What are MSCs - RoosterBio Inc

What are MSCs

What is in a Name?

Adherent bone marrow­‐derived cells that differentiate down various tissue lineages have traditionally been called Mesenchymal Stem Cells, or MSCs. There is great debate in academic circles whether this is an appropriate term, and alternative names such as Multipotent Stromal Cells, Marrow Stromal Cells, and even Multi-factor Secretory Cells have been proposed. While there is debate on the technical name, there is agreement that the “MSC” acronym be maintained. In March of 2013 a workshop was conducted at the National Institutes of Health (NIH) in Bethesda, MD on "Working Together on MSC Standards: A UK/USA/Canada Led Workshop at the NIH" that RoosterBio was lucky enough to be invited to. We are proactively adopting some of the discussed best practices in nomenclature suggested at the NIH MSC meeting: Species-­Source-­Cell, and simply referring to the cells by the “MSC” acronym until there is an agreement on the exact terminology. Thus, you will see human Bone Marrow MSCs, or hBM-­MSC, for sale in our catalog.

A Brief History of MSCs

In the 1950s, it was discovered that the adult bone marrow contained at least two distinct populations of stem cells: Hematopoietic Stem Cells and a rare, plastic‐ adherent stromal cell population, Mesenchymal Stromal Cells (MSCs)1,2. In the bone marrow, MSCs participate in maintaining the blood-­forming, or hematopoietic, niche, and thus, they are implicated in organ homeostasis, wound healing, and aging3. Ex vivo, MSCs grow as long, spindle-­shaped cells with prominent nuclei and are capable of forming single-‐cell colonies on tissue culture plastic4,5. 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 muscle both in vitro and in vivo4-‐7. Thus, these cells are also referred to as Mesenchymal Stem Cells and Mulitpotent Stem/Stromal Cells.

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. In an attempt 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 proposed a set of standards to define human MSCs for laboratory-‐based scientific investigations and for pre-­‐clinical studies8. Three major criteria were established for MSCs in terms of their plastic adherence, surface antigen expression, and multilineage potential. First, MSCs must be plastic-­adherent when maintained in standard culture conditions using tissue culture flasks. Second, ≥95% of the MSC population must express CD105, CD73 and CD90, as measured by flow cytometry. Additionally, these cells must lack expression (≤2% positive) of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA class II. Third, the cells must be able to differentiate to osteoblasts (bone), adipocytes (fat), and chondrocytes (cartilage) under standard in vitro differentiation conditions.

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 application. Tri-­‐lineage differentiation is relevant to less than 20% of all therapeutic applications. Furthermore, flow marker expression has recently been seen as having little relevance to function, and has been referred to as the MSC “identity crisis”. For example, CD markers can stay consistent over multiple passages while MSCs lose their differentiation capability9 or immunomodulatory function10. CD markers can be expressed at lower level when culturing in the presence of growth factors such as bFGF, but regained when cells are cultured in the traditional serum-­‐containing media11. Thus, MSC characterization as it relates to therapeutics development is more focused on cytokine secretion, induction of angiogenesis, influencing 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 literature10.

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 indication10. 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 indolamine-pyrrole 2,3­dioxygenase (IDO) tests MSC immunomodulatory function. In summation, in vitro potency assays could be 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. It is for this reason that RoosterBio characterizes each and every lot of hBM-MSCs for a panel of angiogenic cytokines and induced IDO activity.

Therapeutic Use of MSCs

MSCs have been studied in a number of tissue regeneration and wound healing applications. 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. A more recent paradigm shift, however, has lead to widespread acceptance that positive outcomes following MSC (and most stem cell) transplantation are as much a function of the plethora of biomolecules secreted by these cells as a function of cell survival and differentiation8,11. In addition to the secretion of trophic factors, MSCs possess the ability to modulate the body’s immune response when transplanted alone or with another cell population, thereby providing for their off-the-shelf cell administration13,14. Given their immense therapeutic potential, relative ease of isolation, and the lack of ethical concerns surrounding use of these cells, it is no wonder that the bulk of increases in novel clinical trials since 2006 has been due to MSCs14. There are currently over 400 novel global clinical trials investigating MSCs as therapies across a host of diseases15, 16. Results of clinical trials have thus far been encouraging with both allogeneic and autologous MSCs demonstrating excellent safety profiles17. Thus, MSCs will continue to be key components of future therapeutic agents, engineered tissues, and medical devices.

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 marrow5. Since a typical adult bone marrow aspirate yields very few MSCs (roughly 1 out of every 104 cells)18, prolonged in vitro expansion is typically necessary before clinical use. However, MSCs will often senesce (i.e. stop growing) in culture before adequate cell numbers for transplantation (~109 cells) can be obtained19,21. In addition, prolonged in vitro culture of MSCs has been shown to diminish their multilineage potential and impair their immunosuppressive activity19, 22. The aforementioned challenges associated with MSC culture currently limit their therapeutic potential, and a significant need remains for methods to efficiently expand multipotent MSCs ex vivo. Therefore, extensive effort has been put into developing methods to expand MSCs while maintaining their differentiation potential and paracrine activity. Cell plating density, culture surfaces, and the addition of growth factor supplements have all been investigated. Of these variables, the use of growth factor and cytokine supplements has proven to effectively modulate MSC growth and self-­renewal12.

There are three major challenges that cell and tissue engineering technology efforts face today. These challenges are:

  1. The cost of today’s primary cells is prohibitively high,
  2. 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
  3. 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 is taking 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 paired with our proprietary high‐productivity media drastically reduces total labor hours for MSC culture and dramatically reduces the cost per cell. We can get customers to biomass needs 3-­4 times faster, at about 40-­75% of the overall cost. Thus, the pace of product development should accelerate, 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 products will be produced with GMP-­compatible and scalable manufacturing processes, with standardized procedures and under solid Quality Systems. Thus, RoosterBio will offer its customers ease of translation into first-­in-­man studies once they are ready.

References

  1. National Institutes of Health Stem Cell Primer; 2009.
  2. Friedenstein AJ, et al. Cell Tissue Kinet. 3:393-403; 1970.
  3. Friedenstein AJ, et al. Transplantation. 17:331-340; 1974.
  4. Caplan AI. Prog Clin Biol Res. 217B: 307-318; 1986.
  5. Pittenger MF, et al. Science. 284:143-­147; 1999.
  6. Caplan AI. J Orthop Res. 9:641– 650; 1991. 7. Wakitani S, et al. Muscle Nerve. 18:1417–1426; 1995.
  7. Dominici M, et al. Cytotherapy. 8(4):315-­‐317; 2006.
  8. LoSurdo JL, et al. Cytotherapy, 15(12):1527-1540; 2013.
  9. Carmen J, et al. Regen Med. 7(1):85‐100; 2012.
  10. von Bahr L, et al. Stem Cells. 30(7):1575-­1578; 2012.
  11. Gharibi B and Hughes FJ. Stem Cells Trans Med. 1:771-­782; 2012.
  12. Baraniak PR and McDevitt TC. Regen Med. 5(1):121–143; 2010.
  13. Bernardo ME and Fibbe WE. Cell Stem Cell. 13(4):392‐402; 2013.
  14. US Food and Drug Administration: www.clinicaltrials.gov
  15. Li MD, et al. Regen Med. 9(1); 2014. doi:10.2217/RME.13.80
  16. Lalu MM, et al. PLoS One. 7(10):e47559; 2012.
  17. Warnke PH, et al. J Craniomaxillofac Surg. 41:153-­161; 2013.
  18. Li XY, et al. Mol Med Rep. 6:1183‐1189; 2012.
  19. Crisostomo PR, et al. Shock. 26:575-­580; 2006.
  20. Wagner W, et al. PLoS One. 3:e2213; 2008.
  21. Binato R et al. Cell Prolif. 46(1):10­‐22; 2013.