Effect Of Endothelial Progenitor Cells On Differentiation Potential Of Mesenchymal Stem Cells

Objective1. To investigate the effect and mechanisms of EPCs on the osteogenic differentiation of MSCs in vitro.2. To explore the influence of EPCs on the chondrogenic differentiation of MSCs in vitro.Methods1. MSCs were isolated by the whole bone marrow adherent method from C57BL/6 mice bone marrow; to identify the adherent cells, which of the third passage were used to assay cell surface markers by flow cytometry. To assess the differentiation capacity of MSCs, osteogenic and chondrogenic inductions were performed in vitro.2. EPCs were isolated by anchorage velocity-dependent separation method. The third passage of EPCs was used to assay cell surface markers by flow cytometry. And the cultured cells were further identified by tube formation experiment in vitro.3. Alizarin Red staining was used to examine the effects of EPCs and EPC-CM on MSCs-osteogenic differentiation.4. EPC-conditioned medium(CM) were extracted to detect the concentration of VEGF, TGFβ1,PDGF, IGF-1, SDF-1, b FGF by ELISA.5. On the basis of 50% EPC-CM, above mentioned antibodies of six cytokines were added respectively to investigate the cytokines which could affect the osteogenic differentiation of MSCs.6. Alcian blue staining was used to examine the effects of EPCs and EPC-CM on MSCs-chondrogenic differentiation.Results1. Obtained cells were immunopositive for surface markers of mesenchymal, but were negative for the hematopoietic markers. Osteogenic and chondrogenic induction results showed that the cells could differentiate into chondrocytes and osteoblasts when cultured in induction medium.2. Cultured cells expressed surface markers of haematopoietic stem cell, and they could also expressed endotheliocyte marker. EPCs could form tube-like structure on Matrigel.3. Alizarin staining results showed MSCs/EPCs group formed more mineralized nodules than MSCs group, semi-quantitative results showed the optical density in MSCs/EPCs group was higher than that in MSCs group(P<0.001). The number of formed mineralized nodules in50% EPC-CM group were more than those in 25% EPC-CM group, semi-quantitative results showed that the optical density in 50% EPC-CM group was higher than that in 25% EPC-CM group(P<0.01), the number of formed mineralized nodules in 25% EPC-CM group were more than those in 0% EPC-CM group, semi-quantitative results showed that the optical density in 25% EPC-CM group was higher than that in 0% EPC-CM group(P<0.01).4. EPCs secreted VEGF, TGFβ1, PDGF, IGF-1, SDF-1, b FGF.5. On the basis of 50% EPC-CM, the number of formed mineralized nodules were significantly reduced in the presence of VEGF antibody, TGFβ1 antibody, IGF-1 antibody(P<0.01).6. When Alcian blue was extracted from the stained cultures, glycosaminoglycan deposition in MSCs/EPCs group was showed to be increase significantly compared with MSCs group(P <0.001), no significant differences in glycosaminoglycan deposition were found in 50%EPC-CM group, 25% EPC-CM group, 0% EPC-CM group(P>0.05).Conclusion1. EPCs promote the osteogenic differentiation of MSCs maybe through EPCs paracrine effects of VEGF,TGFβ1 and IGF-1.2. EPCs promote the chondrogenic differentiation of MSCs in direct contact manner.