Isolation, cultivation and identification of endothelial progenitor cells from human peripheral blood

Objective To explore the procedures of isolation, cultivation and identification of endothelial progenitor cells (EPCs) from human peripheral blood, and to provide theoretical foundation for the application in nervous system diseases. Methods Ten milliliters of peripheral blood were collected from healthy male adult volunteers. The mononuclear cells were isolated from human peripheral blood by density gradient centrifugation and were cultured in vitro. The growth and morphological changes were observed by inverted phase-contrast fluorescence microscope. The expressions of EPCs specific markers such as CD133, CD34 and coagulation factor Ⅷ were assessed by immunofluorescent staining. The ratio of fluorescein isothio- cyanate labeled CD133 (CD133-FITC) and phycoerythrin labeled CD34 (CD34-PE) double-positive cells and the ratio of CD34-PE single-positive cells were determined by flow cytometry. Results Under the observation of inverted phase-contrast fluorescence microscope, the cells attached to the wall after 2 d cultivation, ex- hibited the colony after 4-6 d cultivation. There were endothelium-like cells around the colony. The cytoplasm and cell membrane presented red and green bicolor fluorescence, and the cell nuclear presented blue fluorescence. The cells specifically absorbed Dil labeled acetyl-low density lipoprotein (Dil-Ac-LDL) and FITC labeled ulex europaeus agglutinin Ⅰ (FITC-UEA-Ⅰ), and presented epithelial cells function. Immunofluorescent staining showed that the cultivated cells were positive for CD34, CD133 and coagulation factor Ⅷ. The flow cytometry indicated that the ratio of CD133-FITC and CD34-PE double-positive cells was 38.30%, and the ratio of CD34-PE single-positive cells was 82.60%. Conclusion The primary establishment of isolation, cultivation and identification of EPCs from human peripheral blood, indicates that EPCs from human peripheral blood can be isolated, cultivation and identification in vitro. It will be the basis for the re- search on EPCs transplantation.