Transfusion. Author manuscript; available in PMC 2019 Feb 6.
Published in final edited form as:
PMCID: PMC6364749
NIHMSID: NIHMS1005897
1Department of Transfusion Medicine, Post graduate Institute of Medical Education and Research, Chandgarh, India
2Department of Biomedical Engineering, University of Minnesota, Saint Paul, Minnesota
3Department of Mechanical Engineering, University of Minnesota, Saint Paul, Minnesota
4Department of Laboratory Medicine & Pathology, University of Minnesota, Saint Paul, Minnesota.
1Department of Transfusion Medicine, Post graduate Institute of Medical Education and Research, Chandgarh, India
2Department of Biomedical Engineering, University of Minnesota, Saint Paul, Minnesota
3Department of Mechanical Engineering, University of Minnesota, Saint Paul, Minnesota
4Department of Laboratory Medicine & Pathology, University of Minnesota, Saint Paul, Minnesota.
Mesenchymal stem cells (MSCs) have recently generated great interest in the fields of regenerative medicine and immunotherapy due to their unique biologic properties. In this review we attempt to provide an overview of the current clinical status of MSC therapy, primarily focusing on immunomodulatory and regenerative or tissue repair applications of MSCs. In addition, current manufacturing is reviewed with attention to variation in practices (e.g., starting material, approach to culture and product testing). There is considerable variation among the 218 clinical trials assessed here; variations include proposed mechanisms of action, optimal dosing strategy, and route of administration. To ensure the greatest likelihood of success in clinical trials as the field progresses, attention must be given to the optimization of MSC culture.
Cellular therapy has evolved quickly over the past decade with valuable experience gained in both preclinical research and clinical trials. Both embryonic and nonembryonic stem cells have been explored as potential therapeutic strategies for a number of diseases. One group of adult stem cells, mesenchymal stem or stromal cells (MSCs), has generated great interest in the fields of regenerative medicine and immunotherapy due to their unique biologic properties. MSCs were first discovered in 1968 by Friedenstein and colleagues1 as adherent fibroblast-like cells in the bone marrow (BM) capable of differentiating into bone. It was subsequently shown that MSCs could be isolated from various tissues such as BM, adipose tissue (AT),2 and umbilical cord blood (UCB).3 These cells can be expanded in vitro, which allows them to rapidly reach the desired cell counts for use in vivo. Using somewhat different strategies, several laboratories have identified, isolated, and cultured MSCs with specific properties.46
In an effort to better characterize MSCs, the International Society for Cellular Therapy defined MSCs by the following three criteria:7
MSCs must be adherent to plastic under standard tissue culture conditions;
MSCs must express certain cell surface markers such as CD73, CD90, and CD105, and lack expression of other markers including CD45, CD34, CD14, CD11b, CD79, or CD19 and HLA-DR surface molecules;
MSCs must have the capacity to differentiate into osteoblasts, adipocytes, and chondroblasts under defined in vitro conditions.
This definition is fairly nonspecific and does little to distinguish MSCs from the classical fibroblasts.8 In this review we attempt to provide an overview of the current clinical status of MSC therapy, primarily focusing on immunomodulatory and regenerative or tissue repair applications of MSCs. In addition, current manufacturing is reviewed with attention to variation in practices (e.g., starting material, approach to culture and product testing).
Based on current literature,9 it is thought that MSCs exert their therapeutic effects by several mechanisms including:
The ability to home to sites of inflammation after tissue injury;
The ability to differentiate into various cell types;
The ability to secrete multiple bioactive molecules capable of stimulating recovery of injured cells and inhibiting inflammation;
The lack of immunogenicity and the ability to perform immunomodulatory functions.
These four potential modes of therapeutic efficacy have been demonstrated in various preclinical animal model studies.10 However, this review focuses primarily on clinical applications of MSCs in humans.
The first clinical trial using culture-expanded MSCs was carried out in 1995; in this study, 15 hematooncology patients received injections of autologous (BM-MSCs) cells as part of a safety and feasibility study.11 Since then, the use of MSCs has been further explored. As of October 2012, the clinical trials database (http://www.clinicaltrials.gov) showed 218 clinical trials using MSCs for a wide range of therapeutic applications () internationally. Most of these trials are in Phase I (safety studies, n = 42), Phase II (proof of concept for efficacy in human patients, n = 57), or combined Phases I and II studies (n = 105). Only a small number of these trials are in Phase III (comparing a newer treatment to the standard or best known treatment, n = 8) or combined Phases II and III (n = 6). The disease conditions and phase of trials are listed in and their sources are summarized in . In general, MSCs appear to be well tolerated; most trials report a lack of any adverse effects except for mild or transient peri-injection effects.10 Encouraging results from these clinical trials have increased research into MSC therapy for a variety of clinical disorders such as acute myocardial infarction, stroke, liver cirrhosis, amyotrophic lateral sclerosis, graft-versus-host disease (GVHD), solid organ transplant rejection, and autoimmune disorders.
Summary of tissue sources for MSCs currently being used in clinical trials. BM is the most common source of MSCs (n = 121), followed by UCB (n = 37) and AT (n = 26).The Trademarked product category members are commercial products and do not disclose their cell sources (n = 25). Others include menstrual blood, placenta, and endometrial cells (n = 5).The not specified category members did not clearly state the source tissue used to isolate MSCs (n = 4).
Current status and enrollment of MSC clinical trials*
MSCs have unique immunologic characteristics, which promote their survival and growth in allogeneic or xenogeneic environments.12,13 They express very low levels of major histocompatibility complex (MHC) Class I antigens and do not express MHC Class II antigens or costimulatory molecules such as CD40, CD80, and CD86.14 These features protect them from alloreactive natural killer (NK) cellmediated lysis.15 In addition, human MSCs express HLA-G, a nonclassical MHC Class I antigen, which may prevent the immune response against MSCs (as shown by blocking experiments), although its expression seems to decrease in culture.16 Culture conditions may also affect MSC immunogenicity due to internalization of certain protein molecules of the culture medium.17 However, patients receiving treatment with allogeneic human MSCs did not show antiallogeneic MSC antibody production or T-cell priming.18 The precise mechanisms underlying MSC immunomodulation are still not fully understood, although direct cell-to-cell contact and/or release of soluble immunosuppressive factors may play major roles. MSCs can potentially interact with a wide range of immune cells, including T lymphocytes, B lymphocytes, NK cells, and dendritic cells. MSCs act on both the adaptive and the innate immune systems by suppressing T cells,17 suppressing dendritic cell maturation,19,20 reducing B-cell activation and proliferation,21,22 inhibiting proliferation and cytotoxicity of NK cells,23 and promoting the generation of regulatory T cells via an interleukin (IL)-10 mechanism.24,25 Secretion of prostaglandins and growth factors such as vascular endothelial growth factor, keratinocyte growth factor, and hepatocyte growth factors is also thought to influence immunomodulation and repair of various tissues.26
When influenced by inflammatory cytokines, MSCs are capable of migrating to inflamed tissues and modulating the local inflammatory reactions at two levels via their effects on both innate and adaptive immunity.24,27 One level occurs locally via the secretion of mediators that inhibit the proliferation of immune cells in the vicinity of MSCs. The second induces a systemic responseeither an anti-inflammatory Th-2 immune activation or in some instances, the generation of regulatory T cells. In addition, MSCs may recruit and support growth of local autologous stem cells inside the injured tissues, thus promoting cell survival and tissue repair.28
Clinical applications of MSCs are growing rapidly as research progresses and the wide range of MSC-dependent influences on the immune system are further delineated. summarizes the current cellcell interactions of MSCs with the immune system. Clinical-grade ex vivo expanded MSCs have been used to treat BM and organ transplant rejection and inflammatory and auto- and alloimmune diseases (such as systemic collagen abnormalities and GVHD).
Schematic representation of the interactions between MSCs and immune cells. After activation, MSCs secrete soluble mediatorssuch as nitric oxide (NO), prostaglandin (PGE2), indoleamine 2,3-dioxygenase (IDO), IL-6, IL-10, and human leukocyte antigen (HLA)-G. Production of these mediators regulates the proliferation and function of a variety of immune cells as well as the induction of regulatory T (TREG) cells either directly or indirectly through the generation of immature dendritic cells (DC).
The most significant results on the immunosuppressive effects of MSCs so far have been observed in the treatment of acute GVHD after allogeneic stem cell transplantation. The first case of ex vivo expanded haploidentical MSC infusion in a patient with severe Grade IV GVHD of the gut and liver resulted in a striking improvement of the disease.29 A Phase II study reported that 30 of 55 patients had a complete response and nine patients showed improvement indicating that irrespective of the donor, MSC infusion might be an effective therapy for patients with steroid-resistant acute GVHD.30 Since these studies were performed, several others have produced encouraging responses, both in acute and in chronic GVHD refractory to standard steroid treatment.3138 Recently, cotransplantation of MSCs3950 either from the same hematopoietic stem cell (HSC) donor or from a third party has shown rapid engraftment and less severe acute GVHD in most clinical trials. However, a higher incidence of relapse has been reported in a few.43 Cord blood unit cotransplantation or coculture expansion with MSCs has been shown to overcome the limitation posed by low cellularity of cord blood units for unrelated transplants in adults.44,51 The possibility of eliminating this obstacle in transplant would be a major accomplishment and this has opened new avenues of research for studying the properties of MSCs obtained from different sources.
Based on their ability to moderate T-cell proliferation and function, MSCs have also been proposed as a therapeutic option in the treatment of autoimmune diseases,52,53 renal transplantation rejection,54,55 and various immune-mediated neurodegenerative disorders.5660 The initial Phase I and II clinical trials are summarized in and have shown encouraging results to stimulate further research in these areas and the scope of their immunomodulatory and regenerative potential will further expand with better understanding of the underlying mechanism.
Summary of the immunomodulatory role of MSCs in clinical trials
MSCs have a unique characteristic of selectively homing to the sites of tissue injury and/or inflammation after systemic administration.27 Once located at an inflammation site, MSCs can exert local functional effects in the resident tissue.27,28 Ortiz and coworkers64 showed that murine MSCs home to the lung in response to injury, adopt an epithelium-like phenotype, and reduce inflammation in lung tissue of mice challenged with bleomycin. Cell migration is dependent on a multitude of signals ranging from growth factors to chemokines secreted by injured cells and/or respondent immune cells;65 migration of MSCs may also be regulated by such signals. Studies have demonstrated that MSC migration is influenced by a range of growth factors such as platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) and chemokines such as CCR2, CCR3, CCR4, or CCL5 as assessed by in vitro migration assays.66
Since the 1990s, the differentiation potential of MSCs has attracted much attention. Experimental data have demonstrated that MSCs can differentiate into mesodermal lineages such as bone, cartilage, adipocytes, and connective stromal cells.61 It has also been suggested that MSCs might be capable of differentiating into not only ectodermal lineage cells (e.g., neurons and epithelium), but also endodermal lineage cells (e.g., hepatocytes).6769 Although these results come from in vitro experiments, they provide exciting indications of how MSCs may differentiate in vivo. Regulated by the subtle microenvironment of local tissue, the differentiation of engrafted MSCs in vivo, of course, may be more complex and much remains unresolved in this regard.70
Based on current knowledge, when induced by a series of signals at the local tissue, engrafted MSCs appear to be capable of differentiating into at least three types of cells in vivo:
The molecular and environmental mechanisms that control MSC differentiation are not fully understood, and no unique phenotype marker has yet been associated with predictable differentiation potential of MSCs. There are currently several hypotheses to explain the differentiation potential for MSCs. For instance, Dennis and colleagues76 suggested that in MSCs, there are storage genes that can express and adjust differentiation into various lineages when exposed to different conditions. Phinney and Prockop28 proposed that MSCs are equipped with motor proteins and a proteolytic arsenal that enables them to interact with and respond to signals from the extracellular matrix and differentiate into unique structures such as muscle, bone, cartilage, or other connective tissues.
Recently, the trophic effects of MSCs have been identified to be of great significance in tissue regeneration. After engraftment, MSCs can contribute to tissue repair by secreting a number of trophic molecules that include soluble extracellular matrix glycoproteins (collagen types I and II, osteopontin), cytokines (transforming growth factor [TGF]-, IL-10, IL-6), and growth factors (vascular endothelial growth factor, hepatocyte growth factor, keratinocyte growth factor).75 These trophic molecules promote cellcell connections.77 It has been observed that these trophic molecules can not only reduce inflammation, apoptosis, and fibrosis of damaged tissues, but also stimulate tissue cell regeneration. Although there is evidence that MSCs and certain tissue cells such as cardiomyocytes can interact with one another via small-diameter nanotubes, the underlying mechanism of cellcell connection and its possible roles during tissue regeneration remains to be further investigated.77,78
Thus, in the acute phase of injury, MSC differentiation does not seem likely. However, MSCs do seem to play a role in regeneration via their trophic function.79 The regenerative role of MSCs in various disease conditions such as myocardial infarction,8086 ischemic cardiomyopathy,8790 end-stage liver disease,9194 peripheral vascular disease with ischemic ulcers,9598 neurologic stroke,99101 spinal cord injury,102 cartilage regeneration in degenerative arthritis,103105 intraosseous bone defects,106 and rare genetic disorders107 are summarized in .
Summary of the regenerative role of MSCs in clinical trials
An effective dose without adverse side effects has not yet been optimized and likely differs between diseases, route of administration, frequency of dosing, and other variables. Based on the review of currently applied doses in various clinical trials,2961,70,80107,111113 the clinical dose typically ranges from 0.5 106 to 5 106 MSCs/kg body weight of the recipient. Testing of high (8 106 MSCs/kg) as well as low doses (2 106 MSCs/kg) in patients with steroid-refractory acute GVHD37 did not reveal significant differences in response rate or relapse of the primary disease. Similarly, the MSC dose did not affect platelet (PLT) and neutrophil engraftment in post-BMT hematooncology patients.47 However, repeated infusion of MSCs at certain intervals seems to influence the outcome in some studies.30,32,33,36,38,53 Larger randomized trials are needed to determine therapeutic doses and dosing regimens for MSCs in various clinical settings.
With MSCs entering into the clinical arena, the development of production methods in accordance with current Good Manufacturing Practices (GMP) and current Good Tissue Practices is required in the United States. Similar regulations are in place in other countries around the world. Pamphilon and Szczepiorkowski114 and others115 have provided a thorough summary of these regulatory requirements.
MSCs have been derived from several tissue sources (BM, AT, and UCB) listed in and applied in both autologous and allogeneic settings. With evidence suggesting immune-privileged status, a single allogeneic MSC donor may serve for multiple recipients, raising the demand for well-characterized and even qualified donors.116 The screening and testing of donors for MSCs (e.g., health questionnaire, viral testing) is similar to that for other cell- or tissue-based products. The age of the donor seems to be important, with BM from children containing a higher concentration of colony-forming unit fibroblast precursors (CFU-Fs) than that from adults.117 Moreover, increased donor age seems to be directly correlated to detrimental effects in terms of proliferation and multipotency of MSCs.118 The donor should have no abnormalities or risk of abnormalities possibly involving MSCs, which may currently be difficult to assess. No specific regulatory requirement exists for this matter, but the issue should be considered carefully particularly when a single or few universal donors are used for many patients.
The majority of MSC clinical trials published to date (n = 121) have used BM as the source for the MSCs. BM is removed from the donors posterior superior iliac spine or crest using an Illinois needle, or equivalent aspiration needle, in a heparin-containing syringe.119 The sample is subsequently processed by density gradient centrifugation, direct plating, or different enrichment strategies.118 Numerous attempts to enrich MSCs from BM by other methods such as immunomagnetic-based depletion or enrichment strategies have been performed. Selection markers include STRO-1, CD49a, CD105, CD133, CD146, CD271, SSEA-4, antifebrin microbeads, aptamers, and aldehyde dehydrogenase activity.120123 However, no marker has proven capable of discriminating multipotent, highly proliferating MSCs from other less potent lineage-committed cells. Thus, the most common procedures for obtaining MSCs in clinical-scale numbers utilize density gradient centrifugation for isolation or direct plating to separate mesenchymal and hematopoietic cells by their adhesion to plastic cell culture surfaces.
Donor age, as mentioned earlier, and aspirate quality have been shown to influence MSC numbers.117,118 The frequency of MSCs is approximately 1 per 1 106 nucleated cells in adult BM and 1 per 1 104 nucleated cells in UCB.120124 The number of MSCs has been noted to decrease with age, with a 10-fold decrease from birth to teenage and another 10-fold decrease from teenage to elderly.121125
The discovery of multipotent MSCs within AT has established a second major source of MSCs (n = 26).126 Besides a comparable degree of mesodermal differentiation potential, AT-derived MSCs also appear to have higher frequencies (1001000 BM) and a high potential for angiogenesis or vasculogenesis compared to that of BM.127 In most cases, lipoaspirates have been used as starting material. Liposuction procedures may yield volumes ranging from milliliters to liters of tissue.128 The most commonly employed procedure, tumescent liposuction, involves the preprocedure infusion of saline solutions containing anesthetics and adrenaline as vasoconstrictors. This approach gives better cell yields than ultrasound-assisted liposuction, which has been shown to compromise recovery as well as expansion capacity of MSCs.129 For obtaining smaller volumes of tissue, machine and syringe aspiration as well as excision can be used instead.130 Further processing steps include removal of cellular debris, oil, excessive blood cells, proteins, and components of the extracellular matrix followed by extensive washing to obtain higher purity of the desired fraction.131 To isolate MSCs from the other tissues, enzymatic treatment is used. Subsequently, centrifugation is performed to remove the adipocyte fraction and pellet the preadipocyte stromal vascular fraction. This fraction is a heterogeneous mixture of cells, including MSCs as well as endothelial, muscle, fibroblastic and mast cells, pericytes, and preadipocytes. After the initial adherence step, all nonadherent cells are discarded by extensive washing, and the remaining adherent cells appear as fibroblastoid cells. These are cultured for approximately 10 days until a 60% to 70% confluent monolayer has developed. Cells can then be split to initiate subsequent culture passage.
To standardize the process, automated devices have been developed to assist in separation and culture. A bag within a bag device, composed of an inner mesh and an outer sealed bag, assists to separate the tissue fraction from the contaminating fluid fraction.132 A completely closed system (Celuton system, Cytori Therapeutics, San Diego, CA), which can be used at the patients bedside, performs the aspiration, washing, and concentration of the stromal vascular fraction.133 Cells resulting from this process, however, can only be regarded as enriched with MSCs. Only a proportion of approximately 1:1000 cells within the stromal vascular fraction will give rise to CFUs, equivalent to MSCs.5 Admittedly, most studies have used specimens obtained from young and healthy subjects undergoing aesthetic liposuction.To address the effects of age and comorbidity on stem cell frequencies, DiMuzio and Tulenko134 correlated factors such as advanced age (>70 years), obesity, renal failure, and vascular disease and found no significant differences.
Fresh (i.e., not frozen and thawed) UCB is the third common source for isolating MSCs for clinical use (n = 37). The standard process employed for obtaining UCB is gravity-assisted collection after cannulation of one of the umbilical veins (after delivery of the placenta) under aseptic conditions. This product is then typically processed within 24 hours of collection in a similar manner to BM. Various collection methods result in variable cell yield and viability of MSCs obtained; the success rate in isolating and further expanding MSCs depends on the volume of blood collected, the cell content, and the time between collection and processing,135 which highlights the need for minimal delay between delivery and harvesting. Related cell sources envisioned for clinical applications include neonatal tissues such as the amniotic membrane, the placenta, and Whartons jelly of the umbilical cord.136 These sources, like UCB, are of interest due to their relatively unlimited supply of more primitive MSCs with minimal ethical or legal concerns related to tissue sourcing.
The optimal basal medium for culturing MSCs has not yet been determined. Whereas some investigators favor using -minimum essential medium,136139 others favor Dulbeccos modified Eagles medium.61,135,140 The critical ingredient in MSC expansion medium seems to be serum as a source of nutrients, hormones, and growth factors.
Fetal bovine serum (FBS) has historically been considered essential for obtaining high-quantity and quality MSCs withexvivoexpansion.141 However, concerns for the use of FBS do exist and include risk of transfer of immunogenic xenoproteins as well as transmission of infectious agents, especially transmissible spongiform encephalopathy.142 Accordingly, the European Medicines Agency (EMEA) recommends that when manufacturers have a choice the use of materials from non transmissible spongiform encephalopathy relevant animal species or non-animal origin is preferred.143,144 If FBS is deemed necessary for culture, extensively tested FBS can be sourced from qualified herds (i.e., animals from countries considered free of risk of variant Creutzfeldt-Jakob disease). Interestingly, FBS-derived proteins have been shown to be internalized by MSCs145 and to be immunogenic, possibly compromising the clinical effectiveness of MSCs.17,146 Accordingly, many in the cell therapy community have already begun implementing non-FBS supplements for large-scale production of MSCs. However, the role of culture ingredients (including FBS) in maintaining MSC immunomodulatory and regenerative properties is still poorly understood, and thus it may be too premature to exclude FBS from MSC culture.
Although acceptable FBS batches are available and are being used for clinical-grade manufacturing of MSCs, the concern outlined above has paved the way for alternative supplements, including human-derived supplements. Of course, a completely chemically defined medium would be optimal for clinical-scale expansion,147 but this has yet to be achieved or implemented. Several working groups have tried to optimize culture media by adding human serum, plasma, or PLT-derived factors. Pooled human PLT lysate (obtained from buffy coatderived PLT-rich plasma) has growth factors and mitogens released from alpha granules of PLTs during PLT activation either by thrombin or by cell fragmentation during repeated freeze-thaw cycles. Among these potent mediators released from PLTs are epidermal growth factors, basic fibroblast growth factor, PDGFs, TGF-1, and IGF. These factors enhance proliferation of bone cells and chondrocytes, as well as MSCs, highlighting the role of PLTs in processes such as wound healing and tissue repair.148151 However, Marx and colleagues152 have observed that regenerative effects of PLT derivatives show extensive variation due to the dependence of growth factor concentration on PLT content, preparation method, white blood cell (WBC) contamination, and mechanisms of PLT growth factor release.153 Recent literature shows a definite advantage of PLT lysate over FBS with regard to MSC proliferation and cloning efficiency and a similar MSC immunophenotype.154 Thus, human PLT lysate may replace FBS in many cell culture systems previously thought to strictly depend on the presence of FBS due to better reproducibility of the lysate preparation protocol without considerable lot-to-lot variation.
Other alternatives to FBS include pooled human serum, blood group AB human serum, and PLT-derived factors, which have been developed by a variety of protocols.155 If an allogeneic source were to be used, large-scale clinical production involving pooled human blood derivatives may require several donors (i.e., to neutralize donor-specific variations and to mimic an off-the-shelf batch). Both blood group AB human serum and thrombin-activated PLT releasate in plasma compared to FBS have been found to be superior in expanding AT-MSCs. Some studies using allogeneic human serum have reported success in isolating and expanding MSCs from BM with preserved differentiation and immune-suppressive properties;156158 others have observed reduced growth associated with advanced senescence, concluding that autologous serum would be favorable.159,160
The growth factor requirements of MSCs have not been defined. However, some growth factors, such as PDGF, epidermal growth factor, TGF-, and IGF have been tested in culture.161,162 A variety of protocols describe adding fibroblast growth factors to FBS-supplemented medium for expanding MSCs to increase their proliferation rate and maintain multilineage differentiation potential.163 Others indicate that factors like dexamethasone164 or lithium, which both stimulate Wnt signaling, can enhance proliferation of MSCs.165
Oxidative stress can impair MSC qualities. Enhancing the concentration of selenium or selenite has been shown to reduce cell damage induced by reactive oxygen species.166 Likewise, caloric restriction mimicked in vitro by lowering the glucose content has been shown to accelerate MSC proliferation while preventing senescence.167 Contradicting these results, telomerase-immortalized MSCs respond to higher glucose concentrations with enhanced proliferation and osteogenic differentiation.168 Finally, Sotiropoulou and colleagues139 indicate that using astabilized dipeptide form of L-glutamine (GlutaMAX, Life Technologies, Carlsbad, CA) supports better cell growth compared to using L-glutamine.
Plating density has emerged as a critical issue for MSC expansion. Due to the adherent nature of MSCs, plating density is an important variable to ensure a good expansion rate and to maintain necessary cellular functions. The initial mononuclear cell (MNC) plating density is extremely variable; published clinical trials3061,70,80107,111113 have involved both high densities (e.g., 1.70 105 MNCs/cm2) and lower densities (e.g., 5.0 104 MNCs/cm2). On subsequent passages, the plating density should be decreased.48 The choice of cell density remains critical at this stage, and use of a low (e.g., 1.0 103-5.0 103 MSCs/cm2) or very low (e.g., 1.0 101-5.0 101 MSCs/cm2) plating density may better maintain a high proliferation rate and multipotentiality of MSCs.169
A consequence of seeding density and length of culture is the proliferative age of MSCs. MSCs have a restricted lifespan and reach a senescent state in which cellular functions become diminished and the risk for acquiring mutations116,170,171 and inflammatory phenotype increases, making them unfit for therapeutic use. Passage numbers are most commonly used to represent proliferative age; however, passage numbers (in contrast to population doublings) do not describe the critical de facto proliferation history when optimal or maximum length of culture is not yet well defined.171 Closed-system bioreactors may have limitations to scale-up due to size or culture capacity.
MSCs grow as adherent cells until reaching confluency and are then further expanded by serial passaging. Therefore, the number of cells that can be harvested in an ex vivo expansion culture is determined by the surface area of the culture platform. Typically MSCs are cultivated in conventional monolayer cultures. To achieve a large surface area, multilayered cell factories are used.172,173 This approach is labor-intensive and cost-consuming. Alternatively, it is possible to expand MSCs by using bioreactors.174,175 As closed systems should be preferred in a GMP setting, Rojewski and colleagues176 report a fully automated bioreactor allowing GMP-compliant manufacturing.
It has been shown that the most primitive stem cells proliferate and maintain stemness under low O2 concentration (e.g., 5%), which is closer to physiologic values.177 Low O2 conditions limit oxidative damage and, thus, may reduce cytogenetic abnormalities.178 Most MSC trials have not involved cells expanded under low O2 culture conditions. However, a trial involving ischemia-tolerant MSCs for treatment of lung injury is in the planning stage.179
MSCs for clinical use are most commonly frozen in 10% dimethyl sulfoxide within an electrolyte solution (e.g., PlasmaLyteA) and a protein source (e.g., human serum albumin). The freezing rate is typically 1C/min through phase change, followed by 2 to 3C/min until roughly 100C, at which point the cells are placed in liquid nitrogen or vapor-phase liquid nitrogen. This procedure is based on cryopreservation of HSCs and lymphoid cells and is not optimized for MSCs. MSCs in the frozen state can be transported in liquid nitrogen dry shippers (or equivalent).180
Quality control testing usually includes viability, immunophenotyping, sterility and mycoplasma testing, and endotoxin level. Viability can be assessed by a variety of assays, including trypan blue, acridine orange-propidium iodide, and 7-aminoactinomycin-D with an accepted specification of at least 70%. Immunophenotyping typically follows the International Society for Cellular Therapy criteria,7 which includes CD73, CD90, and CD105 as positive markers. Samples for sterility and mycoplasma testing are drawn at various time points in manufacturing, such as before culture (i.e., from starting material), during culture, and after culture and before freeze. Automated methods are often used for sterility (bacterial and fungal culture), and several approaches to mycoplasma testing exist (e.g., culture, polymerase chain reaction). Endotoxin content can be evaluated several ways but most often involves limulus amebocyte lysatebased method (chromogenic, turbidometric, etc.). The upper limit for endotoxin is 5 EU/kg/dose for most modes of administration.181
The determination of assays of function or potency may be guided by the presumed mechanism of action. Several general and mechanism-specific examples are highlighted below.
The standard pathways of MSC differentiation follow osteogenic, chondrogenic, and adipogenic lineages and have been elaborately reported in a large number of publications.61,131,163 This potency assay should be performed if mesenchymal (connective) tissue repair is intended. The lack of evidence of their true biologic role in vivo becomes the limitation of this assay as the hallmark for stem cell characteristics of self-renewal and differentiation has been not accomplished so far.
Human MSC surface molecules such as HLA Class I,Thy-1 (CD90), vascular cell adhesion molecule (CD106), intercellular adhesion molecule-1 and 2, activatedWBC adhesion molecule (ALCAM, CD166), lymphocyte functional antigen-3, and various integrins indicate interaction with cognate ligands on T cells.182 In contrast to an expected induction of T-cell response against allogeneic MSCs, T-cell alloreactivity is inhibited by MSCs in mixed lymphocyte cultures or lymphocyte proliferation induced by mitogens, such as phytohemagglutinin or concanavalin A, and are currently accepted in vitro assays to assess inhibitory effect of MSCs on T-cell proliferation.108,182 Some studies have shown that although MSCs in high concentrations (1040 MSCs per 100 responder lymphocytes) inhibit, low MSC concentrations (1 MSC per responder lymphocyte) may stimulate lymphocyte proliferation in mixed lymphocyte cultures.113 These findings stress the importance of determining an optimal MSC dose to achieve intended outcomes.
The potential benefit of cotransplantation of MSCs with HSCs has been demonstrated.39 This effect can be assayed in vitro in coculture experiments using HSCs and MSCs and thus may be a relevant functional or potency assay for this therapeutic indication.124
MSCs have limited lifespan in vitro and enter senescence after multiple passages (2530 population doublings) in culture. Therefore, for clinical use of MSCs, genomic stability is a major concern during long-term cultures as there is always a risk of cell transformation due to replicative senescence. Cells begin to show telomere shortening, lose a part of their differentiation potential, and exhibit an altered cytokine secretion profile.183,184
All these changes appear to be a continuous process and are hypothesized to start as early as the first passage. However, so far the evidence favoring transformation is low.171 Karyotyping and comparative genomic hybridization have low sensitivity to detect these abnormalities and do not appear to be relevant controls, neither indicating the real risk of MSC transformation or their senescent status. Thus, real relevant controls for transformation and senescence could refer to genes or molecules involved in the senescence and transformation pathways, such as p53, p21, and p16 and may ensure the safety of the product.185
The CFU assay is a suitable tool for evaluating the self-renewal capacity of cells. Friedenstein and colleagues186 were the first to describe an assay system to study CFU-F in various hematopoietic cell populations. Analysis of CFU-Fs frequency in BM aspirates requires adequate dilution, minimal manipulation, and low seeding density to get true colony counts in the sample as has been observed previously.123,187
Continue reading here:
Mesenchymal stem or stromal cells: a review of clinical applications ...
- Mesenchymal stem cell based therapies for uveitis: a systematic review of preclinical studies | Eye - Nature.com - April 10th, 2024
- Development of exosome therapy to treat inflammatory bowel disease by VesiCURE Therapeutics - StreetInsider.com - April 10th, 2024
- Adipose Derived Stem Cell Therapy Market Will Show the Highest Growth Rates, Incredible Demand by 2031 - WhaTech - April 3rd, 2024
- Injectable microspheres adhering to the cartilage matrix promote rapid reconstruction of partial-thickness cartilage defects - ScienceDirect.com - March 30th, 2024
- Safety and Potential Effect of Intrauterine Infusion of Autologous Adipose Tissue-Derived Regenerative Cells in ... - Cureus - March 30th, 2024
- Instructing iPS cell-derived mesenchymal stem cells to inhibit abnormal bone formation in FOP - Medical Xpress - March 28th, 2024
- Clinical application of mesenchymal stem cell in regenerative medicine ... - March 22nd, 2024
- Mesenchymal Stem Cells: The Past Present and Future - March 22nd, 2024
- CytoMed Diversifies into Regenerative Medicine after Research Collaboration with Singapore's Sengkang General ... - Yahoo Finance - March 20th, 2024
- Overexpression of Wnt5a promoted the protective effect of mesenchymal stem cells on Lipopolysaccharide-induced ... - BMC Infectious Diseases - March 20th, 2024
- Mesenchymal Stem Cells: What We Have Learned and How to Manage Them - MDPI - March 17th, 2024
- Mesenchymal stem cells and their microenvironment - March 17th, 2024
- Mesenchymal stem/stromal cells as a valuable source for the treatment ... - March 17th, 2024
- Proteomic characterization of hUC-MSC extracellular vesicles and evaluation of its therapeutic potential to treat ... - Nature.com - March 13th, 2024
- The impact understanding of exosome therapy in COVID-19 and preparations for the future approaches in dealing with ... - Nature.com - March 13th, 2024
- CytoMed Therapeutics Limited Announces Research Collaboration with Singapore Sengkang General Hospital to ... - Yahoo Finance UK - March 7th, 2024
- CytoMed Therapeutics Limited Announces Research Collaboration with Singapore Sengkang General Hospital to ... - Yahoo Finance - March 6th, 2024
- Combatting osteoarthritis with cartilage replacement therapy - Drug Target Review - March 3rd, 2024
- Ethical considerations in Stem Cell therapy for ALS - Cyprus Mail - February 27th, 2024
- Therapeutic Solutions International Files Patent on Facilitating Effects of JadiCells on Gene Therapy Mediated Cell ... - Business Wire - February 27th, 2024
- Stem Cell Therapy for Ulcerative Colitis - Health Central - February 27th, 2024
- Expanding the Horizons of Cell and Gene Therapy - RegMedNet - February 24th, 2024
- Early differentiation of mesenchymal stem cells is reflected in their dielectrophoretic behavior | Scientific Reports - Nature.com - February 22nd, 2024
- Orthopaedics Department's Regenerative Research Highlighted at ORS Annual Meeting - InventUM - University of Miami - February 20th, 2024
- Cellular uptake and in vivo distribution of mesenchymal-stem-cell-derived extracellular vesicles are protein corona ... - Nature.com - February 18th, 2024
- New University spin-out developing novel adult stem cell-based Therapies - News - University of Liverpool - News - February 15th, 2024
- Examining the potential of the common bovine as a potential therapeutic research model - Medical Xpress - February 15th, 2024
- Intentional Interference: Genetic Engineering Medium Aids The Transfection Of MSCs With siRNA - BioProcess Online - February 15th, 2024
- Cellular senescence is associated with osteonecrosis of the femoral head while mesenchymal stem cell conditioned ... - Nature.com - February 9th, 2024
- Stem Cell Therapy for Crohns Disease Shows Promising Results - RegMedNet - February 9th, 2024
- Targeted transcriptomic analysis of synovial tissues from horses with septic arthritis treated with immune-activated ... - American Veterinary Medical... - February 7th, 2024
- Mesenchymal Stem Cell Immunomodulation: Mechanisms and ... - Cell Press - February 7th, 2024
- New Cell Therapy for ARDS: A Groundbreaking Development and Other Respiratory Health Breakthroughs - Medriva - February 7th, 2024
- Effective treatment of optic neuropathies by intraocular delivery of MSC-sEVs through augmenting the G-CSF ... - pnas.org - January 31st, 2024
- Global Stem Cell Therapy Industry Outlook to 2028, Driven by Therapeutic Innovations and Clinical Advancements ... - Yahoo Finance - January 29th, 2024
- Out-of-this-world study will test how stem cells function in space - YP - January 29th, 2024
- The Applications of Cell Therapy - Technology Networks - January 26th, 2024
- STEM CELL THERAPY FOR MS COST: WHAT YOU NEED TO KNOW? - Island Echo - January 24th, 2024
- Understanding Neuromyelitis Optica: Role of NF-B and Therapeutic Potential - Medriva - January 20th, 2024
- Faculty member Arnold Caplan passes away The Daily The Daily - The Daily | Case Western Reserve University - January 18th, 2024
- Effects of fine particulate matter on bone marrow-conserved hematopoietic and mesenchymal stem cells: a systematic ... - Nature.com - January 11th, 2024
- Expression of HLA-DR by mesenchymal stromal cells in the platelet lysate era: an obsolete release criterion for MSCs ... - Journal of Translational... - January 11th, 2024
- NurOwn and its exosomes for ALS given patents in Europe, elsewhere - ALS News Today - January 8th, 2024
- Single-cell analysis reveals the stromal dynamics and tumor-specific characteristics in the microenvironment of ovarian ... - Nature.com - January 8th, 2024
- First in Class Combination Stem Cell and Exosome Therapy to Treat Pulmonary Fibrosis in Long Haul Covid Patients ... - Yahoo Finance - January 4th, 2024
- Choosing the Right Excipients for MSC and iPSC Therapies - Pharmaceutical Technology Magazine - January 4th, 2024
- Mitochondria Transplantation Therapy It's Farther Along Than You Think - BioProcess Online - January 2nd, 2024
- Embryonic-stem-cell-derived mesenchymal stem cells relieve experimental contact urticaria by regulating the functions ... - Nature.com - December 21st, 2023
- Type I collagen and fibromodulin enhance the tenogenic phenotype of hASCs and their potential for tendon ... - Nature.com - December 19th, 2023
- Could the 'central dogma' of biology be misleading bioengineers? - Phys.org - December 13th, 2023
- The Risks of Stem Cell and Exosome Treatments for Long COVID: A Call for Stricter Regulations - Medriva - December 13th, 2023
- Mesenchymal stem cells from biology to therapy - PMC - December 11th, 2023
- The Safety and Efficacy of Human Umbilical Cord-Derived ... - Cureus - December 5th, 2023
- The pivotal role of Nrf2 signal axis in IDD | JIR - Dove Medical Press - December 5th, 2023
- Futuristic Novel Therapeutic Approaches in the Treatment of ... - Cureus - December 3rd, 2023
- Cartilage's Contribution in Otology: A Comprehensive Review of Its ... - Cureus - December 3rd, 2023
- Comparative evaluation of 3D-printed and conventional implants in ... - Nature.com - November 29th, 2023
- Expression of E-cadherin and N-cadherin in Epithelial-to ... - Cureus - November 29th, 2023
- Enhancing the immunosuppressive properties of human umbilical cord mesenchymal stem cells - Phys.org - November 29th, 2023
- 'Great Potential' in Stem Cell Therapies for Knee Osteoarthritis ... - Pain News Network - November 25th, 2023
- Dietary Sources, Bioavailability, and Functions of Ascorbic Acid ... - Cureus - November 25th, 2023
- FY 2024 VA-Funded Projects - VA's Office of Research and Development - November 25th, 2023
- Real-world data suggest effectiveness of the allogeneic ... - Journal of Translational Medicine - November 23rd, 2023
- Mesenchymal Stem Cells: Characteristics, Function, and Application - November 23rd, 2023
- Combined application of therapeutic viruses and nanomaterials to ... - News-Medical.Net - November 23rd, 2023
- Progress in the Development of Stem Cell-Derived Cell-Free ... - Dove Medical Press - November 23rd, 2023
- Improving the therapeutic efficacy of oncolytic viruses for cancer ... - Journal of Translational Medicine - November 23rd, 2023
- 11.22.23 -- How To Lose A Batch In 10 Days - BioProcess Online - November 23rd, 2023
- The Pros and Cons of Mesenchymal Stem Cell-Based Therapies - November 19th, 2023
- Leptin's influence on MMP-1 expression | JIR - Dove Medical Press - November 15th, 2023
- TWIST1 and TSG6 are coordinately regulated and function as ... - Science - November 11th, 2023
- Association between severe acute malnutrition in childhood and ... - BioMed Central - November 11th, 2023
- Global Market for Treatments for Syndromes of Progressive Ataxia and Weakness Disorders - Yahoo Finance - November 11th, 2023
- Stem Cell Restore Reviews - Does It Work? Real Green Valley ... - The Daily World - November 11th, 2023
- Biological and genetic characterization of a newly established ... - Nature.com - November 11th, 2023
- Placental Stem Cell Collection and Storage Industry Scope ... - Argyle Report - November 9th, 2023
- In Vitro Evaluation of Light-Induced Cytotoxic Property: Synergistic ... - Cureus - November 9th, 2023
- UF professor launches nonprofit to equalize cell-based therapy access - Mainstreet Daily News Gainesville - November 7th, 2023
- Who are the leading innovators in cell immunomodulation for the ... - Pharmaceutical Technology - November 7th, 2023
- The Association of Vitamin D, Growth/Differentiation Factor 5 (GDF-5 ... - Cureus - November 7th, 2023
Recent Comments