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

Charles A. Goldthwaite, Jr., Ph.D.

Cardiovascular disease (CVD), which includes hypertension, coronary heart disease (CHD), stroke, and congestive heart failure (CHF), has ranked as the number one cause of death in the United States every year since 1900 except 1918, when the nation struggled with an influenza epidemic.1 In 2002, CVD claimed roughly as many lives as cancer, chronic lower respiratory diseases, accidents, diabetes mellitus, influenza, and pneumonia combined. According to data from the 19992002 National Health and Nutrition Examination Survey (NHANES), CVD caused approximately 1.4 million deaths (38.0 percent of all deaths) in the U.S. in 2002. Nearly 2600 Americans die of CVD each day, roughly one death every 34 seconds. Moreover, within a year of diagnosis, one in five patients with CHF will die. CVD also creates a growing economic burden; the total health care cost of CVD in 2005 was estimated at $393.5 billion dollars.

Given the aging of the U.S. population and the relatively dramatic recent increases in the prevalence of cardiovascular risk factors such as obesity and type 2 diabetes,2,3 CVD will continue to be a significant health concern well into the 21st century. However, improvements in the acute treatment of heart attacks and an increasing arsenal of drugs have facilitated survival. In the U.S. alone, an estimated 7.1 million people have survived a heart attack, while 4.9 million live with CHF.1 These trends suggest an unmet need for therapies to regenerate or repair damaged cardiac tissue.

Ischemic heart failure occurs when cardiac tissue is deprived of oxygen. When the ischemic insult is severe enough to cause the loss of critical amounts of cardiac muscle cells (cardiomyocytes), this loss initiates a cascade of detrimental events, including formation of a non-contractile scar, ventricular wall thinning (see Figure 6.1), an overload of blood flow and pressure, ventricular remodeling (the overstretching of viable cardiac cells to sustain cardiac output), heart failure, and eventual death.4 Restoring damaged heart muscle tissue, through repair or regeneration, therefore represents a fundamental mechanistic strategy to treat heart failure. However, endogenous repair mechanisms, including the proliferation of cardiomyocytes under conditions of severe blood vessel stress or vessel formation and tissue generation via the migration of bone-marrow-derived stem cells to the site of damage, are in themselves insufficient to restore lost heart muscle tissue (myocardium) or cardiac function.5 Current pharmacologic interventions for heart disease, including beta-blockers, diuretics, and angiotensin-converting enzyme (ACE) inhibitors, and surgical treatment options, such as changing the shape of the left ventricle and implanting assistive devices such as pacemakers or defibrillators, do not restore function to damaged tissue. Moreover, while implantation of mechanical ventricular assist devices can provide long-term improvement in heart function, complications such as infection and blood clots remain problematic.6 Although heart transplantation offers a viable option to replace damaged myocardium in selected individuals, organ availability and transplant rejection complications limit the widespread practical use of this approach.

Figure 6.1. Normal vs. Infarcted Heart. The left ventricle has a thick muscular wall, shown in cross-section in A. After a myocardial infarction (heart attack), heart muscle cells in the left ventricle are deprived of oxygen and die (B), eventually causing the ventricular wall to become thinner (C).

2007 Terese Winslow

The difficulty in regenerating damaged myocardial tissue has led researchers to explore the application of embryonic and adult-derived stem cells for cardiac repair. A number of stem cell types, including embryonic stem (ES) cells, cardiac stem cells that naturally reside within the heart, myoblasts (muscle stem cells), adult bone marrow-derived cells, mesenchymal cells (bone marrow-derived cells that give rise to tissues such as muscle, bone, tendons, ligaments, and adipose tissue), endothelial progenitor cells (cells that give rise to the endothelium, the interior lining of blood vessels), and umbilical cord blood cells, have been investigated to varying extents as possible sources for regenerating damaged myocardium. All have been tested in mouse or rat models, and some have been tested in large animal models such as pigs. Preliminary clinical data for many of these cell types have also been gathered in selected patient populations.

However, clinical trials to date using stem cells to repair damaged cardiac tissue vary in terms of the condition being treated, the method of cell delivery, and the primary outcome measured by the study, thus hampering direct comparisons between trials.7 Some patients who have received stem cells for myocardial repair have reduced cardiac blood flow (myocardial ischemia), while others have more pronounced congestive heart failure and still others are recovering from heart attacks. In some cases, the patient's underlying condition influences the way that the stem cells are delivered to his/her heart (see the section, quot;Methods of Cell Deliveryquot; for details). Even among patients undergoing comparable procedures, the clinical study design can affect the reporting of results. Some studies have focused on safety issues and adverse effects of the transplantation procedures; others have assessed improvements in ventricular function or the delivery of arterial blood. Furthermore, no published trial has directly compared two or more stem cell types, and the transplanted cells may be autologous (i.e., derived from the person on whom they are used) or allogeneic (i.e., originating from another person) in origin. Finally, most of these trials use unlabeled cells, making it difficult for investigators to follow the cells' course through the body after transplantation (see the section quot;Considerations for Using These Stem Cells in the Clinical Settingquot; at the end of this article for more details).

Despite the relative infancy of this field, initial results from the application of stem cells to restore cardiac function have been promising. This article will review the research supporting each of the aforementioned cell types as potential source materials for myocardial regeneration and will conclude with a discussion of general issues that relate to their clinical application.

In 2001, Menasche, et.al. described the successful implantation of autologous skeletal myoblasts (cells that divide to repair and/or increase the size of voluntary muscles) into the post-infarction scar of a patient with severe ischemic heart failure who was undergoing coronary artery bypass surgery.8 Following the procedure, the researchers used imaging techniques to observe the heart's muscular wall and to assess its ability to beat. When they examined patients 5 months after treatment, they concluded that treated hearts pumped blood more efficiently and seemed to demonstrate improved tissue health. This case study suggested that stem cells may represent a viable resource for treating ischemic heart failure, spawning several dozen clinical studies of stem cell therapy for cardiac repair (see Boyle, et.al.7 for a complete list) and inspiring the development of Phase I and Phase II clinical trials. These trials have revealed the complexity of using stem cells for cardiac repair, and considerations for using stem cells in the clinical setting are discussed in a subsequent section of this report.

The mechanism by which stem cells promote cardiac repair remains controversial, and it is likely that the cells regenerate myocardium through several pathways. Initially, scientists believed that transplanted cells differentiated into cardiac cells, blood vessels, or other cells damaged by CVD.911 However, this model has been recently supplanted by the idea that transplanted stem cells release growth factors and other molecules that promote blood vessel formation (angiogenesis) or stimulate quot;residentquot; cardiac stem cells to repair damage.1214 Additional mechanisms for stem-cell mediated heart repair, including strengthening of the post-infarct scar15 and the fusion of donor cells with host cardiomyocytes,16 have also been proposed.

Regardless of which mechanism(s) will ultimately prove to be the most significant in stem-cell mediated cardiac repair, cells must be successfully delivered to the site of injury to maximize the restored function. In preliminary clinical studies, researchers have used several approaches to deliver stem cells. Common approaches include intravenous injection and direct infusion into the coronary arteries. These methods can be used in patients whose blood flow has been restored to their hearts after a heart attack, provided that they do not have additional cardiac dysfunction that results in total occlusion or poor arterial flow.12, 17 Of these two methods, intracoronary infusion offers the advantage of directed local delivery, thereby increasing the number of cells that reach the target tissue relative to the number that will home to the heart once they have been placed in the circulation. However, these strategies may be of limited benefit to those who have poor circulation, and stem cells are often injected directly into the ventricular wall of these patients. This endomyocardial injection may be carried out either via a catheter or during open-heart surgery.18

To determine the ideal site to inject stem cells, doctors use mapping or direct visualization to identify the locations of scars and viable cardiac tissue. Despite improvements in delivery efficiency, however, the success of these methods remains limited by the death of the transplanted cells; as many as 90% of transplanted cells die shortly after implantation as a result of physical stress, myocardial inflammation, and myocardial hypoxia.4 Timing of delivery may slow the rate of deterioration of tissue function, although this issue remains a hurdle for therapeutic approaches.

Embryonic and adult stem cells have been investigated to regenerate damaged myocardial tissue in animal models and in a limited number of clinical studies. A brief review of work to date and specific considerations for the application of various cell types will be discussed in the following sections.

Because ES cells are pluripotent, they can potentially give rise to the variety of cell types that are instrumental in regenerating damaged myocardium, including cardiomyocytes, endothelial cells, and smooth muscle cells. To this end, mouse and human ES cells have been shown to differentiate spontaneously to form endothelial and smooth muscle cells in vitro19 and in vivo,20,21 and human ES cells differentiate into myocytes with the structural and functional properties of cardiomyocytes.2224 Moreover, ES cells that were transplanted into ischemically-injured myocardium in rats differentiated into normal myocardial cells that remained viable for up to four months,25 suggesting that these cells may be candidates for regenerative therapy in humans.

However, several key hurdles must be overcome before human ES cells can be used for clinical applications. Foremost, ethical issues related to embryo access currently limit the avenues of investigation. In addition, human ES cells must go through rigorous testing and purification procedures before the cells can be used as sources to regenerate tissue. First, researchers must verify that their putative ES cells are pluripotent. To prove that they have established a human ES cell line, researchers inject the cells into immunocompromised mice; i.e., mice that have a dysfunctional immune system. Because the injected cells cannot be destroyed by the mouse's immune system, they survive and proliferate. Under these conditions, pluripotent cells will form a teratoma, a multi-layered, benign tumor that contains cells derived from all three embryonic germ layers. Teratoma formation indicates that the stem cells have the capacity to give rise to all cell types in the body.

The pluripotency of ES cells can complicate their clinical application. While undifferentiated ES cells may possibly serve as sources of specific cell populations used in myocardial repair, it is essential that tight quality control be maintained with respect to the differentiated cells. Any differentiated cells that would be used to regenerate heart tissue must be purified before transplantation can be considered. If injected regenerative cells are accidentally contaminated with undifferentiated ES cells, a tumor could possibly form as a result of the cell transplant.4 However, purification methodologies continue to improve; one recent report describes a method to identify and select cardiomyocytes during human ES cell differentiation that may make these cells a viable option in the future.26

This concern illustrates the scientific challenges that accompany the use of all human stem cells, whether derived from embryonic or adult tissues. Predictable control of cell proliferation and differentiation requires additional basic research on the molecular and genetic signals that regulate cell division and specialization. Furthermore, long-term cell stability must be well understood before human ES-derived cells can be used in regenerative medicine. The propensity for genetic mutation in the human ES cells must be determined, and the survival of differentiated, ES-derived cells following transplantation must be assessed. Furthermore, once cells have been transplanted, undesirable interactions between the host tissue and the injected cells must be minimized. Cells or tissues derived from ES cells that are currently available for use in humans are not tissue-matched to patients and thus would require immunosuppression to limit immune rejection.18

While skeletal myoblasts (SMs) are committed progenitors of skeletal muscle cells, their autologous origin, high proliferative potential, commitment to a myogenic lineage, and resistance to ischemia promoted their use as the first stem cell type to be explored extensively for cardiac application. Studies in rats and humans have demonstrated that these cells can repopulate scar tissue and improve left ventricular function following transplantation.27 However, SM-derived cardiomyocytes do not function in complete concert with native myocardium. The expression of two key proteins involved in electromechanical cell integration, N-cadherin and connexin 43, are downregulated in vivo,28 and the engrafted cells develop a contractile activity phenotype that appears to be unaffected by neighboring cardiomyocytes.29

To date, the safety and feasibility of transplanting SM cells have been explored in a series of small studies enrolling a collective total of nearly 100 patients. Most of these procedures were carried out during open-heart surgery, although a couple of studies have investigated direct myocardial injection and transcoronary administration. Sustained ventricular tachycardia, a life-threatening arrhythmia and unexpected side-effect, occurred in early implantation studies, possibly resulting from the lack of electrical coupling between SM-derived cardiomyocytes and native tissue.30,31 Changes in preimplantation protocols have minimized the occurrence of arrhythmias in conjunction with the use of SM cells, and Phase II studies of skeletal myoblast therapy are presently underway.

In 2001, Jackson, et.al. demonstrated that cardiomyocytes and endothelial cells could be regenerated in a mouse heart attack model through the introduction of adult mouse bone marrow-derived stem cells.9 That same year, Orlic and colleagues showed that direct injection of mouse bone marrow-derived cells into the damaged ventricular wall following an induced heart attack led to the formation of new cardiomyocytes, vascular endothelium, and smooth muscle cells.11 Nine days after transplanting the stem cells, the newly-formed myocardium occupied nearly 70 percent of the damaged portion of the ventricle, and survival rates were greater in mice that received these cells than in those that did not. While several subsequent studies have questioned whether these cells actually differentiate into cardiomyocytes,32,33 the evidence to support their ability to prevent remodeling has been demonstrated in many laboratories.7

Based on these findings, researchers have investigated the potential of human adult bone marrow as a source of stem cells for cardiac repair. Adult bone marrow contains several stem cell populations, including hematopoietic stem cells (which differentiate into all of the cellular components of blood), endothelial progenitor cells, and mesenchymal stem cells; successful application of these cells usually necessitates isolating a particular cell type on the basis of its' unique cell-surface receptors. In the past three years, the transplantation of bone marrow mononuclear cells (BMMNCs), a mixed population of blood and cells that includes stem and progenitor cells, has been explored in more patients and clinical studies of cardiac repair than any other type of stem cell.7

The results from clinical studies of BMMNC transplantation have been promising but mixed. However, it should be noted that these studies have been conducted under a variety of conditions, thereby hampering direct comparison. The cells have been delivered via open-heart surgery and endomyocardial and intracoronary catheterization. Several studies, including the Bone Marrow Transfer to Enhance ST-Elevation Infarct Regeneration (BOOST) and the Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI) trials, have shown that intracoronary infusion of BMMNCs following a heart attack significantly improves the left ventricular (LV) ejection fraction, or the volume of blood pumped out of the left ventricle with each heartbeat.3436 However, other studies have indicated either no improvement in LV ejection fraction upon treatment37 or an increased LV ejection fraction in the control group.38 An early study that used endomyocardial injection to enhance targeted delivery indicated a significant improvement in overall LV function.39 Discrepancies such as these may reflect differences in cell preparation protocols or baseline patient statistics. As larger trials are developed, these issues can be explored more systematically.

Mesenchymal stem cells (MSCs) are precursors of non-hematopoietic tissues (e.g., muscle, bone, tendons, ligaments, adipose tissue, and fibroblasts) that are obtained relatively easily from autologous bone marrow. They remain multipotent following expansion in vitro, exhibit relatively low immunogenicity, and can be frozen easily. While these properties make the cells amenable to preparation and delivery protocols, scientists can also culture them under special conditions to differentiate them into cells that resemble cardiac myocytes. This property enables their application to cardiac regeneration. MSCs differentiate into endothelial cells when cultured with vascular endothelial growth factor40 and cardiomyogenic (CMG) cells when treated with the dna-demethylating agent, 5-azacytidine.41 More important, however, is the observation that MSCs can differentiate into cardiomyocytes and endothelial cells in vivo when transplanted to the heart following myocardial infarct (MI) or non-injury in pig, mouse, or rat models.4245 Additionally, the ability of MSCs to restore functionality may be enhanced by the simultaneous transplantation of other stem cell types.43

Several animal model studies have shown that treatment with MSCs significantly increases myocardial function and capillary formation.5,41 One advantage of using these cells in human studies is their low immunogenicity; allogeneic MSCs injected into infarcted myocardium in a pig model regenerated myocardium and reduced infarct size without evidence of rejection.46 A randomized clinical trial implanting MSCs after MI has demonstrated significant improvement in global and regional LV function,47 and clinical trials are currently underway to investigate the application of allogeneic and autologous MSCs for acute MI and myocardial ischemia, respectively.

Recent evidence suggests that the heart contains a small population of endogenous stem cells that most likely facilitate minor repair and turnover-mediated cell replacement.7 These cells have been isolated and characterized in mouse, rat, and human tissues.48,49 The cells can be harvested in limited quantity from human endomyocardial biopsy specimens50 and can be injected into the site of infarction to promote cardiomyocyte formation and improvements in systolic function.49 Separation and expansion ex vivo over a period of weeks are necessary to obtain sufficient quantities of these cells for experimental purposes. However, their potential as a convenient resource for autologous stem cell therapy has led the National Heart, Lung, and Blood Institute to fund forthcoming clinical trials that will explore the use of cardiac stem cells for myocardial regeneration.

The endothelium is a layer of specialized cells that lines the interior surface of all blood vessels (including the heart). This layer provides an interface between circulating blood and the vessel wall. Endothelial progenitor cells (EPCs) are bone marrow-derived stem cells that are recruited into the peripheral blood in response to tissue ischemia.4 EPCs are precursor cells that express some cell-surface markers characteristic of mature endothelium and some of hematopoietic cells.19,5153 EPCs home in on ischemic areas, where they differentiate into new blood vessels; following a heart attack, intravenously injected EPCs home to the damaged region within 48 hours.12 The new vascularization induced by these cells prevents cardiomyocyte apoptosis (programmed cell death) and LV remodeling, thereby preserving ventricular function.13 However, no change has been observed in non-infarcted regions upon EPC administration. Clinical trials are currently underway to assess EPC therapy for growing new blood vessels and regenerating myocardium.

Several other cell populations, including umbilical cord blood (UCB) stem cells, fibroblasts (cells that synthesize the extracellular matrix of connective tissues), and peripheral blood CD34+ cells, have potential therapeutic uses for regenerating cardiac tissue. Although these cell types have not been investigated in clinical trials of heart disease, preliminary studies in animal models indicate several potential applications in humans.

Umbilical cord blood contains enriched populations of hematopoietic stem cells and mesencyhmal precursor cells relative to the quantities present in adult blood or bone marrow.54,55 When injected intravenously into the tail vein in a mouse model of MI, human mononuclear UCB cells formed new blood vessels in the infarcted heart.56 A human DNA assay was used to determine the migration pattern of the cells after injection; although they homed only to injured areas within the heart, they were also detected in the marrow, spleen, and liver. When injected directly into the infarcted area in a rat model of MI, human mononuclear UCB cells improved ventricular function.57 Staining for CD34 and other markers found on the cell surface of hematopoietic stem cells indicated that some of the cells survived in the myocardium. Results similar to these have been observed following the injection of human unrestricted somatic stem cells from UCB into a pig MI model.58

Adult peripheral blood CD34+ cells offer the advantage of being obtained relatively easily from autologous sources.59 Although some studies using a mouse model of MI claim that these cells can transdifferentiate into cardiomyocytes, endothelial cells, and smooth muscle cells at the site of tissue injury,60 this conclusion is highly contested. Recent studies that involve the direct injection of blood-borne or bone marrow-derived hematopoietic stem cells into the infarcted region of a mouse model of MI found no evidence of myocardial regeneration following injection of either cell type.33 Instead, these hematopoietic stem cells followed traditional differentiation patterns into blood cells within the microenvironment of the injured heart. Whether these cells will ultimately find application in myocardial regeneration remains to be determined.

Autologous fibroblasts offer a different strategy to combat myocardial damage by replacing scar tissue with a more elastic, muscle-like tissue and inhibiting host matrix degradation.4 The cells may be manipulated to express muscle-specific transcription factors that promote their differentiation into myotubes such as those derived from skeletal myoblasts.61 One month after these cells were implanted into the post-infarction scar in a rat model of MI, they occupied a large portion of the scar but were not functionally integrated.61 Although the effects on ventricular function were not evaluated in this study, authors noted that modified autologous fibroblasts may ultimately prove useful in elderly patients who have a limited population of autologous skeletal myoblasts or bone marrow stem cells.

As these examples indicate, many types of stem cells have been applied to regenerate damaged myocardium. In select applications, stem cells have demonstrated sufficient promise to warrant further exploration in large-scale, controlled clinical trials. However, the current breadth of application of these cells has made it difficult to compare and contextualize the results generated by the various trials. Most studies published to date have enrolled fewer than 25 patients, and the studies vary in terms of cell types and preparations used, methods of delivery, patient populations, and trial outcomes. However, the mixed results that have been observed in these studies do not necessarily argue against using stem cells for cardiac repair. Rather, preliminary results illuminate the many gaps in understanding of the mechanisms by which these cells regenerate myocardial tissue and argue for improved characterization of cell preparations and delivery methods to support clinical applications.

Future clinical trials that use stem cells for myocardial repair must address two concerns that accompany the delivery of these cells: 1) safety and 2) tracking the cells to their ultimate destination(s). Although stem cells appear to be relatively safe in the majority of recipients to date, an increased frequency of non-sustained ventricular tachycardia, an arrhythmia, has been reported in conjunction with the use of skeletal myoblasts.30,6264 While this proarrhythmic effect occurs relatively early after cell delivery and does not appear to be permanent, its presence highlights the need for careful safety monitoring when these cells are used. Additionally, animal models have demonstrated that stem cells rapidly diffuse from the heart to other organs (e.g., lungs, kidneys, liver, spleen) within a few hours of transplantation,65,66 an effect observed regardless of whether the cells are injected locally into the myocardium. This migration may or may not cause side-effects in patients; however, it remains a concern related to the delivery of stem cells in humans. (Note: Techniques to label stem cells for tracking purposes and to assess their safety are discussed in more detail in other articles in this publication).

In addition to safety and tracking, several logistical issues must also be addressed before stem cells can be used routinely in the clinic. While cell tracking methodologies allow researchers to determine migration patterns, the stem cells must target their desired destination(s) and be retained there for a sufficient amount of time to achieve benefit. To facilitate targeting and enable clinical use, stem cells must be delivered easily and efficiently to their sites of application. Finally, the ease by which the cells can be obtained and the cost of cell preparation will also influence their transition to the clinic.

The evidence to date suggests that stem cells hold promise as a therapy to regenerate damaged myocardium. Given the worldwide prevalence of cardiac dysfunction and the limited availability of tissue for cardiac transplantation, stem cells could ultimately fulfill a large-scale unmet clinical need and improve the quality of life for millions of people with CVD. However, the use of these cells in this setting is currently in its infancymuch remains to be learned about the mechanisms by which stem cells repair and regenerate myocardium, the optimal cell types and modes of their delivery, and the safety issues that will accompany their use. As the results of large-scale clinical trials become available, researchers will begin to identify ways to standardize and optimize the use of these cells, thereby providing clinicians with powerful tools to mend a broken heart.

Chapter 5|Table of Contents|Chapter 7

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Mending a Broken Heart: Stem Cells and Cardiac Repair ...

Aqua Botanical Stem Cell Therapy

Ethical concerns have slowed embryonic medical research into applications for stem cells. Also, the embryonic stem cells can unpredictably cause cancer in the treated patient.

New research demonstrate that Stem cell nutrition dereived from aqua botanical source supports the natural role of adult stem cells. These plant stem cell extracts are typically derived from certain edible algae that grows in fresh water.

When there is an injury or a stress to an organ, compounds are released that reach the bone marrow and trigger the release of stem cells. Stem Cells can be thought of as master cells. Stem cells circulate and function to replace dysfunctional cells, thus fulfilling the natural process of maintaining optimal health

Dr. Robert Sampson, MD on stem cell nutrition - "... we have a product that has been shown and demonstrated in the patent to increase the level of adult circulating stem cells by up to 30%. It seems to me we're having a great opportunity here to optimize the body's natural ability to create health."

Stem cell nutrition are typically aquatic botanicals and support wellness by assisting the body in its ability to maintain healthy stem cell physiology, production, and placement. Just as antioxidants are important to protect your cells from free radical damage, stem cell nutrition is equally important to support your stem cells in maintaining proper organ and tissue functioning in your body.

The health benefits of having more stem cells in the blood circulation have been demonstrated by numerous scientific studies. It would be too long here to summarize this vast body of scientific data. I simply suggest you research the work of Dr. Donald Orlic at the National Institute of Health.

The theory that Adult Stem Cells are nothing less than the human body's natural self-renewal system has profound implications for every area of modern medicine. The idea that heart disease, diabetes, liver degeneration, and other conditions could be things of the past is no longer science fiction; because of recent Adult Stem Cell research breakthroughs, these are real possibilities in the short term.

Stem cells are defined as cells with the unique capacity to self-replicate throughout the entire life of an organism and to differentiate into cells of various tissues. Most cells of the body are specialized and play a well-defined role in the body. For example, brain cells respond to electrical signals from other brain cells and release neurotransmitters; cells of the retina are activated by light, and pancreatic -cells produce insulin. These cells, called somatic cells, will never differentiate into other types of cells or even proliferate. By contrast, stem cells are primitive cells that remain undifferentiated until they receive a signal prompting them to become various types of specialized cells.

Dr. Cliff Minter - "Stem cells are the most powerful cells in the body. We know that stem cells, once they're circulating in the bloodstream, will travel to any area of the body that has been compromised or damaged and turn into healthy cells. There have been controversial discussions about the new stem cells found in embryos, but the truth is that everyone has adult stem cells in their own bodies. We are all created from stem cells.

As a child or a young adult, your body automatically releases stem cells whenever you injure yourself. That's why you heal so fast when you are younger. After about age 35, we don't heal as fast anymore, because the stem cells aren't released the same way as when we are younger. Stem cell nutrition helps all of us heal our bodies. If you look at the New England Journal of Medicine, you'll find that the number one indicator of a healthy heart is the number of stem cells circulating in the body. Stem cell nutrition is the organic and all-natural way to stimulate the bone marrow to release adult stem cells into the bloodstream.

By taking stem cell nutrition, you can maintain optimum health and aid your body in healing itself. It's certainly a better way to recuperate from an illness than using prescription drugs, because even when a medication works, it can often be hard on your liver and the rest of your body. Stem cell nutrition has no negative side effects. This makes it a powerful approach to healing and good health in general.

I found out about stem cell nutrition after someone asked for my opinion on it. I did some research and found it to be one of the greatest ways to slow down aging that we have. Aging is nothing more than the breakdown of cells. Stem cell nutrition combats that action. As cells break down, stem cell nutrition replaces them with healthy cells. This is the greatest, most natural anti-aging method I know. I was skeptical at first, but the results I've personally seen in people I've talked with have been wide-ranged. Lots of people have reported an increase in energy and better sleeping patterns.

I've seen people with arthritis in various parts of their bodies reverse the disease, and people with asthma end up with their lungs totally clear. One person that was on oxygen almost 24/7 is now totally off of oxygen. Two ladies who suffered badly from PMS told me they were 100 percent symptom-free within weeks of starting the stem cell nutrition. Two people I know had tennis elbow which usually takes about six to nine months to heal. Within weeks of taking stem cell nutrition, both report their "tennis elbow" is gone. It makes sense, because stem cells go to whatever area is compromised and turn into healthy cells.

I use stem cell nutrition as a preventative. I've noticed an increase in my energy level and an improved sleeping pattern. Stem cell nutrition has zero negative side effects, is very powerful, and we know how it works. It's good for children as well as adults. This is the best, most natural way I know to optimum health. If you just want to use it for prevention, this is the best thing I know for staying healthy. And if you do those and regaining optimum health. I recommend it to everybody."

Dr. Cliff Minter (retired) graduated from Illinois College of Podiatric Medicine. He completed his residency at the Hugar Surgery Center in the Hines Veteran Administration Hospital in Illinois before going into private practice in Ventura, CA. Dr. Minter is a national and international speaker on the subjects of business and nutritional products.

The Stem Cell Theory of Renewal proposes that stem cells are naturally released by the bone marrow and travel via the bloodstream toward tissues to promote the body's natural process of renewal. When an organ is subjected to a process that requires renewal, such as the natural aging process, this organ releases compounds that trigger the release of stem cells from the bone marrow. The organ also releases compounds that attracts stem cells to this organ. The released stem cells then follow the concentration gradient of these compounds and leave the blood circulation to migrate to the organ where they proliferate and differentiate into cells of this organ, supporting the natural process of renewal.

Most of the cells in the human body are specialists assigned to a specific organ or type of tissue, such as the neuronal cells that wire the brain and central nervous system. Stem cells are different. When they divide, they can produce either more stem cells, or they can serve as progenitors that differentiate into specialized cells as they mature. Hence the name, because specialist cells can "stem" from them. The potential to differentiate into specialist cells whose populations in the body have become critically depleted as the result of illness or injury is what makes stem cells so potentially valuable to medical research.

The idea is that if the fate of a batch of stem cells could be directed down specific pathways, they could be grown, harvested, and then transplanted into a problem area. If all went according to plan, these new cells would overcome damaged or diseased cells, leading to healing and recovery. "The life of a stem cell can be viewed as a hierarchical branching process, where the cell is faced with a series of fate switches," Schaffer says. "Our goal is to identify the cell fate switches, and then provide stem cells with the proper signals to guide them down a particular developmental trajectory."

Stem cells have the remarkable potential to develop into many different cell types in the body. Serving as a sort of repair system for the body, they can theoretically divide without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.

When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function. Scientists believe it should be possible to harness this ability to turn stem cells into a super "repair kit" for the body.

Scientist and author Christian Drapeau explains how the Stem Cell enhancers function to maximize human performance - Supporting the release of stem cells from the bone marrow and increasing the number of circulating stem cells improves various aspects of human health. For very active and sports focused people, Stem Cells are the raw materials to repair micro-tears and micro-injuries created during training. The results, according to Drapeau, are that active people, whether former NBA stars or amateur weekenders, can exercise more intensely at each training session with the ultimate consequence of greater performance.

Theoretically, it should be possible to use stem cells to generate healthy tissue to replace that either damaged by trauma, or compromised by disease. Among the conditions which scientists believe may eventually be treated by stem cell therapy are Parkinson's disease, Alzheimer's disease, heart disease, stroke, arthritis, diabetes, burns and spinal cord damage.

Both of my big dogs have gained their youth back. I am a true believer in Stem Cell Nutrition for pets as it has provided a spectacular change in both Ginger and Rowdy. Sonya, IN

Stem cell nutrition for dogs, horses and other animals are specially formulated to be a delectable treat for your animal. The pet chewables and equine blends make it easy to provide your animals with this valuable nutritional supplement. The most common story is that of old, tired and sluggish dogs turned within a week or so into active, alert dogs running around like puppies. The same was observed in horses. Old horses who used to remain standing in the barn or under a tree, sluggish or stricken by too much discomfort to walk around, suddenly began moving about, and at times running and bucking like young colts. One of the most common reports was obvious improvements in hoof health and coat appearance.

times. When there is an injury or a stress to an organ of your beloved pet or horse, compounds are released that reach the bone marrow and trigger the release of stem cells. Stem Cells can be thought of as master cells. Stem cells circulate and function to replace dysfunctional cells, thus fulfilling the natural process of maintaining optimal health.

As they do in humans, adult stem cells reside in animals bone marrow, where they are released whenever there is a problem somewhere in the body. Looking back on stem cell research, we realize that most studies have been done with animals, mostly mice, but also with dogs, horses, pigs, sheep and cattle. These studies have revealed that animal stem cells conduct themselves the same way human stem cells do. When there is an injury or a stress to an organ of your beloved pet or horse, compounds are released that reach the bone marrow and trigger the release of stem cells. The stem cells then travel to tissues and organs in need of help to regain optimal health.

Eve-Marie Lucerne - Eve-Marie keeps nine horses, all older thoroughbreds, and was eager to participate in the trials of a new stem cell enhancer for horses. She shared her allotment of test products with a few large commercial thoroughbred farms, veterinarians and other horse people she knows, and has been pleased with the consistently excellent results she has seen and others have reported to her. This product will help so many animals, she says, adding, People and animals are more alike than we are different. So it makes sense that a stem cell enhancer for animals with promote their health, too.

Eve-Marie's Equine Stem Cells Nutrition show dramatic results. For several horses facing serious physical challenges, cases where the animals might have to be put down, we saw a return to quality of life. This did not happen before Equine Stem Cell Nutrition. Eve-Marie says that this turnaround was quick, less than two weeks in many cases, and that the subject horses were back to health and enjoying pasture life within a month. One of the unofficial trial subjects for the equine stem cell nutrition was a 30-year old donkey who was in bad shape, Eve-Marie reports. He hadchronic respiratory difficulty and could move about only haltingly. His owner had stem cell enhancer supplements to help with her own serious health challenges and shared it with the donkey. The donkey's owner says this is the first time she wasn't sick, and her donkey is walking all around, feeling great an enjoying life again!

Farrier and National Hoof practitioner Stephen Dick received some of the trial product from Eve-Marie, and had good results with the two horses he selected for trial. For a 12-year-old quarterhorse stallion, the equine product brought dramatic results. This horse used to lie down twenty-two hours of the day, because he suffered discomfort whenever he stood, Steve reports, continuing, after a couple of weeks with Equine Stem Cell Nutrition, he was getting up and moving around, showing no discomfort. For a high-spirited mare with a leg problem, the equine product brought about a whole new lease on life, Steve says. This horse had been in a stall for 8 months. After about 6 weeks taking the equine product with her grain, her condition had improved and she was out of the stall, walking around in the pasture again.

Little Joe, a small 18-year-old quarter horse that Judy Fisher bought when he was nearly 400 pounds underweight. You could count his ribs, Judy says, remembering, and his backbone stuck up like a ridge all along his back. He was very, very thin! Little Joe also suffered from breathing problems that kept him lethargic and inactive. Vet-recommended remedies were unsuccessful in changing Little Joe's physical problems, and the vet told Judy he didn't expect Little Joe to live through the winter. I figured Little Joe was in such bad shape that anything was worth a try, she says.

She began giving the horse stem cell nutrition with his feed and grain twice a day. Within a couple of weeks, Judy was surprised to see Little Joe beginning to gain weight and run, buck, snort and kick. His breathing was no longer labored and his skin and coat were improving. Within six weeks Little Joe's overall appearance had changed dramatically. He had put on almost 300 pounds. When his former owner came to visit, Judy says, he didn't recognize Little Joe. That's how different he looked!

Sara participated in the stem cell nutrition product trials with her two horses and her 80-pound mixed-breed dog. She noted significant improvement in the health and quality of life for all three animals during the time of the trials. For JJ, Sara's 18-year old quarterhorse, the equine product brought about improvements in his overall mood, appearance and alertness quickly. He really liked the product from the beginning, Sara reports, pointing out that Hank, her 16-year-old thoroughbred/quarterhorse, had not taken to the taste of it too readily. I was able to slowly wean him on it though, she says. For Hank, the equine product was a balm for the skin problems resulting from his allergy to fly bites.

His skin condition improved dramatically. Sara reports, noting that before the equine product the horse had scratched and bitten himself into ope wounds; after the equine product, the scratching and biting dropped off to almost nothing. Sara also noticed an increase in Hank's energy and liveliness in the first week on the equine product. The horse's foot and hip discomforts also responded well, leading to a noticeable increase in his mobility and an overall improvement in his quality of life throughout the two-month study.

Sara gave the pet product to her dog, Roxy, who had suffered for two years with ear problems that led to scratching, often until her skin was raw. Vet-recommended remedies had been temporary, quick-fixes, Sara says, but the discomfort always returned with a vengeance. For the pet trials, Sara gave Roxy two tabs of the product a day for two months, noting this is the only supplement she was getting. Sara says Roxy's problem with her ears definitely improved, the hair as grown back on her head and ears, and the ear problem has not recurred, adding that Roxy is happier and engaging, more playful.

The National Health Institute lists seventy-four treatable diseases using ASCs in therapy - an invasive and costly procedure of removing the stem cells from one's bone marrow (or a donor's bone marrow) and re-injecting these same cells into an area undergoing treatment. For example, this procedure is sometimes done before a cancer patient undergoes radiation. Healthy stem cells from the bone marrow are removed and stored, only to be re-inserted after radiation into the area of the body in need of repair. This is a complex and expensive procedure, not accessible to the average person. However, there is now a way that every single person, no matter what their health condition, can have access to the benefits of naturally supporting their body's innate ability to repair every organ and tissue using stem cell nutrition.

David A. Prentice, Ph.D. - "Within just a few years, the possibility that the human body contains cells that can repair and regenerate damaged and diseased tissue has gone from an unlikely proposition to a virtual certainty. Adult stem cells have been isolated from numerous adult tissues, umbilical cord, and other non-embryonic sources, and have demonstrated a surprising ability for transformation into other tissue and cell types and for repair of damaged tissues.

A new U.S. study involving mice suggests the brain's own stem cells may have the ability to restore memory after an injury. These neural stem cells work by protecting existing cells and promoting neuronal connections. In their experiments, a team at the University of California, Irvine,were able to bring the rodents' memory back to healthy levels up to three months after treatment. The finding could open new doors for treatment of brain injury, stroke and dementia, experts say.

"This is one of the first reports that you can take a stem cell transplantation approach and restore memory," said lead researcher Mathew Blurton-Jones, a postdoctorate fellow at the university. "There is a lot of awareness that stem cells might be useful in treating diseases that cause loss of motor function, but this study shows that they might benefit memory in stroke or traumatic brain injury, and potentially Alzheimer's disease."

In the study, published in the Oct. 31 issue of the Journal of Neuroscience, Blurton-Jones and his colleagues used genetically engineered mice that naturally develop brain lesions. The researchers destroyed cells in a brain area called the hippocampus. These cells are known to be vital to memory formation and it is in this region that neurons often die after injury, the researchers explained. To test the mice's memory, Blurton-Jones's group conducted place and object recognition tests with both healthy mice and brain-injured mice.

Healthy mice remembered their surroundings about 70 percent of the time, while brain-injured mice remembered it only 40 percent of the time. For objects, healthy mice recalled objects about 80 percent of the time, but injured mice remembered them only 65 percent of the time. The researchers then injected each mouse with about 200,000 neural stem cells. They found that mice with brain injuries that received the stem cells now remembered their surroundings about 70 percent of the time -- the same as healthy mice. However, mice that didn't receive stem cells still had memory deficits.

The researchers also found that in healthy mice injected with stem cells, the stem cells traveled throughout the brain. In contrast, stem cells given to injured mice lingered in the hippocampus. Only about 4 percent of those stem cells became neurons, indicating that the stem cells were repairing existing cells to improve memory, rather than replacing the dead brain cells, Blurton-Jones's team noted. The researchers are presently doing another study with mice stricken with Alzheimer's. "The initial results are promising," Blurton-Jones said. "This has a huge potential, but we have to be cautious about not rushing into the clinic too early."

One expert is optimistic about the findings. "Putting in these stem cells could eventually help in age-related memory decline," said Dr. Paul R. Sanberg, director of the Center of Excellence for Aging and Brain Repair at the University of South Florida College of Medicine. "There is clearly a therapeutic potential to this." Sanberg noted that for the process to work with Alzheimer's it has to work with older brains. "There is clearly therapeutic potential in humans, but there are a lot of hurdles to overcome," he said. "This is another demonstration of the potential for neural stem cells in brain disorders.".

Dr. Nancy White Ph.D.- " I've always been interested in health generally and in particular the brain, focusing on the balance of neurotransmitters. I often do quantitative EEG's for assessment of my patients. I'm impressed with the concept of a natural product like stem cell nutrition that could help release adult stem cells from the bone mass where the body would have no objection and no rejection. I've tried stem cell nutrition for general health anti-aging. After taking it for a time, I fell more agile and my joints are far more flexible. I was astounded while doing yoga that I was suddenly able to bend over and touch my forehead to my knees. I haven't been able to do that comfortably in probably twenty years. I noticed how much better my balance has become. I believe stem cell nutrition is responsible for these effects, because I certainly haven't been trained extensively in yoga. Also since taking stem cell nutrition, I feel better and my skin is more moist and has a finer texture.

A bald friend of mine, who is also taking the stem cell nutrition, had several small cancers on top of his head. His doctor had removed one from his arm already, and his dermatologist set a date to remove those from his scalp. Before the appointment, my friend was shaving one morning and, looking in the mirror, saw that the cancers were all gone. They had disappeared within a few weeks of starting the stem cell nutrition and his skin is better overall. Also, his knee, which he'd strained playing tennis, was like new. Stem cell nutrition seems to go where the body's priority is. You never know what the affect is going to be, but you notice something is changing. Another friend of mine seems to be dropping years. Her skin looks smoother and her face younger. After about six weeks on the stem cell nutrition, she looks like she's ten years younger. A woman who gives her regular facials asked what she was doing, because her skin looked so much different. Stem cell nutrition is remarkable and could help anybody. Everybody should try it, because it's natural and there are no risks. As we grow older in years, we still can have good health. That's the ideal. Even if you don't currently have a problem, stem cell nutrition is a preventative." Dr. White holds a Ph. D. in Clinical Psychology, an MA in Behavioral Science, and a B.F.A. in Fine Arts, Magna Cum Laude. In addition, she is licensed in the State of Texas as a Psychologist , a Marriage and Family Therapist and as a Chemical Dependency Counselor.

Fernando Aguila, M.D. - "Due to a heavy patient load, I have recently found that I tire more easily, my legs are cramping, and by the time I get home, even my shoulders and rib cage hurt. I knew I had to find a way to increase my stamina, energy and vitality. A friend gave me information about stem cell nutrition and how it promotes the release of stem cells in the body. One of the components apparently promotes the migration of the stem cells to tissues or organs where regeneration and repair is needed most. My attention was drawn to the fact that it can increase energy, vitality, wellness, concentration, and much more. It sounded just like what I needed. Since then, I've heard reports of people experiencing excellent results in a number of different areas in their health. The improvements sounded dramatic. Because of all of their testimonies, I was willing to believe it could promote wellness in the human body.

I tried stem cell nutrition myself. After a day, of hard work, I realized I wasn't tired at all, my legs were not aching, and I didn't have any shoulder pain. I decided the stem cell nutrition must be working. I continued to take it, and was able to work so efficiently and steadily that one surgeon commented that I was moving like a ball of fire. Stem cell nutrition gives me support physically and mentally. I look forward to seeing what the major medical journals have to say about the studies being done with this new approach to wellness." Fernando Aguila, M.D., graduated from the University of Santa Thomas in Manila , Philippines. He finished his internship at Cambridge City Hospital, Cambridge, MA and completed his residency at the New England Medical Center in Boston, MA. He obtained a fellowship in OB-GYN anesthesia at the Brigham and Women's Hospital in Boston and a fellowship in cardio-thoracic anesthesia at the Cleveland Clinic Foundation in Cleveland, OH.

Christian Drapeau is America's best known advocate for Adult Stem Cell science health applications and the founder of the field of Stem Cell Nutrition. He holds a BS in Neurophysiology from McGill University and a Master of Science in Neurology and Neurosurgery from the Montreal Neurological Institute.

One particular stem cell enhancers that was studied was found to contain a polysaccharide fraction that was shown to stimulate the migration of Natural Killer (NK) cells out of the blood into tissues. The same polysaccharide fraction was also shown to strongly stimulate the activation of NK cells. NK cells play the very important role in the body of identifying aberrant or defective cells and eliminating them. NK cells are especially known for their ability to detect and destroy virally infected cells and cells undergoing uncontrolled cellular division. The same polysaccharide fraction was also shown to stimulate macrophage activity. Macrophages constitute the front line of the immune system. They first detect an infection or the presence of bacteria or virally infected cells, and they then call for a full immune response. Adult Stem Cell Nutritional Enhancer also contains a significant concentration of chlorophyll and phycocyanin, the blue pigment in AFA. Phycocyanin has strong anti-inflammatory properties and therefore can assist the immune system.

The release of stem cells from the bone marrow and their migration to tissues is a natural process that happens everyday. Stem cell enhancers simply support that natural process and tips the balance toward health everyday. It does not do anything that the body does not already do everyday. So far, no instances of cancer or any similar problem have ever been observed when using in vivo natural release of stem cells from the bone marrow.

Each day, stem cells in the bone marrow evolve to produce red blood cells, white blood cells, and platelets. These mature cells are then released into the bloodstream where they perform their vital life-supporting functions. When bone marrow stem cell activity is interfered with, diseases such as anemia (red blood cell deficit), neutropenia (specialized white blood cell deficit), or thrombocytopenia (platelet deficit) are often diagnosed. Any one of these conditions can cause death if not corrected.

Scientists have long known that folic acid, vitamin B12, and iron are required for bone marrow stem cells to differentiate into mature red blood cells.3-7 Vitamin D has been shown to be crucial in the formation of immune cells,8-11 whereas carnosine has demonstrated a remarkable ability to rejuvenate cells approaching senescence and extend cellular life span.12-28

Other studies of foods such as blueberries show this fruit can prevent and even reverse cell functions that decline as a result of normal aging.29-36 Blueberry extract has been shown to increase neurogenesis in the aged rat brain.37,38 Green tea compounds have been shown to inhibit the growth of tumor cells, while possibly providing protection against normal cellular aging.39,40

Based on these findings, scientists are now speculating that certain nutrients could play important roles in maintaining the healthy renewal of replacement stem cells in the brain, blood, and other tissues. It may be possible, according to these scientists, to use certain nutrient combinations in the treatment of conditions that warrant stem cell replacement

These studies demonstrate for the first time that various natural compounds can promote the proliferation of human bone marrow cells and human stem cells. While these studies were done in vitro, they provide evidence that readily available nutrients may confer a protective effect against today's epidemic of age-related bone marrow degeneration.

Dr. Robert Sampson, MD on stem cell nutrition - "... we have a product that has been shown and demonstrated in the patent to increase the level of adult circulating stem cells by up to 30%. It seems to me we're having a great opportunity here to optimize the body's natural ability to create health." Recent scientific developments have revealed that stem cells derived from the bone marrow, travel throughout the body, and act to support optimal organ and tissue function. Stem cell enhancers supports the natural role of adult stem cells. Stem cell enhancer are typically derived from certain edible algae that grows in fresh water.

The possibility that a decline in the numbers or plasticity of stem cell populations contributes to aging and age-related disease is suggested by recent findings. The remarkable plasticity of stem cells suggests that endogenous or transplanted stem cells can be tweaked' in ways that will allow them to replace lost or dysfunctional cell populations in diseases ranging from neurodegenerative and hematopoietic disorders to diabetes and cardiovascular disease.

As you age, the number and quality of stem cells that circulate in your body gradually decrease, leaving your body more susceptible to injury and other age-related health challenges. Just as antioxidants are important to protect your cells from free radical damage, stem cell nutrition is equally important to support your stem cells in maintaining proper organ and tissue functioning in your body.

A fundamental breakthrough in our understanding of nervous system development was the identification of multipotent neural stem cells (neurospheres) about ten years ago. Dr. Weiss and colleagues showed that EGF (epidermal growth factor) dependent stem cells could be harvested from different brain regions at different developmental stages and that these could be maintained over multiple passages in vitro. This initial finding has lead to an explosion of research on stem cells, their role in normal development and their potential therapeutic uses. Many investigators have entered this field and the progress made has been astounding.

How does an increase in the number of circulating stem cells lead to optimal health? Circulating stem cells can reach various organs and become cells of that organ, helping such organ regain and maintain optimal health. Recent studies have suggested that the number of circulating stem cells is a key factor; the higher the number of circulating stem cells the greater is the ability of the body at healing itself. Scientific interest in adult stem cells has centered on their ability to divide or self-renew indefinitely, and generate all the cell types of the organ from which they originate, potentially regenerating the entire organ from a few cells. Adult stem cells are already being used clinically to treat many diseases. These include as reparative treatments with various cancers, autoimmune disease such as multiple sclerosis, lupus and arthritis, anemias including sickle cells anemia and immunodeficiencies. Adult stem cells are also being used to treat patients by formation of cartilage, growing new corneas to restore sight to blind patients, treatments for stroke, and several groups are using adult stem cells to repair damage after heart attacks. Early clinical trials have shown initial success in patient treatments for Parkinsons disease and spinal cord injury. The first FDA approved trial to treat juvenile diabetes in human patients is ready to begin at Harvard Medical School, using adult stem cells. An advantage of using adult stem cells is that in most cases, the patients own stem cells can be used for the treatment, circumventing the problems of immune rejection, and without tumor formation.

Why do we hear much in the news about embryonic stem cells and very little about adult stem cells? The first human embryonic stem cells were grown in vitro, in a petri dish, in the mid 1990s. Rapidly, scientists were successful at growing them for many generations and to trigger their differentiation into virtually any kind of cells, i.e. brain cells, heart cells, liver cells, bone cells, pancreatic cells, etc. When scientists tried growing adult stem cells, the endeavor was met with less success, as adult stem cells were difficult to grow in vitro for more than a few generations. This led to the idea that embryonic stem cells have more potential than adult stem cells. In addition, the ethical concerns linked to the use of embryonic stem cells have led to a disproportionate representation of embryonic stem cells in the media. But recent developments over the past 2-3 years have established that adult stem cells have capabilities comparable to embryonic stem cells in the human body, not in the test tube. Many studies have indicated that simply releasing stem cells from the bone marrow can help support the body's natural process for renewal of tissues and organs.

The bone marrow constantly produces stem cells for the entire life of an individual. Stem cells released by the bone marrow are responsible for the constant renewal of red blood cells and lymphocytes (immune cells). A 25-30% increase in the number of circulating stem cells is well within physiological range and does not constitute stress on the bone marrow environment. The amount of active bone marrow amounts to about 2,600 g (5.7 lbs), with about 1.5 trillion marrow cells. Stem cells that do not reach any tissue or become blood cells return to the bone marrow.

Effectiveness of stem cell "enhancers" was demonstrated in a triple-blind study. Volunteers rested for one hour before establishing baseline levels. After the first blood samples, volunteers were given stem cell "enhancers"or placebo. Thereafter, blood samples were taken at 30, 60 and 120 minutes after taking the consumables. The number of circulating stem cells was quantified by analyzing the blood samples using Fluorescence-Activated Cell Sorting (FACS). Consumption of stem cell "enhancers" triggered a significant 25-30% increase in the number of circulating stem cells.

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stem cells - Shirley's Wellness Cafe

Californias stem cell agency is on the hunt for a new president and CEO after the surprise announcement this week that C. Randal Mills will be departing the California Institute for Regenerative Medicine. He will leave at the end of June.

Mills, who has headed the agency for three years, will become the next president and CEO of the National Marrow Donor Program. CIRM is replacing him on an interim basis with Maria Millan, M.D., the agencys vice president of therapeutics.

The state agency will soon begin a search for a permanent replacement, said Jonathan Thomas, CIRMs chairman. Millan is a candidate to fill that position, with Mills strong endorsement.

Mills is noted for reorganizing CIRM to provide greater systemic support for translating basic research into clinical science, and to provide quicker and more helpful responses to researchers seeking funding.

His initiative, called CIRM 2.0, was a response to criticism that the agency, funded with $3 billion in California bond money in 2004, has been too slow in getting treatments to patients.

Agency-supported treatments are now being tested in medical centers throughout the state, including San Diego County. Most prominently, CIRM has established an alpha stem cell clinic at UC San Diego. It is the cell therapy arm of UCSDs Sanford Stem Cell Clinical Center.

Mills said he decided to leave because the National Marrow Donor Program, which he was familiar with, resonated with his own goals of making personal connections with patients.

Before joining CIRM in 2014, Mills was president and CEO of Osiris Therapeutics, developer of a pediatric stem cell drug called Prochymal, used to treat a complication of bone marrow transplants called graft vs. host disease.

If you look at my office, the walls are covered with pictures of the children that we treated who went through bone marrow transplantation, Mills said. Getting to know them, and getting to know their families that had a tremendous effect.

The unexpected announcement drew surprise and concern from stem cell researchers and observers. As admirers of CIRM 2.0, they expressed uncertainty about what direction the agency would take. And with the $3 billion beginning to run out, looking for a new source of funding will be a top concern of Mills successor.

Confidence

But Mills said Wednesday the agency will do well.

If me leaving CIRM is a problem, then I didnt do a good job at CIRM, Mills said. Whether its because Im going to be the head of the National Marrow Donor Program or I get hit by a car, the success of this organization, or any organization thats healthy and functional, should never pivot on one person, Mills said. Ive assembled a team at CIRM that I have absolute, absolute confidence in.

Mills said he would be surprised if Millan didnt turn out to be the agency boards overwhelming choice to be his permanent successor. She assisted in developing the agencys strategic plan and helped it run smoothly, he said.

In 2015, Mills named Millan as senior director of medical affairs and stem cell centers, one of three appointments to CIRMs leadership team. Before joining CIRM, she was vice president and acting chief medical officer at StemCells, Inc. Before that, Millan was director of the Pediatric Liver and Kidney Transplant Program at Stanford University School of Medicine.

Millan said the agencys strategic plan is working, and taking the agency where it needs to go. That plan was developed to guide researchers, doctors and companies over the predictable hurdles they encounter in translating basic research into therapies testable in the clinic and that companies would want to commercialize.

Weve already done the challenging piece of identifying the how how to get to the mission, which is to accelerate these stem cell treatments to those with unmet medical needs, Millan said. Team members are all aligned in accomplishing these goals One cant help but be more energized and motivated to execute on the strategic plan.

About 30 stem cell clinical trials are under way that the agency has funded at one stage or another in research and development.

Jonathan Thomas, the CIRM chairman, said Mills has done what he promised when joining CIRM, and the agency is operating markedly better, in productivity, speed and efficiency.

He has made it, through CIRM 2.0 and beyond, a humming machine that is operating on all cylinders, Thomas said. In doing that, hes worked extensively and highly collaboratively with Maria (Millan) and the rest of the team. That has made CIRM an even better operation than it ever was. So we are in extremely good shape right now to go forward.

Goals accomplished

Jeanne Loring, a CIRM-funded stem cell scientist at The Scripps Research Institute, said Mills made the agency friendlier and more predictable for the scientists it funds.

The first and most dramatic thing he did was to end the process of independent grants, Loring said. Under that process, each grant proposal was considered on its own, with no consideration for success under a previous grant for an earlier stage of the research.

It was always very troubling to people, I think, that they could do very well with CIRM money on an early-stage grant, and that would earn them nothing in a further application to continue the work, Loring said.

As part of CIRM 2.0, Mills emphasized that once projects were accepted for funding, CIRM would become a partner with the scientists to help them accelerate research and development, and ultimately commercialization.

Loring leads a team researching the use of stem cells for Parkinsons therapy. The cells are collected from the patients to be treated, making them a genetic match. They are then genetically reprogrammed to resemble embryonic stem cells, and then matured into the brain cells destroyed in Parkinsons.

Lorings team was awarded $2.4 million in 2016 from CIRM to advance its research. A next-stage grant to translate the research to a clinically ready approach would need about $7 million, Loring said. The work is part of Summit for Stem Cell, a nonprofit alliance of scientists, doctors, patients and Parkinsons disease community supporters.

Veteran stem cell watcher David Jensen praised Mills on his blog, California Stem Cell Report.

"Dr. Mills made substantial contributions to the agency during his tenure, improving both efficiency of the grant making process and transparency of CIRM's operations, Jensen quoted stem cell observer John M. Simpson of Consumer Watchdog as saying.

Simpson added that as CIRM draws down the rest of its $3 billion with no new funding in sight, its not surprising that Mills would accept another job.

Paul Knoepfler, a CIRM-funded stem cell scientist and blogger, wrote Tuesday that Mills had a big positive impact on CIRM and helped it go to the next level.

About the only thing I wasnt a fan of in terms of his leadership was my perception of his negativity toward the FDA and toward FDA oversight of stem cells, and how that manifested at CIRM during his time there, Knoepfler wrote. But good people can strongly disagree on policy.

bradley.fikes@sduniontribune.com

(619) 293-1020

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Amid uncertain future, state's stem cell agency loses transformational leader - The San Diego Union-Tribune

by Jos Domen*, Amy Wagers** and Irving L. Weissman***

Blood and the system that forms it, known as the hematopoietic system, consist of many cell types with specialized functions (see Figure 2.1). Red blood cells (erythrocytes) carry oxygen to the tissues. Platelets (derived from megakaryocytes) help prevent bleeding. Granulocytes (neutrophils, basophils and eosinophils) and macrophages (collectively known as myeloid cells) fight infections from bacteria, fungi, and other parasites such as nematodes (ubiquitous small worms). Some of these cells are also involved in tissue and bone remodeling and removal of dead cells. B-lymphocytes produce antibodies, while T-lymphocytes can directly kill or isolate by inflammation cells recognized as foreign to the body, including many virus-infected cells and cancer cells. Many blood cells are short-lived and need to be replenished continuously; the average human requires approximately one hundred billion new hematopoietic cells each day. The continued production of these cells depends directly on the presence of Hematopoietic Stem Cells (HSCs), the ultimate, and only, source of all these cells.

Figure 2.1. Hematopoietic and stromal cell differentiation.

2001 Terese Winslow (assisted by Lydia Kibiuk)

The search for stem cells began in the aftermath of the bombings in Hiroshima and Nagasaki in 1945. Those who died over a prolonged period from lower doses of radiation had compromised hematopoietic systems that could not regenerate either sufficient white blood cells to protect against otherwise nonpathogenic infections or enough platelets to clot their blood. Higher doses of radiation also killed the stem cells of the intestinal tract, resulting in more rapid death. Later, it was demonstrated that mice that were given doses of whole body X-irradiation developed the same radiation syndromes; at the minimal lethal dose, the mice died from hematopoietic failure approximately two weeks after radiation exposure.1 Significantly, however, shielding a single bone or the spleen from radiation prevented this irradiation syndrome. Soon thereafter, using inbred strains of mice, scientists showed that whole-body-irradiated mice could be rescued from otherwise fatal hematopoietic failure by injection of suspensions of cells from blood-forming organs such as the bone marrow.2 In 1956, three laboratories demonstrated that the injected bone marrow cells directly regenerated the blood-forming system, rather than releasing factors that caused the recipients' cells to repair irradiation damage.35 To date, the only known treatment for hematopoietic failure following whole body irradiation is transplantation of bone marrow cells or HSCs to regenerate the blood-forming system in the host organisms.6,7

The hematopoietic system is not only destroyed by the lowest doses of lethal X-irradiation (it is the most sensitive of the affected vital organs), but also by chemotherapeutic agents that kill dividing cells. By the 1960s, physicians who sought to treat cancer that had spread (metastasized) beyond the primary cancer site attempted to take advantage of the fact that a large fraction of cancer cells are undergoing cell division at any given point in time. They began using agents (e.g., chemical and X-irradiation) that kill dividing cells to attempt to kill the cancer cells. This required the development of a quantitative assessment of damage to the cancer cells compared that inflicted on normal cells. Till and McCulloch began to assess quantitatively the radiation sensitivity of one normal cell type, the bone marrow cells used in transplantation, as it exists in the body. They found that, at sub-radioprotective doses of bone marrow cells, mice that died 1015 days after irradiation developed colonies of myeloid and erythroid cells (see Figure 2.1 for an example) in their spleens. These colonies correlated directly in number with the number of bone marrow cells originally injected (approximately 1 colony per 7,000 bone marrow cells injected).8 To test whether these colonies of blood cells derived from single precursor cells, they pre-irradiated the bone marrow donors with low doses of irradiation that would induce unique chromosome breaks in most hematopoietic cells but allow some cells to survive. Surviving cells displayed radiation-induced and repaired chromosomal breaks that marked each clonogenic (colony-initiating) hematopoietic cell.9 The researchers discovered that all dividing cells within a single spleen colony, which contained different types of blood cells, contained the same unique chromosomal marker. Each colony displayed its own unique chromosomal marker, seen in its dividing cells.9 Furthermore, when cells from a single spleen colony were re-injected into a second set of lethally-irradiated mice, donor-derived spleen colonies that contained the same unique chromosomal marker were often observed, indicating that these colonies had been regenerated from the same, single cell that had generated the first colony. Rarely, these colonies contained sufficient numbers of regenerative cells both to radioprotect secondary recipients (e.g., to prevent their deaths from radiation-induced blood cell loss) and to give rise to lymphocytes and myeloerythroid cells that bore markers of the donor-injected cells.10,11 These genetic marking experiments established the fact that cells that can both self-renew and generate most (if not all) of the cell populations in the blood must exist in bone marrow. At the time, such cells were called pluripotent HSCs, a term later modified to multipotent HSCs.12,13 However, identifying stem cells in retrospect by analysis of randomly chromosome-marked cells is not the same as being able to isolate pure populations of HSCs for study or clinical use.

Achieving this goal requires markers that uniquely define HSCs. Interestingly, the development of these markers, discussed below, has revealed that most of the early spleen colonies visible 8 to 10 days after injection, as well as many of the later colonies, visible at least 12 days after injection, are actually derived from progenitors rather than from HSCs. Spleen colonies formed by HSCs are relatively rare and tend to be present among the later colonies.14,15 However, these findings do not detract from Till and McCulloch's seminal experiments to identify HSCs and define these unique cells by their capacities for self-renewal and multilineage differentiation.

While much of the original work was, and continues to be, performed in murine model systems, strides have been made to develop assays to study human HSCs. The development of Fluorescence Activated Cell Sorting (FACS) has been crucial for this field (see Figure 2.2). This technique enables the recognition and quantification of small numbers of cells in large mixed populations. More importantly, FACS-based cell sorting allows these rare cells (1 in 2000 to less than 1 in 10,000) to be purified, resulting in preparations of near 100% purity. This capability enables the testing of these cells in various assays.

Figure 2.2. Enrichment and purification methods for hematopoietic stem cells. Upper panels illustrate column-based magnetic enrichment. In this method, the cells of interest are labeled with very small iron particles (A). These particles are bound to antibodies that only recognize specific cells. The cell suspension is then passed over a column through a strong magnetic field which retains the cells with the iron particles (B). Other cells flow through and are collected as the depleted negative fraction. The magnet is removed, and the retained cells are collected in a separate tube as the positive or enriched fraction (C). Magnetic enrichment devices exist both as small research instruments and large closed-system clinical instruments.

Lower panels illustrate Fluorescence Activated Cell Sorting (FACS). In this setting, the cell mixture is labeled with fluorescent markers that emit light of different colors after being activated by light from a laser. Each of these fluorescent markers is attached to a different monoclonal antibody that recognizes specific sets of cells (D). The cells are then passed one by one in a very tight stream through a laser beam (blue in the figure) in front of detectors (E) that determine which colors fluoresce in response to the laser. The results can be displayed in a FACS-plot (F). FACS-plots (see figures 3 and 4 for examples) typically show fluorescence levels per cell as dots or probability fields. In the example, four groups can be distinguished: Unstained, red-only, green-only, and red-green double labeling. Each of these groups, e.g., green fluorescence-only, can be sorted to very high purity. The actual sorting happens by breaking the stream shown in (E) into tiny droplets, each containing 1 cell, that then can be sorted using electric charges to move the drops. Modern FACS machines use three different lasers (that can activate different set of fluorochromes), to distinguish up to 8 to 12 different fluorescence colors and sort 4 separate populations, all simultaneously.

Magnetic enrichment can process very large samples (billions of cells) in one run, but the resulting cell preparation is enriched for only one parameter (e.g., CD34) and is not pure. Significant levels of contaminants (such as T-cells or tumor cells) remain present. FACS results in very pure cell populations that can be selected for several parameters simultaneously (e.g., Linneg, CD34pos, CD90pos), but it is more time consuming (10,000 to 50,000 cells can be sorted per second) and requires expensive instrumentation.

2001 Terese Winslow (assisted by Lydia Kibiuk)

Assays have been developed to characterize hematopoietic stem and progenitor cells in vitro and in vivo (Figure 2.3).16,17In vivo assays that are used to study HSCs include Till and McCulloch's classical spleen colony forming (CFU-S) assay,8 which measures the ability of HSC (as well as blood-forming progenitor cells) to form large colonies in the spleens of lethally irradiated mice. Its main advantage (and limitation) is the short-term nature of the assay (now typically 12 days). However, the assays that truly define HSCs are reconstitution assays.16,18 Mice that have been quot;preconditionedquot; by lethal irradiation to accept new HSCs are injected with purified HSCs or mixed populations containing HSCs, which will repopulate the hematopoietic systems of the host mice for the life of the animal. These assays typically use different types of markers to distinguish host and donor-derived cells.

For example, allelic assays distinguish different versions of a particular gene, either by direct analysis of dna or of the proteins expressed by these alleles. These proteins may be cell-surface proteins that are recognized by specific monoclonal antibodies that can distinguish between the variants (e.g., CD45 in Figure 2.3) or cellular proteins that may be recognized through methods such as gel-based analysis. Other assays take advantage of the fact that male cells can be detected in a female host by detecting the male-cell-specific Y-chromosome by molecular assays (e.g., polymerase chain reaction, or PCR).

Figure 2.3. Assays used to detect hematopoietic stem cells. The tissue culture assays, which are used frequently to test human cells, include the ability of the cells to be tested to grow as quot;cobblestonesquot; (the dark cells in the picture) for 5 to 7 weeks in culture. The Long Term Culture-Initiating Cell assay measures whether hematopoietic progenitor cells (capable of forming colonies in secondary assays, as shown in the picture) are still present after 5 to 7 weeks of culture.

In vivo assays in mice include the CFU-S assay, the original stem cell assay discussed in the introduction. The most stringent hematopoietic stem cell assay involves looking for the long-term presence of donor-derived cells in a reconstituted host. The example shows host-donor recognition by antibodies that recognize two different mouse alleles of CD45, a marker present on nearly all blood cells. CD45 is also a good marker for distinguishing human blood cells from mouse blood cells when testing human cells in immunocompromised mice such as NOD/SCID. Other methods such as pcr-markers, chromosomal markers, and enzyme markers can also be used to distinguish host and donor cells.

Small numbers of HSCs (as few as one cell in mouse experiments) can be assayed using competitive reconstitutions, in which a small amount of host-type bone marrow cells (enough to radioprotect the host and thus ensure survival) is mixed in with the donor-HSC population. To establish long-term reconstitutions in mouse models, the mice are followed for at least 4 months after receiving the HSCs. Serial reconstitution, in which the bone marrow from a previously-irradiated and reconstituted mouse becomes the HSC source for a second irradiated mouse, extends the potential of this assay to test lifespan and expansion limits of HSCs. Unfortunately, the serial transfer assay measures both the lifespan and the transplantability of the stem cells. The transplantability may be altered under various conditions, so this assay is not the sine qua non of HSC function. Testing the in vivo activity of human cells is obviously more problematic.

Several experimental models have been developed that allow the testing of human cells in mice. These assays employ immunologically-incompetent mice (mutant mice that cannot mount an immune response against foreign cells) such as SCID1921 or NOD-SCID mice.22,23 Reconstitution can be performed in either the presence or absence of human fetal bone or thymus implants to provide a more natural environment in which the human cells can grow in the mice. Recently NOD/SCID/c-/- mice have been used as improved recipients for human HSCs, capable of complete reconstitution with human lymphocytes, even in the absence of additional human tissues.24 Even more promising has been the use of newborn mice with an impaired immune system (Rag-2-/-C-/-), which results in reproducible production of human B- and T-lymphoid and myeloerythroid cells.25 These assays are clearly more stringent, and thus more informative, but also more difficult than the in vitro HSC assays discussed below. However, they can only assay a fraction of the lifespan under which the cells would usually have to function. Information on the long-term functioning of cells can only be derived from clinical HSC transplantations.

A number of assays have been developed to recognize HSCs in vitro (e.g., in tissue culture). These are especially important when assaying human cells. Since transplantation assays for human cells are limited, cell culture assays often represent the only viable option. In vitro assays for HSCs include Long-Term Culture-Initializing Cell (LTC-IC) assays2628 and Cobble-stone Area Forming Cell (CAFC) assays.29 LTC-IC assays are based on the ability of HSCs, but not more mature progenitor cells, to maintain progenitor cells with clonogenic potential over at least a five-week culture period. CAFC assays measure the ability of HSCs to maintain a specific and easily recognizable way of growing under stromal cells for five to seven weeks after the initial plating. Progenitor cells can only grow in culture in this manner for shorter periods of time.

While initial experiments studied HSC activity in mixed populations, much progress has been made in specifically describing the cells that have HSC activity. A variety of markers have been discovered to help recognize and isolate HSCs. Initial marker efforts focused on cell size, density, and recognition by lectins (carbohydrate-binding proteins derived largely from plants),30 but more recent efforts have focused mainly on cell surface protein markers, as defined by monoclonal antibodies. For mouse HSCs, these markers include panels of 8 to 14 different monoclonal antibodies that recognize cell surface proteins present on differentiated hematopoietic lineages, such as the red blood cell and macrophage lineages (thus, these markers are collectively referred to as quot;Linquot;),13,31 as well as the proteins Sca-1,13,31 CD27,32 CD34,33 CD38,34 CD43,35 CD90.1(Thy-1.1),13,31 CD117(c-Kit),36 AA4.1,37 and MHC class I,30 and CD150.38 Human HSCs have been defined with respect to staining for Lin,39 CD34,40 CD38,41 CD43,35 CD45RO,42 CD45RA,42 CD59,43 CD90,39 CD109,44 CD117,45 CD133,46,47CD166,48 and HLA DR(human).49,50 In addition, metabolic markers/dyes such as rhodamine123 (which stains mitochondria),51 Hoechst33342 (which identifies MDR-type drug efflux activity),52 Pyronin-Y (which stains RNA),53 and BAAA (indicative of aldehyde dehydrogenase enzyme activity)54 have been described. While none of these markers recognizes functional stem cell activity, combinations (typically with 3 to 5 different markers, see examples below) allow for the purification of near-homogenous populations of HSCs. The ability to obtain pure preparations of HSCs, albeit in limited numbers, has greatly facilitated the functional and biochemical characterization of these important cells. However, to date there has been limited impact of these discoveries on clinical practice, as highly purified HSCs have only rarely been used to treat patients (discussed below). The undeniable advantages of using purified cells (e.g., the absence of contaminating tumor cells in autologous transplantations) have been offset by practical difficulties and increased purification costs.

Figure 2.4. Examples of Hematopoietic Stem Cell staining patterns in mouse bone marrow (top) and human mobilized peripheral blood (bottom). The plots on the right show only the cells present in the left blue box. The cells in the right blue box represent HSCs. Stem cells form a rare fraction of the cells present in both cases.

HSC assays, when combined with the ability to purify HSCs, have provided increasingly detailed insight into the cells and the early steps involved in the differentiation process. Several marker combinations have been developed that describe murine HSCs, including [CD117high, CD90.1low, Linneg/low, Sca-1pos],15 [CD90.1low, Linneg, Sca-1pos Rhodamine123low],55 [CD34neg/low, CD117pos, Sca-1pos, Linneg],33 [CD150 pos, CD48neg, CD244neg],38 and quot;side-populationquot; cells using Hoechst-dye.52 Each of these combinations allows purification of HSCs to near-homogeneity. Figure 2.4 shows an example of an antibody combination that can recognize mouse HSCs. Similar strategies have been developed to purify human HSCs, employing markers such as CD34, CD38, Lin, CD90, CD133 and fluorescent substrates for the enzyme, aldehyde dehydrogenase. The use of highly purified human HSCs has been mainly experimental, and clinical use typically employs enrichment for one marker, usually CD34. CD34 enrichment yields a population of cells enriched for HSC and blood progenitor cells but still contains many other cell types. However, limited trials in which highly FACS-purified CD34pos CD90pos HSCs (see Figure 2.4) were used as a source of reconstituting cells have demonstrated that rapid reconstitution of the blood system can reliably be obtained using only HSCs.5658

The purification strategies described above recognize a rare subset of cells. Exact numbers depend on the assay used as well as on the genetic background studied.16 In mouse bone marrow, 1 in 10,000 cells is a hematopoietic stem cell with the ability to support long-term hematopoiesis following transplantation into a suitable host. When short-term stem cells, which have a limited self-renewal capacity, are included in the estimation, the frequency of stem cells in bone marrow increases to 1 in 1,000 to 1 in 2,000 cells in humans and mice. The numbers present in normal blood are at least ten-fold lower than in marrow.

None of the HSC markers currently used is directly linked to an essential HSC function, and consequently, even within a species, markers can differ depending on genetic alleles,59 mouse strains,60 developmental stages,61 and cell activation stages.62,63 Despite this, there is a clear correlation in HSC markers between divergent species such as humans and mice. However, unless the ongoing attempts at defining the complete HSC gene expression patterns will yield usable markers that are linked to essential functions for maintaining the quot;stemnessquot; of the cells,64,65 functional assays will remain necessary to identify HSCs unequivocally.16

More recently, efforts at defining hematopoietic populations by cell surface or other FACS-based markers have been extended to several of the progenitor populations that are derived from HSCs (see Figure 2.5). Progenitors differ from stem cells in that they have a reduced differentiation capacity (they can generate only a subset of the possible lineages) but even more importantly, progenitors lack the ability to self-renew. Thus, they have to be constantly regenerated from the HSC population. However, progenitors do have extensive proliferative potential and can typically generate large numbers of mature cells. Among the progenitors defined in mice and humans are the Common Lymphoid Progenitor (CLP),66,67 which in adults has the potential to generate all of the lymphoid but not myeloerythroid cells, and a Common Myeloid Progenitor (CMP), which has the potential to generate all of the mature myeloerythroid, but not lymphoid, cells.68,69 While beyond the scope of this overview, hematopoietic progenitors have clinical potential and will likely see clinical use.70,71

Figure 2.5. Relationship between several of the characterized hematopoietic stem cells and early progenitor cells. Differentiation is indicated by colors; the more intense the color, the more mature the cells. Surface marker distinctions are subtle between these early cell populations, yet they have clearly distinct potentials. Stem cells can choose between self-renewal and differentiation. Progenitors can expand temporarily but always continue to differentiate (other than in certain leukemias). The mature lymphoid (T-cells, B-cells, and Natural Killer cells) and myeloerythroid cells (granulocytes, macrophages, red blood cells, and platelets) that are produced by these stem and progenitor cells are shown in more detail in Figure 2.1.

HSCs have a number of unique properties, the combination of which defines them as such.16 Among the core properties are the ability to choose between self-renewal (remain a stem cell after cell division) or differentiation (start the path towards becoming a mature hematopoietic cell). In addition, HSCs migrate in regulated fashion and are subject to regulation by apoptosis (programmed cell death). The balance between these activities determines the number of stem cells that are present in the body.

One essential feature of HSCs is the ability to self-renew, that is, to make copies with the same or very similar potential. This is an essential property because more differentiated cells, such as hematopoietic progenitors, cannot do this, even though most progenitors can expand significantly during a limited period of time after being generated. However, for continued production of the many (and often short-lived) mature blood cells, the continued presence of stem cells is essential. While it has not been established that adult HSCs can self-renew indefinitely (this would be difficult to prove experimentally), it is clear from serial transplantation experiments that they can produce enough cells to last several (at least four to five) lifetimes in mice. It is still unclear which key signals allow self-renewal. One link that has been noted is telomerase, the enzyme necessary for maintaining telomeres, the DNA regions at the end of chromosomes that protect them from accumulating damage due to DNA replication. Expression of telomerase is associated with self-renewal activity.72 However, while absence of telomerase reduces the self-renewal capacity of mouse HSCs, forced expression is not sufficient to enable HSCs to be transplanted indefinitely; other barriers must exist.73,74

It has proven surprisingly difficult to grow HSCs in culture despite their ability to self-renew. Expansion in culture is routine with many other cells, including neural stem cells and ES cells. The lack of this capacity for HSCs severely limits their application, because the number of HSCs that can be isolated from mobilized blood, umbilical cord blood, or bone marrow restricts the full application of HSC transplantation in man (whether in the treatment of nuclear radiation exposure or transplantation in the treatment of blood cell cancers or genetic diseases of the blood or blood-forming system). Engraftment periods of 50 days or more were standard when limited numbers of bone marrow or umbilical cord blood cells were used in a transplant setting, reflecting the low level of HSCs found in these native tissues. Attempts to expand HSCs in tissue culture with known stem-cell stimulators, such as the cytokines stem cell factor/steel factor (KitL), thrombopoietin (TPO), interleukins 1, 3, 6, 11, plus or minus the myeloerythroid cytokines GM-CSF, G-CSF, M-CSF, and erythropoietin have never resulted in a significant expansion of HSCs.16,75 Rather, these compounds induce many HSCs into cell divisions that are always accompanied by cellular differentiation.76 Yet many experiments demonstrate that the transplantation of a single or a few HSCs into an animal results in a 100,000-fold or greater expansion in the number of HSCs at the steady state while simultaneously generating daughter cells that permitted the regeneration of the full blood-forming system.7780 Thus, we do not know the factors necessary to regenerate HSCs by self-renewing cell divisions. By investigating genes transcribed in purified mouse LT-HSCs, investigators have found that these cells contain expressed elements of the Wnt/fzd/beta-catenin signaling pathway, which enables mouse HSCs to undergo self-renewing cell divisions.81,82 Overexpression of several other proteins, including HoxB48386 and HoxA987 has also been reported to achieve this. Other signaling pathways that are under investigation include Notch and Sonic hedgehog.75 Among the intracellular proteins thought to be essential for maintaining the quot;stem cellquot; state are Polycomb group genes, including Bmi-1.88 Other genes, such as c-Myc and JunB have also been shown to play a role in this process.89,90Much remains to be discovered, including the identity of the stimuli that govern self-renewal in vivo, as well as the composition of the environment (the stem cell quot;nichequot;) that provides these stimuli.91 The recent identification of osteoblasts, a cell type known to be involved in bone formation, as a critical component of this environment92,93 will help to focus this search. For instance, signaling by Angiopoietin-1 on osteoblasts to Tie-2 receptors on HSCs has recently been suggested to regulate stem cell quiescence (the lack of cell division).94 It is critical to discover which pathways operate in the expansion of human HSCs to take advantage of these pathways to improve hematopoietic transplantation.

Differentiation into progenitors and mature cells that fulfill the functions performed by the hematopoietic system is not a unique HSC property, but, together with the option to self-renew, defines the core function of HSCs. Differentiation is driven and guided by an intricate network of growth factors and cytokines. As discussed earlier, differentiation, rather than self-renewal, seems to be the default outcome for HSCs when stimulated by many of the factors to which they have been shown to respond. It appears that, once they commit to differentiation, HSCs cannot revert to a self-renewing state. Thus, specific signals, provided by specific factors, seem to be needed to maintain HSCs. This strict regulation may reflect the proliferative potential present in HSCs, deregulation of which could easily result in malignant diseases such as leukemia or lymphoma.

Migration of HSCs occurs at specific times during development (i.e., seeding of fetal liver, spleen and eventually, bone marrow) and under certain conditions (e.g., cytokine-induced mobilization) later in life. The latter has proven clinically useful as a strategy to enhance normal HSC proliferation and migration, and the optimal mobilization regimen for HSCs currently used in the clinic is to treat the stem cell donor with a drug such as cytoxan, which kills most of his or her dividing cells. Normally, only about 8% of LT-HSCs enter the cell cycle per day,95,96 so HSCs are not significantly affected by a short treatment with cytoxan. However, most of the downstream blood progenitors are actively dividing,66,68 and their numbers are therefore greatly depleted by this dose, creating a demand for a regenerated blood-forming system. Empirically, cytokines or growth factors such as G-CSF and KitL can increase the number of HSCs in the blood, especially if administered for several days following a cytoxan pulse. The optimized protocol of cytoxan plus G-CSF results in several self-renewing cell divisions for each resident LT-HSC in mouse bone marrow, expanding the number of HSCs 12- to 15-fold within two to three days.97 Then, up to one-half of the daughter cells of self-renewing dividing LT-HSCs (estimated to be up to 105 per mouse per day98) leave the bone marrow, enter the blood, and within minutes engraft other hematopoietic sites, including bone marrow, spleen, and liver.98 These migrating cells can and do enter empty hematopoietic niches elsewhere in the bone marrow and provide sustained hematopoietic stem cell self-renewal and hematopoiesis.98,99 It is assumed that this property of mobilization of HSCs is highly conserved in evolution (it has been shown in mouse, dog and humans) and presumably results from contact with natural cell-killing agents in the environment, after which regeneration of hematopoiesis requires restoring empty HSC niches. This means that functional, transplantable HSCs course through every tissue of the body in large numbers every day in normal individuals.

Apoptosis, or programmed cell death, is a mechanism that results in cells actively self-destructing without causing inflammation. Apoptosis is an essential feature in multicellular organisms, necessary during development and normal maintenance of tissues. Apoptosis can be triggered by specific signals, by cells failing to receive the required signals to avoid apoptosis, and by exposure to infectious agents such as viruses. HSCs are not exempt; apoptosis is one mechanism to regulate their numbers. This was demonstrated in transgenic mouse experiments in which HSC numbers doubled when the apoptosis threshold was increased.76 This study also showed that HSCs are particularly sensitive and require two signals to avoid undergoing apoptosis.

The best-known location for HSCs is bone marrow, and bone marrow transplantation has become synonymous with hematopoietic cell transplantation, even though bone marrow itself is increasingly infrequently used as a source due to an invasive harvesting procedure that requires general anesthesia. In adults, under steady-state conditions, the majority of HSCs reside in bone marrow. However, cytokine mobilization can result in the release of large numbers of HSCs into the blood. As a clinical source of HSCs, mobilized peripheral blood (MPB) is now replacing bone marrow, as harvesting peripheral blood is easier for the donors than harvesting bone marrow. As with bone marrow, mobilized peripheral blood contains a mixture of hematopoietic stem and progenitor cells. MPB is normally passed through a device that enriches cells that express CD34, a marker on both stem and progenitor cells. Consequently, the resulting cell preparation that is infused back into patients is not a pure HSC preparation, but a mixture of HSCs, hematopoietic progenitors (the major component), and various contaminants, including T cells and, in the case of autologous grafts from cancer patients, quite possibly tumor cells. It is important to distinguish these kinds of grafts, which are the grafts routinely given, from highly purified HSC preparations, which essentially lack other cell types.

In the late 1980s, umbilical cord blood (UCB) was recognized as an important clinical source of HSCs.100,101 Blood from the placenta and umbilical cord is a rich source of hematopoietic stem cells, and these cells are typically discarded with the afterbirth. Increasingly, UCB is harvested, frozen, and stored in cord blood banks, as an individual resource (donor-specific source) or as a general resource, directly available when needed. Cord blood has been used successfully to transplant children and (far less frequently) adults. Specific limitations of UCB include the limited number of cells that can be harvested and the delayed immune reconstitution observed following UCB transplant, which leaves patients vulnerable to infections for a longer period of time. Advantages of cord blood include its availability, ease of harvest, and the reduced risk of graft-versus-host-disease (GVHD). In addition, cord blood HSCs have been noted to have a greater proliferative capacity than adult HSCs. Several approaches have been tested to overcome the cell dose issue, including, with some success, pooling of cord blood samples.101,102 Ex vivo expansion in tissue culture, to which cord blood cells are more amenable than adult cells, is another approach under active investigation.103

The use of cord blood has opened a controversial treatment strategyembryo selection to create a related UCB donor.104 In this procedure, embryos are conceived by in vitro fertilization. The embryos are tested by pre-implantation genetic diagnosis, and embryos with transplantation antigens matching those of the affected sibling are implanted. Cord blood from the resulting newborn is then used to treat this sibling. This approach, successfully pioneered at the University of Minnesota, can in principle be applied to a wide variety of hematopoietic disorders. However, the ethical questions involved argue for clear regulatory guidelines.105

Embryonic stem (ES) cells form a potential future source of HSCs. Both mouse and human ES cells have yielded hematopoietic cells in tissue culture, and they do so relatively readily.106 However, recognizing the actual HSCs in these cultures has proven problematic, which may reflect the variability in HSC markers or the altered reconstitution behavior of these HSCs, which are expected to mimic fetal HSC. This, combined with the potential risks of including undifferentiated cells in an ES-cell-derived graft means that, based on the current science, clinical use of ES cell-derived HSCs remains only a theoretical possibility for now.

An ongoing set of investigations has led to claims that HSCs, as well as other stem cells, have the capacity to differentiate into a much wider range of tissues than previously thought possible. It has been claimed that, following reconstitution, bone marrow cells can differentiate not only into blood cells but also muscle cells (both skeletal myocytes and cardiomyocytes),107111 brain cells,112,113 liver cells,114,115 skin cells, lung cells, kidney cells, intestinal cells,116 and pancreatic cells.117 Bone marrow is a complex mixture that contains numerous cell types. In addition to HSCs, at least one other type of stem cell, the mesenchymal stem cell (MSC), is present in bone marrow. MSCs, which have become the subject of increasingly intense investigation, seem to retain a wide range of differentiation capabilities in vitro that is not restricted to mesodermal tissues, but includes tissues normally derived from other embryonic germ layers (e.g., neurons).118120MSCs are discussed in detail in Dr. Catherine Verfaillie's testimony to the President's Council on Bioethics at this website: refer to Appendix J (page 295) and will not be discussed further here. However, similar claims of differentiation into multiple diverse cell types, including muscle,111 liver,114 and different types of epithelium116 have been made in experiments that assayed partially- or fully-purified HSCs. These experiments have spawned the idea that HSCs may not be entirely or irreversibly committed to forming the blood, but under the proper circumstances, HSCs may also function in the regeneration or repair of non-blood tissues. This concept has in turn given rise to the hypothesis that the fate of stem cells is quot;plastic,quot; or changeable, allowing these cells to adopt alternate fates if needed in response to tissue-derived regenerative signals (a phenomenon sometimes referred to as quot;transdifferentiationquot;). This in turn seems to bolster the argument that the full clinical potential of stem cells can be realized by studying only adult stem cells, foregoing research into defining the conditions necessary for the clinical use of the extensive differentiation potential of embryonic stem cells. However, as discussed below, such quot;transdifferentiationquot; claims for specialized adult stem cells are controversial, and alternative explanations for these observations remain possible, and, in several cases, have been documented directly.

While a full discussion of this issue is beyond the scope of this overview, several investigators have formulated criteria that must be fulfilled to demonstrate stem cell plasticity.121,122 These include (i) clonal analysis, which requires the transfer and analysis of single, highly-purified cells or individually marked cells and the subsequent demonstration of both quot;normalquot; and quot;plasticquot; differentiation outcomes, (ii) robust levels of quot;plasticquot; differentiation outcome, as extremely rare events are difficult to analyze and may be induced by artefact, and (iii) demonstration of tissue-specific function of the quot;transdifferentiatedquot; cell type. Few of the current reports fulfill these criteria, and careful analysis of individually transplanted KTLS HSCs has failed to show significant levels of non-hematopoietic engraftment.123,124In addition, several reported trans-differentiation events that employed highly purified HSCs, and in some cases a very strong selection pressure for trans-differentiation, now have been shown to result from fusion of a blood cell with a non-blood cell, rather than from a change in fate of blood stem cells.125127 Finally, in the vast majority of cases, reported contributions of adult stem cells to cell types outside their tissue of origin are exceedingly rare, far too rare to be considered therapeutically useful. These findings have raised significant doubts about the biological importance and immediate clinical utility of adult hematopoietic stem cell plasticity. Instead, these results suggest that normal tissue regeneration relies predominantly on the function of cell type-specific stem or progenitor cells, and that the identification, isolation, and characterization of these cells may be more useful in designing novel approaches to regenerative medicine. Nonetheless, it is possible that a rigorous and concerted effort to identify, purify, and potentially expand the appropriate cell populations responsible for apparent quot;plasticityquot; events, characterize the tissue-specific and injury-related signals that recruit, stimulate, or regulate plasticity, and determine the mechanism(s) underlying cell fusion or transdifferentiation, may eventually enhance tissue regeneration via this mechanism to clinically useful levels.

Recent progress in genomic sequencing and genome-wide expression analysis at the RNA and protein levels has greatly increased our ability to study cells such as HSCs as quot;systems,quot; that is, as combinations of defined components with defined interactions. This goal has yet to be realized fully, as computational biology and system-wide protein biochemistry and proteomics still must catch up with the wealth of data currently generated at the genomic and transcriptional levels. Recent landmark events have included the sequencing of the human and mouse genomes and the development of techniques such as array-based analysis. Several research groups have combined cDNA cloning and sequencing with array-based analysis to begin to define the full transcriptional profile of HSCs from different species and developmental stages and compare these to other stem cells.64,65,128131 Many of the data are available in online databases, such as the NIH/NIDDK Stem Cell Genome Anatomy Projects. While transcriptional profiling is clearly a work in progress, comparisons among various types of stem cells may eventually identify sets of genes that are involved in defining the general quot;stemnessquot; of a cell, as well as sets of genes that define their exit from the stem cell pool (e.g., the beginning of their path toward becoming mature differentiated cells, also referred to as commitment). In addition, these datasets will reveal sets of genes that are associated with specific stem cell populations, such as HSCs and MSCs, and thus define their unique properties. Assembly of these datasets into pathways will greatly help to understand and to predict the responses of HSCs (and other stem cells) to various stimuli.

The clinical use of stem cells holds great promise, although the application of most classes of adult stem cells is either currently untested or is in the earliest phases of clinical testing.132,133 The only exception is HSCs, which have been used clinically since 1959 and are used increasingly routinely for transplantations, albeit almost exclusively in a non-pure form. By 1995, more than 40,000 transplants were performed annually world-wide.134,135 Currently the main indications for bone marrow transplantation are either hematopoietic cancers (leukemias and lymphomas), or the use of high-dose chemotherapy for non-hematopoietic malignancies (cancers in other organs). Other indications include diseases that involve genetic or acquired bone marrow failure, such as aplastic anemia, thalassemia sickle cell anemia, and increasingly, autoimmune diseases.

Transplantation of bone marrow and HSCs are carried out in two rather different settings, autologous and allogeneic. Autologous transplantations employ a patient's own bone marrow tissue and thus present no tissue incompatibility between the donor and the host. Allogeneic transplantations occur between two individuals who are not genetically identical (with the rare exceptions of transplantations between identical twins, often referred to as syngeneic transplantations). Non-identical individuals differ in their human leukocyte antigens (HLAs), proteins that are expressed by their white blood cells. The immune system uses these HLAs to distinguish between quot;selfquot; and quot;nonself.quot; For successful transplantation, allogeneic grafts must match most, if not all, of the six to ten major HLA antigens between host and donor. Even if they do, however, enough differences remain in mostly uncharacterized minor antigens to enable immune cells from the donor and the host to recognize the other as quot;nonself.quot; This is an important issue, as virtually all HSC transplants are carried out with either non-purified, mixed cell populations (mobilized peripheral blood, cord blood, or bone marrow) or cell populations that have been enriched for HSCs (e.g., by column selection for CD34+ cells) but have not been fully purified. These mixed population grafts contain sufficient lymphoid cells to mount an immune response against host cells if they are recognized as quot;non-self.quot; The clinical syndrome that results from this quot;non-selfquot; response is known as graft-versus-host disease (GVHD).136

In contrast, autologous grafts use cells harvested from the patient and offer the advantage of not causing GVHD. The main disadvantage of an autologous graft in the treatment of cancer is the absence of a graft-versusleukemia (GVL) or graft-versus-tumor (GVT) response, the specific immunological recognition of host tumor cells by donor-immune effector cells present in the transplant. Moreover, the possibility exists for contamination with cancerous or pre-cancerous cells.

Allogeneic grafts also have disadvantages. They are limited by the availability of immunologically-matched donors and the possibility of developing potentially lethal GVHD. The main advantage of allogeneic grafts is the potential for a GVL response, which can be an important contribution to achieving and maintaining complete remission.137,138

Today, most grafts used in the treatment of patients consist of either whole or CD34+-enriched bone marrow or, more likely, mobilized peripheral blood. The use of highly purified hematopoietic stem cells as grafts is rare.5658 However, the latter have the advantage of containing no detectable contaminating tumor cells in the case of autologous grafts, therefore not inducing GVHD, or presumably GVL,139141in allogeneic grafts. While they do so less efficiently than lymphocyte-containing cell mixtures, HSCs alone can engraft across full allogeneic barriers (i.e., when transplanted from a donor who is a complete mismatch for both major and minor transplantation antigens).139141The use of donor lymphocyte infusions (DLI) in the context of HSC transplantation allows for the controlled addition of lymphocytes, if necessary, to obtain or maintain high levels of donor cells and/or to induce a potentially curative GVL-response.142,143 The main problems associated with clinical use of highly purified HSCs are the additional labor and costs144 involved in obtaining highly purified cells in sufficient quantities.

While the possibilities of GVL and other immune responses to malignancies remain the focus of intense interest, it is also clear that in many cases, less-directed approaches such as chemotherapy or irradiation offer promise. However, while high-dose chemotherapy combined with autologous bone marrow transplantation has been reported to improve outcome (usually measured as the increase in time to progression, or increase in survival time),145154 this has not been observed by other researchers and remains controversial.155161 The tumor cells present in autologous grafts may be an important limitation in achieving long-term disease-free survival. Only further purification/ purging of the grafts, with rigorous separation of HSCs from cancer cells, can overcome this limitation. Initial small scale trials with HSCs purified by flow cytometry suggest that this is both possible and beneficial to the clinical outcome.56 In summary, purification of HSCs from cancer/lymphoma/leukemia patients offers the only possibility of using these cells post-chemotherapy to regenerate the host with cancer-free grafts. Purification of HSCs in allotransplantation allows transplantation with cells that regenerate the blood-forming system but cannot induce GVHD.

An important recent advance in the clinical use of HSCs is the development of non-myeloablative preconditioning regimens, sometimes referred to as quot;mini transplants.quot;162164 Traditionally, bone marrow or stem cell transplantation has been preceded by a preconditioning regimen consisting of chemotherapeutic agents, often combined with irradiation, that completely destroys host blood and bone marrow tissues (a process called myeloablation). This creates quot;spacequot; for the incoming cells by freeing stem cell niches and prevents an undesired immune response of the host cells against the graft cells, which could result in graft failure. However, myeloablation immunocompromises the patient severely and necessitates a prolonged hospital stay under sterile conditions. Many protocols have been developed that use a more limited and targeted approach to preconditioning. These nonmyeloablative preconditioning protocols, which combine excellent engraftment results with the ability to perform hematopoietic cell transplantation on an outpatient basis, have greatly changed the clinical practice of bone marrow transplantation.

FACS purification of HSCs in mouse and man completely eliminates contaminating T cells, and thus GVHD (which is caused by T-lymphocytes) in allogeneic transplants. Many HSC transplants have been carried out in different combinations of mouse strains. Some of these were matched at the major transplantation antigens but otherwise different (Matched Unrelated Donors or MUD); in others, no match at the major or minor transplantation antigens was expected. To achieve rapid and sustained engraftment, higher doses of HSCs were required in these mismatched allogeneic transplants than in syngeneic transplants.139141,165167 In these experiments, hosts whose immune and blood-forming systems were generated from genetically distinct donors were permanently capable of accepting organ transplants (such as the heart) from either donor or host, but not from mice unrelated to the donor or host. This phenomenon is known as transplant-induced tolerance and was observed whether the organ transplants were given the same day as the HSCs or up to one year later.139,166Hematopoietic cell transplant-related complications have limited the clinical application of such tolerance induction for solid organ grafts, but the use of non-myeloablative regimens to prepare the host, as discussed above, should significantly reduce the risk associated with combined HSC and organ transplants. Translation of these findings to human patients should enable a switch from chronic immunosuppression to prevent rejection to protocols wherein a single conditioning dose allows permanent engraftment of both the transplanted blood system and solid organ(s) or other tissue stem cells from the same donor. This should eliminate both GVHD and chronic host transplant immunosuppression, which lead to many complications, including life-threatening opportunistic infections and the development of malignant neoplasms.

We now know that several autoimmune diseasesdiseases in which immune cells attack normal body tissuesinvolve the inheritance of high risk-factor genes.168 Many of these genes are expressed only in blood cells. Researchers have recently tested whether HSCs could be used in mice with autoimmune disease (e.g., type 1 diabetes) to replace an autoimmune blood system with one that lacks the autoimmune risk genes. The HSC transplants cured mice that were in the process of disease development when nonmyeloablative conditioning was used for transplant.169 It has been observed that transplant-induced tolerance allows co-transplantation of pancreatic islet cells to replace destroyed islets.170 If these results using nonmyeloablative conditioning can be translated to humans, type 1 diabetes and several other autoimmune diseases may be treatable with pure HSC grafts. However, the reader should be cautioned that the translation of treatments from mice to humans is often complicated and time-consuming.

Banking is currently a routine procedure for UCB samples. If expansion of fully functional HSCs in tissue culture becomes a reality, HSC transplants may be possible by starting with small collections of HSCs rather than massive numbers acquired through mobilization and apheresis. With such a capability, collections of HSCs from volunteer donors or umbilical cords could be theoretically converted into storable, expandable stem cell banks useful on demand for clinical transplantation and/or for protection against radiation accidents. In mice, successful HSC transplants that regenerate fully normal immune and blood-forming systems can be accomplished when there is only a partial transplantation antigen match. Thus, the establishment of useful human HSC banks may require a match between as few as three out of six transplantation antigens (HLA). This might be accomplished with stem cell banks of as few as 4,00010,000 independent samples.

Leukemias are proliferative diseases of the hematopoietic system that fail to obey normal regulatory signals. They derive from stem cells or progenitors of the hematopoietic system and almost certainly include several stages of progression. During this progression, genetic and/or epigenetic changes occur, either in the DNA sequence itself (genetic) or other heritable modifications that affect the genome (epigenetic). These (epi)genetic changes alter cells from the normal hematopoietic system into cells capable of robust leukemic growth. There are a variety of leukemias, usually classified by the predominant pathologic cell types and/or the clinical course of the disease. It has been proposed that these are diseases in which self-renewing but poorly regulated cells, so-called "leukemia stem cells" (LSCs), are the populations that harbor all the genetic and epigenetic changes that allow leukemic progression.171176 While their progeny may be the characteristic cells observed with the leukemia, these progeny cells are not the self-renewing "malignant" cells of the disease. In this view, the events contributing to tumorigenic transformation, such as interrupted or decreased expression of "tumor suppressor" genes, loss of programmed death pathways, evasion of immune cells and macrophage surveillance mechanisms, retention of telomeres, and activation or amplification of self-renewal pathways, occur as single, rare events in the clonal progression to blast-crisis leukemia. As LT HSCs are the only selfrenewing cells in the myeloid pathway, it has been proposed that most, if not all, progression events occur at this level of differentiation, creating clonal cohorts of HSCs with increasing malignancy (see Figure 2.6). In this disease model, the final event, explosive selfrenewal, could occur at the level of HSC or at any of the known progenitors (see Figures 2.5 and 2.6). Activation of the -catenin/lef-tcf signal transduction and transcription pathway has been implicated in leukemic stem cell self-renewal in mouse AML and human CML.177 In both cases, the granulocyte-macrophage progenitors, not the HSCs or progeny blast cells, are the malignant self-renewing entities. In other models, such as the JunB-deficient tumors in mice and in chronic-phase CML in humans, the leukemic stem cell is the HSC itself.90,177 However, these HSCs still respond to regulatory signals, thus representing steps in the clonal progression toward blast crisis (see Figure 2.6).

Figure 2.6. Leukemic progression at the hematopoietic stem cell level. Self-renewing HSCs are the cells present long enough to accumulate the many activating events necessary for full transformation into tumorigenic cells. Under normal conditions, half of the offspring of HSC cell divisions would be expected to undergo differentiation, leaving the HSC pool stable in size. (A) (Pre) leukemic progression results in cohorts of HSCs with increasing malignant potential. The cells with the additional event (two events are illustrated, although more would be expected to occur) can outcompete less-transformed cells in the HSC pool if they divide faster (as suggested in the figure) or are more resistant to differentiation or apoptosis (cell death), two major exit routes from the HSC pool. (B) Normal HSCs differentiate into progenitors and mature cells; this is linked with limited proliferation (left). Partially transformed HSCs can still differentiate into progenitors and mature cells, but more cells are produced. Also, the types of mature cells that are produced may be skewed from the normal ratio. Fully transformed cells may be completely blocked in terminal differentiation, and large numbers of primitive blast cells, representing either HSCs or self-renewing, transformed progenitor cells, can be produced. While this sequence of events is true for some leukemias (e.g., AML), not all of the events occur in every leukemia. As with non-transformed cells, most leukemia cells (other than the leukemia stem cells) can retain the potential for (limited) differentiation.

Many methods have revealed contributing protooncogenes and lost tumor suppressors in myeloid leukemias. Now that LSCs can be isolated, researchers should eventually be able to assess the full sequence of events in HSC clones undergoing leukemic transformation. For example, early events, such as the AML/ETO translocation in AML or the BCR/ABL translocation in CML can remain present in normal HSCs in patients who are in remission (e.g., without detectable cancer).177,178 The isolation of LSCs should enable a much more focused attack on these cells, drawing on their known gene expression patterns, the mutant genes they possess, and the proteomic analysis of the pathways altered by the proto-oncogenic events.173,176,179 Thus, immune therapies for leukemia would become more realistic, and approaches to classify and isolate LSCs in blood could be applied to search for cancer stem cells in other tissues.180

After more than 50 years of research and clinical use, hematopoietic stem cells have become the best-studied stem cells and, more importantly, hematopoietic stem cells have seen widespread clinical use. Yet the study of HSCs remains active and continues to advance very rapidly. Fueled by new basic research and clinical discoveries, HSCs hold promise for such indications as treating autoimmunity, generating tolerance for solid organ transplants, and directing cancer therapy. However, many challenges remain. The availability of (matched) HSCs for all of the potential applications continues to be a major hurdle. Efficient expansion of HSCs in culture remains one of the major research goals. Future developments in genomics and proteomics, as well as in gene therapy, have the potential to widen the horizon for clinical application of hematopoietic stem cells even further.

Notes:

* Cellerant Therapeutics, 1531 Industrial Road, San Carlos, CA 94070. Current address: Department of Surgery, Arizona Health Sciences Center, 1501 N. Campbell Avenue, P.O. Box 245071, Tucson, AZ 857245071,e-mail: jdomen@surgery.arizona.edu.

** Section on Developmental and Stem Cell Biology, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215, E-mail: Amy_Wagers@harvard.edu

*** Director, Institute for Cancer/Stem Cell Biology and Medicine, Professor of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305, Irv@stanford.edu.

Chapter1|Table of Contents|Chapter3

See original here:
Bone Marrow (Hematopoietic) Stem Cells | stemcells.nih.gov

Cell therapies present unique challenges when complying with this paradigm for several reasons only two of which I will mention here.  Firstly, it is not possible to achieve the level of product purification as one might with other therapeutic products.  Secondly, the product characterization is at a cellular rather than molecular level.

Autologous cell therapies present another set of unique challenge in this paradigm because of the notable patient-to-patient variability where the patient is also the donor of the raw material.  This often means there is a wider tolerance of heterogeneity in the product but it still must be within what has been proven to the regulatory agency as a safe and effective range.  


In cases where an autologous cell therapy is centrally manufactured, they are most often subjected to product release testing similar to that described above.  One notable difference, particularly for fresh products, is that the products may be shipped to the clinic and even administered before the full panel of test results are obtained.  This wold be considered highly unusual (if ever acceptable) with other types of products but is tolerated because of the time-sensitivity of these products and their high safety profile.


In the case of autologous cell therapy products produced at the bedside there is often not the same kind of product release discipline.  Often the regulatory agencies deal with the product consistency and specification compliance issue by ensuring that the cell processing device used point-of-care is validated to ensure the cellular product output is always within a specified range shown to be clinically safe and effective.


The Varying Degree of Product Characterization/Specification of Autologous GTP Cell Therapy Products


However - and now I get to the point of this blog post - for cell-based products, procedures and/or devices/kits which are not mandated to be formally approved by a regulatory agency before they can be commercially marketed, there is no product specification rigor.  Compliance with the Good Tissue Practice regulations and guidance is deemed to ensure safety.  In the United States, cell-based products which are deemed to be "minimally manipulated" and intended for "homologous use" are typically allowed to go straight to market with no formal approval.  Safety and clinical data is not required but is practically necessary to support physician adoption and, where applicable, reimbursement.  


This means that for these products there is a great deal of variability in terms of how much rigor companies apply in characterizing their product and then ensuring that each batch complies with the specifications they themselves have determined to be safe and effective. Again, where such products are manufactured in a centralized facility the likelihood of some release testing is greater.  However, those companies relying on a point-of-care processing kit or device business model that has not been deemed to require formal market approval, rarely (if ever) include product release testing.


The common criticism of these companies is that they simply do not know what they are injecting into patients because of the combination of the patient-to-patient donor variability, the lack of any disciplined product characterization or dosing studies, and the absence of any product release testing.  


This criticism is not equally levied at all autologous GTP products or companies - even those relying on point-of-care processing.  Of course some companies care and do a lot to try to ensure their product is well-characterized and that each batch complies with product specifications. This may involve the use of product release tests but can also involve the combination of pre-market research into the product characterization, safety, and dosing along with validation of the device/kit output.  In this way a company can say that within a very small margin, the output will be within the product specifications the company knows is safe and efficacious.


However, in a rush to get their device/kit to market some companies appear to care very little about the cell product characterization, validation of the output of their device/kit, or tying this data to optimal dose.


More concerning are those companies that appear to provide such data but it is wrong or meaningless.  What follows appears to potentially be a case study of precisely this problem. 


The INCELL Study 


This week I came across a fascinating white paper from Incell Corporation analyzing the output of adipose tissue processing kits of MediVet-America apparently demonstrating the inaccuracy of their cell counts (a common type of cell therapy product characterization) and calling into the question the cell count claims of Intellicell Biosciences (New York, NY) and Adistem (Hong Kong).


At the heart of the critique is the claim that the cell counting (product characterization) techniques employed by these companies counts as cells things (namely acellular micelles) which are not cells.

I encourage you to read the white paper in its entirety.  They corresponding author told me to watch for one or more papers which they are preparing for submission to peer-reviewed publications shortly.  Presumably these will rely on a larger data set and perhaps test other methodologies or technologies.


For the purposes of this blog, I've pulled what I believe are the most salient excerpts below:

Intrigued by the high cell numbers  (5 to 20 million cells/gram)  reported by kit/device manufacturers such as MediVet-America (Lexington, KY), Intellicell  Biosciences (New York, NY), and Adistem, Ltd. (Hong Kong) in adipose stem cell therapy compared to other methods (e.g., 
Chung,Vidal, and Yoshimura), INCELL staff conducted a research study to  investigate the high apparent yield of stem cells.  This initial work was focused  on SVF cells from the MediVet Kit, which is marketed to isolate adiposederived canine SVF and stem cells.

The cell yields reported for the Medivet Kits are five to more than ten times higher than the yields routinely obtained by INCELL from freshly harvested human or animal adipose tissue using our adipose tissue processing methods.  These yields are also tenfold or higher than those reported in the literature by most academic researchers (Chung-canine, Vidal–equine, Yoshimura–human).  Since these  cell counts are used to support stem cell dosing recommendations and cell banking, it is important to better understand why the cell numbers are higher.

...

A comparative analytical study of three dog donors of adipose tissue was designed to evaluate the cell yields using the MediVet Kit as an example of this class of isolation system. All  kit procedures were followed as per the instructions provided.  A brief overview of the different cell counting methods used, and the resultant cell counts, observations and explanations of the results observed, are described below

....

This study shows that incorrect counting of adipose derived SVF cells and the subset of regenerative stem cells can subsequently result in inaccurate dosing, both in direct therapeutic applications and in cryostorage of cells for future use.  The DAPI-hemocytometer cell count (manual) was considered the most accurate, but there are various sources of technical difficulties that  can lead to incorrect  cell numbers. The nature of adipose tissue itself with variability in dissociation by enzymatic digestion can all contribute to the outcomes. Fat tissue has a propensity to form acellular micelles and oils upon tissue disruption. Processing methods or reagents (e.g., Solution E or lecithins) can generate micelles that may be  erroneously  counted as cells. Autofluorescence and dye trapping or uptake by the micelles can lead to very high inaccurate cell counts when automated cell counting is used. 

In this study the most inaccurate counting came from the Cellometer. When used according to kitrecommended guidelines and on-site training provided by Nexelcom for counting  cells by the MediVet procedure, the Cellometer overstated the DAPI-hemocytometer cell count by up to 20X or more. The Coulter Counter protocols also led to incorrect, high cell numbers. Although the cell counts were still a bit high, the authors recommend the NucleoCounter, or similar equipment, as more acceptable for automated counting.  The manual hemocytometer-DAPI method is the most accurate, but requires a highly experienced cell biologist or technician to make accurate counts and  is not suitable for routine clinical use. 

...

Other companies also have claims of very high cell numbers when their processes are used. Adistem, like MediVet, states they add an emulsifying agent to their kits to assist in cell release, and they also use a light activation system. Their kits were not tested in this study but it is possible that the high cell numbers reported by Adistem are also incorrect and result from the same problems highlighted in this paper for the MediVet procedure. Ultrasonic energy, which is commonly used to manufacture micellular  liposome  structures and to disrupt and lyse cells, is  another potentially problematic procedure for counting and verifying viable, regenerative cells.  Intellicell 3uses ultrasonic energy to release cells from adipose tissue, and it is possible that resultant micelles or cell fragments contribute to the higher than expected cell numbers.  This assumption could be verified with additional studies.  

In summary, the authors caution that great care must be taken when using kits and automated cell counting for stem cell dosing and cryobanking of cells intended for clinical use. Overestimated  cell numbers would be a major confounding source of variation when efficacy of stem cells injected are compared as doses based on cell number and when cryostored cells are aliquoted for use based on 

specific cell numbers as a treatment dose.  Hopefully, this study will lead to more  reproducible counting and processing methods being reported in the literature, more inter-study comparability of cell doses to clinical outcomes,  more industry diligence to support claims, and more accurate counting for dosing stem cell therapies to patients.

...

Chung D, Hayashi K, Toupadakis A, et al.  Osteogenic proliferation and differentiation of canine bone marrow and adipose tissue derived mesenchymal stromal cells and the influence of hypoxia.  Res Vet Sci, 2010; 92(1):66-75. Vidal MA, Kilroy GE, Lopez MJ, Johnson JR, Moore RM, Gimble JM. Characterization of equine adipose tissue-derived stromal cells: adipogenic and osteogenic capacity and comparison with bone marrow-derived mesenchymal stromal cells. Vet Surg, 2007; 36:613–622.  Yoshimura K, Shigeura T, Matsumoto D, et al:  Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirate.  J Cell Phys, 2006; 205:64-76.

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