In vitro induction and differentiation of human endothelial progenitor cells from peripheral blood and their characteristics

Objective To study the methods of isolation, culture, differentiation and identification of endothelial progenitor cells(EPC) from peripheral blood. Methods Total hPBMNCs were isolated from peripheral blood of human volunteers by Histopaque-1077 density-gradient centrifu-gation. The cells were suspended in endothelial basal medium supplemented with EGM-2-MV-SingleQuots. Cultured cells were subjected to immunohistochemistry and immunofluorescence to analyze the expression of CD31, CD34, VEGFR-2 and vWF. FACS was performed to count the number of CD34+/AC133+ cells. Results EPCs exhibited clonal morphology and cobblestone morphology after 7 days or 4 weeks culture respectively. They were positive stain for CD31, CD34, vascular endothelia growth factor receptor 2 and vWF. The number of CD34+/AC133+ cells increased significantly after 2 weeks culture. Conclusion EPCs can be obtained from hPBMNCs and expanded enough for harvesting in vitro under certain condition.