Steven D. Garner First
Published: 17 January 2018
Key Clinical Message
Mesenchymal Stem Cells (MSC) in dogs are easily obtained from many tissues of the body in the bedside clinical setting and are poised to revolutionize how veterinary medicine is practiced. MSC are a powerful, safe and effective mode of regenerative therapy that has exploded on the scene of medicine. This clinical report is designed to give the clinical practitioner a working vocabulary and knowledge of this new mode of therapy.
Stem cell science has evolved rapidly creating its own language and methods along the way. As such, stem cell literature can be daunting even to the most up to date scientist. Mesenchymal stem cells of bone marrow origin have been used for therapy since the first trials in humans in 1995, nevertheless, adipose derived stem cells such as those used in many clinical applications today were not discovered until 2001 . The past 15 years have had huge advances in knowledge of the therapeutic advantages that stem cell therapy offers. This brief description hopes to bring the medical professional up to speed in the opportunities offered by stem cell therapy. Mesenchymal Stem Cells can be derived from many tissues including bone marrow, adipose tissue, umbilical cord, blood, nasal tissue, tooth pulp and even urine. Mesenchymal Stem Cells or Multipotent Stromal Cells, (MSCs) are a group of fibroblast-like self-renewing, non-hematopoietic, multipotent progenitor cells, and are ontogenically derived from the embryonic layer of the mesoderm.
Identification of MSCs
These cells cannot be distinguished from fibroblasts based on microscopic morphology alone. Without advanced techniques for cell surface molecular identification, the identity of these cells remained obscure until relatively recently. The International Society for Cellular Therapy has suggested 3 minimum physical criteria to define MSC:
- They are plastic-adherent – MSCs stick to plastic laboratory dishes! This characteristic allows for an easy first step in the separation of MSCs from other tissue cells. This characteristic alone brings stem cell therapy and culture to the bedside of the patient by enabling culture and rapid access of the patient to cellular therapy.
- MSCs must present a certain surface molecule profile. The cluster of differentiation (also known as cluster of designation or classification determinant and often abbreviated as CD) represents the designation of a protocol for the identification of cell surface molecules. There are over 370 different CDs recognized on human cells. When a cell has a marker present it is designated with a (+) and when the marker is not present it is designated as a (-). The function of these cell surface molecules helps indicate the function of the cell and the CD designation indicates which genes are activated and being expressed on the surface of the cell. Two commonly used CD molecules are CD4+ and CD8+, which are, in general, used as markers for helper and cytotoxic T cells, respectively. Stem cells are identified as having CD34 but not CD31 markers; so, they would be denoted as CD34+ and CD31-. In addition, Stem cells have CD117+ (stem cell growth factor). CD 34 is an adhesion factor necessary for stem cell function and CD31 is found on platelets and white blood cells and mature endothelial cells and would not be present on a stem cell. Identification of stem cells is essential prior to therapy. Newly developed fluorescent immunocytochemical stains are available for us to perform CD characterization in our laboratory so that we are certain of the cell type and qualities used for therapy. Without this utility, there would be no other way to determine consistent therapeutic strategies and outcomes.
|Type of cell CD markers|
|Stem Cells||CD34+, CD31-, CD117+|
|Granulocyte||CD45+, CD11b, CD15+, CD24+, CD114+,|
|All Leukocyte Groups||CD45+|
|Monocyte||CD4, CD45+, CD14+, CD114+, CD11a, CD11b,|
|T lymphocyte||CD45+, CD3+|
|T helper cell||CD45+, CD3+, CD4+|
|CD4, CD25, FOXP3 (a transcription factor)|
|Cytotoxic T cell||CD45+, CD3+, CD8+|
|CD45+, CD19+, CD20+, CD24+, CD38, CD22|
|CD16+, CD56+, CD3-, CD31, CD30, CD38|
- MSCs can differentiate to at least three mesenchymal lineages (osteogenesis, adipogenesis, and chondrogenesis) . MSCs are mesodermal cells and the mesoderm forms connective tissue such as a bone, fat, cartilage and muscle. If the MSCs by virtue of their ex-vivo culture medium constituents can be coerced to transform themselves into bone, fat and cartilage, they have demonstrated their multipotency. This characteristic of chondrogenesis for example enables healing of cartilage when MSCs are used in treating arthritis.
In addition, to be effective therapeutically, MSCs must maintain their immunomodulatory potential . MSCs lack the major histocompatibility complex II (MHC-II) expression and co-
stimulatory molecules such as CD40+ (antigen presenting cells), CD80+ (activated B cells), and CD86+ (antigen presenting cells), which would cause their recipient to react to them immunologically. MSCs can be used allogenetically (within the same species) since they escape the recognition and action of T cells and natural killer (NK) cells .
Mode of Action
These multipotent cells are physically separate extraembryonic tissues  . In the body they fulfill a function as a reservoir of undifferentiated cells waiting for instructions in the regeneration of the tissues where they are located. They have migration and homing capacity to
relocate themselves to the site of the lesion in response to cellular signals such as cytokines, chemokines, and growth factors released  . MSCs do not raise the ethical issues associated with embryonic stem cells and have very low tumorigenesis potential   .
It is well established that the ability to modulate the immune system plays a fundamental role in almost all the therapeutic effects attributed to MSC cells, rather than their capacity of differentiation into different cell lineages   . This property is carried out through the release of a large variety of bioactive substances with autocrine and paracrine effects,
called the secretome  . The secretome includes a huge variety of molecules, including proteins, growth factors, antioxidants, proteasomes, microvesicles, and exosomes, which target a multitude of biological targets   responsible for: production of extracellular matrix as well as anti- apoptotic, anti-fibrotic, chemo-attractive, neuroprotective, morphogenic, angiogenic, antimicrobial, immunomodulatory effects etc.   . The immunomodulatory effect of MSCs is exerted through different mechanisms such as direct cell-to-cell contact and the secretion of different soluble substances in their secretome . It has been shown that the exosomes excreted by MSCs modulate the inflammatory response, via direct action on
resident cell targets   . MSCs have demonstrated the ability to modulate different types of cells that constitute this first line of defense, such as macrophages, neutrophils, dendritic cells, and NK cells    . MSCs also demonstrate a very interesting aspect of immunomodulation, due to their broad action capacity on Helper T Cells Type 1, and Helper T Cells Type 2 responses    . In fact, it is considered that one of the major MSCs’ immunomodulation mechanisms is the regulation of T cells—both CD4+ and CD8+—by cell-to-cell contact and inhibitory molecules of their secretome   . They are also able to act on B cells by modifying their activation, proliferation, chemotactic response, and differentiation to becoming antibody-secreting plasma cells    . The immunomodulatory capacity of MSCs is also complemented by their important potential to promote the generation and maintenance of the activity of different types of regulatory T cells   . Regulatory T cells are cell mediators of peripheral immunological tolerance, and their absence results in excessive multisystem autoimmunity.
Thus,MSCs help to modulate diseases that are immune mediated in nature. The list of these diseases is very long in both human and veterinary medicine. For example, keratoconjunctivitis sicca or dry eye is an autoimmune syndrome where the body’s T cells are attacking the tear producing glands. Using MSCs resets these cells, stops the reaction and promotes normal tear production. A single injection of MSCs into the tear glands has resulted in resolution of the
disease. Similar diseases of the blood, the intestines and heart and lungs occur. Even Type II diabetes has an immune component that can be treated with MSC therapy.
Dr. Garner is the owner and Chief of Staff at Safari Animal Care Centers where he has practiced for over 30 years. He hopes to educate clients, pet owners, and veterinarians alike about the
regenerative, holistic opportunities offered by stem cell therapy.
Dr. Garner can be reached at the hospital on 281.332.5612 or mobile 281.455.2356, or you can email him at firstname.lastname@example.org .
- A. Bajek, N. Gurtowska, J. Olkowska, L. Kazmierski, M. Maj and T. Drewa, “Adipose-derived stem cells as a tool in cell-based therapies.,” Arch Immunol Ther Exp (Warsz), vol. 64, p. 443–454,2016.
- M. Dominici, K. Le Blanc, I. Mueller, I. Slaper- Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. Prockop and E. Horwitz, “Minimal criteria for defining multipotent mesenchymal stromal cells.The International Society for Cellular Therapy position statement.,” Cytotherapy, vol. 8, pp. 315-317, 2006.
- J. Galipeau, M.Krampera, J. Barrett, F. Dazzi, R. Deans, J. DeBruijn, M. Dominici, W. Fibbe,A. Gee and J. Gimble, “International Society for Cellular Therapy perspective on immune functional assays for mesenchymal stromal cells as potency release criterion for advanced phase clinical trials.,” Cytotherapy, vol. 18, pp.151-159, 2016.
- J. Ankrum, J. Ong and J. Karp, “Mesenchymalstem cells: Immune evasive, not immune privileged.,” Nat. Biotechnol., vol.32, pp. 252-260, 2014.
- P. Mattar and K. Bieback, “Comparingthe Immunomodulatory Properties of Bone Marrow, Adipose Tissue, and Birth-Associated Tissue Mesenchymal Stromal Cells.,” Front. Immunol., vol. 6, pp.85-86, 2015.
- Q. Wang, Q. Yang, Z. Wang, H. Tong, L. Ma, Y. Zhang, F. Shan, Y. Meng and Z. Yuan, “Comparative analysis of human mesenchymal stem cells from fetal-bone marrow, adipose tissue, and Warton’s jelly as sources of cell immunomodulatory therapy.,” Hum. Caccin. Immunother., vol. 12, pp. 85-96, 2016.
- L. Sánchez-Abarca, E. Hernández-Galilea, R. Lorenzo, C. Herrero, A. Velasco, S. Carrancio, T. Caballero-Velázquez, J. Rodríguez-Barbosa, M. Parrilla and C. Del Cañizo, “Human bone marrow stromal cells differentiate into corneal tissue and prevent ocular graft-versus-host disease in mice.,” Cell Transpl., vol. 24, pp. 2423-2433, 2015.
- V. Konala, M. Mamidi, R. Bhonde, A. Das, R. Pochampally and R. Pal, “The current landscape of the mesenchymal stromal cell secretome: A new paradigm for cell-free regeneration.,” Cytotherapy, vol. 18, pp. 13-24,2016.
- C. Li, X. Wu, J. Tong, X. Yang, J. Zhao, Q. Zheng, G.Zhao and Z. Ma, “Comparative analysis of human mesenchymal stem cells from bone marrow and adipose tissue under xeno-free conditions for cell therapy.,” Stem Cell Res. Ther., vol. 6, p. 55, 2015.
- S.-F. Hsiao, A. Asgari, Z. Lokmic, R. Sinclair, G. Dusting, S. Lim and R. Dilley, “Comparative analysis of paracrine factor expression in human adult mesenchymal stem cells derived from bone marrow, adipose, and dermal tissue.,” Stem Cells Dev., vol.21, pp. 2189-2303, 2012.
- M. Murphy, K. Moncivais and A. Caplan, “Mesenchymal stem cells: Environmentally responsive therapeutics for regenerative medicine.,” Exp. Mol. Med., vol. 45, p. e54, 2013.
- S. Ma, N. Xie, W. Li, B. Yuan, Y. Shi and Y. Wang, “Immunobiology of mesenchymal stem cells.,” Cell Death Differ., vol. 21, pp. 216-225, 2014.
- J. Glenn and K. Whartenby, “Mesenchymal stem cells: emerging mechanisms of immunomodulation and therapy.,” World J. Stem Cells, vol. 6, pp.526-539, 2014.
- J. Spees, R. Lee and C. Gregory, “Mechanisms of mesenchymal stem/stromal cell function.,” Stem Cell Res. Ther., vol. 7, p.125, 2016.
- J. Lavoie and M. Rosu-Myles, “Uncovering the secretes of mesenchymal stem cells.,” Biochimie., vol. 95, pp. 2212-2221,2013.
- M. Makridakis, M. Roubelakis and A. Vlahou, “Stem cells: Insights into the secretome.,” Biochm. Biophys. Acta, vol. 1834, pp.2380-2384, 2013.
- H. Kupcova Skalnikova, “Proteomic techniques for characterisation of mesenchymal stem cell secretome.,”
Biochimie, vol. 95, p. 2196–2211, 2013.
- D. Phinney and M.Pittenger, “Concise Review: MSC-derived exosomes for cell-free therapy.,” Stem Cells, vol. 35, p. 851–858., 2017.
- R. Screven, E. Kenyon, M. Myers, H. Yancy, M. Skasko, L. Boxer, E. Bigley, D. Borjesson and M. Zhu, “Immunophenotype and gene expression profile of mesenchymal stem cells derived from canine adipose tissue and bone marrow.,” Vet. Immunol. Immunopathol, vol.161, pp. 21-31, 2014.
- V. Coulson-Thomas, Y. Coulson-Thomas, T. Gesteira and W.-Y. Kao, “Extrinsic and intrinsic mechanisms by which mesenchymal stem cells suppress the immune system.,” Ocul. Surf., vol. 14, pp.121-134, 2016.
- T. Lener, M. Gimona, L. Aigner, V. Börger, E. Buzas, G. Camussi, N. Chaput, D. Chatterjee, F. Court and H. Del Portillo, “; et al. Applying extracellular vesicles based therapeutics in clinicaltrials—An ISEV position paper.,” J. Extracell. Vesicles, vol. 4, p. 30087,2015.
- J. Kim and P. Hematti, “Mesenchymal stem cell-educated macrophages: A novel type of alternatively activated macrophages.,” Exp.Hematol., vol. 37, p. 1445–1453., 2009.
- W. Cao, K. Cao, J. Cao, Y. Wang and Y. Shi, “Mesenchymal stem cells and adaptive immune responses.,” Immunol. Lett., vol. 168, p. 147–153., 2015.
- M. Gazdic, V. Volarevic, N. Arsenijevic and M. Stojkovic, “Mesenchymal stem cells: A friend or foe in immune-mediated diseases.,” Stem Cell Rev., vol. 11, p. 280–287, 2015.
- E. Ghazaryan, Y. Zhang, Y. He, X. Liu, Y. Li, J. Xie and G. Su, “Mesenchymal stem cells in corneal neovascularization: Comparison of different application routes.,” Mol. Med. Rep., vol. 14, pp. 3104-3112, 2016.
- H. Aluri, M. Samizadeh, M. Edman, D. Hawley, H. Armaos, S. Janga, Z. Meng, V. Sendra, P. Hamrah and C. Kublin,”Delivery of bone marrow-derived mesenchymal stem cells improves tear production in a mouse model of Sjögren’s syndrome.,” Stem Cells Int.,