8. Isolation and culturing

All MSCs, despite the protocol used for isolation, characterization and expansion, show the minimum criteria suggested by International Society for Cellular Therapy.

hMSCs are isolated based on their adherence ability to plastic surfaces, however, this method leads to the formation of a diversity of cells (stem cells as well as their progenitor cells) [41]. Considered as the best cell source, Bone marrow-derived MSCs (BM-MSCs) are taken as a standard to compare MSCs from other sources.

To ensure the success of the usage of these cells as a dependable source for regenerative medicine, a complete procedure should be established for MSCs isolation, characterization and expansion [42]. Contrary to bone marrow, MSCs from other tissues can be easily collected through non-invasive methods and their proliferation could be sustained up to many passages. Ficoll density gradient method with small modifications is utilized for isolation of MSCs from bone marrow, peripheral blood and synovial fluid [13] and seeded into culture plates. During isolation of MSCs from bone marrow, some hematopoietic cells also adhere to the plastic plate but they are washed away during sub-culturing, leaving only adherent fibroblast like cells [43]. MSCs from various tissue sources (adipose, dental, endometrium, foreskin, placenta, Wharton's Jelly) were isolated after digestion with collagenase and then cultured at varying densities [42]. Novel marrow filter device is recently explored as an efficient method for isolation of BM-MSCs [16], avoiding the risk of external contamination and saving time. Following their isolation from different sources, MSCs were cultured in condition media such as Dulbecco's modified Eagle's media (DMEM), DMEM-F12, DMEM-LG, DMED-HG, αMEM and RPMI (Roswell Park Memorial Institute medium) [44]. The primary culture medium was supplemented with fetal calf serum (FCS), new-born calf serum (NBCS) or10% FBS [45]. Besides the culture media and supplementation, the oxygen concentration is very important in the expansion and proliferation of MSCs [46]. It is also documented when cultured in DMEM culture with low glucose enriched with growth factors like fibroblast growth factor (FGF), epidermal growth factor (EGF) and B27 also leads to MSCs expansion [47]. But most commonly DMEM with 10% FBS is vastly employed in culturing and expanding MSCs in vitro, on the other hand, the use of exogenous FBS is highly debated.

#### 8.1. Expression of cell surface markers

IL-10 which inhibit neutrophil invasion into the wound. TNF-stimulated gene/protein-6 (TSG-6) is secreted by MSCs, interacts with protein ligands to inhibit rolling and transendothelial migration of neutrophils. Dyer [34] found that TSG-6 interacts with the glycosaminoglycan binding site of CXCL8 (IL-8), a chemokine produced by macrophages and transported to the surface of

Macrophages that arrive at the injury site hours later than neutrophils, are phagocytes that cleanse the wound of matrix and cell debris. They typically classified into two main groups: classically activated macrophages (M1) and alternatively activated macrophages (M2). M1 macrophages generally carry antimicrobial characteristics and stimulate a Th1 type response while M2 macrophages stimulate Th2 type responses. In general, M2 macrophages secrete less proinflammatory cytokines, have high production of anti-inflammatory cytokines such as IL-10, and induce resolution of the inflammatory phase. M2b macrophages show the reverse, as they maintain high levels of inflammatory cytokines [35]. Many studies explain the ability of autologous or allogeneic MSCs to polarize macrophages toward an M2 phenotype in vitro mediated by paracrine mechanisms, enhancing expression of M2 associated macrophage genes. Kim and Hematti [36] have suggested a separate definition for MSC-educated macrophages that secrete high IL-10 and IL-6 and low IL-12 and TNF-α, to call them M2 m, differing them from other subcategories. They suggest the possibility of collecting monocytes through leukapheresis and coculturing these mononuclear cells with allogeneic MSCs to provide MSC-educated macro-

B lymphocytes produce antibodies on exposure for antigens. MSCs may arrest B cell proliferation in the G0/G1 phase of the cell cycle without enhancing apoptosis [37]. IFN-γ inhibits the proliferation, which is probably mediated by MSC production of IDO. IDO is the first and ratelimiting enzyme of the essential amino acid tryptophan catabolism to kynurenine pathway, producing depletion and therefore halting growth. IFN-γ has IDO inducing effects [38].

Inhibitory effects of T cell proliferation by MSCs are mediated by both cell-to-cell contact and soluble factors. T cell proliferation was suppressed by TGF-β1 and HGF [39]. MSCs secrete PGE2 which prevents differentiation of CD4+ T cells into Th17 cells. MSCs also release IDO

All MSCs, despite the protocol used for isolation, characterization and expansion, show the

and enhance secretion of IL-10, which also inhibit cell proliferation [40].

minimum criteria suggested by International Society for Cellular Therapy.

the endothelium, impairing neutrophil adhesion and migration.

10 Stromal Cells - Structure, Function, and Therapeutic Implications

phages prepared for repair of wounds [36].

7.4. Macrophages

7.5. B cells

7.6. T cells

8. Isolation and culturing

One of the essential characteristics of hMSCs is expression of specific set of cell surface markers. According to the International Society for Cellular Therapy standard criteria, MSCs are positive for CD73, D90, CD105 but negative for CD14, CD34, CD45 and HLA-DR [8]. MSCs can be isolated from various human tissues, which express cell surface markers mentioned above along with positive expression of CD29, CD44, CD146, CD140 b specific to tissue origin. The expression of CD34, which is a negative marker, is still controversial [48]. Stage-specific embryonic antigen (SSEA)-4 [49], stromal precursor antigen-1 (Stro-1) and CD146 are reported as are stemness markers for MSCs [50]. MSCs isolated from the human amniotic fluid express HLA-ABC [major histocompatibility complex class I (MHC I)], CD29, CD44, CD90, CD105, as well as SH2 (Src homology 2), SH3 (Src homology 3) and SH4 (Src homology 4). On the other hand, they lack the expression of HLA-DR (MHC II) [51]. Stro-1, a stemness marker for MSCs, is reported positive in dental and bone marrow MSCs, while reported negative in human adipose-derived MSCs (AD-MSCs) [52].
