Focus on cell culture methods of bone marrowª²derived endothelial progenitor cells

    HAN Shuª²Fang, JIA Guoª²Liang, WANG Haiª²Yan, ZHANG Rongª²Qing

    Department of Cardiovasology, Xijing Hospital, Fourth Military Medical University, Xi¬ðan 710033, China

    ¡¾Abstract¡¿ AIM: To examine whether bone marrow (BM) mononuclear cells (BMª²MNCs) can give rise to functional EPCs and whether the proliferation and differentation of EPCs are various using different methods. METHODS: BMª²MNCs were isolated from boneª²marrow blood of rat femur by density gradient centrifugation, then divided into 3 groups, plate on culture dishes and maintained EC basal medium: A: Supplement with VEGF/bFGF; B: Coculture with human umbilical vein endothelial cell (HUVEC); C: control group. After 3, 7, 11, 15 d in culture respectively, the cells were used to fluorescent staining and fluorescenceª²activated cell sorting to determine the differentiation of the BMª²MNCs. RESULTS: BMª²MNCs were isolated and cultured for 7 days resulted in a spindleª²shaped, ECª²like morphology. These differentiating cells could be shown to be positive immunostaining for ¢ø factor relative antigen and NOS3. FACS disclosed that ,the amount of cell and positive immunostaining for factor VIII or NOS3 of B group were significant higher than the other groups (P=0). CONCLUSION: Our findings suggest that BMª²MNCs isolated from boneª²marrow have functional EPCs and can differentiate into ECs in vitro. The adherent cells may contribute to neoangiogenesis in vitro, Bcoculturied with HUVEC can supply cell growth factor at some degree, but can not replace VEGF and bFGF completely. ......