1. Techniques for isolation and propagation of Mesenchymal stem cells and their characteristic features:Mesenchymal Stem cells (MSCs) are multipotent cells capable of self-renewal and can differentiate into various specialised cells 1. Mesenchymal stem cells can be isolated from various sources and are characterised by different surface markers depending on the source 2.1.1. Isolation of Mesenchymal stem cells:MSCs are found associated with Marrow and non-marrow sources and can be found in:• Adipose Tissue• Amniotic fluid• Bone Marrow• Dental tissues• Synovial Fluid• Umbilical cord (blood)• Wharton’s Jelly• Peripheral blood and blood vesselsThere are various isolation techniques for obtaining MSCs and the techniques widely used are listed below:A) Density gradient centrifugation: Density gradient centrifugation is a most widely used technique for isolation of MSCs from biological fluids such as blood- Bone marrow aspirate and umbilical cord blood, Amniotic fluid, Synovial fluid etc., 1,3. Reagents such as Ficoll 3, Hyaluronic acid hydrogel 4, etc., are used to mixed with the blood samples and are centrifuged to obtain layers of cells separated by the density gradient and the Mesenchymal cells are isolated.B) Enzymatic degradation: Enzymatic degradation plays an important role in the isolation of MSCs and used widely to obtain MSCs from Umbilical cord, Adipose tissue, Wharton’s jelly, Skin and dental tissues 1,2. Enzymes like Collagenase I, Trypsin-EDTA, Collagenase V, Dispase etc., are used for digestion and separation of the cells from the extracellular matrix (ECM) 5.1.2. Propagation of MSCs:MSCs are plastic adherent cells and can be easily propagated if sourced from blood samples such as bone marrow aspirate and umbilical cord blood. The samples from various sources can be grown in culture flasks and MSCs can be selected using following techniques 1,5,6:1) Plastic non-adherent, floating cells can be washed off after culturing for 24-48 hrs. However, high specificity cannot be guaranteed.2) Positive or negative selection can be performed to exclude nonspecific cells,3) Magnetic activated cell sorting (MACS) where immunomagnetic beads which have specific antibodies bind to MSCs and are subjected to magnetic field and unwanted cells are washed off.4) Flow cytometric separation like Fluorescence activated cell sorting (FACS) 7.The obtained MSCs are then grown in Dulbecco’s modified eagle media (DMEM) with 10-30% foetal bovine serum (FBS), with or without the use of antibiotics in a 5% CO2 incubator at 37o C 5.1.3. Characteristics of MSCs from various sources:Morphologically MSCs appear to be small cell bodies with thin, long cellular processes resembling fibroblasts and contains a large, round nucleus 8. Irrespective of source the MSCs express three surface markers namely, CD73, CD90, CD105, that are useful in detection of the cells. However, lack surface markers namely, CD11b, CD14, CD19, CD34, CD45, CD79a and Human Leukocyte Antigen – antigen D related (HLA-DR) 9. Depending on the source of the MSCs, the differentiation potential varies. Evidently, Bone Marrow derived MSCs (bMSCs) have high osteogenic and chondrogenic potential compared to Adipose derived MSCs (aMSCs). While Adipogenesis is similar both in bMSCs and aMSCs 1.1.4. Bibliography:1. Mushahary D, Spittler A, Kasper C, Weber V, Charwat V. Isolation, cultivation, and characterization of human mesenchymal stem cells. Cytometry A. 2017 Oct;2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science (80- ) . 1999;284(5411):143–7. http://science.sciencemag.org/content/284/5411/1433. Meppelink AM, Wang X-H, Bradica G, Barron K, Hiltz K, Liu X-H, et al. Rapid isolation of bone marrow mesenchymal stromal cells using integrated centrifuge-based technology.Cytotherapy.2016;18(6):729–39.4. Lee J-S, Kim S-K, Cha J-K, Jung B-J, Choi S-B, Choi E-Y, et al. Novel Technique for Isolating Human Bone Marrow Stem Cells Using Hyaluronic Acid Hydrogel. Tissue Eng Part C Methods. 2016 Oct;22(10):941–51.5. Busser H, Najar M, Raicevic G, Pieters K, Velez Pombo R, Philippart P, et al. Isolation and Characterization of Human Mesenchymal Stromal Cell Subpopulations: Comparison of Bone Marrow and Adipose Tissue. Stem Cells Dev. 2015 Sep;24(18):2142–57.6. Spiropoulos A, Theodosaki M, Stefanaki K, Paterakis G, Tzetis M, Giannikou K, et al. Rapid clinical-scale propagation of mesenchymal stem cells using cultures initiated with immunoselected bone marrow CD105(+) cells. Vol. 15, Journal of Cellular and Molecular Medicine. Oxford, UK; 2011. p. 1983–8.7. Rojewski MT, Weber BM, Schrezenmeier H. Phenotypic Characterization of Mesenchymal Stem Cells from Various Tissues. Vol. 35, Transfusion Medicine and Hemotherapy.; 2008. p. 168–84.8. Brighton CT, Hunt RM. Early histological and ultrastructural changes in medullary fracture callus. J Bone Joint Surg Am. 1991 Jul;73(6):832–47.9. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini FC, Krause DS, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. Cytotherapy . 2018 Jan 25;8(4):315–7. http://dx.doi.org/10.1080/14653240600855905.2. Lineage potential of Mesenchymal Stem/Stromal Cells: In vitro and in vivo differentiation of MSCs: Several studies have found that the MSCs demonstrated differentiation into Ectodermal, Mesodermal and Endodermal lineages when subjected to specific physicochemical parameters 1. During in vitro MSC differentiation, the growth factors in the culture plays major role in commitment for the MSCs to a lineage 2. But, when subjected to an invivo condition In vitro expansion of MSCs is usually performed in either monolayer cultures (2D) or in Bioreactors/ 3D static cultures. Irrespective of the source of MSCs, there are certain chemical factors that induce differentiation of MSCs to a certain lineage 3 such as: (i) Haematopoiesis: The BM-MSCs when cultured along with the hematopoietic stem cells (HSCs), create a stem cell niche that facilitates and promotes haematopoiesis. It has been show that the MSCs interact with the HSCs via cell-cell attachments via surface adhesion molecules, and cytokines such as IL-6, IL-7 and Macrophage -colony stimulating factor that promote proliferation and differentiation of HSCs 1. (ii) Osteogenesis: The MSCs cultured with media containing ascorbate, Bone morphogenetic protein (BMP), dexamethasone and Vitamin D3, osteogenesis is observed. The MSCs when subjected to the above cocktail of osteogenic growth factors over a 3-week period in vitro, mineralisation of the extracellular matrix takes place inducing conversion of MSCs to osteoblasts 4. For tissue engineering of bone defects such as fractures or deformations, bone matrix like scaffolds are seeded with MSCs and subjected to bioreactors along with the osteogenic cocktail and transplanted in-vivo to facilitate bone environment triggering oseteoblast formation within the defect 5. (iii) Chondrogenesis: For production of chondrocytes from MSCs, the media containing Ascorbate, Bone morphogenetic protein 6, Dexamethasone, transforming growth factor ? (TGF- ?) is used in addition to hypoxic conditions 1. It has been observed that the chondrogenesis is most effective when cultured in a three-dimensional setup rather than 2-D surface. MSCs are usually seeded on a synthetic scaffold such as polycaprolactone and implanted in vivo which induces the MSCs to secrete collagen II and aggrecan resulting in integration of the cartilage with the underlying bone resulting in the repair of damaged cartilage 1. (iv) Adipogenesis: Production of adipocytes from MSCs can be observed when the media containing dexamethasone, indomethacin, insulin, methyl sobutylxanthine, thiazolidinedione is subjected. The cocktail of adipogenic compounds induce the fibroblast-like MSCs to transform to oval, lipid droplet containing cells 5. (v) Myogenesis: The MSCs have shown the ability to produce cardiomyocytes in a low serum media containing 5-azacytadine, leading to expression of Atrial natriuretic peptide (ANP) and the transcription factor GATA4 6, while Low serum concentration, L-glutamine, 5-Azacytadine and Fibroblast growth factor have shown to be favourable for skeletal muscle development 7.The MSCs have also shown to have potential for differentiation into Hepatocytes, pancreatic cells and oligodendrocytes, however in vivo applications for these are still being tested and refined. Bibliography: 1. Gimble JM, Guilak F, Nuttall ME, Sathishkumar S, Vidal M, Bunnell BA. In vitro Differentiation Potential of Mesenchymal Stem Cells. Vol. 35, Transfusion Medicine and Hemotherapy.; 2008. p. 228–38. 2. Pountos I, Corscadden D, Emery P, Giannoudis P V. Mesenchymal stem cell tissue engineering: techniques for isolation, expansion and application. Injury. 2007 Sep;38 Suppl 4:S23-33. 3. Hass R, Kasper C, Böhm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Vol. 9, Cell Communication and Signaling : CCS. 2011. p. 12. 4. Maridas DE, Rendina-Ruedy E, Le PT, Rosen CJ. Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice. J Vis Exp. 2018 Jan;(131). 5. Maher S, Kolieb E, Sabik NA, Abd-Elhalim D, El-Serafi AT, El-Wazir Y. Comparison of the osteogenic differentiation potential of mesenchymal cells isolated from human bone marrow, umbilical cord blood and placenta derived stem cells. Beni-Suef Univ J BasicApplSci.2015;4(1):80–5. http://www.sciencedirect.com/science/article/pii/S2314853515000128 6. Li Q, Qi L-J, Guo Z-K, Li H, Zuo H-B, Li N-N. CD73+ adipose-derived mesenchymal stem cells possess higher potential to differentiate into cardiomyocytes in vitro. J Mol Histol. 2013 Aug;44(4):411–22. 7. Stern-straeter J, Bonaterra GA, Juritz S, Birk R, Goessler UR, Bieback K, et al. Evaluation of the effects of different culture media on the myogenic differentiation potential of adipose tissue- or bone marrow-derived human mesenchymal stem cells. Int J Mol Med . 2014 Jan cited 2018 Jan 26;33(1):160–70. https://www.spandidos-publications.com/10.3892/ijmm.2013.15553. Possible Therapeutic mechanisms using MSCs: 3.1. Therapeutic application of MSCs in Vascular diseases: Cardiovascular diseases (CVDs) are major causes for death in adults and treatment of the cardiovascular defects with the help of MSCs have proven to be a promising approach. Inducing MSCs to differentiate into mesodermal lineage and promote cardio -myogenesis for the treatment of ischemic disease has shown to repair myocardium and angiogenesis in rat models when injected intravenously 1. MSCs have shown paracrine secretions of angiogenic growth factors such as bFGF, VEGF, TGF-?, PDGF, angiopoietin-1, placental growth factor (PGF), IL-6, and monocyte chemotactic protein-1 (MCP-1) 1. 3.2. Application of MSCs for Inflammatory Bowel disease: Ulcerative colitis (UC) and Crohn’s disease (CD), are chronic gastrointestinal diseases commonly called as Inflammatory bowel disease (IBD) characterised by dysregulated immune response in the gastrointestinal tract causes inflamed tissue damage 2. In IBD, an increase in Th (T helper) cells is observed with secretion of proinflammatory responses and activated T cells. The T cells are resistant to apoptosis due to the imbalance of apoptotic factors. Several in vivo experiments have shown that implantation of MSCs induced the proliferation of intestinal epithelial cells resulting in repair of damaged tissue and also intravenous administration has shown to induce T-cell apoptosis and increase in IL-10 down regulating the inflammatory response 3. 3.3. Anti-scarring effect of MSCs as therapeutic application: Fibrosis is an excessive wound healing response to an injured or damaged tissue that interferes with the normal organ function. Most of the fibrotic tissues are fatal in vital organs and cause diseases like Endocardial fibrosis, Myocardial infarction, Liver cirrhosis, Cystic fibrosis etc.,4 MSCs have been shown to have anti-fibrotic activity and promotion of native tissue growth in a natural healing process. The exact molecular mechanisms are still unclear. However, MSCs have shown to produce Hepatocyte growth factor (HGF) in response to basic fibroblast growth factor (bFGF), resulting in impaired proliferation of host perivascular cells and decreased fibrogenesis and less fibrosis.4 3.4 Immunomodulatory effect of MSCs as therapeutic application: Auto-immune diseases and graft rejection have been complex challenges and MSCs being immunoprivilaged, provides a great potential in treatment of such diseases. MSCs have shown the ability to modulate the cytokine secretion from the immune cells reducing the immunological response, inhibit or promote B-cell proliferation, Suppress the activation of NK cells and neutralise the antibodies against TGF-?1 5. 3.5. Cell proliferative and adaptive effect of MSCs in Osteochondral defects: MSCs have excellent wound healing effects in adults and several studies and clinical trials are being conducted for repairing damaged tissues and promote bone formation in critical size defects of bone in fracture or surgical removal of osteosarcoma. MSCs seeded on Bone likenatural/ synthetic scaffolds have been used where the MSCs in a osteo-environment and growth factors such as TGF-?1 promote bone formation 6. In osteoporosis, injected cultured MSCs have shown to promote osteoblasts formation and reconstitute the porous bone 7. Bibliography: 1. Tao H, Han Z, Han ZC, Li Z. Proangiogenic Features of Mesenchymal Stem Cells and Their Therapeutic Applications. Stem Cells Int . 2016 Jan 6 cited 2018 Jan 26;2016:1–11. http://www.hindawi.com/journals/sci/2016/1314709/ 2. de Mattos BRR, Garcia MPG, Nogueira JB, Paiatto LN, Albuquerque CG, Souza CL, et al. Inflammatory Bowel Disease: An Overview of Immune Mechanisms and Biological Treatments. Mediators Inflamm . 2015 Aug 3 cited 2018 Jan 26;2015:1–11. http://www.hindawi.com/journals/mi/2015/493012/ 3. Mao F, Tu Q, Wang L, Chu F, Li X, Li HS, et al. Mesenchymal stem cells and their therapeutic applications in inflammatory bowel disease. Oncotarget . 2017 Jun 6 cited 2018 Jan 26;8(23):38008–21. http://www.ncbi.nlm.nih.gov/pubmed/28402942 4. da Silva Meirelles L, Fontes AM, Covas DT, Caplan AI. Mechanisms involved in the therapeutic properties of mesenchymal stem cells. Cytokine Growth Factor Rev . 2009 Oct;20(5–6):419–27. http://linkinghub.elsevier.com/retrieve/pii/S135961010900077X 5. Najar M, Krayem M, Merimi M, Burny A, Meuleman N, Bron D, et al. Insights into inflammatory priming of mesenchymal stromal cells: functional biological impacts. Inflamm Res . 2018 Jan 23 cited 2018 Jan 26; http://link.springer.com/10.1007/s00011-018-1131-1 6. Amini AR, Laurencin CT, Nukavarapu SP. Bone Tissue Engineering: Recent Advances and Challenges. Crit Rev Biomed Eng . 2012;40(5):363–408. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3766369/ 7. Hu L, Yin C, Zhao F, Ali A, Ma J, Qian A. Mesenchymal Stem Cells: Cell Fate Decision to Osteoblast or Adipocyte and Application in Osteoporosis Treatment. Int J Mol Sci . 2018 Jan 25 cited 2018 Jan 26;19(2):360. http://www.mdpi.com/1422-0067/19/2/3604. Privilege of MSCs to evade host immune response: 4.1. Immune-tolerogenic property of MSCs: Cells express a set of surface markers called Major histocompatibility complex (MHC) -I &II, which helps in determination of histocompatibility and recognition of foreign bodies. However, MSCs lack the MHC-II on the surface and express only few MHC I 1. Additionally, MSCs express Programmed death ligand 1 (PD-L1) which inhibits the activity of Programmed cell death protein 1 (PD-1) thus downregulating T-cell inflammatory activity 2. 4.2. Immunomodulation by Cell contact: (i) B-Lymphocytes: MSCs can inhibit the proliferation of B-Lymphocytes by secretion of indoleamine-pyrrole 2,3-dioxygenase (IDO) and few unknown factors. Studies have shown that co-culture of MSCs with B-Lymphocytes supressed the production of immunoglobulins – IgA, IgG and IgM 3. (ii) Dendritic cells: MSCs have shown to interfere with the differentiation and maturation of Dendritic cells which are antigen presenting cells (APC) downregulation of inflammatory cytokines such as Interleukin 12 (IL-12), Tumour necrosis factor-? (TNF- ?), and Interferon-gamma (IFN-?) 2. (iii) T- lymphocytes: MSCs interfere with the differentiation of a naïve T cell (CD4+) into a T-helper cell (Th1) and decrease proliferation, cytokines by secreting indoleamine-pyrrole 2,3-dioxygenase (IDO), Transforming Growth factor- ? (TGF- ?), etc., 3. T-Helper cells are responsible for cytotoxic T-cell activation (Tc) and production of inflammatory cytokines Interleukin 2 (IL-2) and Interferon-gamma (IFN-?). It has been observed that MSCs co-cultured with the T-lymphocytes showed a decrease in the production of Interferon-gamma (IFN-?) 4. 4.3. Immunomodulation by secretion of soluble factors: MSCs tend to secrete soluble factors that have immunosuppressive activity in presence of proinflammatory cytokines such as Interferon-gamma (IFN-?) 5. (i) Transforming Growth factor- ? (TGF- ?): TGF- ? is immunosuppressive protein that controls cell proliferation and differentiation. It inhibits the secretion of inflammatory cytokines such as interferons, Interleukin and Tumour necrosis factor in T-cells and controls the expression of MHC II on B-lymphocytes.3. (ii) Indoleamine-pyrrole 2,3-dioxygenase (IDO): IDO is an enzyme responsible for tryptophan catabolism. Reduction in Tryptophan ceases the growth of T-cells. IDO also promotes development of regulatory T cells (Treg cells) which are responsible for regulation of activated T-effector cells. MSCs secrete IDO as a mechanism to downregulate the maturation of T-cells 2. (iii) Interleukin 10 (IL-10): IL-10 is an anti-inflammatory cytokine also known as “Human cytokine synthesis inhibitory factor (CSIF)”. IL-10 down regulates the expression of MHC-II antigens, Th1 cytokines and block the nuclear factor kappa-light-chain enhancer of activated B lymphocytes (NF-kB). MSCs secrete 6. MSCs use IL-10 to downregulate the inflammatory cytokines to avoid the immunological response.(iv) Human Leukocyte antigen- G5 (HLA-G5): HLA is responsible for encoding MHC proteins and HLA-G5 is a form of HLA class I responsible for immune tolerance in pregnancy. When the MSCs have contact with activated T-cells from allogenic response, MSCs, release an isoform of HLA-G5 which downregulates the T-cell immune activity. It is also observed that the molecule can suppress T-cell proliferation and promote Treg cell proliferation. NK cell mediated cell lysis is also inhibited by the HLA-G5 complex 3.