The aim of this study was to establish the method of isolating and culturing endothelial progenitor cells (EPCs) from human umbilical cord blood. Mononuclear cells (MNCs) from human umbilical cord blood were cultured by using culture system supplemented with endothelial cell-conditioned medium. The obtained two types of cells were purified by picking up colonies, identified by uptake of acetylated low-density lipoprotein (Ac-LDL) and binding to lectin [Ulex European Agglatinin (UEA-1)], and were analyzed for the expression of markers by flow cytometry. The results showed that there were significant differences between two types of cells in proliferation, so they were referred as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs), respectively. They were in accordance with the standards of EPCs, could uptake DiI-Ac-LDL and bind to UEA-1, and expressed the markers of endothelial cells, such as CD31, CD144 and vWF detected by immunocytochemistry. The transcription of CD31, KDR, CD144 and ENOS in both of them could be detected by RT-PCR, but FACS analysis showed significant differences of surface marker expression between them. In conclusion, two types of EPCs are successfully obtained by culturing MNCs isolated from human umbilical cord blood using endothelial cell-conditioned medium.
Cell Separation ; Cells, Cultured ; Culture Media, Conditioned ; metabolism ; Endothelial Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Neovascularization, Physiologic ; physiology ; Stem Cells ; cytology