cytochalasin-D causes rapid transportation of G-actin, leading to osteogenic transformation of MSCs. Furthermore, injection of cytochalasin-D to mice tibia also promotes bone formation within a wk time frame [55]. The bone formation processes in mice, dog, and human are fundamentally similar, so outcomes of research on mice and dogs can be directional for regenerative application to human. Injection of MSCs to femur head of Legg-Calve-Perthes suffering dog heals the bone very fast and reduces the injury associated pain [55]. Degeneration of skeletal muscle and muscle cramps are very common to sledge dogs, animals, and individuals involved in adventurous athletics activities. Direct injection of adipose tissue derived MSCs to tear-site of semitendinosus muscle in dogs heals injuries much faster than traditional therapies [56]. Damage effect treatment for heart muscle regeneration is much more complex than regeneration of skeletal muscles, which needs high grade fine-tuned coordination of neurons with muscles. Coaxing of MSCs into alginate gel increases cell retention time that leads to releasing of tissue repairing factors in controlled manner. Transplantation of alginate encapsulated cells to mice heart reduces scar size and increases vascularisation, which leads to restoration of heart functions. Furthermore, transplanted MSCs face host inhospitable inflammatory immune responses and other mechanical forces at transplantation site, where encapsulation of cells keeps them away from all sorts of mechanical forces and enables sensing of host tissue microenvironment, and respond accordingly [57]. Ageing, disease, and medicine consumption can cause hair loss, known as alopecia. Although alopecia has no life threatening effects, emotional catchments can lead to psychological disturbance. The available treatments for alopecia include hair transplantation and use of drugs, where drugs are expensive to afford and generation of new hair follicle is challenging. Dermal papillary cells (DPCs), the specialized MSCs localized in hair follicle, are responsible for morphogenesis of hair follicle and hair cycling. The layer-by-layer coating of DPCs, called GAG coating, consists of coating of geletin as outer layer, middle layer of fibroblast growth factor 2 (FGF2) loaded alginate, and innermost layer of geletin. GAG coating creates tissue microenvironment for DPCs that can sustain immunological and mechanical obstacles, supporting generation of hair follicle. Transplantation of GAG-coated DPCs leads to abundant hair growth and maturation of hair follicle, where GAG coating serves as ECM, enhancing intrinsic therapeutic potential of DPCs [58]. During infection, the inflammatory cytokines secreted from host immune cells attract MSCs to the site of inflammation, which modulates inflammatory responses, representing MSCs as key candidate of regenerative medicine for infectious disease therapeutics. Coculture of macrophages (M) and adipose derived MSCs from Leishmania major (LM) susceptible and resistant mice demonstrates that AD-MSCs educate M against LM infection, differentially inducing M1 and M2 phenotype that represents AD-MSC as therapeutic agent for leishmanial therapy [93]. In summary, the multilineage differentiation potential of MSCs, as well as adoption of next-generation organoid culture system, avails MSCs as ideal regenerative medicine candidate.

Umbilical cord, generally thrown at the time of child birth, is the best known source for stem cells, procured in noninvasive manner, having lesser ethical constraints than ESCs. Umbilical cord is rich source of hematopoietic stem cells (HSCs) and MSCs, which possess enormous regeneration potential [94] (; ). The HSCs of cord blood are responsible for constant renewal of all types of blood cells and protective immune cells. The proliferation of HSCs is regulated by Musashi-2 protein mediated attenuation of Aryl hydrocarbon receptor (AHR) signalling in stem cells [95]. UCSCs can be cryopreserved at stem cells banks (; ), in operation by both private and public sector organization. Public stem cells banks operate on donation formats and perform rigorous screening for HLA typing and donated UCSCs remain available to anyone in need, whereas private stem cell banks operation is more personalized, availing cells according to donor consent. Stem cell banking is not so common, even in developed countries. Survey studies find that educated women are more eager to donate UCSCs, but willingness for donation decreases with subsequent deliveries, due to associated cost and safety concerns for preservation [96]. FDA has approved five HSCs for treatment of blood and other immunological complications [97]. The amniotic fluid, drawn during pregnancy for standard diagnostic purposes, is generally discarded without considering its vasculogenic potential. UCSCs are the best alternatives for those patients who lack donors with fully matched HLA typing for peripheral blood and PBMCs and bone marrow [98]. One major issue with UCSCs is number of cells in transplant, fewer cells in transplant require more time for engraftment to mature, and there are also risks of infection and mortality; in that case ex vivo propagation of UCSCs can meet the demand of desired outcomes. There are diverse protocols, available for ex vivo expansion of UCSCs, reviewed elsewhere [99]. Amniotic fluid stem cells (AFSCs), coaxed to fibrin (required for blood clotting, ECM interactions, wound healing, and angiogenesis) hydrogel and PEG supplemented with vascular endothelial growth factor (VEGF), give rise to vascularised tissue, when grafted to mice, suggesting that organoid cultures of UCSCs have promise for generation of biocompatible tissue patches, for treating infants born with congenital heart defects [59]. Retroviral integration of OCT4, KLF4, cMYC, and SOX2 transforms AFSCs into pluripotency stem cells known as AFiPSCs which can be directed to differentiate into extraembryonic trophoblast by BMP2 and BMP4 stimulation, which can be used for regeneration of placental tissues [60]. Wharton's jelly (WJ), the gelatinous substance inside umbilical cord, is rich in mucopolysaccharides, fibroblast, macrophages, and stem cells. The stem cells from UCB and WJ can be transdifferentiated into -cells. Homogeneous nature of WJ-SCs enables better differentiation into -cells; transplantation of these cells to streptozotocin induced diabetic mice efficiently brings glucose level to normal [7]. Easy access and expansion potential and plasticity to differentiate into multiple cell lineages represent WJ as an ideal candidate for regenerative medicine but cells viability changes with passages with maximum viable population at 5th-6th passages. So it is suggested to perform controlled expansion of WJ-MSCS for desired regenerative outcomes [9]. Study suggests that CD34+ expression leads to the best regenerative outcomes, with less chance of host-versus-graft rejection. In vitro expansion of UCSCs, in presence of StemRegenin-1 (SR-1), conditionally expands CD34+ cells [61]. In type I diabetic mellitus (T1DM), T-cell mediated autoimmune destruction of pancreatic -cells occurs, which has been considered as tough to treat. Transplantation of WJ-SCs to recent onset-T1DM patients restores pancreatic function, suggesting that WJ-MSCs are effective in regeneration of pancreatic tissue anomalies [62]. WJ-MSCs also have therapeutic importance for treatment of T2DM. A non-placebo controlled phase I/II clinical trial demonstrates that intravenous and intrapancreatic endovascular injection of WJ-MSCs to T2DM patients controls fasting glucose and glycated haemoglobin through improvement of -cells functions, evidenced by enhanced c-peptides and reduced inflammatory cytokines (IL-1 and IL-6) and T-cells counts [63]. Like diabetes, systematic lupus erythematosus (SLE) also can be treated with WJ-MSCs transplantation. During progression of SLE host immune system targets its own tissue leading to degeneration of renal, cardiovascular, neuronal, and musculoskeletal tissues. A non-placebo controlled follow-up study on 40 SLE patients demonstrates that intravenous infusion of WJ-MSC improves renal functions and decreases systematic lupus erythematosus disease activity index (SLEDAI) and British Isles Lupus Assessment Group (BILAG), and repeated infusion of WJ-MSCs protects the patient from relapse of the disease [64]. Sometimes, host inflammatory immune responses can be detrimental for HSCs transplantation and blood transfusion procedures. Infusion of WJ-MSC to patients, who had allogenic HSCs transplantation, reduces haemorrhage inflammation (HI) of bladder, suggesting that WJ-MSCs are potential stem cells adjuvant in HSCs transplantation and blood transfusion based therapies [100]. Apart from WJ, umbilical cord perivascular space and cord vein are also rich source for obtaining MSCs. The perivascular MSCs of umbilical cord are more primitive than WJ-MSCs and other MSCs from cord suggest that perivascular MSCs might be used as alternatives for WJ-MSCs for regenerative therapeutics outcome [101]. Based on origin, MSCs exhibit differential in vitro and in vivo properties and advocate functional characterization of MSCs, prior to regenerative applications. Emerging evidence suggests that UCSCs can heal brain injuries, caused by neurodegenerative diseases like Alzheimer's, Krabbe's disease, and so forth. Krabbe's disease, the infantile lysosomal storage disease, occurs due to deficiency of myelin synthesizing enzyme (MSE), affecting brain development and cognitive functions. Progression of neurodegeneration finally leads to death of babies aged two. Investigation shows that healing of peripheral nervous system (PNS) and central nervous system (CNS) tissues with Krabbe's disease can be achieved by allogenic UCSCs. UCSCs transplantation to asymptomatic infants with subsequent monitoring for 46 years reveals that UCSCs recover babies from MSE deficiency, improving myelination and cognitive functions, compared to those of symptomatic babies. The survival rate of transplanted UCSCs in asymptomatic and symptomatic infants was 100% and 43%, respectively, suggesting that early diagnosis and timely treatment are critical for UCSCs acceptance for desired therapeutic outcomes. UCSCs are more primitive than BMSCs, so perfect HLA typing is not critically required, representing UCSCs as an excellent source for treatment of all the diseases involving lysosomal defects, like Krabbe's disease, hurler syndrome, adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), Tay-Sachs disease (TSD), and Sandhoff disease [65]. Brain injuries often lead to cavities formation, which can be treated from neuronal parenchyma, generated ex vivo from UCSCs. Coaxing of UCSCs into human originated biodegradable matrix scaffold and in vitro expansion of cells in defined culture conditions lead to formation of neuronal organoids, within threewks' time frame. These organoids structurally resemble brain tissue and consisted of neuroblasts (GFAP+, Nestin+, and Ki67+) and immature stem cells (OCT4+ and SOX2+). The neuroblasts of these organoids further can be differentiated into mature neurons (MAP2+ and TUJ1+) [66]. Administration of high dose of drugs in divesting neuroblastoma therapeutics requires immediate restoration of hematopoiesis. Although BMSCs had been promising in restoration of hematopoiesis UCSCs are sparely used in clinical settings. A case study demonstrates that neuroblastoma patients who received autologous UCSCs survive without any associated side effects [12]. During radiation therapy of neoplasm, spinal cord myelitis can occur, although occurrence of myelitis is a rare event and usually such neurodegenerative complication of spinal cord occurs 624 years after exposure to radiations. Transplantation of allogenic UC-MSCs in laryngeal patients undergoing radiation therapy restores myelination [102]. For treatment of neurodegenerative disease like Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), traumatic brain injuries (TBI), Parkinson's, SCI, stroke, and so forth, distribution of transplanted UCSCs is critical for therapeutic outcomes. In mice and rat, injection of UCSCs and subsequent MRI scanning show that transplanted UCSCs migrate to CNS and multiple peripheral organs [67]. For immunomodulation of tumor cells disease recovery, transplantation of allogenic DCs is required. The CD11c+DCs, derived from UCB, are morphologically and phenotypically similar to those of peripheral blood derived CTLs-DCs, suggesting that UCB-DCs can be used for personalized medicine of cancer patient, in need for DCs transplantation [103]. Coculture of UCSCs with radiation exposed human lung fibroblast stops their transdifferentiation, which suggests that factors secreted from UCSCs may restore niche identity of fibroblast, if they are transplanted to lung after radiation therapy [104]. Tearing of shoulder cuff tendon can cause severe pain and functional disability, whereas ultrasound guided transplantation of UCB-MSCs in rabbit regenerates subscapularis tendon in fourwks' time frame, suggesting that UCB-MSCs are effective enough to treat tendons injuries when injected to focal points of tear-site [68]. Furthermore, transplantation of UCB-MSCs to chondral cartilage injuries site in pig knee along with HA hydrogel composite regenerates hyaline cartilage [69], suggesting that UCB-MSCs are effective regenerative medicine candidate for treating cartilage and ligament injuries. Physiologically circulatory systems of brain, placenta, and lungs are similar. Infusion of UCB-MSCs to preeclampsia (PE) induced hypertension mice reduces the endotoxic effect, suggesting that UC-MSCs are potential source for treatment of endotoxin induced hypertension during pregnancy, drug abuse, and other kinds of inflammatory shocks [105]. Transplantation of UCSCs to severe congenital neutropenia (SCN) patients restores neutrophils count from donor cells without any side effect, representing UCSCs as potential alternative for SCN therapy, when HLA matched bone marrow donors are not accessible [106]. In clinical settings, the success of myocardial infarction (MI) treatment depends on ageing, systemic inflammation in host, and processing of cells for infusion. Infusion of human hyaluronan hydrogel coaxed UCSCs in pigs induces angiogenesis, decreases scar area, improves cardiac function at preclinical level, and suggests that the same strategy might be effective for human [107]. In stem cells therapeutics, UCSCs transplantation can be either autologous or allogenic. Sometimes, the autologous UCSCs transplants cannot combat over tumor relapse, observed in Hodgkin's lymphoma (HL), which might require second dose transplantation of allogenic stem cells, but efficacy and tolerance of stem cells transplant need to be addressed, where tumor replace occurs. A case study demonstrates that second dose allogenic transplants of UCSCs effective for HL patients, who had heavy dose in prior transplant, increase the long term survival chances by 30% [10]. Patients undergoing long term peritoneal renal dialysis are prone to peritoneal fibrosis and can change peritoneal structure and failure of ultrafiltration processes. The intraperitoneal (IP) injection of WJ-MSCs prevents methylglyoxal induced programmed cell death and peritoneal wall thickening and fibrosis, suggesting that WJ-MSCs are effective in therapeutics of encapsulating peritoneal fibrosis [70]. In summary, UCB-HSCs, WJ-MSCs, perivascular MSCs, and UCB-MSCs have tissue regeneration potential.

Bone marrow found in soft spongy bones is responsible for formation of all peripheral blood and comprises hematopoietic stem cells (producing blood cells) and stromal cells (producing fat, cartilage, and bones) [108] (; ). Visually bone marrow has two types, red marrow (myeloid tissue; producing RBC, platelets, and most of WBC) and yellow marrow (producing fat cells and some WBC) [108]. Imbalance in marrow composition can culminate to the diseased condition. Since 1980, bone marrow transplantation is widely accepted for cancer therapeutics [109]. In order to avoid graft rejection, HLA typing of donors is a must, but completely matched donors are limited to family members, which hampers allogenic transplantation applications. Since matching of all HLA antigens is not critically required, in that case defining the critical antigens for haploidentical allogenic donor for patients, who cannot find fully matched donor, might relieve from donor constraints. Two-step administration of lymphoid and myeloid BMSCs from haploidentical donor to the patients of aplastic anaemia and haematological malignancies reconstructs host immune system and the outcomes are almost similar to fully matched transplants, which recommends that profiling of critically important HLA is sufficient for successful outcomes of BMSCs transplantation. Haploidentical HLA matching protocol is the major process for minorities and others who do not have access to matched donor [71]. Furthermore, antigen profiling is not the sole concern for BMSCs based therapeutics. For example, restriction of HIV1 (human immune deficiency virus) infection is not feasible through BMSCs transplantation because HIV1 infection is mediated through CD4+ receptors, chemokine CXC motif receptor 4 (CXCR4), and chemokine receptor 5 (CCR5) for infecting and propagating into T helper (Th), monocytes, macrophages, and dendritic cells (DCs). Genetic variation in CCR2 and CCR5 receptors is also a contributory factor; mediating protection against infection has been reviewed elsewhere [110]. Engineering of hematopoietic stem and progenitor cells (HSPCs) derived CD4+ cells to express HIV1 antagonistic RNA, specifically designed for targeting HIV1 genome, can restrict HIV1 infection, through immune elimination of latently infected CD4+ cells. A single dose infusion of genetically modified (GM), HIV1 resistant HSPCs can be the alternative of HIV1 retroviral therapy. In the present scenario stem cells source, patient selection, transplantation-conditioning regimen, and postinfusion follow-up studies are the major factors, which can limit application of HIV1 resistant GM-HSPCs (CD4+) cells application in AIDS therapy [72, 73]. Platelets, essential for blood clotting, are formed from megakaryocytes inside the bone marrow [74]. Due to infection, trauma, and cancer, there are chances of bone marrow failure. To an extent, spongy bone marrow microenvironment responsible for lineage commitment can be reconstructed ex vivo [75]. The ex vivo constructed 3D-scaffolds consisted of microtubule and silk sponge, flooded with chemically defined organ culture medium, which mimics bone marrow environment. The coculture of megakaryocytes and embryonic stem cells (ESCs) in this microenvironment leads to generation of functional platelets from megakaryocytes [75]. The ex vivo 3D-scaffolds of bone microenvironment can stride the path for generation of platelets in therapeutic quantities for regenerative medication of burns [75] and blood clotting associated defects. Accidents, traumatic injuries, and brain stroke can deplete neuronal stem cells (NSCs), responsible for generation of neurons, astrocytes, and oligodendrocytes. Brain does not repopulate NSCs and heal traumatic injuries itself and transplantation of BMSCs also can heal neurodegeneration alone. Lipoic acid (LA), a known pharmacological antioxidant compound used in treatment of diabetic and multiple sclerosis neuropathy when combined with BMSCs, induces neovascularisation at focal cerebral injuries, within 8wks of transplantation. Vascularisation further attracts microglia and induces their colonization into scaffold, which leads to differentiation of BMSCs to become brain tissue, within 16wks of transplantation. In this approach, healing of tissue directly depends on number of BMSCs in transplantation dose [76]. Dental caries and periodontal disease are common craniofacial disease, often requiring jaw bone reconstruction after removal of the teeth. Traditional therapy focuses on functional and structural restoration of oral tissue, bone, and teeth rather than biological restoration, but BMSCs based therapies promise for regeneration of craniofacial bone defects, enabling replacement of missing teeth in restored bones with dental implants. Bone marrow derived CD14+ and CD90+ stem and progenitor cells, termed as tissue repair cells (TRC), accelerate alveolar bone regeneration and reconstruction of jaw bone when transplanted in damaged craniofacial tissue, earlier to oral implants. Hence, TRC therapy reduces the need of secondary bone grafts, best suited for severe defects in oral bone, skin, and gum, resulting from trauma, disease, or birth defects [77]. Overall, HSCs have great value in regenerative medicine, where stem cells transplantation strategies explore importance of niche in tissue regeneration. Prior to transplantation of BMSCs, clearance of original niche from target tissue is necessary for generation of organoid and organs without host-versus-graft rejection events. Some genetic defects can lead to disorganization of niche, leading to developmental errors. Complementation with human blastocyst derived primary cells can restore niche function of pancreas in pigs and rats, which defines the concept for generation of clinical grade human pancreas in mice and pigs [111]. Similar to other organs, diaphragm also has its own niche. Congenital defects in diaphragm can affect diaphragm functions. In the present scenario functional restoration of congenital diaphragm defects by surgical repair has risk of reoccurrence of defects or incomplete restoration [8]. Decellularization of donor derived diaphragm offers a way for reconstruction of new and functionally compatible diaphragm through niche modulation. Tissue engineering technology based decellularization of diaphragm and simultaneous perfusion of bone marrow mesenchymal stem cells (BM-MSCs) facilitates regeneration of functional scaffolds of diaphragm tissues [8]. In vivo replacement of hemidiaphragm in rats with reseeded scaffolds possesses similar myography and spirometry as it has in vivo in donor rats. These scaffolds retaining natural architecture are devoid of immune cells, retaining intact extracellular matrix that supports adhesion, proliferation, and differentiation of seeded cells [8]. These findings suggest that cadaver obtained diaphragm, seeded with BM-MSCs, can be used for curing patients in need for restoration of diaphragm functions (; ). However, BMSCs are heterogeneous population, which might result in differential outcomes in clinical settings; however clonal expansion of BMSCs yields homogenous cells population for therapeutic application [8]. One study also finds that intracavernous delivery of single clone BMSCs can restore erectile function in diabetic mice [112] and the same strategy might be explored for adult human individuals. The infection of hepatitis C virus (HCV) can cause liver cirrhosis and degeneration of hepatic tissue. The intraparenchymal transplantation of bone marrow mononuclear cells (BMMNCs) into liver tissue decreases aspartate aminotransferase (AST), alanine transaminase (ALT), bilirubin, CD34, and -SMA, suggesting that transplanted BMSCs restore hepatic functions through regeneration of hepatic tissues [113]. In order to meet the growing demand for stem cells transplantation therapy, donor encouragement is always required [8]. The stem cells donation procedure is very simple; with consent donor gets an injection of granulocyte-colony stimulating factor (G-CSF) that increases BMSCs population. Bone marrow collection is done from hip bone using syringe in 4-5hrs, requiring local anaesthesia and within a wk time frame donor gets recovered donation associated weakness.

The field of iPSCs technology and research is new to all other stem cells research, emerging in 2006 when, for the first time, Takahashi and Yamanaka generated ESCs-like cells through genetic incorporation of four factors, Sox2, Oct3/4, Klf4, and c-Myc, into skin fibroblast [3]. Due to extensive nuclear reprogramming, generated iPSCs are indistinguishable from ESCs, for their transcriptome profiling, epigenetic markings, and functional competence [3], but use of retrovirus in transdifferentiation approach has questioned iPSCs technology. Technological advancement has enabled generation of iPSCs from various kinds of adult cells phasing through ESCs or direct transdifferentiation. This section of review outlines most recent advancement in iPSC technology and regenerative applications (; ). Using the new edge of iPSCs technology, terminally differentiated skin cells directly can be transformed into kidney organoids [114], which are functionally and structurally similar to those of kidney tissue in vivo. Up to certain extent kidneys heal themselves; however natural regeneration potential cannot meet healing for severe injuries. During kidneys healing process, a progenitor stem cell needs to become 20 types of cells, required for waste excretion, pH regulation, and restoration of water and electrolytic ions. The procedure for generation of kidney organoids ex vivo, containing functional nephrons, has been identified for human. These ex vivo kidney organoids are similar to fetal first-trimester kidneys for their structure and physiology. Such kidney organoids can serve as model for nephrotoxicity screening of drugs, disease modelling, and organ transplantation. However generation of fully functional kidneys is a far seen event with today's scientific technologies [114]. Loss of neurons in age-related macular degeneration (ARMD) is the common cause of blindness. At preclinical level, transplantation of iPSCs derived neuronal progenitor cells (NPCs) in rat limits progression of disease through generation of 5-6 layers of photoreceptor nuclei, restoring visual acuity [78]. The various approaches of iPSCs mediated retinal regeneration including ARMD have been reviewed elsewhere [79]. Placenta, the cordial connection between mother and developing fetus, gets degenerated in certain pathophysiological conditions. Nuclear programming of OCT4 knock-out (KO) and wild type (WT) mice fibroblast through transient expression of GATA3, EOMES, TFAP2C, and +/ cMYC generates transgene independent trophoblast stem-like cells (iTSCs), which are highly similar to blastocyst derived TSCs for DNA methylation, H3K7ac, nucleosome deposition of H2A.X, and other epigenetic markings. Chimeric differentiation of iTSCs specifically gives rise to haemorrhagic lineages and placental tissue, bypassing pluripotency phase, opening an avenue for generation of fully functional placenta for human [115]. Neurodegenerative disease like Alzheimer's and obstinate epilepsies can degenerate cerebrum, controlling excitatory and inhibitory signals of the brain. The inhibitory tones in cerebral cortex and hippocampus are accounted by -amino butyric acid secreting (GABAergic) interneurons (INs). Loss of these neurons often leads to progressive neurodegeneration. Genomic integration of Ascl1, Dlx5, Foxg1, and Lhx6 to mice and human fibroblast transforms these adult cells into GABAergic-INs (iGABA-INs). These cells have molecular signature of telencephalic INs, release GABA, and show inhibition to host granule neuronal activity [81]. Transplantation of these INs in developing embryo cures from genetic and acquired seizures, where transplanted cells disperse and mature into functional neuronal circuits as local INs [82]. Dorsomorphin and SB-431542 mediated inhibition of TGF- and BMP signalling direct transformation of human iPSCs into cortical spheroids. These cortical spheroids consisted of both peripheral and cortical neurons, surrounded by astrocytes, displaying transcription profiling and electrophysiology similarity with developing fetal brain and mature neurons, respectively [83]. The underlying complex biology and lack of clear etiology and genetic reprogramming and difficulty in recapitulation of brain development have barred understanding of pathophysiology of autism spectrum disorder (ASD) and schizophrenia. 3D organoid cultures of ASD patient derived iPSC generate miniature brain organoid, resembling fetal brain few months after gestation. The idiopathic conditions of these organoids are similar with brain of ASD patients; both possess higher inhibitory GABAergic neurons with imbalanced neuronal connection. Furthermore these organoids express forkhead Box G1 (FOXG1) much higher than normal brain tissue, which explains that FOXG1 might be the leading cause of ASD [84]. Degeneration of other organs and tissues also has been reported, like degeneration of lungs which might occur due to tuberculosis infection, fibrosis, and cancer. The underlying etiology for lung degeneration can be explained through organoid culture. Coaxing of iPSC into inert biomaterial and defined culture leads to formation of lung organoids that consisted of epithelial and mesenchymal cells, which can survive in culture for months. These organoids are miniature lung, resemble tissues of large airways and alveoli, and can be used for lung developmental studies and screening of antituberculosis and anticancer drugs [87]. The conventional multistep reprogramming for iPSCs consumes months of time, while CRISPER-Cas9 system based episomal reprogramming system that combines two steps together enables generation of ESCs-like cells in less than twowks, reducing the chances of culture associated genetic abrasions and unwanted epigenetic [80]. This approach can yield single step ESCs-like cells in more personalized way from adults with retinal degradation and infants with severe immunodeficiency, involving correction for genetic mutation of OCT4 and DNMT3B [80]. The iPSCs expressing anti-CCR5-RNA, which can be differentiated into HIV1 resistant macrophages, have applications in AIDS therapeutics [88]. The diversified immunotherapeutic application of iPSCs has been reviewed elsewhere [89]. The -1 antitrypsin deficiency (A1AD) encoded by serpin peptidase inhibitor clade A member 1 (SERPINA1) protein synthesized in liver protects lungs from neutrophils elastase, the enzyme causing disruption of lungs connective tissue. A1AD deficiency is common cause of both lung and liver disease like chronic obstructive pulmonary disease (COPD) and liver cirrhosis. Patient specific iPSCs from lung and liver cells might explain pathophysiology of A1AD deficiency. COPD patient derived iPSCs show sensitivity to toxic drugs which explains that actual patient might be sensitive in similar fashion. It is known that A1AD deficiency is caused by single base pair mutation and correction of this mutation fixes the A1AD deficiency in hepatic-iPSCs [85]. The high order brain functions, like emotions, anxiety, sleep, depression, appetite, breathing heartbeats, and so forth, are regulated by serotonin neurons. Generation of serotonin neurons occurs prior to birth, which are postmitotic in their nature. Any sort of developmental defect and degeneration of serotonin neurons might lead to neuronal disorders like bipolar disorder, depression, and schizophrenia-like psychiatric conditions. Manipulation of Wnt signalling in human iPSCs in defined culture conditions leads to an in vitro differentiation of iPSCs to serotonin-like neurons. These iPSCs-neurons primarily localize to rhombomere 2-3 segment of rostral raphe nucleus, exhibit electrophysiological properties similar to serotonin neurons, express hydroxylase 2, the developmental marker, and release serotonin in dose and time dependent manner. Transplantation of these neurons might cure from schizophrenia, bipolar disorder, and other neuropathological conditions [116]. The iPSCs technology mediated somatic cell reprogramming of ventricular monocytes results in generation of cells, similar in morphology and functionality with PCs. SA note transplantation of PCs to large animals improves rhythmic heart functions. Pacemaker needs very reliable and robust performance so understanding of transformation process and site of transplantation are the critical aspect for therapeutic validation of iPSCs derived PCs [28]. Diabetes is a major health concern in modern world, and generation of -cells from adult tissue is challenging. Direct reprogramming of skin cells into pancreatic cells, bypassing pluripotency phase, can yield clinical grade -cells. This reprogramming strategy involves transformation of skin cells into definitive endodermal progenitors (cDE) and foregut like progenitor cells (cPF) intermediates and subsequent in vitro expansion of these intermediates to become pancreatic -cells (cPB). The first step is chemically complex and can be understood as nonepisomal reprogramming on day one with pluripotency factors (OCT4, SOX2, KLF4, and hair pin RNA against p53), then supplementation with GFs and chemical supplements on day seven (EGF, bFGF, CHIR, NECA, NaB, Par, and RG), and two weeks later (Activin-A, CHIR, NECA, NaB, and RG) yielding DE and cPF [86]. Transplantation of cPB yields into glucose stimulated secretion of insulin in diabetic mice defines that such cells can be explored for treatment of T1DM and T2DM in more personalized manner [86]. iPSCs represent underrated opportunities for drug industries and clinical research laboratories for development of therapeutics, but safety concerns might limit transplantation applications (; ) [117]. Transplantation of human iPSCs into mice gastrula leads to colonization and differentiation of cells into three germ layers, evidenced with clinical developmental fat measurements. The acceptance of human iPSCs by mice gastrula suggests that correct timing and appropriate reprogramming regime might delimit human mice species barrier. Using this fact of species barrier, generation of human organs in closely associated primates might be possible, which can be used for treatment of genetic factors governed disease at embryo level itself [118]. In summary, iPSCs are safe and effective for treatment of regenerative medicine.

The unstable growth of human population threatens the existence of wildlife, through overexploitation of natural habitats and illegal killing of wild animals, leading many species to face the fate of being endangered and go for extinction. For wildlife conservation, the concept of creation of frozen zoo involves preservation of gene pool and germ plasm from threatened and endangered species (). The frozen zoo tissue samples collection from dead or live animal can be DNA, sperms, eggs, embryos, gonads, skin, or any other tissue of the body [119]. Preserved tissue can be reprogrammed or transdifferentiated to become other types of tissues and cells, which opens an avenue for conservation of endangered species and resurrection of life (). The gonadal tissue from young individuals harbouring immature tissue can be matured in vivo and ex vivo for generation of functional gametes. Transplantation of SSCs to testis of male from the same different species can give rise to spermatozoa of donor cells [120], which might be used for IVF based captive breeding of wild animals. The most dangerous fact in wildlife conservation is low genetic diversity, too few reproductively capable animals which cannot maintain adequate genetic diversity in wild or captivity. Using the edge of iPSC technology, pluripotent stem cells can be generated from skin cells. For endangered drill, Mandrillus leucophaeus, and nearly extinct white rhinoceros, Ceratotherium simum cottoni, iPSC has been generated in 2011 [121]. The endangered animal drill (Mandrillus leucophaeus) is genetically very close to human and often suffers from diabetes, while rhinos are genetically far removed from other primates. The progress in iPSCs, from the human point of view, might be transformed for animal research for recapturing reproductive potential and health in wild animals. However, stem cells based interventions in wild animals are much more complex than classical conservation planning and biomedical research has to face. Conversion of iPSC into egg or sperm can open the door for generation of IVF based embryo; those might be transplanted in womb of live counterparts for propagation of population. Recently, iPSCs have been generated for snow leopard (Panthera uncia), native to mountain ranges of central Asia, which belongs to cat family; this breakthrough has raised the possibilities for cryopreservation of genetic material for future cloning and other assisted reproductive technology (ART) applications, for the conservation of cat species and biodiversity. Generation of leopard iPSCs has been achieved through retroviral-system based genomic integration of OCT4, SOX2, KLF4, cMYC, and NANOG. These iPSCs from snow leopard also open an avenue for further transformation of iPSCs into gametes [122]. The in vivo maturation of grafted tissue depends both on age and on hormonal status of donor tissue. These facts are equally applicable to accepting host. Ectopic xenografts of cryopreserved testis tissue from Indian spotted deer (Moschiola indica) to nude mice yielded generation of spermatocytes [123], suggesting that one-day procurement of functional sperm from premature tissue might become a general technique in wildlife conservation. In summary, tissue biopsies from dead or live animals can be used for generation of iPSCs and functional gametes; those can be used in assisted reproductive technology (ART) for wildlife conservation.

The spectacular progress in the field of stem cells research represents great scope of stem cells regenerative therapeutics. It can be estimated that by 2020 or so we will be able to produce wide array of tissue, organoid, and organs from adult stem cells. Inductions of pluripotency phenotypes in terminally differentiated adult cells have better therapeutic future than ESCs, due to least ethical constraints with adult cells. In the coming future, there might be new pharmaceutical compounds; those can activate tissue specific stem cells, promote stem cells to migrate to the side of tissue injury, and promote their differentiation to tissue specific cells. Except few countries, the ongoing financial and ethical hindrance on ESCs application in regenerative medicine have more chance for funding agencies to distribute funding for the least risky projects on UCSCs, BMSCs, and TSPSCs from biopsies. The existing stem cells therapeutics advancements are more experimental and high in cost; due to that application on broad scale is not feasible in current scenario. In the near future, the advancements of medical science presume using stem cells to treat cancer, muscles damage, autoimmune disease, and spinal cord injuries among a number of impairments and diseases. It is expected that stem cells therapies will bring considerable benefits to the patients suffering from wide range of injuries and disease. There is high optimism for use of BMSCs, TSPSCs, and iPSCs for treatment of various diseases to overcome the contradictions associated with ESCs. For advancement of translational application of stem cells, there is a need of clinical trials, which needs funding rejoinder from both public and private organizations. The critical evaluation of regulatory guidelines at each phase of clinical trial is a must to comprehend the success and efficacy in time frame.

Dr. Anuradha Reddy from Centre for Cellular and Molecular Biology Hyderabad and Mrs. Sarita Kumari from Department of Yoga Science, BU, Bhopal, India, are acknowledged for their critical suggestions and comments on paper.

There are no competing interests associated with this paper.

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Stem Cells Applications in Regenerative Medicine and ...

Curr Cardiol Rev. 2013 Feb; 9(1): 6372.

1Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA

2Stem Cell Institute, University of Minnesota Medical School, Minneapolis, Minnesota, USA

1Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA

1Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA

2Stem Cell Institute, University of Minnesota Medical School, Minneapolis, Minnesota, USA

3Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, USA

1Department of Medicine, University of Minnesota Medical School, Minneapolis, Minnesota, USA

2Stem Cell Institute, University of Minnesota Medical School, Minneapolis, Minnesota, USA

3Department of Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota, USA

Received 2012 Jun 11; Revised 2012 Jul 31; Accepted 2012 Aug 27.

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 . 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 ).

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 ). Besides mouse and human somatic cells, iPS cells from other species have been successfully generated (Table ).

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 ). 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 ). 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 ...

Researchers have been looking for something that can help the body heal itself. Although studies are ongoing, stem cell research brings this notion of regenerative medicine a step closer. However, many of its ideas and concepts remain controversial. So, what are stem cells, and why are they so important?

Stem cells are cells that can develop into other types of cells. For example, they can become muscle or brain cells. They can also renew themselves by dividing, even after they have been inactive for a long time.

Stem cell research is helping scientists understand how an organism develops from a single cell and how healthy cells could be useful in replacing cells that are not working correctly in people and animals.

Researchers are now studying stem cells to see if they could help treat a variety of conditions that impact different body systems and parts.

This article looks at types of stem cells, their potential uses, and some ethical concerns about their use.

The human body requires many different types of cells to function, but it does not produce every cell type fully formed and ready to use.

Scientists call a stem cell an undifferentiated cell because it can become any cell. In contrast, a blood cell, for example, is a differentiated cell because it has already formed into a specific kind of cell.

The sections below look at some types of stem cells in more detail.

Scientists extract embryonic stem cells from unused embryos left over from in vitro fertilization procedures. They do this by taking the cells from the embryos at the blastocyst stage, which is the phase in development before the embryo implants in the uterus.

These cells are undifferentiated cells that divide and replicate. However, they are also able to differentiate into specific types of cells.

There are two main types of adult stem cells: those in developed bodily tissues and induced pluripotent stem (iPS) cells.

Developed bodily tissues such as organs, muscles, skin, and bone include some stem cells. These cells can typically become differentiated cells based on where they exist. For example, a brain stem cell can only become a brain cell.

On the other hand, scientists manipulate iPS cells to make them behave more like embryonic stem cells for use in regenerative medicine. After collecting the stem cells, scientists usually store them in liquid nitrogen for future use. However, researchers have not yet been able to turn these cells into any kind of bodily cell.

Scientists are researching how to use stem cells to regenerate or treat the human body.

The list of conditions that stem cell therapy could help treat may be endless. Among other things, it could include conditions such as Alzheimers disease, heart disease, diabetes, and rheumatoid arthritis. Doctors may also be able to use stem cells to treat injuries in the spinal cord or other parts of the body.

They may do this in several ways, including the following.

In some tissues, stem cells play an essential role in regeneration, as they can divide easily to replace dead cells. Scientists believe that knowing how stem cells work can help treat damaged tissue.

For instance, if someones heart contains damaged tissue, doctors might be able to stimulate healthy tissue to grow by transplanting laboratory-grown stem cells into the persons heart. This could cause the heart tissue to renew itself.

One study suggested that people with heart failure showed some improvement 2 years after a single-dose administration of stem cell therapy. However, the effect of stem cell therapy on the heart is still not fully clear, and research is still ongoing.

Another investigation suggested that stem cell therapies could be the basis of personalized diabetes treatment. In mice and laboratory-grown cultures, researchers successfully produced insulin-secreting cells from stem cells derived from the skin of people with type 1 diabetes.

Study author Jeffrey R. Millman an assistant professor of medicine and biomedical engineering at the Washington University School of Medicine in St. Louis, MO said, What were envisioning is an outpatient procedure in which some sort of device filled with the cells would be placed just beneath the skin.

Millman hopes that these stem cell-derived beta cells could be ready for research in humans within 35 years.

Stem cells could also have vast potential in developing other new therapies.

Another way that scientists could use stem cells is in developing and testing new drugs.

The type of stem cell that scientists commonly use for this purpose is the iPS cell. These are cells that have already undergone differentiation but which scientists have genetically reprogrammed using genetic manipulation, sometimes using viruses.

In theory, this allows iPS cells to divide and become any cell. In this way, they could act like undifferentiated stem cells.

For example, scientists want to grow differentiated cells from iPS cells to resemble cancer cells and use them to test anticancer drugs. This could be possible because conditions such as cancer, as well as some congenital disabilities, happen because cells divide abnormally.

However, more research is taking place to determine whether or not scientists really can turn iPS cells into any kind of differentiated cell and how they can use this process to help treat these conditions.

In recent years, clinics have opened that offer different types of stem cell treatments. One 2016 study counted 570 of these clinics in the United States alone. They appear to offer stem cell-based therapies for conditions ranging from sports injuries to cancer.

However, most stem cell therapies are still theoretical rather than evidence-based. For example, researchers are studying how to use stem cells from amniotic fluid which experts can save after an amniocentesis test to treat various conditions.

The Food and Drug Administration (FDA) does allow clinics to inject people with their own stem cells as long as the cells are intended to perform only their normal function.

Aside from that, however, the FDA has only approved the use of blood-forming stem cells known as hematopoietic progenitor cells. Doctors derive these from umbilical cord blood and use them to treat conditions that affect the production of blood. Currently, for example, a doctor can preserve blood from an umbilical cord after a babys birth to save for this purpose in the future.

The FDA lists specific approved stem cell products, such as cord blood, and the medical facilities that use them on its website. It also warns people to be wary of undergoing any unproven treatments because very few stem cell treatments have actually reached the earliest phase of a clinical trial.

Historically, the use of stem cells in medical research has been controversial. This is because when the therapeutic use of stem cells first came to the publics attention in the late 1990s, scientists were only deriving human stem cells from embryos.

Many people disagree with using human embryonic cells for medical research because extracting them means destroying the embryo. This creates complex issues, as people have different beliefs about what constitutes the start of human life.

For some people, life starts when a baby is born, while for others, it starts when an embryo develops into a fetus. Meanwhile, other people believe that human life begins at conception, so an embryo has the same moral status and rights as a human child.

Former U.S. president George W. Bush had strong antiabortion views. He believed that an embryo should be considered a life and not be used for scientific experiments. Bush banned government funding for human stem cell research in 2001, but former U.S. president Barack Obama then revoked this order. Former U.S. president Donald Trump and current U.S. president Joe Biden have also gone back and forth with legislation on this.

However, by 2006, researchers had already started using iPS cells. Scientists do not derive these stem cells from embryonic stem cells. As a result, this technique does not have the same ethical concerns. With this and other recent advances in stem cell technology, attitudes toward stem cell research are slowly beginning to change.

However, other concerns related to using iPS cells still exist. This includes ensuring that donors of biological material give proper consent to have iPS cells extracted and carefully designing any clinical studies.

Researchers also have some concerns that manipulating these cells as part of stem cell therapy could lead to the growth of cancerous tumors.

Although scientists need to do much more research before stem cell therapies can become part of regular medical practice, the science around stem cells is developing all the time.

Scientists still conduct embryonic stem cell research, but research into iPS cells could help reduce some of the ethical concerns around regenerative medicine. This could lead to much more personalized treatment for many conditions and the ability to regenerate parts of the human body.

Learn more about stem cells, where they come from, and their possible uses here.

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Stem cells: Therapy, controversy, and research

Introduction

Extracellular vesicles (EVs) including exosomes, microvesicles, and apoptotic bodies are produced and released by almost all types of cell. EVs vary in size, properties, and secretion pathway depending on the originating cell.1,2 Exosomes are small EVs (sEVs) which are formed by a process of inward budding in early endosomes to form multivesicular bodies (MVBs) with an average size of 100 nm, and released into the extracellular microenvironment to transfer their components.3,4 Microvesicles are composed of lipid components of the plasma membrane and their sizes range from 1001000 nm, whereas apoptotic bodies result from programmed cell death.5 Initially, EVs were considered to maintain cellular waste through release of unwanted proteins and biomolecules; later, these organelles were considered important for intercellular communications through various cargo molecules such as lipids, proteins, DNA, RNA, and microRNAs (miRNAs).6 Previously, it was suggested that EVs play a critical role in normal cells to maintain homeostasis and prevent cancer initiation. Inhibition of EVs secretion causes accumulation of nuclear DNA in the cytoplasm, leading to apoptosis.1 The induction of apoptosis is the principal event of the reactive oxygen species (ROS) dependent DNA damage response.7,8

Several studies reported that exosomes are synthesized by means of two major pathways, the endosomal sorting complexes required for transport (ESCRT)-dependent and ESCRT-independent, and the processes are highly regulated by multiple signal transduction cascades.18 Exosomes released from the cell through normal exocytosis mechanisms are characterized by vesicular docking and fusion with the aid of SNARE complexes. Exosomes are considered to be organelle responsible for garbage disposal agents. However, at a later stage, these secretory bodies play a critical role in maintaining the physiological and pathological conditions of the surrounding cells by transferring information from donor cells to recipient cells. Exosome development begins with endocytosis to form early endosomes, later forming multivesicular endosomes (MVEs), and finally generating late endosomes by inward budding. MVEs merge with the cell membrane and release intraluminal endosomal vesicles that become exosomes into the extracellular space.9,10 Exosome biogenesis is dependent on various critical factors including the site of biogenesis, protein sorting, physicochemical aspects, and transacting mediators.11

Exosomes contain various types of cargo molecules including lipids, proteins, DNAs, mRNAs, and miRNAs. Most of the cargo is involved in the biogenesis and transportation ability of exosomes.12,13 Exosomes are released by fusion of MVBs with the cell membrane via activation of Rab-GTPases and SNAREs. Exosomes are abundant and can be isolated from a wide variety of body fluids and also cell culture medium.14 Exosomes contain tetraspanins that are responsible for cell penetration, invasion, and fusion events. Exosomes are released onto the external surface by the MVB formation proteins Alix and TSG101. Exosome-bound proteins, annexins and Rab protein, govern membrane transport and fusion whereas Alix, flotillin, and TSG101 are involved in exosome biogenesis.15,16 Exosomes contain various types of proteins such as integral exosomal membrane proteins, lipid-anchored outer and inner membrane proteins, peripheral surface and inner membrane proteins, exosomal enzymes, and soluble proteins that play critical roles in exosome functions.11

The functions of exosomes depend on the origin of the cell/tissue, and are involved in the immune response, antigen presentation programmed cell death, angiogenesis, inflammation, coagulation, and morphogen transporters in the creation of polarity during development and differentiation.1720 Ferguson and Nguyen reported that the unique functions of exosomes depend on the availability of unique and specific proteins and also the type of cell.21 All of these categories influence cellular aspects of proteins such as the cell junction, chaperones, the cytoskeleton, membrane trafficking, structure, and transmembrane receptor/regulatory adaptor proteins. The role of exosomes has been explored in different pathophysiological conditions including metabolic diseases. Exosomes are extremely useful in cancer biology for the early detection of cancer, which could increase prognosis and survival. For example, the presence of CD24, EDIL3, and fibronectin proteins on circulating exosomes has been proposed as a marker of early-stage breast cancer.22 Cancer-derived exosomes promoted tumor growth by directly activating the signaling pathways such as P13K/AKT or MAPK/ERK.23 Tumor-derived exosomes are significantly involved in the immune system, particularly stimulating the immune response against cancer and delivering tumor antigens to dendric cells to produce exosomes, which in turn stimulates the T-cell-mediated antitumor immune response.24 Exosomal surface proteins are involved in immunotherapies through the regulation of the tumor immune microenvironment by aberrant cancer signaling.25 A study demonstrated that exosomes have the potential to affect health and pathology of cells through contents of the vesicle.26 Exosomes derived from mesenchymal stem cells exhibit protective effects in stroke models following neural injury resulting from middle cerebral artery occlusion.27 The structural region of the exosome facilitate the release of misfolded and prion proteins, and are also involved in the propagation of neurodegenerative diseases such as Huntington disease, Alzheimers disease (AD), and Parkinsons disease (PD).28,29

Exosomes serve as novel intercellular communicators due to their cell-specific cargo of proteins, lipids, and nucleic acids. In addition, exosomes released from parental cells may interact with target cells, and it can influence cell behavior and phenotype features30 and also it mediate the horizontal transfer of genetic material via interaction of surface adhesion proteins.31 Exosomes are potentially serving as biomarkers due to the wide-spread and cell-specific availability of exosomes in almost all body fluids.13 Therefore, exosomes are exhibited as delivery vehicles for the efficient delivery of biological therapeutics across different biological barriers to target cells.3234

In this review, first, we comprehensively describe the factors involved in exosome biogenesis and the role of exosomes in intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases. In addition, we discuss the role of exosomes as diagnostic markers, and the therapeutic and clinical implications. Finally, we discuss the challenges and outstanding developments in exosome research.

The extracellular vesicles play critical role in inter cellular communication by serving as vehicles for transfer of biomolecules. These vesicles are generally classified into microvesicles, ectosomes, shedding vesicles, or microparticles. MVs bud directly from the plasma membrane, whereas exosomes are represented by small vesicles of different sizes that are formed as the ILV by budding into early endosomes and MVBs and are released by fusion of MVBs with the plasma membrane (Figure 1). Invagination of late endosomal membranes results in the formation of intraluminal vesicles (ILVs) within large MVBs.35 Biogenesis of exosomes occurs in three ways including vesicle budding into discrete endosomes that mature into multivesicular bodies, which release exosomes upon plasma membrane fusion; direct vesicle budding from the plasma membrane; and delayed release by budding at intracellular plasma membrane-connected compartments (IPMCs) followed by deconstruction of IPMC neck(s).11 The mechanisms of biogenesis of exosomes are governed by various types of proteins including the ESCRT proteins Hrs, CHMP4, TSG101, STAM1, VPS4, and other proteins such as the Syndecan-syntenin-ALIX complex, nSMase2, PLD2, and CD9.14,3639 After formation, the MVB can either fuse with the lysosome to degrade its content or fuse with the plasma membrane to release the ILVs as exosomes. The release of exosomes to the extracellular milieu is driven by proteins of the Rab-GTPase family including RAB2B, 5A, 7, 9A, 11, 27, and 35. SNARE family proteins VAMP7 and YKT6 have also been implicated in the release.14,38,4042 Biogenesis of exosomes is influenced by several external factors including cell type, cell confluency, serum conditions, and the presence and absence of cytokines and growth factors. In addition, biogenesis is also regulated by the sites of exosomes, protein sorting, physico-chemical aspects, and trans-acting mediators (Figure 2). For example, THP-1 cells were cultured in RPMI-1640 cell culture medium supplemented with 10% FCS secreted low level of exosomes compared to cells grown on cell culture medium supplemented with 1% FCS (Figure 3). The exogenous factor like serum starvation influences biogenesis and secretion of exosomes.

Figure 1 Biogenesis and cargoes of exosomes.

Figure 2 Effect of various factors on biogenesis of exosomes.

Figure 3 Serum deprivation causes an increase of the number of cellular exosomes in THP-1 cells. Panel (A); 10% FCS. Panel (B); 1% FCS. Panel (C) Quantification of exosomes using DLS and NTA.

Exosome release depends on expression of Rab27 or Ral. For example, exosomes released from the MVB significantly decrease in cells depleted of Rab2741 or Ral.43 The most efficient EV-producing cell types have yet to be determined44 and few reports suggest that immature dendritic cells produce limited amounts of EVs45,46 whereas mesenchymal stem cells secrete vast amounts, relevant for the production of EV therapeutics on a clinical scale.47,48 A few proteins play a critical role in the biogenesis of EVs, such as Rab27a and Rab27b.49 Over expression of Rab27a and Rab27b produce significant amounts of EVs in cancer cells. For example, overexpression of Rab27a and Rab27b in breast cancer cells,50 hepatocellular carcinoma cells,51 glioma cells,52 and pancreas cancer cells53 produces significant levels of EVs. Although all types of cells secrete and release EVs, cancer cells seem to produce higher levels than normal cells.54 Furthermore, the presence of invadopodia that are docking sites for Rab27a-positive MVBs induces secretion of EVs, and also enhances secretion of EVs in cancer cells.55 Thus, inhibition of invadopodia formation greatly reduces exosome secretion into conditioned media. This evidence demonstrates that cancer cells potentially release more EVs than non-cancer cells.

The rate of origin of exosomes from the plasma membrane of stem cells is vigorous, at rates equal to the production of exosomes,56 which is consistent with a report suggesting that stem cells bud ~50100 nm-diameter vesicles directly from the plasma membrane.57 Plasma membrane-derived exosomes contain selectively enriched protein and lipid markers in leukocytes.58 Plasma membrane exosomal budding is also observed for glioblastoma exosomes.59 Conventional transmission electron microscopy revealed that certain cell types contain deep invaginations of the plasma membrane that are indistinguishable from MVBs.6062 Certain cell types secrete exosomes containing cargo proteins, which primarily bud from the plasma membrane, and exosome composition is determined predominantly by intracellular protein trafficking pathways, rather than by the distinct mechanisms of exosome biogenesis.63 Biogenesis of exosomes is regulated by syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin. Syntenin interacts directly with ALIX through LYPX (n) L motifs.64 Glycosylation is an essential factor in the biogenesis of exosomes and N-linked glycosylation directs glycoprotein sorting into EMVs.65 Collectively, these reports suggest that exosomes are made at both plasma and endosome membranes rather than endosome alone. Oligomerization is a critical factor for exosomal protein sorting and it was found to be sufficient to target plasma membrane proteins to exosomes. High-order oligomeric proteins target them to exosomes.66 Further, plasma membrane anchors support exosomal protein budding. For example, budding of CD63 and CD9 from the plasma membrane is much more efficient than endosome-targeted budding of CD63 and CD9.63 Protein clustering is another factor that induces membrane scission.67

Physico-chemical properties determine budding efficiency and are crucial factors of exosome biogenesis, a fundamental process involving the budding of vesicles that are 30200 nm in size. In particular, lipids are critical players in exosome biogenesis, especially those able to form cone and inverse cone shapes. Generally, exosome membranes contain phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositols (PIs), phosphatidic acid (PA), cholesterol, ceramides, sphingomyelin, glycosphingolipids, and a number of lower abundance lipids.68,69 Exosomes have a rich content of PE and PS, which increase budding efficiency and promote exosome genesis and release. PA promotes exosome biogenesis and PLD2 is involved in the budding of certain exosomal cargoes.70 Besides these factors, ceramide is an important lipid molecule regulating exosome biogenesis and facilitating membrane curvature, which is essential for vesicular budding. Inhibition of an enzyme that generates ceramide impairs exosome biogenesis.71

The next critical factor is trans-acting mediators that are involved in the biogenesis of exosomes through regulating plasma membrane homeostasis, intracellular protein trafficking pathways, MVB maturation and trafficking, IPMC biogenesis, vesicle budding, and scission.11 For example, Rab proteins regulate exosome biogenesis via endosomes and the plasma membrane by determining organelle membrane identity, recruiting mechanistic effectors, and mediating organelle dynamics.72 The functions of Rab proteins in the control and biogenesis of exosomes depends on cell type. MVB biogenesis is regulated by Rab27a, Rab27b, their effectors Slp4, Slac2b, and Munc13-4, and also Rab 35 and Rab 11.73 Loss of Rab27 function leads to a ~5075% drop in exosome production, and is also involved in assembling the plasma membrane microdomains involved in plasma membrane vesicle budding, by regulating plasma membrane PIP2 dynamics.74 Overall, Rab27 proteins control exosome biogenesis at both endosomes and plasma membranes. In addition, Rab35 also contributes to exosome biogenesis by regulating PIP2 levels of plasma membrane, and its loss leads to a reduction of exosome release by ~50%.75 Gurunathan et al76 reported that yeast produces two classes of secretory vesicles, low density and high density, and dynamin and clathrin are required for the biogenesis of these two different types of vesicle.

The Ral family is involved in the biogenesis of exosomes, and inhibition of Ral causes an accumulation of MVBs near the plasma membrane and a ~50% decrease in the vesicular secretion of exosomes and exosomal marker proteins.43 Ral GTPases function through various effectors proteins, including Arf6 and the phospholipase PLD2, which are involved in exosomal release of SDCs.37 The ESCRT complex machinery (0 through III) are involved in MVB biogenesis on a major level including membrane deformation, sealing, and repair during a wide array of processes. The major contributions of the ESCRT complex to the biogenesis of vesicles are the recognition and sequestration of ubiquitinated proteins to specific domains of the endosomal membrane via ubiquitin binding subunits of ESCRT-0. After interaction with the ESCRT-I and -II complexes, the total complex will then combine with ESCRT-III, a protein complex that is involved in promoting the budding process. Finally, following cleaving of the buds to form ILVs, the ESCRT-III complex separates from the MVB membrane using energy supplied by the sorting protein Vps4.77 In addition, other proteins such as Alix, which is associated with several ESCRT (TSG101 and CHMP4) proteins, are involved in endosomal membrane budding and abscission, as well as exosomal cargo selection via interaction with syndecan.39 Another important factor, autophagy, is critically involved in exosome secretion. Autophagy related (Atg) proteins coordinate initiation, nucleation, and elongation during autophagosome biogenesis in the presence of ESCRT-III components including CHMP2A and VPS4. For instance, the absence of Atg5 in cancer cells causes a reduction in exosome production.78 Conversely, CRISPR/Cas9-mediated knockout of Atg5 in neuronal cells increases the release of exosomes and exosome-associated prions from neuronal cells.79

Exosomes play a critical role in the physiologic regulation of mammary gland development and are important mediators of breast tumorigenesis.80 Biogenesis of exosomes occurs in all cell types; however, production depends on cell type. For example, breast cancer cells (BCC) produce increased numbers of exosomes compared to normal mammary epithelial cells. Studies revealed that patients with BC have increased numbers of MVs in their blood.81 Kavanagh et al reported that several fold changes were observed from exosomes isolated from triple negative breast cancer (TNBC) chemoresistant therapeutic induced senescent (TIS) cells compared with control EVs.82 TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, and apoptosis, and exhibit the senescence-associated secretory phenotype (SASP). Cannabidiol (CBD), inhibits exosome and microvesicle (EMV) release in three different types of cancer cells including prostate cancer (PC3), hepatocellular carcinoma (HEPG2), and breast adenocarcinoma (MDA-MB-231). All three different cell lines show variability in the release of exosomes in a dose-dependent manner. These variabilities are all due to mitochondrial function, including modulation of STAT3 and prohibitin expression. This study suggests that the anticancer agent CBD plays critical role in EMV biogenesis.83 Sulfisoxazole (SFX) inhibits sEV secretion from breast cancer cells through interference with endothelin receptor A (ETA) through the reduced expression of proteins involved in the biogenesis and secretion of sEV, and triggers co-localization of multivesicular endosomes with lysosomes for degradation.84 Secreted EVs from human colorectal cancer cells contain 957 vesicular proteins. The direct protein interactions between cellular proteins play a critical role in protein sorting during EV formation. SRC signaling plays a major role in EV biogenesis, and inhibition of SRC kinase decreases the intracellular biogenesis and cell surface release of EVs.85 Proteomic analysis revealed that the exosomes released from imatinib-sensitive GIST882 cell line exhibit 764 proteins. The authors found that significant amount of proteins belong to protein release function and involved in the classical pathway and overlap to a high degree with proteins of exosomal origin.86 Exosomes secreted by antigen-presenting cells contain high levels of MHC class II proteins and costimulatory proteins, whereas exosomes released from other cell types lack these proteins.1,87

The biogenesis of exosomes depends on a percentage of confluency of approximately 6090%, which influences the yield and functions of EVs.44 Gal et al88 observed a 10-fold decreased level of cholesterol metabolism in confluent cell cultures compared to cells in the preconfluent state. The high level of cholesterol content in confluent cells leads to a decreased level of EVs in prostate cancer.68 The major reason behind for the reduced level of vesicle production is contact inhibition, which triggers confluent cells to enter quiescence and/or alters their characteristics compared to actively dividing cells.89,90 Exogenous stimulation could influence the condition of the cells including the phenotype and efficacy of secretion. Previously, several studies demonstrated that various external factors increase biogenesis of EVs such as Ca2+ ionophores,91 hypoxia,9294 and detachment of cells,95 whereas lipopolysaccharide reduces biogenesis and release of EVs.96 Furthermore, serum, which supports adherence of the cells, plays a critical role in the biogenesis of EVs.97 For example, FCS has noticeable effects on cultured cells; however, the effects depend on cell type and differentiation status.97,98 To avoid the immense amounts of vesicles present in FCS, the use of conditioned media has been suggested. Culture viability and health status of cells are important aspects for producing an adequate amount of vesicles with proper cargo molecules such as protein and RNA.99,100 Exogenous stress, such as starvation, can induce phenotypic alterations and changes in proliferation. These changes cause alterations in the cells metabolism and eventually lead to low yields.101,102

Cellular stresses, such as hypoxia, inflammation, and hyperglycemia, influence the RNA and protein content in exosomes. To examine these factors, the effects of cellular stresses on endothelial cells were studied.99 Endothelial cells were exposed to different types of cellular stress such as hypoxia, tumor necrosis factor- (TNF-)-induced activation, and high glucose and mannose concentrations. The mRNA and protein content of exosomes produced by these cells were compared using microarray analysis and a quantitative proteomics approach. The results indicated that endothelial cell-derived exosomes contain 1354 proteins and 1992 mRNAs. Several proteins and mRNAs showed altered levels after exposure of their producing cells to cellular stress. Interestingly, cells exposed to high sugar concentrations had altered exosome protein composition only to a minor extent, and exosome RNA composition was not affected. Low-intensity ultrasound-induced (LIUS) anti-inflammatory effects have been achieved by upregulation of extracellular vesicle/exosome biogenesis. These exosomes carry anti-inflammatory cytokines and anti-inflammatory microRNAs, which inhibit inflammation of target cells via multiple shared and specific pathways. A study suggested that exosome-mediated anti-inflammatory effects of LIUS are feasible and that these techniques are potential novel therapeutics for cancers, inflammatory disorders, tissue regeneration, and tissue repair.103 Another factor, called manumycin-A (MA), a natural microbial metabolite, was analyzed in exosome biogenesis and secretion in castration-resistant prostate cancer (CRPC) C4-2B, cells. The effect of MA on cell growth was observed, and the results revealed that there was no effect on cell growth. However, MA attenuated the ESCRT-0 proteins Hrs, ALIX, and Rab27a, and exosome biogenesis and secretion by CRPC cells. The inhibitory effect of MA on exosome biogenesis and secretion was primarily mediated via targeted inhibition of Ras/Raf/ERK1/2 signaling. These findings suggest that MA is a potential drug candidate for the suppression of exosome biogenesis and secretion by CRPC cells.104

Methods of isolation of exosomes play critical roles in functions and delivery. Although several methods such as ultracentrifugation, density gradient centrifugation, chromatography, filtration, polymer-based precipitation, and immunoaffinity have been adopted to isolate pure exosomes without contamination, there is still a lack of consistency and agreement.105 Isolation of exosomes along with non-exosomal materials and damaged exosomal membranes creates artifacts and alters the protein and RNA profiles. Since exosomes are obtained from a variety of sources, the composition of proteins/lipids influences the sedimentation properties and isolation. Thus, precise and consistent techniques are warranted for the isolation, purification, and application of exosomes.

Although several functions of exosomes have been explored, the precise function of exosomes remains a mystery. Historically, exosomes have been known to function as cellular garbage bags, recyclers of cell surface proteins, cellular signalers, intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases.106 (Figure 4) ECVs are secreted cell-derived membrane particles involved in intercellular signaling and cell-cell communications, and contain immense bioactive information. Most cell types produce exosomes and release these into the extracellular environment, circulating through different bodily fluids such as urine, blood, and saliva and transferring their cargo to recipient cells. These vesicles play a significant role in various pathological conditions, such as different types of cancer, neurodegenerative diseases, infectious diseases, pregnancy complications, obesity, and autoimmune diseases, as reviewed elsewhere.107 Exosomes play a significant role in intercellular communication between cells by interacting with target cells via endocytosis.108 More specifically, exosomes are involved in cancer development, survival and metastasis of tumors, drug resistance, remodeling of the extracellular matrix, angiogenesis, thrombosis, and proliferation of tumor cells.94,109111 Exosomes contribute significantly to tumor vascularization and hypoxia-mediated inter-tumor communication during cancer progression, and premetastatic niches, which are significant players in cancer.16,94,109,112 Exosomes derived from hepatic epithelial cells increase the expression of enhancer zeste homolog 2 (EZH2) and cyclin-D1, and subsequently promotes G1/S transition.113

Figure 4 Multifunctional aspects biological functions of exosomes.

Conventionally, cells communicate with adjacent cells through direct cell-cell contact through gap junctions and cell surface protein/protein interactions, whereas cells communicating with distant cells do so through secreted soluble factors, such as hormones and cytokines, to facilitate signal propagation.114 Cells also communicate through electrical and chemical signals.115 Several studies have suggested that exosomes play vital roles in intercellular communication by serving as vehicles for transferring various cellular constituents, such as proteins, lipids, and nucleic acids, between cells.6,116118 Exosomes function as exosomal shuttle RNAs in which some exosomal RNAs from donor cells functions in recipient cells,6 a form of genetic exchange. Recently, researchers found that cells communicating with other cells through exosomes carrying cell-specific cargoes of proteins, lipids, and nucleic acids may employ novel intercellular communication mechanisms.30 Exosomes exert influences through various mechanistic approaches, such as direct stimulation of target cells via surface-bound ligands; transfer of activated receptors to recipient cells; and epigenetic reprogramming of recipient cells.119,120 Exosomes play critical roles in immunoregulation, including antigen presentation, immune activation, immune suppression, and immune tolerance via exosome-mediated intercellular communication. Mesenchymal stem cell (MSC)-derived exosomes play significant roles in wound healing processes.121 Exosomes from platelet-rich plasma (PRP) inhibit the release of TNF-. PRP-Exos significantly decreases the apoptotic rate of osteoarthritis (OA) chondrocytes compared with activated PRP (PRP-As).122 Extracellular vesicle (ECV)-modified polyethylenimine (PEI) complexes enhance short interfering RNA (siRNA) delivery by forming non-covalent complexes with small RNA molecules, including siRNAs and anti-miRs, in both conditions, in vitro and in vivo.123 Non-GSC glioma cells were treated with GSC-released exosomes. The results showed that GSC-released exosomes increase proliferation, neurosphere formation, invasive capacities, and tumorigenicity of non-GSC glioma cells through the Notch1 signaling pathway and stemness-related protein expressions.124

Exosomal miR-1910-3p promotes proliferation and migration of breast cancer cells in vitro and in vivo through downregulation of myotubularin-related protein 3 and activation of the nuclear factor-B (NF-B) and wnt/-catenin signaling pathway, and promotes breast cancer progression.125 Human hepatic progenitor cell (CdH)-derived exosomes (EXOhCdHs) play a crucial role in maintaining cell viability and inhibit oxidative stress-induced cell death. Experimental evidence suggests that inhibition of exosome secretion treatment with GW4869 results in the acceleration of reactive oxygen species (ROS) production, thereby causing a decrease in cell viability.126 Tumor-derived EXs (TDEs) are vehicles that enable communication between cells by transferring bioactive molecules, also delivering oncogenic molecules and containing different molecular cargoes compared to EXs delivered from normal cells. They can therefore be used as non-invasive biomarkers for the early diagnosis and prognosis of most cancers, including breast and ovarian cancers.127 Exosomes released by ER-stressed HepG2 cells significantly enhance the expression levels of several cytokines, including IL-6, monocyte chemotactic protein-1, IL-10, and tumor necrosis factor- in macrophages. ER stress-associated exosomes mediate macrophage cytokine secretion in the liver cancer microenvironment, and also indicate the potential of treating liver cancer via an ER stress-exosomal-STAT3 pathway.128 Mesenchymal stem cell-derived exosomal miR-223 protects neuronal cells from apoptosis, enhances cell migration and increases miR-223 by targeting PTEN, thus activating the PI3K/Akt pathway. In addition, exosomes isolated from the serum of AD patients promote cell apoptosis through the PTEN-PI3K/Akt pathway and these studies indicate a potential therapeutic approach for AD.129 A mouse model of diabetes demonstrated that mesenchymal stromal cell-derived exosomes ameliorate peripheral neuropathy through increased nerve conduction velocity. In addition, MSC-derived exosomes substantially suppress proinflammatory cytokines.130

Exosomes derived from activated astrocytes promote microglial M2 phenotype transformation following traumatic brain injury (TBI). miR-873a-5p significantly inhibits LPS-induced microglial M1 phenotype transformation.131 Several studies reported that exosomes are involved in cancer progression and metastasis; however, this depends on the type of cells the exosomes were derived from. For example, human umbilical vein endothelial cells (HUVEC) were treated with exosomes derived from HeLa cells (ExoHeLa), and the expression of tight junctions (TJ) proteins, such as zonula occludens-1 (ZO-1) and Claudin-5, was significantly reduced compared with exosomes from human cervical epithelial cells. Thus, permeability of the endothelial monolayer was increased after the treatment with ExoHeLa. Mice studies have shown that injection of ExoHeLa into mice increased vascular permeability and tumor metastasis. The results from this study demonstrated that HeLa cell-derived exosomes promote metastasis by triggering ER stress in endothelial cells and break down endothelial integrity. Such effects of exosomes are microRNA-independent.132 Exosomes mediate the gene expression of target cells and regulate pathological and physiological processes including promoting angiogenesis, inhibiting ventricular remodeling and improving cardiac function, as well as inhibiting local inflammation and regulating the immune response. Accumulating evidence shows that exosomes possess therapeutic potential through their anti-apoptotic and anti-fibrotic roles.

The functions of exosomes in immune responses are well established and do not cause any severe immune responses. A mouse study demonstrated that administration of a low dose of mouse or human cell-derived exosomes for extended periods of time caused no severe immune reactions.133 The function of exosomes in immune regulation is regulated by the transfer and presentation of antigenic peptides. Exosomes contain antigen-presenting cells (APCs) carrying peptide MHC-II and costimulatory signals and directly present the peptide antigen to specific T cells to induce their activation.134 For example, intradermal injection of APC-derived exosomes with MHC-II loaded with tumor peptide delayed tumor progression and growth.135 Exosome-derived immunogenic peptides activate immature mouse dendritic cells and indirectly activate APCs, and induce specific CD4+ T cell proliferation.136 Exosomes containing IFNa and IFNg, tumor necrosis factor a (TNFa), and IL from macrophages promoted dendritic cell maturation, CD4+ and CD8+ T cell activation, and the regulation of macrophage IL expression.137 The cargo of exosomes, such as DNA and miRNA, regulate the innate and adaptive immune responses. Exosomes are able to regulate the immune response by controlling gene expression and signaling pathways in recipient cells through transfer of miRNAs, and eventually control dendritic cell maturation.138 Exosomes containing miR-212-3p derived from tumors down-regulate the MHC-II transcription factor RFXAP (regulatory factor X associated protein) in dendritic cells, possibly promoting immune evasion by cancer cells.139 Exosomes containing miR-222-3p down regulate expression of SOCS3 (suppressor of cytokine signaling 3) in monocytes, which is involved in STAT3-mediated M2 polarization of macrophages.140 In mice, exosomes stimulate adaptive immune responses, including the activation of dendritic cells, with the uptake of breast cancer cell-derived exosomal genomic DNA and activation of cGAS-STING signaling and antitumor responses.141 The priming of dendritic cells is associated with the uptake of exosomal genomic and mitochondrial DNA (mtDNA) from T cells, inducing type I IFN production by cGAS-STING signaling.142 Inhibition of EGFR leads to increased levels of DNA in the exosomes and induces cGAS-STING signaling in dendritic cells, contributing to the overall suppression of tumor growth.143 Conversely, uptake of tumor-derived exosomal DNA by circulating neutrophils was shown to enhance the production of tissue factor and IL-8, which play a role in promoting tumor inflammation and paraneoplastic events.144 Melanoma-derived exosomes containing PD-L1 (programmed cell death ligand 1) suppress CD8+ T cell antitumor function and cancer cell-derived exosomes block dendritic cell maturation and migration in a PD-L1-dependent manner. Engineered cancer cell-derived exosomes promote dendritic cell maturation, resulting in increased proliferation of T cells and antitumor activity.145147

Inflammation is an important process for maintaining homeostasis in cellular systems. Systemic inflammation is an essential component in the pathogenesis of several diseases.148,149 Exosomes seem to play a crucial role in inflammation processes through cargo molecules, such as miRNA and proteins, which act on nearby as well as distant target tissues. Exosomes play a vital role in intercellular communication between cells via endocytosis and are associated with modulation of inflammation, coagulation, angiogenesis, and apoptosis.20,150153 Exosomes derived from dendritic cells, B lymphocytes, and tumor cells release exosomes that can regulate immunological memory through the surface expression of antigen-presenting MHC I and MHC II molecules, and subsequently elicit T cell activation and maturation.134,137,154156 Exosomes play a crucial role in carrying and presenting functional MHC-peptide complexes to modulate antigen-specific CD8+ and CD4+ responses.157,158 Exosomes containing miR-Let-7d influence the growth of T helper 1 (Th1) cells and inhibit IFN- secretion.159 Exosomes derived from choroid plexus epithelial cells containing miR-146a and miR-155 upregulate the expression of inflammatory cytokines in astrocytes and microglia.160 Exosomes containing miR-181c suppress the expression of Toll-like receptor 4 (TLR-4) and subsequently lower TNF- and IL-1 levels in burn-induced inflammation.161 Exosomal miR-155 from bone marrow cells (BMCs) increases the level of TNF- and subsequently enhances innate immune responses in chronic inflammation.162 Exosomes containing miR-150-5p and miR-142-3p derived from dendritic cells (DCs) increase expression of interleukin 10 (IL-10) and a decrease in IL-6 expression.163 Exosomal miR-138 can protect against inflammation by decreasing the expression level of NF-B, a transcription factor that regulates inflammatory cytokines such as TNF- and IL-18.164 HIF-1-inducing exosomal microRNA-23a expression from tubular epithelial cells mediates the cross talk between tubular epithelial cells and macrophages, promoting macrophage activation and triggering tubulointerstitial inflammation.165 A rat model study demonstrated that bone marrow mesenchymal stem cell (BMSC)-derived exosomes reduced inflammatory responses by modulating microglial polarization and maintaining the balance between M2-related and M1-related cytokines.165 Melatonin-stimulated mesenchymal stem cell (MSC)-derived exosomes improve diabetic wound healing through regulating macrophage M1 and M2 polarization by targeting the PTEN/AKT pathway, and significantly suppressed the pro-inflammatory factors IL-1 and TNF- and reduced the relative gene expression of IL-1, TNF-, and iNOS. Increasing levels of anti-inflammatory factor IL-10 are associated with increasing relative expression of Arg-1.166

Immunomodulators are essential factors for the prevention and treatment of disorders occurring due to an over high-spirited immune response, such as the SARS-CoV-2-triggered cytokine storm leading to lung pathology and mortality seen during the ongoing viral pandemic.167 MSC-secreted extracellular vesicles exhibit immunosuppressive capacity, which facilitates the regulation of the migration, proliferation, activation, and polarization of various immune cells, promoting a tolerogenic immune response while inhibiting inflammatory responses.168 Collagen scaffold umbilical cord-derived mesenchymal stem cell (UC-MSC)-derived exosomes induce collagen remodeling, endometrium regeneration, increasing the expression of the estrogen receptor /progesterone receptor, and restoring fertility. Furthermore, exosomes modulate CD163+ M2 macrophage polarization, reduce inflammation, increase anti-inflammatory responses, facilitate endometrium regeneration, and restore fertility through the immunomodulatory functions of miRNAs.169 Exosomes released into the airways during influenza virus infection trigger pulmonary inflammation and carry viral antigens and it facilitate the induction of a cellular immune response.170 Shenoy et al171 reported that exosomes derived from chronic inflammatory microenvironments contribute to the immune suppression of T cells. These exosomes arrest the activation of T cells stimulated via the T cell checkpoint (TCR). Exosomes secreted by normal retinal pigment epithelial cells (RPE) by rotenone-stimulated ARPE-19 cells induce apoptosis, oxidative injury, and inflammation in ARPE-19 cells. Exosomes secreted under oxidative stress induce retinal function damage in rats and upregulate expression of Apaf1. Overexpression of Apaf1 in exosomes secreted under oxidative stress (OS) can cause the inhibition of cell proliferation, increase in apoptosis, and elicitation of inflammatory responses in ARPE-19 cells. Exosomes derived from ARPE-19 cells under OS regulate Apaf1 expression to increase apoptosis and to induce oxidative injury and inflammatory response through a caspase-9 apoptotic pathway.172 Collectively, these findings highlight the critical role of exosomes in inflammation and suggest the possibility of utilizing exosomes as an inducer to attenuate inflammation and restore impaired immune responses in various diseases including cancer.

The endomembrane system of eukaryotic cells is a complex series of interconnected membranous organelles that play vital roles in protecting cells from adverse conditions, such as stress, and maintaining cell homeostasis during health and disease.173 To preserve cellular homeostasis, higher eukaryotic cells are equipped with various potent self-defense mechanisms, such as cellular senescence, which blocks the abnormal proliferation of cells at risk of neoplastic transformation and is considered to be an important tumor-suppressive mechanism.174,175 Exosomes contribute to reduce intracellular stress and preservation of cellular homeostasis through clearance of damaged or toxic material, including proteins, lipids, and even nucleic acids. Therefore, exosomes serve as quality controller in cells.176 The vesicular transport system plays pivotal roles in the maintenance of cell homeostasis in eukaryote cells, which involves the cytoplasmic trafficking of biomolecules inside and outside of cells. Several types of membrane-bound organelles, such as the Golgi apparatus, endoplasmic reticulum (ER), endosomes and lysosomes, in association with cytoskeleton elements, are involved in the intracellular vesicular system. Molecules are transported through exocytosis and endocytosis to maintain homeostasis through the intracellular vesicular system and regulate cells responses to the internal and external environment. To maintain homeostasis and protect cells from various stress conditions, autophagy is an intracellular vesicular-related process that plays an important role through the endocytosis/lysosomal/exocytosis pathways through degradation and expulsion of damaged molecules out of the cytoplasm.177179 Autophagy, as an intracellular waste elimination system, is a synchronized process that actively participates in cellular homeostasis through clearance and recycling of damaged proteins and organelles from the cytoplasm to autophagosomes, and then to lysosomes.38,180182 Cells maintain homeostasis by autophagosomes, which are vesicles derived from autophagic and endosomal compartments. These processes are involved in adaption to nutrient deprivation, cell death, growth, and tumor progression or suppression. Autophagy flux contributes to maintaining homeostasis in the tumor microenvironment of endothelial cells. To support this concept, a study provided evidence suggesting that depletion of Atg5 in ECs could intensify the abnormal function of tumor vessels.183 Exosome secretion plays a crucial role in maintaining cellular homeostasis in exosome-secreting cells. As a consequence of blocking exosome secretion, nuclear DNA accumulates in the cytoplasm, thereby causing the activation of cytoplasmic DNA sensing machinery. Blocking exosome secretion aggravates the innate immune response, leading to ROS-dependent DNA damage responses and thus inducing senescence-like cell-cycle arrest or apoptosis in normal human cells. Thus, cells remove harmful cytoplasmic DNA, protecting them from adverse effects.182 Salomon and Rice reported that the involvement of exosomes in placental homeostasis and pregnancy disorders. EVs of placental origin are found in a variety of body fluids including urine and blood. Moreover, the number of exosomes throughout gestation is higher in complications of pregnancy, such as preeclampsia and gestational diabetes mellitus, compared to normal pregnancies.184

The endolysosomal system is critically involved in maintaining homeostasis through the highly regulated processes of internalization, sorting, recycling, degradation, and secretion. For example, endocytosis allows the internalization of various receptor proteins into cells, and vesicles formed from the plasma membrane fuse and deliver their membrane and protein content to early endosomes. Similarly, significant amounts of internalized content are recycled back to the plasma membrane via recycling endosomes,76 while the remaining material is sequestered in ILVs in late endosomes, also known as multivesicular bodies.185,186 Tetraspanin proteins, such as CD63 and CD81, are regulators of ILV formation. Once ILVs are formed, MVBs can degrade their cargo by fusing with lysosomes or, alternatively, MVBs can secrete their ILVs by fusing with the plasma membrane and release their content into extracellular milieu.187190 Exosomes play an important role in regulating intracellular RNA homeostasis by promoting the release of misfolded or degraded RNA products, and toxic RNA products. Y RNAs are involved in the degradation of structured and misfolded RNAs. Further studies have demonstrated that proteins involved in RNA processing are abundant in exosomes, and the half-lives of secreted RNAs are almost twice as short as those of intracellular mRNAs. These studies suggest that cells maintain intracellular RNA homeostasis through the release of distinct RNA species in extracellular vesicles.191193 Exosomes reduce cholesterol accumulation in Niemann-Pick type C disease, a lysosomal storage disease in which cells accumulate unesterified cholesterol and sphingolipids within the endosomal and lysosomal compartment.194

Autophagy is the intracellular vesicular-related process that regulates the cell environment against pathological and stress conditions. In order to maintain homeostasis and protect the cells against stress conditions, internal vesicles or secreted vesicles serve as a canal to degrade and expel damaged molecules out of the cytoplasm.38,181,182 Autophagy protects the cell from various stress conditions and maintains cellular homeostasis, regulating cell survival and differentiation through clearance and recycling of damaged proteins and organelles from the cytoplasm to autophagosomes, and then to lysosomes.180 Several studies have demonstrated that proteins are involved in controlling tumor cell function and fate, and mediate crosstalk between exosome biogenesis and autophagy. Coordination between exosome-autophagy networks serves as a tool to conserve cellular homeostasis via the lysosomal degradative pathway and/or secretion of cargo into the extracellular milieu.176,195 Autophagy is a multi-step process that occurs by initiation, membrane nucleation, maturation and finally the fusion of autophagosomes with lysosomes. The autophagy process is not only linked with endocytosis but is also linked with the biogenesis of exosomes. For example, subsets of the autophagy machinery involved in the biogenesis of exosomes and the autophagic process itself appear dispensable.78,196 Crosstalk between exosomal and autophagic pathways has been reported in a growing number of diseases. Proteomic studies were performed to analyze the involvement of key proteins in the interconnection between exosome and autophagy pathways. They found that almost all proteins were identified; however, their involvement differed between them. Among 100 proteins, four proteins were highly ranked including HSPA8 (3/100), HSP90AA1 (8/100), VCP (24/100), and Rab7A (81/100). These data suggest an interconnection between the exosome and autophagy.197,198 Endosomal autophagy plays a significant role in the interconnection between exosomes and autophagy. Stress is a major factor for autophagy. In particular, the starvation of cells is a key inducer of autophagy, and induces enlargement of MVB structures and a co-localization of Rab11 and LC3 in these structures, an indication that autophagy-related processes are associated with the MVB.199 The sorting of autophagy-related cargo into MVBs is dependent on Hsc70 (HSPA8), VPS4, and TSG101, and independent on LAMP-2A, thereby excluding a role for, the lysosome.200 Several proteins are involved in the regulation and biogenesis of secretory autophagy compartments such as GRASPs, LC3, Rab8a, ESCRTs, and SNAREs, along with several Atg proteins.181,201,202 Autophagosomes could fuse with MVBs to form amphisomes and release vesicles to the external environment.203

Autophagy and exosome biogenesis and function are interconnected by microRNA. Over-expression of miR-221/222 inhibits the level of PTEN and activates Akt signaling, and subsequently reduces the expression of hallmarks that positively relate to autophagy including LC3, ATG5 and Beclin1, and increases the expression of SQSTM1/p62.204 MiR-221/222 from human aortic smooth muscle cell (HAoSMC)-derived exosomes inhibit autophagy in HUVECs by modulating the PTEN/Akt signaling pathway. miRNA-223 attenuates hypoxia-induced apoptosis and excessive autophagy in neonatal rat cardiomyocytes and H9C2 cells via the Akt/mTOR pathway, by targeting poly(ADP-ribose) polymerase 1 (PARP-1) through increased autophagy via the AMPK/mTOR and Akt/mTOR pathways205 ATG5 mediates the dissociation of vacuolar proton pumps (V1Vo-ATPase) from MVBs, which prevents acidification of the MVB lumen and allows MVB-PM fusion and exosome release. Accordingly, knockout of ATG5 or ATG16L1 significantly reduces exosome release and attenuates the exosomal enrichment of lipidated LC3B. These findings demonstrate that autophagic mechanisms possibly regulate the fate of MVBs and subsequent exosome biogenesis.78 Bone marrow MSC (BMMSC)-derived exosomes contain a high level of miR-29c, which regulates autophagy under hypoxia/reoxygenation (H/R) conditions.206 Human umbilical cord MSC-derived exosomes (HucMDEs) promote hepatic glycolysis, glycogen storage, and lipolysis, and reduce gluconeogenesis. Additionally, autophagy potentially contributes to the effects of HucMDE treatment and increases formation of autophagosomes and the autophagy marker proteins BECN1, MAP, and 1LC3B. These findings suggest that HucMDEs improve hepatic glucose and lipid metabolism in T2DM rats by activating autophagy via the AMPK pathway.207 Liver fibrosis is a serious disorder caused by prolonged parenchymal cell death, leading to the activation of fibrogenic cells, extracellular matrix accumulation, and eventually liver fibrosis. Exosomes derived from adipose-derived mesenchymal stem cells (ADSCs) have been used to deliver circular RNAs mmu_circ_0000623 to treat liver fibrosis. The findings from this study suggest that Exos from ADSCs containing mmu_circ_0000623 significantly suppress CCl4-induced liver fibrosis by promoting autophagy activation. Autophagy inhibitor treatment significantly reverses the treatment effects of Exos.208 Inhibition of autophagy by PDGF and its downstream molecule SHP2 (Src homology 2-containing protein tyrosine phosphatase 2) increased hepatic stellate cell (HSC)-derived EV release. Disruption of mTOR signaling abolishes PDGF-dependent EV release. Activation of mTOR signaling induces the release of MVB-derived exosomes by inhibiting autophagy, as well as microvesicles, through activation of ROCK1 signaling. Furthermore, deletion of SHP2 attenuates CCl4 or BDL-induced liver fibrosis.209 The therapeutic effects of exosomes containing high concentrations of mmu_circ_0000250 were analyzed in diabetic mice. The findings indicated that a high concentration of mmu_circ_0000250 had a better therapeutic effect on wound healing when compared with wild-type exosomes from ADSCs. The results also showed that exosome treatment with mmu_circ_0000250 increased angiopoiesis in wounded skin and suppressed apoptosis by inducing miR-128-3p/SIRT1-mediated autophagy.210 A study showed that mice treated with differentiated cardiomyocyte (iCM) exosomes exhibited significant cardiac improvement post-myocardial infarction, with significantly reduced apoptosis and fibrosis. Apoptosis was associated with reduced levels of hypoxia and inhibition of exosome biogenesis. iCM-exosome-treated groups showed upregulation of autophagosome production and autophagy flux. Hence, these findings indicate that iCM-Ex can improve post-myocardial infarction cardiac function by regulating autophagy in hypoxic cardiomyocytes.211 Exosomes of hepatocytes play a crucial role in inhibiting hepatocyte apoptosis and promoting hepatocyte regeneration. Mesenchymal stem cell-derived hepatocyte-like cell exosomes (MSC-Heps-Exo) were injected into a mouse hepatic Ischemia/reperfusion (I/R) I/R model through the tail. The results demonstrated that MSC-Heps-Exo effectively relieve hepatic I/R damage, reduce hepatocyte apoptosis, and decrease liver enzyme levels. A possible mechanism of reduced hepatic ischemia/reperfusion injury is the enhancement of autophagy.212

Exosomes play a critical role in viral infections, particularly of retroviruses and retroviruses, and use preexisting pathways for intracellular protein trafficking and formation of infectious particles. Exosomes and viruses share several features including biogenesis, uptake by cells, and the intracellular transfer of RNAs, mRNAs, and cellular proteins. Some features are different, including self-replication after infection of new cells, regulation of viral expression, and complex viral entry mechanisms.213,214 Exosomes secreted from virus-infected cells carry mostly cargo molecules such as viral proteins, genomic RNA, mRNA, miRNA, and genetic regulatory elements.215218 These cargo molecules are involved in the alteration of recipient cell behavior, regulating cellular responses, and enabling infection by various types of viruses such as human T-cell lymphotropic virus (HTLV), hepatitis C virus (HCV), dengue virus, and human immunodeficiency virus (HIV).215 Exosomes communicate with host cells through contact between exosomes and their recipient cells, via different kinds of mechanisms. Initially, the transmembrane proteins of exosomes build a network directly with the signaling receptors of target cells and then join with the plasma membrane of recipient cells to transport their content to the cytosol. Finally, the exosomes are incorporated into the recipient cells.219221 A report suggested that disruption of exosomal lipid rafts leads to the inhibition of internalization of exosomes.95 Exosomes derived from HIV-infected patients contain the trans-activating response element, which is responsible for HIV-1 replication in recipient cells through downregulation of apoptosis.222 While exosomes serving as carrier molecules, exosomes contain miRNAs that induce viral replication and immune responses either by direct targeting of viral transcripts or through indirect modulation of virus-related host pathways. In addition, exosomes have been found to act as nanoscale carriers involved in HIV pathogenesis. For example, exosomes enhance HIV-1 entry into human monocytic and T cell lines through the exosomal tetraspanin proteins CD9 and CD81.223 Influenza virus infection causes accumulation of various types of microRNAs in bronchoalveolar lavage fluid, which are responsible for the potentiation of the innate immune response in mouse type II pneumocytes. Serum of influenza virus-infected mice show significant levels of miR-483-3p, which increases the expression of proinflammatory cytokine genes and inflammatory pathogenesis of H5N1 influenza virus infection in vascular endothelial cells.224 Exosomes are involved in the transmission of inflammatory, apoptotic, and regenerative signals through RNAs. Chen et al investigated the potential functions of exosomal RNAs by RNA sequencing analysis in exosomes derived from clinical specimens of healthy control (HC) individuals and patients with chronic hepatitis B (CHB) and acute-on-chronic liver failure caused by HBV (HBV-ACLF). The results revealed that the samples contained unique and distinct types of RNAs in exosomes.225 Zika virus (ZIKV) infection causes severe neurological malfunctions including microcephaly in neonates and other complications associated with Guillain-Barr syndrome in adults. Interestingly, ZIKV uses exosomes as mediators of viral transmission between neurons and increases production of exosomes from neuronal cells. Exosomes derived from ZIKV-infected cells contained both ZIKV viral RNA and protein(s) which are highly infectious to nave cells. ZIKV uses neutral Sphingomyelinase (nSMase)-2/SMPD3 to regulate production and release of exosomes.226

During infections, viruses replicate in host cells through vesicular trafficking through a sequence of complexes known as ESCRT, and assimilate viral constituents into exosomes. Exosomes encapsulate viral antigens to maximize infectivity by hiding viral genomes, entrapping the immune system, and maximizing viral infection in uncontaminated cells. Exosomes can be used as a source of viral antigens that can be targeted for therapeutic use. A Variety of infectious diseases caused by viruses such as HCV, ZIKV, West Nile virus (WNV), and DENV enter into the host cells using clathrin-mediated or receptor-mediated endocytosis. For example, HCV infects host cells by specific targeting of cells through cellular contact, and hepatocyte-derived exosomes that contain HCV RNA can stimulate innate immune cells.217,227230 Exosomes show structural and molecular similarity to HIV-1 and HIV-2, which are enclosed by a lipid bilayer, and in the vital features of size and density, RNA species, and macro biomolecules including carbohydrates, lipids, and proteins. HIV-infected cells release enriched viral RNAs containing exosomes derived from HIV-infected cells and are enhanced with viral RNAs and Nef protein.6,38,231236 Izquierdo-Useros et al reported that both exosomes and HIV-1 express sialyllactose-containing gangliosides and interact with each other via sialic-acid-binding immunoglobulin-like lectins (Siglecs)-1. Siglecs-1 stimulates mature dendritic cell (mDC) capture and storage of both exosomes and HIV-1 in mDCs.237 Exosomes released from HIV-infected T cells contain transactivation response (TAR) element RNA, which stimulate proliferation, migration, and invasion of oral/oropharyngeal and lung cancer cells.238 Nuclear VP40 from Ebola virus VP40 upregulates cyclin D1 levels, resulting in dysregulated cell cycle and EV biogenesis. Synthesized extracellular vesicles contain cytokines and EBOV proteins from infected cells, which are responsible for the destruction of immune cells during EBOV pathogenesis.239 HIV enters into the host cells through human T-cell immunoglobin mucin (TIM) proteins. TIMs are a group of proteins (TIM-1, TIM-3, and TIM-4) that promote phagocytosis of apoptotic cells.240 TIM-4 is involved in HIV-1 exosome-dependent cellular entry mechanisms. Substantiating this hypothesis, neural stem cell (NSC)-derived exosomes containing TIM-4 protein increase HIV-1 exosome-dependent cellular entry into host cells, and antibody against TIM4 inhibits exosome-mediated entry of HIV in various types of cell.241

Exosomes show immense promise in biomedical applications due to their potential in drug delivery, the carriage of biomolecular markers of many diseases, and cellular protection. In addition, they can be used in non-invasive diagnostics or minimum invasive diagnostics.150 Detection of biomarkers is vital for early diagnosis of cancer and also critical for treatment. Several studies have documented the importance of exosomes in a variety of diseases, although further examination of the biology and functions of exosomes is warranted due to the continuing emergence of new diseases in the present world. The complex cargo of exosomes facilitates the exploration of a variety of diagnostic windows into disease detection, monitoring, and treatment. Exosomes are found in all biological fluids and are secreted by all cells, rendering them attractive for use through minimally invasive liquid biopsies, and they have the potential for use in longitudinal sampling to follow disease progression.242 Exosomes are produced and secreted by almost all body fluids, including blood, urine, saliva, breast milk, cerebrospinal fluid, semen, amniotic fluid, and ascites. These exosomes contain micro RNAs, proteins, and lipids serving as diagnostic markers.120 Exosomes are used in diagnostic applications in various kinds of diseases, such as cardiovascular diseases (CVDs),243 diseases of the central nervous system (CNS),244 cancer,245 and other prominent diseases including in the liver,246 kidney,247 and lung.248 Exosomes are potentially used to detect cancer-associated mutations in serum and also for the transfer of genomic DNA from donor cells to recipient cells.249 Exosomes carrying specific miRNAs or groups of miRNAs can be used as diagnostic markers to detect cancer. For example, exosomes containing oncogenic Kras, which have tumor-suppressor miRNAs-100, seem to have high diagnostic value, which could facilitate the differentiation of the expression pattern between cancer cells and normal cells.250,251 Similarly, miR-21 is considered to be diagnostic marker for various types of cancer including glioblastomas and pancreatic, colorectal, colon, liver, breast, ovarian, and esophageal cancers.252 Tumor suppressor miRNAs, such as miR-146a and miR-34a, function as diagnostic tools to detect liver, breast, colon, pancreatic, and hematologic malignancies.251 Exosomes containing GPC1 (glypican 1) are used as diagnostic markers to detect pancreatic, breast, and colon cancer.253,254

Exosomes play critical roles in various types of disease, and particularly in cancer progression and resistance to therapy. The unique biogenesis of exosomes and their biological features have generated excitement for their potential use as biomarkers for cancer.255 Generally, exosomes are produced and secreted by most cells and contain all the biological components of a cell. Hence, exosomes are found in all biological fluids and provide excellent opportunities for use as biomarkers.242 Surface proteins of exosomes are involved in the regulation of the tumor immune microenvironment and the monitoring of immunotherapies. Hence, exosome proteins play a critical role in cancer signaling.256 Exosomes from patients with metastatic pancreatic cancer show a higher mutant Kras allele frequency than exosomes from patients with local disease. In addition, the exosomes also accumulate a significantly higher level of cancer cell-specific DNA such as cytoplasmic DNA.8,257 Exosomes protect DNA and RNA from enzymatic degradation by encapsulation and stability in exosomes. The enhanced stability and retention of exosomes in liquid biopsies increases the availability and performance of exosomes as cancer biomarkers.258 Cancer cells contain cargo molecules, such as nucleic acid, proteins, metabolites, and lipids that are relatively different from normal cells, which is a contributing factor for their candidacy as cancer biomarkers. Exosomes isolated and purified from patient plasma samples enriched for miR-10b-5p, miR-101-3p, and miR-143-5p have been identified as potential diagnostic markers for gastric cancer with lymph node metastasis, gastric cancer with ovarian metastasis, and gastric cancer with liver metastasis, respectively.259 Kato et al analyzed the expression of CD44 protein and mRNA from cell lysates and exosomes from prostate cancer cells.260 Exosomes from serum containing CD44v8-10 mRNA was used as a diagnostic marker for docetaxel resistance in prostate cancer patients. The study was performed to evaluate plasma exosomal mRNA-125a-5p and miR-141-5p miRNAs as biomarkers for the diagnosis of prostate cancer from 19 healthy individuals and 31 prostate cancer patients. In comparing the miR-125a-5p/miR-141-5p level ratio, prostate cancer patients had significantly higher levels of miR-125a-5p/miR-141-5p. The findings from this study demonstrated that plasma exosomal expression of miR-141-3p and miR-125a-5p are markers of specific tumor traits associated with prostate cancer.261 Serum samples from 81 patients with gastric cancer showed that exosomes contained significant levels of long non-coding RNA (lncRNA) H19, which could be a diagnostic marker for gastric cancer.262 Plasma exosomes are suitable candidates as biomarkers for various diseases. For instance, plasma exosome lncRNA expression profiles were examined in esophageal squamous cell carcinoma (ESCC) patients. The findings suggest that five different types of lncRNAs were at significantly higher levels in exosomes from ESCC patients than in non-cancer controls. These lncRNAs may serve as highly effective, noninvasive biomarkers for ESCC diagnosis.263 Differential expression of lncRNAs, such as LINC00462, HOTAIR, and MALAT1, are significantly upregulated in hepatocellular carcinoma (HCC) tissues. The exosomes of the control group had a larger number of lncRNAs with a high amount of alternative splicing compared to hepatic disease patients.264 To demonstrate exosomes as a non-invasive cancer diagnostic tool, RNA-sequencing analysis was performed between three pairs of non-small-cell lung cancer (NSCLC) patients and controls from Chinese populations. The results show that circ_0047921, circ_0056285, and circ_0007761 were significantly expressed and that these exosomal circRNAs are promising biomarkers for NSCLC diagnosis.265 Exosomes were isolated from the serum of 34 patients with acute myocardial infarction (AMI), 31 patients with unstable angina (UA), and 22 healthy controls. The isolated exosomes exhibited higher levels of miR-126 and miR-21 in the patients with UA and AMI than in the healthy controls.266 Xu et al designed a study to examine tumor-derived exosomes as diagnostic biomarkers. In this study exosome miRNA microarray analysis was performed in the peripheral blood from four lung adenocarcinoma patients, including two with metastasis and two without metastasis. The results found that miR-4436a and miR-4687-5p were upregulated in the metastasis and non-metastasis group, while miR-22-3p, miR-3666, miR-4448, miR-4449, miR-6751-5p, and miR-92a-3p were downregulated. Exosomes containing miR-4448 have served as a diagnostic marker of patients with adenocarcinoma metastasis. Increased understanding of exosome biogenesis, structure, and function would enhance the performance of biomarkers in various kinds of disease diagnosis, prognosis, and surveillance.267

Exosomes have unique features such as ease of handling, molecular composition, and critical immunogenicity, and it is particularly easy to use them to transfer genes and proteins into cells. These unique characteristic features can inhibit angiogenesis and cancer metastasis, which are the two main targets of cancer therapy.268,269 Exosomes have potential therapeutic applications in a variety of diseases due to their potential capacity as vehicles for the delivery of therapeutic agents (Figure 5). Exosomes from colon cancer cells contain the highly immunogenic antigens MelanA/Mart-1 and gp100, serving as an indicator of tumor origin in particular organelles. Animal studies have demonstrated that tumor-derived antigen-containing exosomes induce potent antitumor T-cell responses and tumor regression.270 Exosomes containing tumor antigens are able to stimulate CD4+and CD8+T cells, and antigen-presenting exosomes inhibit tumor growth.135,271,272 MSC-derived exosomes exhibit the immunomodulatory and cytoprotective activities of their parent cells.273,274 Similarly, exosomes derived from bone marrow show protective roles in myocardial ischemia/reperfusion injury,109 hypoxia-induced pulmonary hypertension,275 and brain injury,276,277 and inhibit breast cancer growth via vascular endothelial growth factor down-regulation and miR-16 transfer in mice.278 Mesenchymal cell- and epithelial cell-derived exosomes exhibit tolerance and without any undesired side effects in patients and also act as therapeutic agents themselves.48,279 Exosomes engineered with ligands containing RGD peptide are used to induce signaling in specific cell types, and doxorubicin-loaded exosomes derived from dendritic cells show therapeutic responses in mammary tumor-bearing mice.46 Exosomal microRNAs are able to control other cells, and the delivery of miRNA or siRNA payload promotes anticancer activity in mammary carcinoma and glioma.280,281 Rabies virus glycoprotein (RVG)-modified dendritic cell-derived exosomes suppress the expression of BACE1 in the brain, which indicates the therapeutic potential of exosomes to target AD.282 Furthermore, these exosomes stimulated neurite outgrowth in cultured astrocytes by transferring miR-133b between cells.27 Immunotherapy is able to induce tumor-targeting immunity or an antitumor host immune response. For example, tumor-associated antigen-loaded mature autologous dendritic cells increase survival of metastatic castration-resistant patients.283 Exosome therapy induces upregulation of CD122 molecules in CD4+ T cells, whereas the lymphocyte pool is stable. Multiple vaccinations with exosomes increase circulating CD3-/CD56+ natural killer (NK) cells.284 An in vitro study demonstrated that adipose stem cell-derived exosomes up-regulate the peroxisome proliferator-activated receptor gamma coactivator 1, phosphorylate the cyclic AMP response element binding protein, and ameliorate abnormal apoptotic protein levels.285 Exosomes are used as potential carriers to carry anti-inflammatory drugs. Curcumin-encapsulated exosomes show significant anti-inflammatory activity, and exosomes are also used to deliver anti-inflammatory drugs to the brain through a noninvasive intranasal route.286,287 Turturici et al reported that specific progenitor cell-derived EVs contain biological cargo that promotes angiogenesis and tissue repair, and modulates immune functions.288

Figure 5 Therapeutic potential and versatile clinical implications of exosomes.

Generally, exosomes serve as vehicles for the delivery of drugs and are also actively involved as therapeutic agents. Conversely, injected exosomes enter into other cells and deliver functional cargo molecules very efficiently and rapidly, with minimal immune clearance and are well tolerated.16,21,245,289,290 Intravenous administration of human MSC-derived exosomes supports neuroprotection in a swine model of traumatic brain injury.291 In vitro and in vivo models demonstrate that exosomes from human-induced pluripotent stem cell-derived mesenchymal stromal Cells (hiPSC-MSCs) protect the liver against hepatic ischemia/reperfusion injury through increasing the level of proliferation of primary hepatocytes, activity of sphingosine kinase, and synthesis of sphingosine-1-phosphate (S1P).292 Exosomes derived from macrophages show potential for use in neurological diseases because of their easy entry into the brain by crossing the blood-brain barrier (BBB). Catalase-loaded exosomes displayed a neuroprotective effect in a mouse model of PD and exosomes loaded with dopamine entered into the brain better in comparison to free dopamine.33,293 Treatment of tumor-bearing mice with autologous exosomes loaded with gemcitabine significantly suppressed tumor growth and increase longevity, and caused only minimal damage to normal tissues. The study demonstrated that autologous exosomes are safe and effective vehicles for targeted delivery of GEM against pancreatic cancer.294

Generally, lipid-based nanoparticles such as liposomes or micelles, or synthetic delivery systems have been adopted to transport active molecules. However, the merits of synthetic systems are limited due to various factors including inefficiency, cytotoxicity and/or immunogenicity. Therefore, the development of natural carrier systems is indispensable. One of the most prominent examples of such natural carriers are exosomes, which are used to transport drug and active biomolecules. Exosomes are more compatible with other cells because they carry various targeting molecules from their cells of origin. Exosomes are nano-sized membrane vesicles derived from almost all cell types, which carry a variety of cargo molecules from their parent cells to other cells. Due to their natural biogenesis and unique qualities, including high biocompatibility, enhanced stability, and limited immunogenicity, they have advantages as drug delivery systems (DDSs) compared to traditional synthetic delivery vehicles. For instance, extracellular vesicles, including exosomes, carry and protect a wide array of nucleic acids and can potentially deliver these into recipient cells.6 EVs possess inherent targeting properties due to their lipid composition and protein content enabling them to cross biological barriers, and these salient features exploit endogenous intracellular trafficking mechanisms and trigger a response upon uptake by recipient cells.45,295297 The lipid composition and protein content of exocytic vesicles have specific tropism to specific organs.296 The integrin of exosomes determines the ability to alter the pharmacokinetics of EVs and increase their accumulation in various type of organs including brain, lungs, or liver.117 For example, EVs containing Tspan8 in complex with integrin alpha4 were shown to be preferentially taken up by pancreatic cells.298 Similarly, the lipid composition of EVs influences the cellular uptake of EVs by macrophages.299 EVs derived from dendritic cell achieved targeted knockdown by fusion between expression of Lamp2b and neuron-specific RVG peptide by using siRNA in neuronal cell.45 EVs loaded with Cre recombinase protein were able to deliver functional CreFRB to recipient cells through active and passive mechanisms in the presence of endosomal escape, enhancing the compounds chloroquine and UNC10217832A.300 EVs from cardiosphere-derived cells achieved targeted delivery by fusion of the N-terminus of Lamp2b to a cardiomyocyte-specific peptide (CMP).301 RVG-exosomes were used to deliver anti-alpha-synuclein shRNA minicircle (shRNA-MC) therapy to the alpha-synuclein preformed-fibril-induced mouse model of parkinsonism. This therapy decreased alpha-synuclein aggregation, reduced the loss of dopaminergic neurons, and improved clinical symptoms. RVG exosome-mediated therapy prolonged the effectiveness and was specifically delivered into the brain.302 Zhang et al evaluated the effects of umbilical cord-derived macrophage exosomes loaded with cisplatin on the growth and drug resistance of ovarian cancer cells. High loading efficiency of cisplatin was achieved by membrane disruption of exosomes by sonication.303 Incorporation of cisplatin into umbilical cord blood-derived M1 macrophage exosomes increased cytotoxicity 3.3-fold in drug-resistant A2780/DDP cells and 1.4-fold in drug-sensitive A2780 cells, compared to chemotherapy alone. Loading of cisplatin into M2 exosomes increased cytotoxicity by nearly 1.7-fold in drug-resistant A2780/DDP cells and 1.4-fold in drug-sensitive A2780 cells. The findings suggest that cisplatin-loaded M1 exosomes are potentially powerful tools for the delivery of chemotherapeutics to treat cancers regardless of drug resistance. Shandilya et al developed a chemical-free and non-mechanical method for the encapsulation and intercellular delivery of siRNA using milk-derived exosomes through conjugation between bovine lactoferrin with poly-L-lysine, wherein lactoferrin as a ligand was captured by the GAPDH present in exosomes, loading siRNA in an effortless manner.304 Targeted drug delivery was achieved with low immunogenicity and toxicity using exosomes derived from immature dendritic cells (imDCs) from BALB/c mice by expressing the fusion protein RGD. Recombinant methioninase (rMETase) was loaded into tumor-targeting iRGD-Exos. The findings suggest that the iRGD-Exos-rMETase group exhibited significant antitumor activity compared to the rMETase group.305 Several diseases show high inflammatory responses; therefore, amelioration of inflammatory responses is a critical factor. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IB (srIB), which is the dominant active form of IB, and can inhibit translocation of nuclear factor B into the nucleus. Intraperitoneal injection of purified srIB-loaded exosomes (Exo-srIBs) showed diminished mortality and systemic inflammation in septic mouse models.306 Systemic administration of macrophage-derived exosomes modified with azide and conjugated with dibenzocyclooctyne-modified antibodies of CD47 and SIRP (aCD47 and aSIRP) through pH-sensitive linkers can actively and specifically target tumors through distinguishing between aCD47 and CD47 on the tumor cell surface.307 SPION-decorated exosomes prepared using fusion proteins of cell-penetrating peptides (CPP) and TNF- (CTNF-)-anchored exosomes coupled with superparamagnetic iron oxide nanoparticles (CTNF--exosome-SPIONs) significantly enhanced tumor cell growth inhibition via induction of the TNFR I-mediated apoptotic pathway. Furthermore, in vivo studies in murine melanoma subcutaneous cancer models showed that TNF--loaded exosome-based vehicle delivery enhanced cancer targeting under an external magnetic field and suppressed tumor growth with mitigating toxicity.308 Yu et al309 developed a formulation of erastin-loaded exosomes labeled with folate (FA) to form FA-vectorized exosomes loaded with erastin (erastin@FA-exo) to target triple-negative breast cancer (TNBC) cells with overexpression of FA receptors. Erastin@FA-exo increased the uptake efficiency of erastin and also significantly inhibited the proliferation and migration of MDA-MB-231 cells compared with erastin@exo and free erastin. Interestingly, erastin@FA-exo promoted ferroptosis with intracellular depletion of glutathione and ROS generation. Plasma exosomes (Exo) loaded with quercetin (Exo-Que) improved the drug bioavailability, enhanced the brain targeting of Que and potently ameliorated cognitive dysfunction in okadaic acid (OA)-induced AD mice compared to free quercetin by inhibiting phosphorylated tau-mediated neurofibrillary tangles.310 Spinal cord injury (SCI) causes paralysis of the limbs. To determine the role of resveratrol in SCI, exosomes derived from resveratrol-treated primary microglia were used as carriers which are able to enhance the solubility of resveratrol and enhance penetration of the drug through the BBB, thereby increasing its concentration in the CNS. The findings demonstrated that Exo + Res are highly effective at crossing the BBB with good stability, suggesting they have potential for enhancing targeted drug delivery and recovering neuronal function in SCI therapy, and is likely associated with the induction of autophagy and inhibition of apoptosis via the PI3K signaling pathway.311 Delivery of miR-204-5p by exosomes inhibits cancer cell proliferation and tumor growth, and induces apoptosis and chemoresistance by specifically suppressing the target genes of miR-204-5p in human cancer cells.312 Engineered exosomes with RVG peptide on the surface for neuron targeting and NGF-loaded exosomes (NGF@ExoRVG) were efficiently delivered into ischemic cortex, with a burst release of encapsulated NGF protein and de novo NGF protein translated from the delivered mRNA. The delivered NGF protein showed high stability and a long retention time, and also reduced inflammation by reshaping microglia polarization, promoted cell survival, and increased the population of double cortin-positive cells, a neuroblast marker.313 Intranasal delivery of mesenchymal stem cell-derived extracellular vesicles exerts immunomodulatory and neuroprotective effects in a 3xTg model of AD by activation of microglia cells and increased dendritic spine density.314 Exosome-encapsulated paclitaxel showed efficacy in the treatment of multi-drug resistant cancer cells and it overcomes MDR in cancer cells.315,316 Saari et al found that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect.316 Exosome loaded doxorubicin (exoDOX) avoids undesired and unnecessary heart toxicity by partially limiting the crossing of DOX through the myocardial endothelial cells.317 Studies from in vitro and in vivo demonstrate that exosome loaded doxorubicin showed that exosomes did not decrease the efficacy of DOX and there is no cardiotoxicity in DOX-treated mice.318

The intrinsic properties of exosomes have been exploited to control various types of diseases, including neurodegenerative conditions and cancer, through promoting or restraining the delivery of proteins, metabolites, and nucleic acids into recipient cells effectively, eventually altering their biological response. Furthermore, exosomes can be engineered to deliver diverse therapeutic payloads to the target site, including siRNAs, antisense oligonucleotides, chemotherapeutic agents, and immune modulators. The natural lipid and protein composition of exosomes increases bioavailability and minimizes undesirable side effects to the recipients. Due to the availability of exosomes in biological fluid, they can be easily used as potential biomarkers for diagnosis of diseases. Exosomes are naturally decorated with numerous ligands on the surface that can be beneficial for preferential tumor targeting.282 Due to their unique properties, including superior targeting capabilities and safety profile, exosomes are the subject of clinical trials as cancer therapeutic agents.284 Exosomes derived from DCs loaded with tumor antigens have been used to vaccinate cancer patients with the goal of enhancing anti-tumor immune responses.284,319,320

Due to the potential level of various types of cargoes and salient features, exosomes are involved in intercellular messaging and disease diagnosis. As a result of dedicated studies, exosomes have been identified as natural drug delivery vehicles. However, we still face challenges regarding the purity of exosomes due to the lack of standardized techniques for their isolation and purification, inefficient separation methods, difficulties in characterization, and lack of specific biomarkers.321 The first challenge is the use of conventional methods, which are laborious for isolation and purification, time consuming, and vulnerable to contamination by other impurities, which will affect drug delivery processes. The second challenge is the various cellular origins of exosomes, which could affect specific applications. For example, in the application of exosomes in cancer therapy, we should avoid the use of exosomes derived from cancer cells, due to their oncogenic properties. Finally, exosomes have variable properties due to extraction from different types of cell and different cell culture techniques. Therefore, there is a necessity to address and overcome the challenges. There is also a need for an exosome consortium to develop common protocols for the development of rapid and precise methods of exosome isolation, and to assist the selection of sources that are dependent upon the specific therapeutic application. The most important challenge of exosome biology is the clinical translation of exosome-based research using different cell sources. Further characterization studies based on therapeutic applications are needed. Finally, important steps need to be taken to purify exosomes in a feasible, rapid, cost-effective, and scalable manner, which are free from downstream processing and have minimal processing times, that are specifically targeted to therapeutic applications and clinical settings.

The achievement of exosome therapy is based on success rate of clinical trials. Exosomes with size ranges from 60 to 200nm have been used as an active pharmaceutical ingredient or drug carrier in disease treatment. Exosomes derived from human and plant-derived exosomes are registered in clinical trials, but more complete reports are available for humanderived exosomes.322 There are two major exosomes from DCs and MSCs are frequently used in clinical trials, which potentially induce inflammation response and inflammation treatment. The more crucial aspect of exosomes in clinical trials needs to comply with good manufacturing practice (GMP) including upstream, downstream and quality control. Recently, France and USA conducted clinical trials using EVs containing MHCpeptide complexes derived from dendritic could alter tumor growth in immune competent mice and a Phase I anti-non-small cell lung cancer319,320 and several other clinical trial studies are shown in Table 1. Recent clinical case shows promising results with MSC-EVs derived from unrelated bone marrow donors for the treatment of a steroid-refractory graft-vs-host disease patient.279 Similarly, exosomes were used for the treatment of various types of diseases such as melanoma, non-small-cell-lung cancer, colon cancer and chronic kidney disease.284,319,320,323,324

Table 1 Summary of the Exosome Used in Clinical Trials (Source: clinicaltrials.com)

Exosomes are nano-sized membrane vesicles released by the fusion of an organelle of the endocytic pathway, a multivesicular body, with the plasma membrane. Since the last decade, exosomes have played a critical role in nanomedicine and studies related to exosome biology have increased immensely. Exosomes are secreted by almost all cell types and they are found in almost all types of body fluids. They function as mediators of cell-cell communications and play a significant role in both physiological and pathological processes. Exosomes carry a wide range of cargoes including proteins, lipids, RNAs, and DNA, which mediate signaling to recipient cells or tissues, making them a promising diagnostic biomarker and therapeutic tool for the treatment of cancers and other pathologies. In this review, we summarized what is known to date about the factors involved in exosome biogenesis and the role of exosomes in intercellular signaling and cell-cell communications, immune responses, cellular homeostasis, autophagy, and infectious diseases. Further, we reviewed the role of exosomes as diagnostic markers, and their therapeutic and clinical implications. Furthermore, we highlighted the challenges and outstanding developments in exosome research. The clinical application of exosomes is inevitable and they represent multicomponent biomarkers for several diseases including cancer and neurological diseases, etc. Recently, the mortality rate due to various types of cancers has increased. Therefore, therapies are essential to reduce mortality rates. At this juncture, we need sensitive, rapid, cost-effective, and large-scale production of exosomes to use as cancer biomarkers in diagnosis, prognosis, and surveillance. Furthermore, novel technologies are required for further tailoring exosomes as drug delivery vesicles with high drug pay loads, high specificity and low immunogenicity, and free of toxicity undesired side-effects. In addition, standardized and uniform protocols are necessary to isolate and purify exosomes for clinical applications, and more precise isolation and characterization procedures are required to increase understanding of the heterogeneity of exosomes, their cargo, and functions. There is an urgent need for information regarding the composition and mechanisms of action of the various substances in exosomes and to determine how to obtain highly purified exosomes at the right dosage for their clinical use. Currently, exosomes represent a promising tool in the field of nanomedicine and may provide solutions to a variety of todays medical mysteries.

The future direction of exosome research must focus on addressing the differential responses of communication between normal cells and cancer cells, how normal cells rapidly become cancerous, and how exosomes plays critical role in cancer progression via cell-cell communications. In vivo studies need to urgently address the critical factors such as biogenesis, trafficking, and cellular entry of exosomes originating from unmanipulated exosomes that control regulatory pathological functions. Further studies are required to decipher the mechanism of the cell-specific secretion and transport of exosomes, and the biological controls exerted by target cells. Exosomes represent a clinically significant nanoplatform. To substantiate this idea, numerous systematic in vivo studies are necessary to demonstrate the potency and toxicology of exosomes, which could help bring this novel idea a step closer to clinical reality. The most vital part of the system is to optimize the conditions for the engineering of exosomes that are non-toxic, for use in clinical trials. Furthermore, the translation of exosomes into clinical therapies requires their categorization as active drug components or drug delivery vehicles. Finally, future research should focus on the nanoengineering of exosomes that are tailored specifically for drug delivery and clinical efficacy.

Although we are the authors of this review, we would never have been able to complete it without the great many people who have contributed to the field of exosomes biogenesis, functions, therapeutic and clinical implications of exosomes aspects. We owe our gratitude to all those researchers who have made this review possible. We have cited as many references as permitted and apologize to the authors of those publications that we have not cited due to the limitation of references. We apologize to other authors who have worked on these aspects but whom we have unintentionally overlooked.

This study was supported by the KU-Research Professor Program of Konkuk University.

This work was supported by a grant from the Science Research Center (2015R1A5A1009701) of the National Research Foundation of Korea.

The authors report no conflicts of interest related to this work..

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[Full text] A Comprehensive Review on Factors Influences Biogenesis, Functions, Th | IJN - Dove Medical Press

This article was reviewed by Russell N. Van Gelder, MD, PhD

Neuronal cell replacement therapies remain a challenge in retinal diseases. Some fish and salamanders have the innate ability to regenerate retinal tissue after injuries and, as Russell N. Van Gelder, MD, PhD, pointed out, if researchers could harness this ability in humans, the possibilities would be great for repairing or replacing damaged tissue in a wide variety of retinal diseases. Stem cells are the key to cell replacement therapies.

Stem cells are cells that have not terminally differentiated and still have the potential to become many types of terminal cells, said Van Gelder, from the Department of Ophthalmology at the University of Washington in Seattle. We all started as embryonic stem cells in the earliest phases of development.

Related: Retinal pathologies challenging to image with current technologies

Van Gelder went on to explain that there are now methods to create equivalently totipotent stem cells from individual induced progenitor stem cells derived from an individuals blood or epithelial cells.

The overarching goal is to create a cell type that needs replacement from a stem cell precursor, he said.

A major achievement in this quest for regenerative ability occurred in 2014 when an entire eye cup was grown from progenitor stem cells.

Van Gelder also described a study1 in which green fluorescent proteinlabeled retinal precursors derived from embryonic stem cells were transplanted into the subretinal space of macaques. Three months after the procedure, the researchers demonstrated that the bolus of cells persisted and had outgrowth of axons that were seen going to the optic nerve and on to the brain.

This result establishes the validity of a stem cell-based approach for doing regenerative medicine in primates, he said.

Related: Persistent retinal detachment associated with retinoblastoma

Replacement therapy hurdlesAs of now, however, no stem cell-based replacement treatment has received FDA approval. The problems preventing establishment of a treatment have been technical in nature and include correct cellular differentiation as well as generating adequate numbers of cells for large transplantation experiments, establishing correct cell polarity and connectivity, and ensuring the safety of these approaches regarding tumor or hamartoma formation, Van Gelder explained.

Managing inflammatory responses is a problem after cell transplantation. He cited a Japanese study2 of individual progenitor cell-derived retinal progenitor cells transplanted subretinally in monkey models.

Even with an immune HLA-matched donor, there was still a marked inflammatory response at the site of the transplantation, Van Gelder said. This and other inflammatory responses will have to be managed for cell transplantation to be successful.Related: Intravitreally injected hRPCs improve vision in retinitis pigmentosa cases

There are regulatory hurdles to clear. The FDA Center for Biologics Evaluation and Research regulates cellular therapy products, human gene therapy products, and certain devices related to cell and gene therapy.

Van Gelder recalled the well-publicized case of transplantation of fat-derived mesenchymal cells into patients eyes, resulting in loss of vision bilaterally. He pointed out that it is important to temper patient expectations regarding these therapies and to ensure that the work is being done with the highest degree of ethical integrity.

While great progress has been made in this field, significant barriers remain to the successful adoption in the clinical setting in the coming years, Van Gelder concluded. The barriers to cell replacement should be overcome.

Read more by Lynda Charters

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Russell N. Van Gelder, MD, PhDe: russvg@uw.edu Van Gelder has no financial interests in this subject matter. He serves on the advisory committee for the National Eye Institute Audacious Goals Initiative.

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References

1. Chao JR, Lamba DA, Kiesert TR, et al. Transl Vis Sci Technol. 2017;6:4; doi:10.1167/tvst/6/3/4

2. Fujii S, Sugita S, Futatsugi Y, et al. A strategy for personalized treatment of iPS-retinal immune rejections assessed in cynomolgus monkey models. Int J Mol Sci. 2020;21(9):3077. doi:10.3390/ijms21093077

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Harnessing regeneration of retinal tissues: An option almost within reach - Ophthalmology Times

In a surgical procedure last month, neurosurgeons from Kyoto University implanted 2.4 million cells into the brain of a patient with Parkinsons disease. The cellsderived from peripheral blood cells of an anonymous donorhad been reprogrammed into induced pluripotent stem cells (iPSCs) and then into dopaminergic precursor cells, which researchers hope will boost dopamine levels and ameliorate the patients symptoms.

The procedure is the most recent attempt by clinicians to test whether iPSCs can treat disease. In recent years, Japanese scientists have launched several clinical studies to examine their efficacy in heart disease and macular degeneration of the eye. And other researchers around the globe are exploring ways to turn the cells into treatments for everything from endometriosis to spinal cord injury. The initial foray into clinical trials raises hopes that the technology will bear fruit 12 years after its Nobel Prizewinning discovery.

Im excited that theyre trying to move it to the clinical level, because the iPS field does at some point need to start demonstrating that [these cells] have regenerative potential, says Jalees Rehman of the University of Illinois at Chicago. But the move towards clinical work is also revealing the difficulties of developing therapies. Its a learning curve, he adds.

So far, only a handful of patients have undergone iPSC-based treatments. In 2014, a woman with macular degeneration of the eye received a transplant of iPSC-based retinal cells derived from her own cells. The woman treated showed no apparent improvement in her vision, but the safety of the iPSC-derived cells was confirmed, writes Jun Takahashi, a stem cell biologist at Kyoto University who helped derive the dopaminergic precursor cells implanted into the Parkinsons disease patient. It was his wife, Masayo Takahashi of the RIKEN Center for Developmental Biology, who created the retinal cells used in that trial.

Last year, five patients were treated for the same eye condition with iPSC-derived retinal cells, which were taken from different donors. One of them patients developed a serious, but non-lifethreatening, reaction to the transplant, forcing doctors to remove it, according to the Japan Times.

More clinical studies are underway: Next year, heart surgeons plan to implant sheets of iPSC-derived cardiomyocytes into the hearts of three patients with heart disease, and Takahashi hopes to treat six more patients with Parkinsons disease by 2022. These are all in the earliest phases of testing. It is too early to say something [about the cells efficacy] in our trial, he adds.

While some researchers are waiting for the results of clinical studies to determine whether iPSCs have regenerative potential, others are racing ahead with preclinical studies presenting ever more ways on how to use them therapeutically. For instance, April Pyle, a stem cell biologist at the University of California, Los Angeles, recently developed an approach she believes is promising in treating Duchenne muscular dystrophy, a devastating disease caused by a mutation in the gene encoding the muscle-strengthening protein dystrophin. She and her colleagues used CRISPR-Cas9 to repair the gene in human iPSCs, turned them into skeletal muscle cells, and injected them into the muscle of dystrophin-deficient mice. We [could] actually see that weve restored dystrophin in pockets of the muscle, she explains.

I think its really just the beginning, she says. I think that were finally seeing the payoff for all of the hard work . . . and there will be many more trials to follow from these initial studies.

By now, researchers have figured out how to coax iPSCs to grow into most known cell types, Rehman says. But to get these cells to take on the roles of mature cells in a new tissue environment is another issue. In the heart, for instance, researchers have found that new stem cells have to be electrically aligned with the other cells. Experiments on human iPSC-derived heart muscle cells in culture show that by subjecting them to electrically induced contractions as they develop, the cells mature faster, suggesting that they become more able to handle the adult workload in vivo. How to integrate the new cells so they will survive in injured or diseased tissue is another question. Do you need a special matrix, a gel, a patch, an organoid, to ensure the success of these cells long term? Rehman asks. These challenges are faced in all the organs.

Researchers have been relying on monkey models to evaluate the efficacy of engraftment procedures before testing them in human patients, explains Takahashi. Last year, his team demonstrated on monkeys that human iPSCderived dopaminergic neurons stably integrated into existing brain tissue, where they produced dopamine and ultimately improved Parkinsonian symptoms.

The closer we get to [clinical] applications, the more we obviously realize the challenges that lie ahead.

Jalees Rehman, University of Illinois at Chicago

Another challenge with the implantation of iPSC-derived tissue is the ever-present risk that the cells might trigger cancer, because they stem from a cell type that is by nature highly proliferative. To avoid this, Takahashi and his colleagues filter the implanted cells to eliminate undifferentiated ones that are most prone to overgrowth, and also test the cell lines for tumorgenicity by implanting a sample into mice.

Still, we cannot completely eliminate the possibility of tumor formation, notes Tetsuo Maruyama, an associate professor of obstetrics and gynecology from Keio University School of Medicine. He thinks that such procedures should focus on non-essential organs, such as the eye or the uterus, for instance. He recently succeeded in deriving healthy uterine cells from iPSCs and plans to use these to study how endometriosis occurs, and also to generate human endometrium that could eventually be used clinically.

Another concern researchers have frequently raised are the immunosuppressive drugs that patients require if the iPSCs are derived from cells other than the patients own. Takahashis patient with Parkinsons, for instance, will be on immunosuppressants for a year, possibly making the patient less able to fight off infections and cancer. But despite the risks, many researchers have opted to use allogeneic stem cellsthose from a donorforemost because the approach will save time, cost, and labor when the time comes to scale up such treatments for commercialization. It is important when you think about industrialization, Takahashi writes in an email.

The possibility to create off the shelf iPSC therapies has also attracted industry, not just academics. For instance, Australia-based biotech company Cynata Therapeutics recently concluded a Phase I trial using iPSC-derived mesenchymal stem cells to treat graft-versus-host disease (GVHD). The condition occurs after bone marrow transplants when immune cells of the donor recognize cells in the recipients body as foreign and attack them, often resulting in death. But mesenchymal stem cells, which can mature into a variety of cell types, suppress the proliferation and activation of the donors T cells, explains Kilian Kelly, the companys vice president for product development. The company produced these cells by starting from iPSCs, reprogramming them in to an intermediary cell called a mesenchymoangioblast, and then directing them to become mesenchymal cells.

The trial, which the company claims is the worlds first to use iPSCs, administered the cells intravenously to 15 patients with GVHD who had previously failed to respond to steroid treatment and as such faced a grim prognosis. Although its too soon to evaluate efficacy, Kelly says, he sees it as a positive sign that 14 of them showed a notable improvement in their condition. And conveniently, immune rejection isnt an issue with mesenchymal stem cells because they dont express the donor-specific antigens that trigger rejection. So that means that we can use cells from a single iPS [cell] bank to treat essentially anybody, says Kelly.

Developing off-the-shelf treatments is also vastly more cost effective than maturing iPSC-derived cells for individual patients, adds Ross McDonald, the companys CEO. He points to personalized T-cell immunotherapiestwo of which have been recently FDA-approvedwhich can nearly$500,000 per patient. Its too soon to predict how much his product might cost, he adds.

This is one reason why several groups are developing banks of iPSCs that can be used to develop regenerative therapies at scale. For instance, the Japanese government decided to put around $250 million towards developing an iPSC stock for biomedical research. The donors from whom these cells are derived were carefully selected with immune compatibility in mind: the bank is designed to encompass a diverse set of commonly present human leukocyte antigen (HLA) types, so that they are broadly representative of the majority of the population. Then, implantation will require only a minimum amount of immune suppression. This is kind of a middle ground between using patient-specific cells and cells chosen at random, explains Amanda Mack, director of iPSC reprogramming at Fujifilm Cellular Dynamics, a Wisconsin-based company that grows human cells for biomedical research.

Together, the cells will be immunocompatible with almost 70 percent of the Japanese population, says Maruyama. This might be more difficult for countries such as the US, where the genetic makeup is more diverse, but similar efforts are also underway there. For instance, Macks company aims to develop a bank of iPSCs that are matched to a majority of the US population.

While efforts like these continue, researchers around the world are still figuring out the nuts and bolts of applying these cells therapeutically. The closer we get to [clinical] applications, the more we obviously realize the challenges that lie ahead, says Rehman. I think thats a very normal process for scientific discovery.

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Increasing Number of iPS Cell Therapies Tested in Clinical ...

Houston has played a significant role in boosting the nations biotech industry. While Houston is still a hotspot for energy and oil, the city is steadily becoming a burgeoning life sciences hub. In fact, the city boasted the third fastest-growing biotech community in the nation between 2014 and 2017, according to a CBRE report. Houstons biotech industry is gaining momentum due to an increase in funding as well. According to the Greater Houston Partnership, nearly $180 million in VC funding was allocated to the citys ecosystem of life sciences-related companies in 2019 alone.

Like many startups and tech companies across Houston, the citys life sciences leaders have been tackling some of the worlds most pressing issues. Whether theyre developing oncology drug candidates or advancing genomic medicine through the creation of sequencing technologies, the citys biotech organizations are pulling on decades of research and determination to transform the medical landscape on a global scale. Heres a look at 15 biotech companies in Houston making a major impact on medical research and discovery.

Founded: 2015

Focus: Canine Cancer Treatment

What they do:CAVU Biotherapiesprovides immune-based solutions to treat cancer and autoimmune diseases in dogs. The company offers an immune health monitoring service, which describes a dogs immune system through the use of a blood sample, as well as an autologous prescription product that retrains and expands a dogs T cells to recognize and fight cancer. CAVU Biotherapies ultimate aim is to use its immune-guided medicine to treat horses, cats, andeventually, humans.

Founded: 2006

Focus: Stem Cell Banking + Therapy

What they do: Founded by David Eller and Dr. Stanley Jones, Celltex Therapeutics focuses on developing stem cell therapies for a variety of conditions. The companys stem cell processing and banking methods are designed to ensure the genetic integrity and uniformity of an individuals cells in quantities necessary for therapeutic applications. Using proprietary technology, Celltex Therapeutics enables stem cells to be used for regenerative therapy for conditions like vascular, autoimmune and degenerative diseases.

Founded: 2006

Focus: Cell Therapy

What they do: InGeneron is a clinical stage cell therapy company that specializes in novel, evidence-based regenerative medicine therapies. The companys therapy is designed to repair injured tissue, improve the quality of life for patients and modify the progression of their disease. InGeneron focuses mainly on musculoskeletal indications such as pain management.

Founded: 2006

Focus: Cancer Treatment

What they do: Moleculin Biotech is a pharmaceutical company dedicated to the treatment of highly resistant cancers and viruses. The company develops oncology drug candidates for highly resistant tumors as well as as prodrug to exploit the potential uses of inhibitors of glycolysis. Guided by the aim to provide new hope to cancer patients, Moleculin Biotech focuses on discovering new treatments for acute myeloid leukemia, skin cancer, pancreatic cancer and brain tumors.

Founded: 2001

Focus: Nanomedicine

What they do: Nanospectra Biosciences is spearheading a patient-centric use of nanomedicine for the removal of cancerous tissues. The companys ultra-focal nanoshell technology is designed to thermally destroy solid tumors without damaging adjacent healthy tissue. Nanospectra Biosciences aims to maximize treatment efficacy while minimizing side effects associated with surgery, radiation and traditional focal therapies.

Founded: 2018

Focus: Cell Therapy

What they do: Marker Therapeutics is an immuno-oncology company that focuses on the development of next-generation T cell-based immunotherapies. With the aim of treating hematological malignancies and solid tumor indications, the company uses its own MultiTAA T cell technology, which is based on the selective expansion of non-engineered, tumor-specific T cells. Marker Therapeutics is also working on developing proprietary DNA expression technology that is intended to improve the cellular immune systems ability to recognize and destroy diseased cells.

Founded: 2008

Focus: 3D Cell Culture

What they do: Nano3D Biosciences is dedicated to the development of 3D cell culture solutions. The companys core technology allows them to levitate or bioprint cells, which results in the formation of cultures that are more easily assembled and handled. Nano3D Biosciences products and services are intended for biomedical research, drug discovery, precision medicine, toxicology and regenerative medicine.

Founded: 2017

Focus: Small Molecule Inhibitors

What they do: Tvardi Therapeutics is a clinical-stage biotech company working on a new class of medicines for cancer, chronic inflammation and fibrosis. The company is focusing on the creation of orally delivered, small molecule inhibitors of STAT3, which is a key regulatory protein positioned at the intersection of many disease pathways. Tvardi Therapeutics is dedicated to delivering safe and effective solutions for use in the treatment of numerous diseases.

Founded: 2011

Focus: Targeted Cancer Therapies

What they do: Salarius Pharmaceuticals focuses on developing targeted therapies to treat various types of cancers. The companys lead candidate, Seclidemstat, is intended to treat Ewing sarcoma, a pediatric and young adult bone cancer that currently lacks targeted therapies. Salarius Pharmaceuticals performs clinical trials for the treatment of other advanced solid tumors including prostate, breast and ovarian cancers.

Founded: 2013

Focus: Genomic Medicine

What they do: Founded by Michael Metzker, RedVault Biosciences develops technologies with the aim of advancing genomic medicine. The company is currently working on a variety of projects including the development of sequencing technologies to determine haplotypes and structural variation in complex genomes. RedVault Biosciences is dedicated to identifying technology needs, creating and testing ideas, and transferring deliverables to production and distribution.

Founded: 2010

Focus: DNA Sequencing

What they do: Avance Biosciences focuses on assay development, assay validation and sample testing using next-generation DNA sequencing and other biological methods. The company offers biologics testing, diagnostic assay validation, GMO genomic testing, gene / cell therapy testing, digital and real-time PCR, microbial testing and more. Avance Biosciences aim is to assist its clients in advancing drug development and genomic research.

Founded: 2008

Focus: Bioremediation

What they do: Bionex Technology develops cost-effective, natural solutions for cleaning oil-polluted soil. The companys Super Microbe spill solution is naturally derived from microbes that digest and convert harmful contaminants on the ground and in soil, therefore lowering flammability, suppressing harmful vapors and creating a safer environment for spill responders. Bionex Technology offers a variety of other bioremediation products such as a customizable degreaser and detergent used for cleaning industrial tools.

Founded: 2016

Focus: Stem Cell Research

What they do: Located in nearby Sugar Land, Hope Biosciences is dedicated to developing stem cell-based therapies that are safe, effective and secure. The companys proprietary technology enables patients to make virtually unlimited and identical stem cells from their own tissue. Hope Biosciences offers stem cell banking solutions for both adults and newborns.

Founded: 2013

Focus: Interventional Cardiology

What they do: Saranas has created technology that enables the early detection and monitoring of bleeding complications associated with vascular access procedures. The companys monitoring system checks changes in the blood vessels electrical resistance before monitoring if bleeding has occurred from an unintentionally injured blood vessel. Saranas aims to allow physicians to mitigate downstream consequences by addressing bleeds before they become complications.

Founded: 1984

Focus: Microbiology

What they do: Microbiology Specialists Inc. specializes in microbiology testing, playing a role in microbial investigations and studies. The company also focuses on infectious disease diagnosis, forensic bacteriology and mycology, medical device testing and infection prevention. Microbiology Specialists Inc. is committed to delivering reliable, accurate and cost-effective microbiological results.

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15 Biotech Companies In Houston To Know - Built In

TORONTO, March 31, 2020 /CNW/ - The Gairdner Foundation is pleased to announce the 2020 Canada Gairdner Award laureates, recognizing some of the world's most significant biomedical research and discoveries. During these challenging times, we believe it is important to celebrate scientists and innovators from around the world and commend them for their tireless efforts to conduct research that impacts human health.

2020 Canada Gairdner International AwardThe five 2020 Canada Gairdner International Award laureates are recognized for seminal discoveries or contributions to biomedical science:

Dr. Masatoshi TakeichiSenior Visiting Scientist, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan; Professor Emeritus, Kyoto University, Kyoto, Japan

Dr. Rolf KemlerEmeritus Member and Director, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany

Awarded "For their discovery, characterization and biology of cadherins and associated proteins in animal cell adhesion and signalling."

Dr. Takeichi

The Work: The animal body is made up of numerous cells. Dr. Takeichi was investigatinghow animal cells stick together to form tissues and organs, and identified a key protein which he named 'cadherin'.Cadherin is present on the surface of a cell and binds to the same cadherin protein on the surface of another cell through like-like interaction, thereby binding the cells together. Without cadherin, cell to cell adhesion becomes weakened and leads to the disorganization of tissues. Dr. Takeichi found that there are multiple kinds of cadherin within the body, each of which are made by different cell types, such as epithelial and neuronal cells. Cells with the same cadherins tend to cluster together, explaining the mechanism of how different cells are sorted out and organized to form functional organs.

Further studies by Dr. Takeichi's group showed that cadherin function is supported by a number of cytoplasmic proteins, includingcatenins, and their cooperation is essential for shaping of tissues. His studies also revealed that the cadherin-dependent adhesion mechanism is involved in synaptic connections between neurons, which are important for brain wiring.

Dr. Kemler

The Work: Dr. Kemler, using an immunological approach, developed antibodies directed against surface antigens of early mouse embryos. These antibodies were shown to prevent compaction of the mouse embryo and interfered with subsequent development. Both Dr. Kemler and Dr. Takeichi went on to clone and sequence the gene encoding E-cadherin and demonstrate that it was governing homophilic cell adhesion.

Dr. Kemler also discovered the other proteins that interact with the cadherins, especially the catenins, to generate the machinery involved in animal cell-to-cell adhesion. This provided the first evidence of their importance in normal development and diseases such as cancer. It has been discovered that cadherins and catenins are correlated to the formation and growth of some cancers and how tumors continue to grow. Beta catenin is linked to cell adhesion through interaction with cadherins but is also a key component of the Wnt signalling pathway that is involved in normal development and cancer. There are approximately 100 types of cadherins, known as the cadherin superfamily.

Dr. Takeichi

The Impact: The discovery of cadherins, which are found in all multicellular animalspecies, has allowed us to interpret how multicellular systems are generated and regulated. Loss of cadherin function has been implicated as the cause of certain cancers, as well as in invasiveness of many cancers. Mutations in special types of cadherin result in neurological disorders, such as epilepsy and hearing loss. The knowledge of cadherin function is expected to contribute to the development of effective treatments against such diseases.

Dr. Kemler

The Impact: Human tumors are often of epithelial origin. Given the role of E-cadherin for the integrity of an epithelial cell layer, the protein can be considered as a suppressor of tumor growth. The research on the cadherin superfamily has had great impact on fields as diverse as developmental biology, cell biology, oncology, immunology and neuroscience. Mutations in cadherins/catenins are frequently found in tumors. Various screens are being used to identify small molecules that might restore cell adhesion as a potential cancer therapy.

Dr. Roel NusseProfessor & Chair, Department of Developmental Biology; Member, Institute for StemCell Biology andRegenerativeMedicine, Stanford University, School of Medicine.Virginia and Daniel K. Ludwig Professor of Cancer Research. Investigator, Howard Hughes Medical Institute

Awarded"For pioneering work on the Wnt signaling pathway and its importance in development, cancer and stem cells"

The Work: Dr. Nusse's research has elucidated the mechanism and role of Wnt signaling, one of the most important signaling systems in development. There is now abundant evidence that Wnt signaling is active in cancer and in control of proliferation versus differentiation of adult stem cells, making the Wnt pathway one of the paradigms for the fundamental connections between normal development and cancer.

Among Dr. Nusse's contributions is the original discovery of the first Wnt gene (together with Harold Varmus) as an oncogene in mouse breast cancer. Afterwards Dr. Nusse identified the Drosophila Wnt homolog as a key developmental gene, Wingless. This led to the general realization of the remarkable links between normal development and cancer, now one of the main themes in cancer research. Using Drosophila genetics, he established the function of beta-catenin as a mediator of Wnt signaling and the Frizzleds as Wnt receptors (with Jeremy Nathans), thereby establishing core elements of what is now called the Wnt pathway. A major later accomplishment of his group was the first successful purification of active Wnt proteins, showing that they are lipid-modified and act as stem cell growth factors.

The Impact: Wnt signaling is implicated in the growth of human embryos and the maintenance of tissues. Consequently, elucidating the Wnt pathway is leading to deeper insights into degenerative diseases and the development of new therapeutics. The widespread role of Wnt signaling in cancer is significant for the treatment of the disease as well. Isolating active Wnt proteins has led to the use of Wnts by researchers world-wide as stem cell growth factors and the expansion of stem cells into organ-like structures (organoids).

Dr. Mina J. Bissell Distinguished Senior Scientist, Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory; Faculty; Graduate Groups in Comparative Biochemistry, Endocrinology, Molecular Toxicology and Bioengineering, University of California Berkeley, Berkeley, CA, USA

Awarded "For characterizing "Dynamic Reciprocity" and the significant role that extracellular matrix (ECM) signaling and microenvironment play in gene regulation in normal and malignant cells, revolutionizing the fields of oncology and tissue homeostasis."

The Work: Dr. Mina Bissell's career has been driven by challenging established paradigms in cellular and developmental biology. Through her research, Dr. Bissell showed that tissue architecture plays a dominant role in determining cell and tissue phenotype and proposed the model of 'dynamic reciprocity' (DR) between the extracellular matrix (ECM) and chromatin within the cell nucleus. Dynamic reciprocity refers to the ongoing, bidirectional interaction between cells and their microenvironment. She demonstrated that the ECM could regulate gene expression just as gene expression could regulate ECM, and that these two phenomena could occur concurrently in normal or diseased tissue.

She also developed 3D culture systems to study the interaction of the microenvironment and tissue organization and growth, using the mammary gland as a model.

The Impact:Dr. Bissell's model of dynamic reciprocity has been proven and thoroughly established since its proposal three decades ago and the implications have permeated every area of cell and cancer biology, with significant implications for current and future therapies. Dr. Bissell's work has generated a fundamental and translationally crucial paradigm shift in our understanding of both normal and malignant tissues.

Her findings have had profound implications for cancer therapy by demonstrating that tumor cells can be influenced by their environment and are not just the product of their genetic mutations. For example, cells from the mammary glands grown in two-dimensional tissue cultures rapidly lose their identity, but once placed in proper three-dimensional microenvironments, they regain mammary form and function. This work presages the current excitement about generation of 3D tissue organoids and demonstrates Dr. Bissell's creative and innovative approach to science.

Dr. Elaine FuchsHoward Hughes Medical Institute Investigator and Rebecca C. Lancefield Professor and Head of the Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Cell Biology; The Rockefeller University, New York, NY, USA

Awarded"For her studies elucidating the role of tissue stem cells in homeostasis, wound repair, inflammation and cancer."

The Work: Dr. Fuchs has used skin to study how the tissues of our body are able to replace dying cells and repair wounds. The skin must replenish itself constantly to protect against dehydration and harmful microbes. In her research, Fuchs showed that this is accomplished by a resident population of adult stem cells that continually generates a shell of indestructible cells that cover our body surface.

In her early research, Fuchs identified the proteins---keratinsthat produce the iron framework of the skin's building blocks, and showed that mutations in keratins are responsible for a group of blistering diseases in humans. In her later work, Fuchs identified the signals that prompt skin stem cells to make tissue and when to stop. In studying these processes, Fuchs learned that cancers hijack the fundamental mechanisms that tissue stem cells use to repair wounds. Her team pursued this parallel and isolated and characterized the malignant stem cells that are responsible for propagating a type of cancer called "squamous cell carcinoma." In her most recent work, she showed that these cells can be resistant to chemotherapies and immunotherapies and lead to tumor relapse.

The Impact: All tissues of our body must be able to replace dying cells and repair local wounds. Skin is particularly adept at performing these tasks. The identification and characterization of the resident skin stem cells that make and replenish the epidermis, sweat glands and hair provide important insights into this fountain of youth process and hold promise for regenerative medicine and aging. In normal tissues, the self-renewing ability of stem cells to proliferate is held in check by local inhibitory signals coming from the stem cells' neighbours. In injury, stimulatory signals mobilize the stem cells to proliferate and repair the wound. In aging, these normal balancing cues are tipped in favour of quiescence. In inflammatory disorders, stem cells become hyperactivated. In cancers, the wound mechanisms to mobilize stem cells are hijacked, leading to uncontrolled tissue growth. Understanding the basic mechanisms controlling stem cells in their native tissue is providing new strategies for searching out refractory tumor cells in cancer and for restoring normalcy in inflammatory conditions.

2020 John Dirks Canada Gairdner Global Health AwardThe 2020 John Dirks Canada Gairdner Global Health Award laureate is recognized for outstanding achievements in global health research:

Professor Salim S. Abdool KarimDirector of CAPRISA (Centre for the AIDS Program of Research in South Africa), the CAPRISA Professor in Global Health at Columbia University, New York and Pro Vice-Chancellor (Research) at the University of KwaZulu-Natal, Durban, South Africa

Professor Quarraisha Abdool KarimAssociate Scientific Director of CAPRISA, Professor in Clinical Epidemiology, Columbia University, New York and Professor in Public Health at the Nelson Mandela Medical School and Pro Vice-Chancellor (African Health) at the University of KwaZulu-Natal, Durban, South Africa

Awarded"For their discovery that antiretrovirals prevent sexual transmission of HIV, which laid the foundations for pre-exposure prophylaxis (PrEP), the HIV prevention strategy that is contributing to the reduction of HIV infection in Africa and around the world."

The Work: UNAIDS estimates that 37 million people were living with HIV and 1.8 million people acquired HIV in 2017. In Africa, which has over two thirds of all people with HIV, adolescent girls and young women have the highest rates of new HIV infections. ABC (Abstinence, Be faithful, and use Condoms) prevention messages have had little impact - due to gender power imbalances, young women are often unable to successfully negotiate condom use, insist on mutual monogamy, or convince their male partners to have an HIV test.

In responding to this crisis, Salim and Quarraisha Abdool Karim started investigating new HIV prevention technologies for women about 30 years ago. After two unsuccessful decades, their perseverance paid off when they provided proof-of-concept that antiretrovirals prevent sexually acquired HIV infection in women. Their ground-breaking CAPRISA 004 trial showed that tenofovir gel prevents both HIV infection and genital herpes. The finding was ranked inthe "Top 10 Scientific Breakthroughs of 2010" by the journal, Science. The finding was heralded by UNAIDS and the World Health Organization (WHO) as one of the most significant scientific breakthroughs in AIDS and provided the first evidence for what is today known as HIV pre-exposure prophylaxis (PrEP).

The Abdool Karims have also elucidated the evolving nature of the HIV epidemic in Africa, characterising the key social, behavioural and biological risk factors responsible for the disproportionately high HIV burden in young women. Their identification of the "Cycle of HIV Transmission", where teenage girls acquire HIV from men about 10 years older on average, has shaped UNAIDS policies on HIV prevention in Africa.

The impact: CAPRISA 004 and several clinical trials of oral tenofovir led tothe WHO recommending a daily tenofovir-containing pill for PrEP as a standard HIV prevention tool for all those at high risk a few years later. Several African countries are among the 68 countries across all continents that are currently making PrEP available for HIV prevention. The research undertaken in Africa by this South African couple has played a key role in shaping the local and global response to the HIV epidemic.

2020 Canada Gairdner Wightman AwardThe 2020 Canada Gairdner Wightman Award laureate is a Canadian scientist recognized for outstanding leadership in medicine and medical science throughout their career:

Dr. Guy Rouleau Director of the Montreal Neurological Institute-Hospital (The Neuro); Professor & Chair of the Department of Neurology and Neurosurgery, McGill University; Director of the Department of Neuroscience, McGill University Health Center

Awarded "For identifying and elucidating the genetic architecture of neurological and psychiatric diseases, including ALS, autism and schizophrenia, and his leadership in the field of Open Science."

The Work: Dr. Rouleau has identified over 20 genetic risk factors predisposing to a range of brain disorders, both neurological and psychiatric, involving either neurodevelopmental processes or degenerative events. He has defined a novel disease mechanism for diseases related to repeat expansions that are at play in some of the most severe neurodegenerative conditions. He has significantly contributed to the understanding of the role of de novo variants in autism and schizophrenia. In addition, he has made important advances for various neuropathies, in particular for amyotrophic lateral sclerosis (ALS) where he was involved in the identification of the most prevalent genetic risk factors -which in turn are now the core of innumerable ALS studies worldwide.

Dr. Rouleau has also played a pioneering role in the practice of Open Science (OS), transforming the Montreal Neurological Institute-Hospital (The Neuro) into the first OS institution in the world. The Neuro now uses OS principles to transform research and careand accelerate the development of new treatments for patients through Open Access, Open Data, Open Biobanking, Open Early Drug Discovery and non-restrictive intellectual property.

The Impact: The identification of genetic risk factors has a number of significant consequences. First, allowing for more accurate genetic counselling, which reduces the burden of disease to affected individuals, parents and society. A revealing case is Andermann syndrome, a severe neurodevelopmental and neurodegenerative condition that was once relatively common in the Saguenay-Lac-St-Jean region of Quebec. Now this disease has almost disappeared from that population. Second, identifying the causative gene allows the development of treatments. For instance, his earlier work on a form of ALS linked to the superoxide dismutase-1 gene (SOD1) opened up studies which are now the focal point of phase 2 clinical studies showing great promise.

Byactingasalivinglabforthelast coupleofyears,TheNeuroisspearheading the practice of OpenScience (OS).TheNeurois alsoengagingstakeholdersacross Canadawiththegoal of formalizinganational OSallianceforthe neurosciences.Dr.Rouleau'sworkinOScontributesfundamentallytothetransformationoftheveryecosystemofsciencebystimulatingnewthinkingandfosteringcommunitiesofsharing.InspiredbyTheNeuro'svision,theglobalsciencecommunityisreflecting oncurrentresearchconventionsandcollaborativeprojects,andthemomentumforOSisgainingafootholdinorganizationsandinstitutionsinallcornersoftheearth.

About the Gairdner Foundation:

The Gairdner Foundation was established in 1957 by Toronto stockbroker, James Gairdner to award annual prizes to scientists whose discoveries have had major impact on scientific progress and on human health. Since 1959 when the first awards were granted, 387scientists have received a Canada Gairdner Award and 92 to date have gone on to receive the Nobel Prize.The Canada Gairdner Awards promote a stronger culture of research and innovation across the country through our Outreach Programs including lectures and research symposia. The programs bring current and past laureates to a minimum of 15 universities across Canada to speak with faculty, trainees and high school students to inspire the next generation of researchers. Annual research symposia and public lectures are organized across Canada to provide Canadians access to leading science through Gairdner's convening power.

http://www.gairdner.org

SOURCE Gairdner Foundation

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2020 Canada Gairdner Awards Recognize World-renowned Scientists for Transformative Contributions to Research That Impact Human Health - Benzinga

Heart disease is the primary cause of death worldwide, principally because the heart has minimal ability to regenerate muscle tissue. Myocardial infarction (heart attack) caused by coronary artery disease leads to heart muscle loss and replacement with scar tissue, and the heart's pumping ability is permanently reduced. Breakthroughs in stem cell biology in the 1990s and 2000s led to the hypothesis that heart muscle cells (cardiomyocytes) could be regenerated by transplanting stem cells or their derivatives. It has been 18 years since the first clinical trials of stem cell therapy for heart repair were initiated (1), mostly using adult cells. Although cell therapy is feasible and largely safe, randomized, controlled trials in patients show little consistent benefit from any of the treatments with adult-derived cells (2). In the meantime, pluripotent stem cells have produced bona fide heart muscle regeneration in animal studies and are emerging as leading candidates for human heart regeneration.

In retrospect, the lack of efficacy in these adult cell trials might have been predicted. The most common cell type delivered has been bone marrow mononuclear cells, but other transplanted cell types include bone marrow mesenchymal stromal cells and skeletal muscle myoblasts, and a few studies have used putative progenitors isolated from the adult heart itself. Although each of these adult cell types was originally postulated to differentiate directly into cardiomyocytes, none of them actually do. Indeed, with the exception of skeletal muscle myoblasts, none of these cell types survive more than a few days in the injured heart (see the figure). Unfortunately, the studies using bone marrow and adult resident cardiac progenitor cells were based on a large body of fraudulent work (3), which has led to the retraction of >30 publications. This has left clinical investigators wondering whether their trials should continue, given the lack of scientific foundation and the low but measurable risk of bleeding, stroke, and infection.

Additionally, investigators have struggled to explain the beneficial effects of adult cell therapy in preclinical animal models. Because none of these injected cell types survive and engraft in meaningful numbers or directly generate new myocardium, the mechanism has always been somewhat mysterious. Most research has focused on paracrine-mediated activation of endogenous repair mechanisms or preventing additional death of cardiomyocytes. Multiple protein factors, exosomes (small extracellular vesicles), and microRNAs have been proposed as the paracrine effectors, and an acute immunomodulatory effect has recently been suggested to underlie the benefits of adult cell therapy (4). Regardless, if cell engraftment or survival is not required, the durability of the therapy and need for actual cells versus their paracrine effectors is unclear.

Of particular importance to clinical translation is whether cell therapy is additive to optimal medical therapy. This remains unclear because almost all preclinical studies do not use standard medical treatment for myocardial infarction. Given the uncertainties about efficacy and concerns over the veracity of much of the underlying data, whether agencies should continue funding clinical trials using adult cells to treat heart disease should be assessed. Perhaps it is time for proponents of adult cardiac cell therapy to reconsider the approach.

Pluripotent stem cells (PSCs) include embryonic stem cells (ESCs) and their reprogrammed cousins, induced pluripotent stem cells (iPSCs). In contrast to adult cells, PSCs can divide indefinitely and differentiate into virtually every cell type in the human body, including cardiomyocytes. These remarkable attributes also make ESCs and iPSCs more challenging to control. Through painstaking development, cell expansion and differentiation protocols have advanced such that batches of 1 billion to 10 billion pharmaceutical-grade cardiomyocytes, at >90% purity, can be generated.

Preclinical studies indicate that PSC-cardiomyocytes can remuscularize infarcted regions of the heart (see the figure). The new myocardium persists for at least 3 months (the longest time studied), and physiological studies indicate that it beats in synchrony with host myocardium. The new myocardium results in substantial improvement in cardiac function in multiple animal models, including nonhuman primates (5). Although the mechanism of action is still under study, there is evidence that these cells directly support the heart's pumping function, in addition to providing paracrine factors. These findings are in line with the original hope for stem cell therapyto regenerate lost tissue and restore organ function. Additional effects, such as mechanically buttressing the injured heart wall, may also contribute.

Breakthroughs in cancer immunotherapy have led to the adoption of cell therapies using patient-derived (autologous) T cells that are genetically modified to express chimeric antigen receptors (CARs) that recognize cancer cell antigens. CAR T cells are the first U.S. Food and Drug Administration (FDA)approved, gene-modified cellular pharmaceutical (6). The clinical and commercial success of autologous CAR T cell transplant to treat B cell malignancies has opened doors for other complex cell therapies, including PSC derivatives. There is now a regulatory path to the clinic, private-sector funding is attracted to this field, and clinical investigators in other areas are encouraged to embrace this technology. Indeed, the first transplants of human ESC-derived cardiac progenitors, surgically delivered as a patch onto the heart's surface, have been carried out (7). In the coming years, multiple attempts to use PSC-derived cardiomyocytes to repair the human heart are likely.

What might the first human trials look like? These studies will probably employ an allogeneic (non-self), off-the-shelf, cryopreserved cell product. Although the discovery of iPSCs raised hopes for widespread use of autologous stem cell therapies, the current technology and regulatory requirements likely make this approach too costly for something as common as heart disease, although this could change as technology and regulations evolve. Given that it would take at least 6 months to generate a therapeutic dose of iPSC-derived cardiomyocytes, such cells could only be applied to patients whose infarcts are in the chronic phase where scarring (fibrosis) and ventricular remodeling are complete. Preclinical data indicate that chronic infarcts benefit less from cardiomyocyte transplantation than do those with active wound-healing processes.

Adult cells from bone marrow or the adult heart secrete beneficial paracrine factors but do not engraft in the infarcted heart. Pluripotent stem cells give rise to cardiomyocytes that engraft long term in animal models, beat in synchrony with the heart, and secrete beneficial paracrine factors. Long-term cardiomyocyte engraftment partially regenerates injured heart, which is hypothesized to bring clinical benefits.

The need for allogeneic cells raises the question of how to prevent immune rejection, both from innate immune responses in the acute phase of transplantation or from adaptive immune responses that develop more slowly through the detection of non-self antigens presented by major histocompatibility complexes (MHCs). A current strategy is the collection of iPSCs from patients who have homozygous MHC loci, which results in exponentially more MHC matches with the general population. However, studies in macaque monkeys suggest that MHC matching will be insufficient. In a macaque model of brain injury, immunosuppression was required to prevent rejection of MHC-matched iPSC-derived neurons (8). Similarly, MHC matching reduced the immunogenicity of iPSC-derived cardiomyocytes transplanted subcutaneously or into the hearts of rhesus macaques, but immunosuppressive drugs were still required to prevent rejection (9).

Numerous immune gene editing approaches have been proposed to circumvent rejection, including preventing MHC class I and II molecule expression, overexpressing immunomodulatory cell-surface factors, such CD47 and human leukocyte antigen E (HLA-E) and HLA-G (two human MHC molecules that promote maternal-fetal immune tolerance), or engineering cells to produce immunosuppressants such as programmed cell death ligand 1 (PDL1) and cytotoxic T lymphocyteassociated antigen 4 (CTLA4) (10). These approaches singly or in combination seem to reduce adaptive immune responses in vitro and in mouse models. Overexpressing HLA-G or CD47 also blunts the innate natural killer cellmediated response that results from deleting MHC class I genes (11). However, these manipulations are not without theoretical risks. It could be difficult to clear viral infections from an immunostealthy patch of tissue, and possible tumors resulting from engraftment of PSCs might be difficult to clear immunologically.

Ventricular arrhythmias have emerged as the major toxicity of cardiomyocyte cell therapy. Initial studies in small animals showed no arrhythmic complications (probably because their heart rates are too fast), but in large animals with human-like heart rates, arrhythmias were consistently observed (5, 12). Stereotypically, these arrhythmias arise a few days after transplantation, peak within a few weeks, and subside after 4 to 6 weeks. The arrhythmias were well tolerated in macaques (5) but were lethal in a subset of pigs (12). Electrophysiological studies indicate that these arrhythmias originate in graft regions from a source that behaves like an ectopic pacemaker. Understanding the mechanism of these arrhythmias and developing solutions are major areas of research. There is particular interest in the hypothesis that the immaturity of PSC-cardiomyocytes contributes to these arrhythmias, and that their maturation in situ caused arrhythmias to subside.

A successful therapy for heart regeneration also requires understanding the host side of the equation. PSC-derived cardiomyocytes engraft despite transplantation into injured myocardium that is ischemic with poor blood flow. Although vessels eventually grow in from the host tissue, normal perfusion is not restored. Achieving a robust arterial input will be key to restoring function, which may require cotransplanting other cell populations or tissue engineering approaches (13, 14). Most PSC-mediated cardiac cell therapy studies have been performed in the subacute window, equivalent to 2 to 4 weeks after myocardial infarction in humans. At this point, there has been insufficient time for a substantial fibrotic response. Fibrosis has multiple deleterious features, including mechanically stiffening the tissue and creating zones of electrical insulation that can cause arrhythmias. Extending this therapy to other clinical situations, such as chronic heart failure, will require additional approaches that address the preexisting fibrosis. Cell therapy may again provide an answer because CAR T cells targeted to cardiac fibroblasts reduced fibrosis (15).

Developing a human cardiomyocyte therapy for heart regeneration will push the limits of cell manufacturing. Each patient will likely require a dose of 1 billion to 10 billion cells. Given the widespread nature of ischemic heart disease, 105 to 106 patients a year are likely to need treatment, which translates to 1014 to 1016 cardiomyocytes per year. Growing cells at this scale will require introduction of next generation bioreactors, development of lower-cost media, construction of large-scale cryopreservation and banking systems, and establishment of a robust supply chain compatible with clinical-grade manufacturing practices.

Beyond PSC-cardiomyocytes, other promising approaches include reactivating cardiomyocyte division and reprogramming fibroblasts to form new cardiomyocytes. However, these approaches are at an earlier stage of development, and currently, PSC-derived cardiomyocyte therapy is the only approach that results in large and lasting new muscle grafts. The hurdles to this treatment are known, and likely addressable, thus multiple clinical trials are anticipated.

Acknowledgments: C.E.M. and W.R.M. are scientific founders of and equity holders in Sana Biotechnology. C.E.M. is an employee of Sana Biotechnology. W.R.M. is a consultant for Sana Biotechnology. C.E.M. and W.R.M. hold issued and pending patents in the field of stem cell and regenerative biology.

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Stem cells and the heartthe road ahead - Science Magazine

Testing a new therapeutic in human subjects for the first time is a major step in the translation of any novel treatment from the laboratory bench to clinical use.

When the therapeutic represents a paradigm shift, reaching this milestone is even more significant.

After years of planning, preparation and hard work to establish a base camp, starting human clinical trials is the first step towards the summit itself: gaining regulatory approval for product sales.

Exosomes tiny packets of proteins and nucleic acids (e.g. mRNA and miRNA) released by cells, that have powerful regenerative properties ranging from promoting wound healing to stimulating brain injury recovery following stroke represent just such a paradigm-shifting potential advance in human medicine.

The first commercial exosome therapeutics conference was held in Boston in September 2019 and over 15 companies participated.

This conference signals the emergence of exosomes as a new class of regenerative medicine products.

So far, just one or two of the companies working in the novel field of exosome-based therapies have reached the pivotal point and transitioned into human clinical trials. In this article we survey the field, starting with the pace-setters.

During the past few years, a handful of universities and research hospitals have carried out small scale, first-in-human Phase I clinical trials using exosomes. In each case where the study results are available, the exosome treatment was found to be safe and well-tolerated.

But the field has hotted up in the past few months, with the first companies reaching the pivotal point of testing exosome-based products in people.

On 28th January 2020, Melbourne-based Exopharm announced the first dosing under its first human clinical trial, becoming the first company to test exosomes potential for healing wounds in people.

The PLEXOVAL Phase I study will test Exopharms Plexaris product, a cell-free formulation of exosomes from platelets, which in preclinical animal studies have shown a regenerative effect, improving wound closure and reducing scarring.

The main readouts of the PLEXOVAL study the results of which are expected to be available sometime after mid-2020 will be safety, wound closure and scarring.

Joining Exopharm at the front of the pack is Maryland-based United Therapeutics.

Founded in 1996, United Therapeutics specialises in lung diseases and has a portfolio of FDA-approved conventional small molecule and biologic drugs on the market for a range of lung conditions.

On 26th June 2019, United Therapeutics announced approval for a Phase I trial (NCT03857841) of an exosome-based therapy against bronchopulmonary dysplasia (BDP), a condition common in preterm infants that receive assisted ventilation and supplemental oxygen.

Recruitment has commenced but dosing has not been announced. The study is due to conclude by December 2021. BDP is characterised by arrested lung growth and development, with health implications that can persist into adulthood.

Human clinical trials of a stem cell therapy for BDP, by Korean stem cell company Medipost, are already underway. However as with many stem cell therapies recent animal studies have shown that is the exosomes released by stem cells that are responsible for the therapeutic effect.

United Therapeutics therapy, UNEX-42, is a preparation of extracellular vesicles that are secreted from human bone marrow-derived mesenchymal stem cells. The company has not released any information about how its exosomes are produced or isolated.

A little behind the two leaders, three other companies have announced their aim to initiate their first clinical trials of exosome therapeutics within the next 12 months.

Launched in 2015, Cambridge, Massachusetts-based Codiak has long been considered among the leaders in developing exosome-based therapies.

Rather than exploiting the innate regenerative potential of select exosome populations, Codiak is developing engineered exosomes that feature a defined therapeutic payload. The companys initial focus has been to target immune cells, leveraging the immune system to combat cancer.

The company plans to initiate clinical trials of its lead candidate, exoSTING, in the first half of 2020. The therapeutic is designed to trigger a potent antitumor response from the patients own immune system, mediated by T cells. A second immuno-oncology candidate, exoIL-12, is due to enter clinical trials in the second half of 2020, the company says.

In nearby New Jersey, Avalon Globocare is also developing engineered exosomes. Its lead product, AVA-201, consists of exosomes enriched in the RNA miR-185, which are produced using engineered mesenchymal stem cells.

In animal tests, miR-185 suppressed cancer cell proliferation, invasion and migration in oral cancer. In July 2019, the company announced plans to start its first exosome clinical trial before the close of 2019. As of February 2020, however, no further announcement regarding this clinical trial has been made.

Avalon has also made no further announcement on a second planned clinical trial, also intended to start during the fourth quarter of 2019, of a second exosome candidate, AVA-202.

These angiogenic regenerative exosomes, derived from endothelial cells, can promote wound healing and blood vessel formation, the company says. The planned Phase I trial was to test AVA-202 for vascular diseases and wound healing.

Meanwhile, Miami-based Aegle Therapeutics plans to begin a Phase I/IIa clinical trial of its exosome therapy, AGLE-102, during 2020. AGLE-102 is based on native regenerative exosomes isolated from bone marrow mesenchymal stem cells.

After initially focussing on burns patients, in January 2020 to company announced had raised the funds to commence an FDA-cleared clinical trial of AGLE-102 to treat dystrophic epidermolysis bullosa, a rare paediatric skin blistering disorder. The company says it plans to commence this clinical trial in the first half of 2020.

A number of companies are in the preclinical phase of exosome therapy research.

Some of these companies have been set up specifically to develop exosome-based products. In the UK, Evox co-founded by University of Oxford researcher Matthew Wood in 2016 is developing engineered exosomes to treat rare diseases.

The company has developed or sourced technology that allows it to attach proteins to exosomes surface, or to load proteins or nucleic acids inside the exosome, to deliver a therapeutic cargo to a target organ.

Its lead candidate targets a lysosomal storage disorder called Niemann-Pick Disease type C, using exosomes that carry a protein therapeutic cargo. Evox says it plans to submit the Investigational New Drug (IND) application to the FDA during 2020, paving the way for the first clinical trial. It currently has five other candidates, for various indications, at the preclinical stage of development.

In Korea, Ilias and ExoCoBio are developing exosome therapeutics. Ilias founded by faculty from the Korean Advance Institute of Science and Technology specialises in loading large protein therapeutics into exosomes.

It is currently carrying out preclinical research toward treating sepsis, preterm labour and Gauchers disease. ExoCoBio is focusing on the native regenerative capacity of exosomes derived from mesenchymal stem cells, including to treat atopic dermatitis.

New companies continue to enter the exosome space. In August 2019, Carmine Therapeutics was launched, with the aim to develop gene therapies that utilize exosomes from red blood cells to deliver large nucleic acid cargoes. The company is targeting the areas of haematology, oncology and immunology.

Meanwhile, a wave of companies originally set up to develop live stem cell therapies are diversifying into stem cell derived exosome production and research.

It is now generally acknowledged that stem cell exosomes are the main therapeutically active component of stem cells, and that medical products based on exosomes will be safer to apply, and easier and cheaper to make and transport, than live cell therapies.

Originally established to produce neural stem cells for other research organisations, Aruna Bio has developed proprietary neural exosomes that can cross the blood brain barrier.

The company is now developing an exosome therapy for stroke. In October 2019, the Athens, Georgia-based company said had raised funding to support the research and development to enable its first IND application to the FDA in 2021.

In the UK, ReNeuron has also focussed on stroke, and has several clinical trials underway assessing its CTX stem cells to promote post stroke rehabilitation. The company is also working with third parties to investigate the drug- and gene therapy delivery potential of exosomes derived from CTX stem cells.

Switzerland-based Anjarium is also developing an exosome platform to selectively deliver therapeutics.20 The company is focussing on engineering exosomes loaded with therapeutic RNA cargo and displaying targeting moieties on its surface.

California-based Capricor has commenced clinical trials of a cardiosphere-derived stem cell therapy for the treatment of Duchenne muscular dystrophy (DMD).

At an earlier phase, its regenerative exosome therapy CAP-2003 is in pre-clinical development for a variety of inflammatory disorders including DMD.

A number of other stem cell companies, including TriArm, Creative Medical, AgeX Therapeutics and BrainStorm Cell Therapeutics, are reported to be investigating exosome-based therapies derived from their stem cell lines.

Exopharms position as a frontrunner in bringing exosomes into humans is no lucky accident. The companys operations are based around its unique, proprietary method for manufacturing and isolating exosomes, known as LEAP technology.

As academics and observers of the exosome field have pointed out, reliable and scalable exosome manufacture has threatened to be a major bottleneck that limits the translation of exosome therapeutics into clinical use. The standard laboratory-scale method for collecting the exosomes produced by cultured cells has been to spin the liquid cell culture medium in an ultracentrifuge, or pass it through a fine filter.

The most common technique used so far, the ultracentrifuge, has major scalability limitations. Issues include the high level of skill and manual labour required, the time-intensive nature of the process, and the associated costs of reagents and equipment. It is impossible to imagine collecting enough exosomes for a late stage clinical trial this way.

Another issue is the low purity of the exosomes collected. These techniques sort the contents of cell culture medium by their mass and/or size. Although the exosomes are concentrated, they could be accompanied by other biological components present in the cell culture medium that happen to be a similar size or mass to the exosome.

Importantly, a biotechnology company needs a proprietary step in the process to make a proprietary product over which it has exclusivity. Exopharms LEAP technology is a good example of a proprietary manufacturing step. Ultracentrifuge is not a proprietary process.

So the big players in the emerging exosome field have generally placed a strong emphasis on developing their manufacturing and purification capability.

Exopharm developed a chromatography-based purification method, in which a patent-applied-for inexpensive functionalised polymer a LEAP Ligand is loaded into a chromatography column. The LEAP Ligand sticks to the membrane surface of exosomes passed through the column. Everything else in the cell culture medium mixture is simply washed away. The pure exosome product is then eluted from the column and collected for use. As well as being very scalable, the technique is versatile. LEAP can be used to produce a range of exosome products, by isolating exosomes from different cell sources.

Codiak, similarly, says it has developed scalable, proprietary chromatography-based methods to produced exosomes with comparable identity, purity, and functional properties as exosomes purified using methods such as ultracentrifugation. Chromatography is a flow-based technique for separating mixtures. In an April 2019 SEC filing, the company said it is establishing its own Phase 1/2 clinical manufacturing facility, which it is aiming to have fully-operational by first half 2020.

Avalon GloboCare teamed up with Weill Cornell Medicine to develop a standardised production method for isolating clinical-grade exosomes. Aegle also says it has a proprietary isolation process for producing therapeutic-grade exosomes. And Evox emphasises the GMP compliant, scalable, commercially viable manufacturing platform it has developed.

At Exopharm, the manufacturing technique that has allowed the company to leap ahead of the pack and into human clinical trials is its proprietary LEAP platform. Overcoming the exosome production and isolation bottleneck was exactly the problem the companys scientists set out to solve when Exopharm formed in 2013.

In addition to the Plexaris exosomes, isolated from platelets, currently being tested in human clinical trials, Exopharm is progressing toward human clinical trials of its second product, Cevaris, which are exosomes isolated from stem cells.

Exosomes are now under development by around 20 companies across the world. The leaders in the field are now entering clinical trials with both nave exosome products and engineered exosome products. A number of cell therapy companies are also moving across into the promising exosome product space.

The coming years promise dynamic changes, with partnerships and eventually product commercialization. Exopharm is a clear leader in this emerging field.

(Featured image by Darko Stojanovic from Pixabay)

DISCLAIMER: This article was written by a third party contributor and does not reflect the opinion of Born2Invest, its management, staff or its associates. Please review our disclaimer for more information.

This article may include forward-looking statements. These forward-looking statements generally are identified by the words believe, project, estimate, become, plan, will, and similar expressions. These forward-looking statements involve known and unknown risks as well as uncertainties, including those discussed in the following cautionary statements and elsewhere in this article and on this site. Although the Company may believe that its expectations are based on reasonable assumptions, the actual results that the Company may achieve may differ materially from any forward-looking statements, which reflect the opinions of the management of the Company only as of the date hereof. Additionally, please make sure to read these important disclosures.

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Biotech companies leading the way with exosome human clinical trials - Born2Invest

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REHOVOT, Israel and NEW YORK, Nov. 14, 2019 (GLOBE NEWSWIRE) -- Todos Medical Ltd. (TOMDF), a clinical-stage in-vitro diagnostics company focused on the development of blood tests for the early detection of cancer and neurodegenerative disorders, and Amarantus Bioscience Holdings, Inc. a US-based JLABS-alumnus biotechnology holding company developing proprietary orphan neurologic, regenerative medicine and ophthalmic therapies and diagnostics through its subsidiaries, today announced that their joint venture company, Breakthrough Diagnostics, Inc. has completed enrollment of its ongoing clinical trial evaluating the relationship of Alzheimers blood diagnostic Lymphocyte Proliferation Test (LymPro Test) with amyloid PET neuroimaging at Leipzig University in Germany (the LymPro PET 2). Topline results are expected before the end of the first quarter of 2020.

Breakthrough completed a 20-subject clinical study (LymPro PET 1) in 2018 evaluating the correlation between LymPro scores and the diagnosis of Alzheimers disease, as confirmed with amyloid PET neuroimaging and other Alzheimers disease biomarkers. LymPro measures cell cycle dysregulation in peripheral lymphocytes. The top-line data, announced in July 2019, revealed a strong and statistically significant correlation between LymPro scores and amyloid PET neuroimaging cSUVR scores (r = -0.849; p = 0.00000216). Breakthroughs academic collaborators at the Leipzig University then expanded enrollment of that study to include an additional cohort of 20 subjects (LymPro PET 2) to confirm the strong relationship seen from LymPro PET 1. The data from both LymPro 1 and LymPro 2 will be published together in a peer-reviewed journal in 2020.

LymPro is a unique immune system-based Alzheimers blood test, said Dr. Herman Weiss, President & CEO of Todos. LymPro could prove to be a major breakthrough for Alzheimers disease diagnosis by measuring cell cycle dysregulation and amyloid, together, conveniently as part of a blood workup in routine clinical practice. The therapeutic field in Alzheimers has begun to see some renewed hope based upon recent Aducanumab data announced by Biogen that is directly related to the amyloid hypothesis, as well as conditional approval by the National Medical Products Administration in China for the first new Alzheimers drug in over 20 years, called Oligomannate from Shanghai Green Valley Pharmaceuticals, that is based on gut-brain biology of the microbiome and its effects on the immune system. We believe this renewed optimism and broadening of pathophysiological hypotheses relevant to Alzheimers disease being evaluated in the clinic significantly increases the scope for LymPro pharma services collaborations and begins to refine LymPros clinical utility profile for primary care physicians as strategies to correct cell cycle dysregulation emerge.

About Alzheimer's DiseaseAccording to the Alzheimer's Association, it is estimated that over 5.4 million people in the United States suffer from Alzheimer's disease. Over 500,000 patients are diagnosed annually, with nearly one-in-eight older Americans affected by the disease. Alzheimer's disease is the third leading cause of death in the United States. The cost of unpaid care in the United States is estimated at over $210 billion annually.Total payments for care are estimated at over $200 billion annually, including $140 billion in cost to Medicare and Medicaid. Alzheimer's expenditures in the United States are expected to exceed $1.2 trillion by 2050. There is no cure or effective treatment for Alzheimer's disease. Worldwide, about 35.6 million individuals have the disease and, according to the World Health Organization, the number will double every 20 years to 115.4 million people with Alzheimer's by 2050.

About Dr. Arendt's Research at Leipzig UniversityDr. Thomas Arendt is Professor of Neuroscience at Leipzig University where he runs the Paul Flechsig Institute of Brain Research. He has a 30-year record in R&D of therapeutic and diagnostic strategies of neurodegenerative disorders and made several seminal contributions to therapeutic concepts of Alzheimer's disease, including stem cell therapy and modulating tumor suppressor genes. In the early 1980's, he was involved in identifying the degeneration of the cholinergic system in Alzheimer's disease laying the basis for today's only available treatment. He is one of the pioneers of the "cell-cycle theory" of Alzheimer's disease, which he developed towards a diagnostic and therapeutic concept.

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About Breakthrough Diagnostics, Inc.Breakthrough Diagnostics, Inc. is a joint venture owned by Amarantus Bioscience Holdings, Inc. (AMBS) (80.01%) and Todos Medical Ltd. (19.99%). Breakthrough has been assigned the intellectual property and other rights to the LymPro Test, a diagnostic blood test for Alzheimers disease, as well as rights to other neurological diagnostics testing intellectual property. Todos Medical has provided Amarantus with notice of Todos decision to exercise its exclusive option to acquire the 80.01% of Breakthrough Diagnostics that it currently does not own.

The Lymphocyte Proliferation Test (LymPro Test) determines the ability of peripheral blood lymphocytes (PBLs) and monocytes to withstand an exogenous mitogenic stimulation that induces them to enter the cell cycle. It is believed that certain diseases, most notably Alzheimer's disease, are the result of compromised cellular machinery that leads to aberrant cell cycle re-entry by neurons, which then leads to apoptosis. LymPro is unique in the use of peripheral blood lymphocytes as surrogates for neuronal cell function, suggesting a common relationship between PBLs and neurons in the brain.

About Todos Medical Ltd.Todos Medical Ltd. is an in-vitro diagnostic company engaged in the development of blood tests for the early detection of a variety of cancers, and also has initiated the development of blood tests for neurodegenerative disorders such as Alzheimer's disease through Breakthrough Diagnostics, Inc., its joint venture with Amarantus Bioscience Holdings, Inc. Todos has developed two cancer screening tests based on TBIA (Todos Biochemical Infrared Analyses), a method for cancer screening using peripheral blood analysis. The TBIA screening method is based on the cancers influence on the immune system, which triggers biochemical changes in peripheral blood mononuclear cells and plasma. This proprietary and patented method incorporates biochemistry, physics and signal processing. The companys two cancer screening tests, TM-B1 and TM-B2, have received the CE mark. Breakthrough Diagnostics is developing the LymPro Test, a blood test for diagnosing Alzheimers disease.

For more information, the content of which is not part of this press release, please visithttp://www.todosmedical.com

About Amarantus Bioscience Holdings, Inc.Amarantus Bioscience Holdings (AMBS) is a JLABS alumnus biotechnology company developing treatments and diagnostics for diseases in the areas of neurology, regenerative medicine and orphan diseases through its subsidiaries. The Companys 80.01%-owned subsidiaryBreakthrough Diagnostics, Inc.,currently a joint venture with Todos Medical, Ltd., has licensed intellectual property rights to the Alzheimers blood diagnostic LymPro Test from Leipzig University that was originally developed by Dr. Thomas Arendt, as well as certain rights to multiple sclerosis diagnostic MSPrecise and Parkinsons diagnostic NuroPro. Amarantus entered into a joint venture agreement withTodos Medical, Ltd. to advance diagnostic screening assets and Todos has exercised its exclusive option to acquire Amarantus remaining ownership in Breakthrough in exchange for approximately 50% ownership of Todos. The transaction is expected close before the end of the first quarter of 2020. Amarantus also owns approximately 30% of the common shares of Avant Diagnostics, Inc., a healthcare data-generating technology company that specializes in biomarker assay services that target multiple areas of oncology. Avant provides precision oncology data through its TheraLink assays to assist the biopharmaceutical industry and clinical oncologists in identifying likely responders, initially for breast cancer, to over 70 FDA-approved drug treatments.

AMBS 50%-owned subsidiaryElto Pharma, Inc. has development rights to eltoprazine, a Phase 2b-ready small molecule indicated for Parkinson's disease levodopa-induced dyskinesia, Alzheimers aggression and adult attention deficit hyperactivity disorder, commonly known as ADHD. AMBS acquiredCutanogen Corporationfrom Lonza Group in 2015. Cutanogen is preparing for pivotal studies with Engineered Skin Substitute (ESS) for the treatment of pediatric life-threatening severe burns. ESS is a regenerative medicine-based, autologous full-thickness skin graft technology originally developed by the Shriners Hospital that can be used to treat severe burns, as well as several other catastrophic and cosmetic dermatological indications. AMBS wholly-owned subsidiary,MANF Therapeutics Inc.owns key intellectual property rights and licenses from a number of prominent universities related to the development of the therapeutic protein known as mesencephalic astrocyte-derived neurotrophic factor (MANF). MANF Therapeutics is developing MANF-based products as treatments for ophthalmological disorders such as Wolfram Syndrome, Retinitis Pigmentosa and Glaucoma, as well as neurodegenerative diseases such as Parkinsons disease. MANF was discovered by the Companys Chief Scientific Officer John Commissiong, PhD. Dr. Commissiong discovered MANF from AMBS proprietary discovery engine PhenoGuard, and believes several other neurotrophic factors remain to be discovered. Amarantus has entered into a binding letter of intent to license the therapeutic assets from Elto Pharma, Cutanogen and MANF Therapeutics to Emerald Organic Products.

Forward-looking StatementsCertain statements contained in this press release may constitute forward-looking statements. For example, forward-looking statements are used when discussing our expected clinical development programs and clinical trials. These forward-looking statements are based only on current expectations of management, and are subject to significant risks and uncertainties that could cause actual results to differ materially from those described in the forward-looking statements, including the risks and uncertainties related to the progress, timing, cost, and results of clinical trials and product development programs; difficulties or delays in obtaining regulatory approval or patent protection for product candidates; competition from other biotechnology companies; and our ability to obtain additional funding required to conduct our research, development and commercialization activities. In addition, the following factors, among others, could cause actual results to differ materially from those described in the forward-looking statements: changes in technology and market requirements; delays or obstacles in launching our clinical trials; changes in legislation; inability to timely develop and introduce new technologies, products and applications; lack of validation of our technology as we progress further and lack of acceptance of our methods by the scientific community; inability to retain or attract key employees whose knowledge is essential to the development of our products; unforeseen scientific difficulties that may develop with our process; greater cost of final product than anticipated; loss of market share and pressure on pricing resulting from competition; and laboratory results that do not translate to equally good results in real settings, all of which could cause the actual results or performance to differ materially from those contemplated in such forward-looking statements. Except as otherwise required by law, Todos Medical does not undertake any obligation to publicly release any revisions to these forward-looking statements to reflect events or circumstances after the date hereof or to reflect the occurrence of unanticipated events. For a more detailed description of the risks and uncertainties affecting Todos Medical, please refer to its reports filed from time to time with the U.S. Securities and Exchange Commission.

Todos Investor and Corporate Contact:Kim Sutton GolodetzLHA Investor RelationsSenior Vice President (212) 838-3777kgolodetz@lhai.com

Todos Corporate ContactDaniel HirschTodos MedicalInvestor RelationsEmail:Dan.h@todosmedical.comPhone: (347) 699-0029

Amarantus Investor and Media Contact:Gerald CommissiongPresident & CEOOffice: 650-862-5391Email: gerald@amarantus.com

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Todos and Amarantus JV Announces Full Enrollment for Clinical Trial of LymPro Alzheimers Blood Test Relationship with Amyloid PET - Yahoo Finance