Originally Authored by Jon A. Rowley, PhD, Founder (February 15, 2014), Updated by Iain Farrance, PhD Technical Marketing Associate (January 9, 2020)
What’s in a Name?
MSC is the traditional acronym used in describing Mesenchymal Stem Cells, Mesenchymal Stromal Cells, and even Medicinal Signaling Cells (1, 2). There is discussion in academic circles over which term(s) is appropriate to describe the plastic adherent cells, derived from bone marrow and other tissues, that can, among other things, differentiate down multiple tissue lineages. While there is debate on the technical name, there is agreement that the “MSC” acronym be maintained. Position papers from the ISCT MSC committee in 2005 and 2019 support the use of the acronyms “MSC” and “MSCs” but recommend that MSC be supplemented with the species and tissue of origin (3, 4). So, you will see human Bone Marrow MSCs (hBM-MSC), human adipose MSCs (hAD-MSC), and human umbilical cord MSCs (hUC-MSC) available from RoosterBio, Inc.
A Brief History of MSCs
Adult bone marrow contains at least two distinct populations of stem cells: Hematopoietic Stem Cells and a rare, plastic-adherent mesenchymal/stromal cell population, Mesenchymal Stromal Cells (MSC, 5, 6, 7, 8). In the bone marrow, MSCs participate in maintaining the blood-forming, or hematopoietic, stem cell niche (7). Ex vivo, MSCs grow as long, spindle-shaped cells with prominent nuclei and are capable of forming single-‐cell colonies on tissue culture plastic (5, 6, 7, 9). MSCs have the potential for self-renewal and are multipotent, i.e. they have the ability to differentiate to cells from a number of mesenchymal lineages including fat, bone, cartilage, and skeletal muscle (5, 9, 10, 11). It is this multipotent nature that lead Arnold Caplan in 1991 to propose the term “mesenchymal stem cells” (5). Since their initial discovery in bone marrow, MSCs have been found to reside in many tissues in the body, including fat, umbilical cord blood, dental pulp, and peripheral blood, to name a few.
To clearly define universal criteria to define an MSC, and to move the field forward, the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT) proposed a set of standards to define human MSCs for laboratory‐based scientific investigations and for pre-clinical studies (12, 13):
- MSCs must be plastic-adherent when maintained in standard culture conditions using tissue culture flasks.
- ≥95% of the MSC population must express CD105, CD73 and CD90, as measured by flow cytometry. Additionally, MSCs must lack expression (≤2% positive) of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA class II.
- MSCs must be capable of trilineage differentiation in vitroto osteoblasts (bone), adipocytes (fat), and chondrocytes (cartilage).
- MSCs must have immunomodulatory activity, e.g. T-cell suppression via induction of indoleamine 2,3-dioxygenase (IDO) activity by IFN-γ (±TNF-α).
Contemporary MSC Research and Development
While these criteria were helpful in establishing a framework, these historical criteria have not kept up with how these cells are used in applications (reviewed in 2). For example, trilineage differentiation is relevant to a minority of all therapeutic applications. Furthermore, flow marker expression has recently been seen as having little relevance to function. For example, CD markers can stay consistent over multiple passages while MSCs lose their differentiation capability (14) or immunomodulatory function (15). Current research shows that MSCs achieve their therapeutic effects by secreting a plethora of biomolecules that influence many biologic processes (2). Thus, MSC characterization, as it relates to therapeutics development is more focused on secreted biomolecules affecting tissue regeneration or modulating the immune system, and the most relevant cell characteristics applicable to therapeutics are very different from those characteristics in the early standardization literature (12, 15).
A growing MSC research and therapeutic area is MSC-derived extracellular vesicles (MSC-EV, 16, 17). MSC-EV can mediate paracrine effects by transfer from MSCs to target cells of various macromolecules, including non-secreted proteins, RNAs (e.g. mRNA & miRNA), metabolites, and membrane-associated factors. Importantly, EVs derived from MSCs benefit from MSCs’ well-defined safety profile and are on the rise as a novel clinical therapy for a broad range of applications.
Cell characterization under Good Manufacturing Practices (GMP) must not only confirm MSC identity (what the cell is), but must also specify cell potency (what the cell does). The US FDA defines potency tests as measures of appropriate biological activity, with respect to the clinical indication (15). Since it is hypothesized that many of the therapeutic functions of MSCs are derived from their secreted paracrine factors rather than from their ability to differentiate, relevant measures of biological function involve quantifying factors that MSCs secrete in culture supernatants, most commonly by ELISA. In addition, potency tests may measure an induced activity. For example, following product delivery of MSCs to a site of inflammation, their response may be the secretion of anti‐inflammatory and/or pro-angiogenic cytokines. Thus, measuring the secretion of VEGF by MSCs is relevant for angiogenic activity, and treatment of MSCs with IFN‑γ to stimulate the activity of IDO tests MSC immunomodulatory function. In summation, in vitro potency assays are likely a useful surrogate of in vivo potency for the development of cell‐based therapies and provide a much more clinically-relevant measure of cell function when compared to conventional MSC characterization methods (18). It is for this reason that RoosterBio characterizes each and every lot of hMSCs for a panel of angiogenic cytokines and induced IDO activity.
Therapeutic Use of MSCs
MSCs have been studied in a wide variety of clinical and therapeutic, including tissue regeneration and wound healing. Structural regeneration and functional improvements in damaged and diseased tissues upon therapeutic MSC application were initially attributed to cell engraftment and differentiation within target tissues. Recent results, however, have lead to widespread acceptance that most positive outcomes following MSC treatments are due to secreted biomolecules or MSC-EV affecting target cell survival and differentiation (2, 16, 19). In addition to MSC’s MOA being secretion of trophic factors, the benefits of an established safety profile and lack of engraftment allow for an off-the-shelf cell, administration of allogeneic MSC. Given their immense therapeutic potential, relative ease of isolation, and the lack of ethical concerns surrounding use of these cells, there have been greater than 900 clinical trials initiated (database purchased from celltrials.org), using hMSC from various sources across a host of indications and therapeutic strategies (20, 21). Results of clinical trials have thus far been encouraging with MSCs demonstrating an excellent safety profile. Thus, MSCs will continue to be key components of future therapeutic agents, engineered tissues, and medical devices with a “peak” demand years in the future (22).
Current Bottlenecks in MSC Research and the RoosterBio Solution
Despite their immense therapeutic potential, MSCs are very rare, comprising only 0.001%‐0.01% of the mononuclear cells in the bone marrow (9). Since a typical adult bone marrow aspirate contains many cell types and does not yield enough MSC for a clinical dose, in vitro expansion of hBM-MSC and other MSC types is necessary before use. However, prolonged culture of MSCs will cause senescence and loss of multilineage potential and immunosuppressive activity (23, 24). These challenges associated with prolonged MSC culture means that there is a significant need for methods to efficiently expand MSCs ex vivo. Therefore, extensive effort has been put into developing methods to expand MSCs while maintaining their functionality. Cell plating density, culture surfaces, and the addition of growth factor and cytokine supplements have proven to effectively modulate MSC growth and allow for the expansion of MSCs to clinically relevant lot sizes.
There are three major challenges that cell and tissue engineering product development efforts face today. These challenges are:
- The cost of today’s primary cells is prohibitively high,
- primary cells are not readily available at volumes that support product development efforts. Most cells are offered at less than one million cells per vial at an average cost of over $900 per million cells, and
- most product research is performed with cells produced using traditional small-scale processes that are not directly transferrable into a GMP setting – slowing the translation into First in Man studies, and eventual commercialization.
RoosterBio takes the Innovation Approach of simultaneously implementing a number of bioprocess scale‐up and streamlining improvements that together form a new cost basis for primary cells. Importantly, we do this without sacrificing product quality (i.e. we maintain MSC phenotype and function). RoosterBio’s Process Engineering innovation of well-characterized hMSC, from several tissues, paired with highly engineered media systems dramatically reduces the total labor hours for MSC culture and the cost per 1M cells. We get customers to the required cell numbers 3-4 times faster, at about 40-75% of the overall cost. Thus, the pace of product development accelerates, propelling the field into a new era of productivity, and opening up cell- and tissue-based technology development to a much broader market. RoosterBio’s research use products are produced with standardized, GMP-compatible and scalable manufacturing processes, facilitating RoosterBio’s customers translation into the clinic using our cGMP CliniControl™ hMSCs and media.
- Caplan AI (2017) Mesenchymal Stem Cells: Time to Change the Name! Stem cells translational medicine 6(6):1445-1451. https://www.ncbi.nlm.nih.gov/pubmed/28452204
- Pittenger MF, et al. (2019) Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen Med 4:22. https://www.ncbi.nlm.nih.gov/pubmed/31815001
- Horwitz EM, et al. (2005) Clarification of the nomenclature for MSC: The International Society for Cellular Therapy position statement. Cytotherapy 7(5):393-395. https://www.ncbi.nlm.nih.gov/pubmed/16236628
- Viswanathan S, et al. (2019) Mesenchymal stem versus stromal cells: International Society for Cell & Gene Therapy (ISCT) Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy 21(10):1019-1024. https://www.ncbi.nlm.nih.gov/pubmed/31526643
- Caplan AI (1991) Mesenchymal stem cells. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 9(5):641-650. https://www.ncbi.nlm.nih.gov/pubmed/1870029
- Friedenstein AJ, et al. (1970) The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 3(4):393-403. https://www.ncbi.nlm.nih.gov/pubmed/5523063
- Friedenstein AJ, et al. (1974) Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo. Transplantation 17(4):331-340. https://www.ncbi.nlm.nih.gov/pubmed/4150881
- Friedenstein AJ, et al. (1968) Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6(2):230-247. https://www.ncbi.nlm.nih.gov/pubmed/5654088
- Pittenger MF, et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143-147. http://www.ncbi.nlm.nih.gov/pubmed/10102814
- Caplan AI (1986) Molecular and cellular differentiation of muscle, cartilage, and bone in the developing limb. Prog Clin Biol Res 217B:307-318. https://www.ncbi.nlm.nih.gov/pubmed/3092248
- Wakitani S, et al. (1995) Myogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine. Muscle Nerve 18(12):1417-1426. https://www.ncbi.nlm.nih.gov/pubmed/7477065
- Dominici M, et al. (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315-317. http://www.ncbi.nlm.nih.gov/pubmed/16923606
- Krampera M, et al. (2013) Immunological characterization of multipotent mesenchymal stromal cells–The International Society for Cellular Therapy (ISCT) working proposal. Cytotherapy 15(9):1054-1061. http://www.ncbi.nlm.nih.gov/pubmed/23602578
- Lo Surdo JL, et al. (2013) Automated microscopy as a quantitative method to measure differences in adipogenic differentiation in preparations of human mesenchymal stromal cells. Cytotherapy 15(12):1527-1540. http://www.ncbi.nlm.nih.gov/pubmed/23992827
- Carmen J, et al. (2012) Developing assays to address identity, potency, purity and safety: cell characterization in cell therapy process development. Regenerative medicine 7(1):85-100. http://www.ncbi.nlm.nih.gov/pubmed/22168500
- Elahi FM, et al. (2019) Preclinical translation of exosomes derived from mesenchymal stem/stromal cells. Stem cells. https://www.ncbi.nlm.nih.gov/pubmed/31381842
- Thery C, et al. (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles 7(1):1535750. https://www.ncbi.nlm.nih.gov/pubmed/30637094
- Galipeau J, et al. (2016) International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials. Cytotherapy 18(2):151-159. https://www.ncbi.nlm.nih.gov/pubmed/26724220
- von Bahr L, et al. (2012) Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation. Stem cells 30(7):1575-1578. http://www.ncbi.nlm.nih.gov/pubmed/22553154
- Mendicino M, et al. (2014) MSC-based product characterization for clinical trials: an FDA perspective. Cell stem cell 14(2):141-145. http://www.ncbi.nlm.nih.gov/pubmed/24506881
- Moll G, et al. (2019) Intravascular Mesenchymal Stromal/Stem Cell Therapy Product Diversification: Time for New Clinical Guidelines. Trends Mol Med 25(2):149-163. https://www.ncbi.nlm.nih.gov/pubmed/30711482
- Olsen TR, et al. (2018) Peak MSC-Are We There Yet? Front Med (Lausanne) 5:178. https://www.ncbi.nlm.nih.gov/pubmed/29977893
- Binato R, et al. (2013) Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy. Cell Prolix 46(1):10-22. https://www.ncbi.nlm.nih.gov/pubmed/23163975
- Li XY, et al. (2012) Long-term culture in vitro impairs the immunosuppressive activity of mesenchymal stem cells on T cells. Mol Med Rep 6(5):1183-1189. https://www.ncbi.nlm.nih.gov/pubmed/22923041