Research Of Endothelial Progenitor Cells On Regulating The Proliferation Of Mesenchymal Stem Cells

Objective To establish a method for simultaneously isolate,culture and identify of C57BL/6 murine Mesenchymal Stem Cells(MSCs) and Endothelial Progenitor Cells(EPCs) from bone marrow and to investigate the effects of EPCs conditioned medium(EPC-CM) and co-cultured in non-contact co-culture systems on the proliferation of MSCs.Methods 1. The cells were isolated by modified differential adhesion method from murine bone marrow and cultured for 48 h. Then the primary adherent cells at 48 h were cultured in LG-DMEM and the non-adhered cells were collected and induced by EGM-2MV complete medium in human fibronectin-coated dishes. Osteogenic, chondrogenic,and adipogenic induced multi-directional differentiation potentials were performed on the primary adherent cells, their immune phenotypes were detected by flow cytometry(FCM).Tube formation experiment on the matrigel in vitro and the expression of specific surface marker CD31 determined by immunofluoresence cell staining were identified for the subsequent adherent cells, and their immune phenotypes were detected by FCM. 2.The MSCs were divided into 0 EPC-CM group, 50% EPC-CM group and 100% EPC-CM group(they were all cultured by LG-DMEM).The effect of different concentration of EPC-CM on the proliferation of MSCs was detected by MTT method. 3.The passage 3 of EPCs and MSCs were harvested and placed into Transwell co-culture systems at the ratio of 1:1,MSCs were placed in the bottom and EPCs in the upper served as experimental group. Meanwhile, only MSCs were placed in the bottom at the same concentration served as control group. MTT assay and Ed U fluorescent assay were applied to detect MSCs proliferation co-cultured with EPCs.Results 1.FCM results showed that the third passage of MSCs were strongly positive for Sca-1,CD29 and negative for CD45,CD11 b and had the potentials to be induced differentiation into osteoblasts and adipocytes and chondrocytes. The third passage of EPCs were cultured on Matrigel, resulted in the formation of tube-like structures and positive express CD34,CD133 and VEGFR2. The expression of the passage 5 of the subsequent adherent cells specific surface marker CD31 was positive. 2.Compared with control group, the abilities of proliferation of MSCs 50% EPC-CM group and 100% EPC-CM group were increased(P<0.05), which presented a percentage-depended manner. 3.Compared with the control group, the ability of proliferation of MSCs in experimental group was significantly enhanced after 3d(P<0.05) and the proportion of MSCs in the stage of DNA synthesis was higher as evaluated with Ed U fluorescent assay( P<0.01).Conclusion The method of modify differential adhesion can simultaneously isolate,purify and amplify mouse bone marrow MSCs and EPCs.The ability of proliferation of MSCs is promoted by not only EPC-CM but also co-cultured with EPCs in non-contact co-culture systems.