The Isolation and Culture of Endothelial Progenitor cells in Umbilical Blood

Objective:To investigate how to isolate and culture endothelial progenitor cells and how to make it differentiate into endothelial cells. Methods: mononuclear cells were isolated from umbilical blood with Ficoll-paque by density gradient centrifu gation. MNCs were cultured in M199 with the supplement of VEGF,b-FGF,IGF-1. Every 3-4 days culture media was changed. After 16 days of culture,attached cells were detached, stained with DiI-LDL and FITC-UEA and were be observed under flu romicroscope. Simultaneously,MNCs were analyzed with flow cytometer. Results: Attached cells were observed after 3 or 4 days of culture and cord-like structure were noticed after 16 days or so. According to immunofluorescence,more than 90% attached cells were double positive fluorescence. Analyzed by flowcytometry,70% attached cells bound FITC-UEA and more than 90% attached cells uptook Dii-LDL. Conclusion:Endothelial progenitor cells enriched in umbilical blood and can differentiate into endothelial cells under certain conditions.