Phenotypic and Functional Characterization of Mesenchymal Stromal Cells Derived from CD271⁺- Bone Marrow Mononuclear Cells and Mesenchymal Stromal Cells Derived through Plastic Adherence.

Mesenchymal stromal cells (MSCs) are non- hematopoietic multipotent cells which can be derived from bone marrow mononuclear cells either by plastic adherence (PA-MSCs) or by a positive selection with antibodies against cell surface antigens expressed by MSC-progenitor cells (CD271, CD73, CD146, CD105, CD166, SSEA-4 and recently GD2). In this study, we compare the phenotype, proliferation potential, differentiation potential, the cytokine expression pattern and inhibitory potential of MSC- derived by plastic adherence and MSCs derived from positively selected CD271⁺ bone marrow mononuclear cells (BM-MNCs). According to CFU-F assay, the enriched CD271⁺ BM cells possess a significantly higher frequency (2952 per 1×10⁶ BM cells) compared to PA-MSCs (21 per 1×10⁶ BM cells). Phenotypically, both populations expressed high levels of common MSC antigens such as CD73, CD105, CD44, CD166, CD90, HLA-Class I and were negative for CD34, CD133, and CD14. Compared to PA-MSCs, CD271⁺ BM cells after the isolation express high levels of the hematopoietic antigen CD45 which is down- regulated within the first passage and HLA-DR, which remains constant through many passages. These cells have a 10- to 1000-fold higher proliferation capacity compared to PA- MSCs. However, both populations differentiated in vitro along adipogenic, chondrogenic and osteogenic lineage. In MLR, both populations significantly suppressed the proliferation of PHA- stimulated allogeneic T- lymphocytes at the ratio 10: 1 (MSCs:T-cells). However, CD271⁺ BM- derived MSCs were more efficient in secreting IFN-γ, IL-1β, IL-2, IL-4, GM-CSF and TNF-α, whereas PA- MSCs secreted significantly more IL-6 and IL-8 than CD271⁺ BM- derived MSCs. Ongoing in vivo studies with immunodeficient NOD/SCID mice will show the whole potential of both population in the improvement of engraftment of mobilized peripheral blood hemaatopoietic CD133⁺ cells. Based on their higher frequency and proliferation capacity we suggest that CD271⁺ BM- cells may represent a better source than PA- MSCs in order to generate bulk quantities of MSCs for clinical applications.