Stromal cells – also known as mesenchymal stem cells (MSCs) – are non-hematopoietic, multipotent, self-renewable cells that are capable of trilineage differentiation (mesoderm, ectoderm, and endoderm). The pluripotency and immunomodulatory features of MSCs makes them an effective tool in cell therapy and tissue repair.
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MSCs are easy to isolate and culturally expandable in vitro for long periods of time without losing their characteristics. They are able to trans-differentiate into ectodermal cells and endodermal cells. Moreover, due to their abundance in the adult body, research on these cells does not require ethical approval. MSCs are also safer than iPSCs, with no risk of teratoma formation. This makes them ideal candidates for cell therapy.
What defines a stromal cell?
The International Society for Cellular Therapy provides the following guidelines on MSCs:
- The cells should demonstrate plastic adherence.
- They should express specific cell surface markers, such as cluster of differentiation (CD) 73, D90, CD105, and lack the expression of CD14, CD34, CD45 and human leukocyte antigen-DR (HLA-DR).
- They should be able to differentiate in vitro into adipocytes, chondrocytes, and osteoblasts.
Sources of MSCs
MSCs are present in almost all tissues. A significant population of MSCs has been derived from the bone marrow. Cells exhibiting properties of MSCs have also been isolated from adipose tissue, dental tissues, amniotic membrane and fluid, placenta and fetal membrane, endometrium, menstrual blood, peripheral blood, synovial fluid, salivary gland, limb bud, skin and foreskin, sub-amniotic umbilical cord lining membrane and Wharton’s jelly.
Isolation and culture of MSCs
Despite relatively low numbers of MSCs in bone marrow aspirates, there is a keen interest in these cells as they can be easily isolated and expanded in culture through approximately 40 population doublings in 8 – 10 weeks.
Bone marrow is considered to be the best source for MSCs and used as a benchmark for comparison of MSCs obtained from other sources.
MSCs obtained from bone marrow, peripheral blood and synovial fluid are obtained using Ficoll density gradient method. MSCs obtained from other tissue sources, such as adipose, dental, endometrium, placenta, skin, and foreskin, and Wharton’s Jelly are obtained after digestion with collagenase.
MSCs isolated from different sources are cultured in Dulbecco’s modified Eagle’s medium (DMEM), DMEM-F12, a-MEM (minimal essential medium), DMEM supplemented with low or high concentration of glucose and RPMI (Rosewell Park Memorial Institute medium). The culture medium was supplemented with either 10% fetal bovine serum (FBS), new-born calf serum (NBCS) or fetal calf serum (FCS).
Expression of cell surface markers
The cells showing positive expression for CD63, D90 and CD105, and lack of expression of CD14, CD34, CD45 and HLA-DR are considered as MSCs. In addition to the above-mentioned markers, MSCs also express CD29, CD44, CD146 and CD140b, depending on the tissue of origin.
Stage-specific embryonic antigen (SSEA)-4, CD146 and stromal precursor antigen-1 (Stro-1) are the hallmarks of MSCs. Stro-1 is positively expressed in bone marrow and dental tissue, but negative in human adipose-derived MSCs.
Capacity for long-term in vitro culturing of MSCs
It is a challenge to obtain adequate number of cells for clinical applications as they tend to lose their potency during sub-culturing and at higher passages.
Early MSCs show high differentiation potential into chondrocytes, osteocytes, and adipocytes. However, long-term culture and higher passages cause senescence characterized by a decrease in differentiation ability, shortening of telomere length and an increased probability of malignant transformation.
Serum and growth factors impact the properties of MSCs during in vitro culturing. MSCs culturing requires 10% FCS, but MSCs retain FCS proteins that may trigger an immunologic response in vivo.
When MSCs are expanded in serum-free media, there is a gradual decline in differentiation potential and telomerase activity. However, the cells are resistant to malignant transformation and can be expanded at higher passages.
Immunomodulatory effects of MSCs
MSCs have been shown to suppress the excessive immune response of T and B cells, as well as dendritic cells, macrophages and natural killer (NK) cells by a mechanism that involves the combined effect of many immunosuppressive mediators. Most of the mediators, such as nitric oxide (NO), indoleamine 2,3-dioxygenase (IDO), prostaglandin E2 (PGE2), tumor necrosis factor-inducible gene 6 protein (TSG6), CCL-2, and programmed death ligand 1 (PD-L1) are inducible by inflammatory stimuli.
Although these factors show minimal expression in inactivated MSCs, they can be stimulated by inflammatory cytokines, such as interferon gamma (IFN-g), Tumor necrosis factor alpha (TNF-a) and interleukin -1 (IL-1). MSCs expressing IDO following stimulation with IFN-g catalyze the conversion of tryptophan to kynurenine, which causes the inhibition of the pathway for T-cell proliferation.
Production of NO by MSCs also inhibits T-cell proliferation. MSCs inhibit the maturation of monocytes to dendritic cells leading to reduced T-cell activation. MSCs also inhibit the upregulation of CD1a, CD40, CD80, and CD86 during DC maturation. MSCs inhibit the secretion of TNF-a, IFN-g, and IL-12 in dendritic cells and increase the levels of IL-10, inducing a more anti-inflammatory dendritic cell phenotype.
The secretion of soluble factors such as transforming growth factor (TGF-b) and prostaglandin E2 (PGE2) and direct cell-cell contact between MSCs and natural killer (NK) cells suppress the proliferation of NK cells. Cell-cell contact of MSCs through PD-1 binding to its ligand may also be responsible for inhibition of T-cell proliferation.