Isolation,expansion and characterizations of endothelial progenitor cells from human umbilical cord blood

Objective:To isolate and culture endothelial progenitor cells(EPCs)from human umbilical cord blood(HUCB)and study their biological characterizations in vitro.Method:Mononuclear cells(MNCs)were isolated from HUCB by density gradient centrifugation,and detected for the percentage of CD133/CD34 with flow cytometry.MNCs were seeded in gelatin-coated culture dishes,cultured in the medium containing growth factors combining different speed adherence.Cell morphology and the capability of forming colony units were investigated by phase-contrast microscopy.The differentiation of the EPCs was determined by immunocytochemistry.Result:The percentage of CD34+CD133+ in MNCs isolated from HUCB was 1.06%.We obtained two types of EPCs using endothelial cell growth medium(EGM),referred to as early EPCs and late outgrow EPCs.The adherent cells after 48 hours were early EPCs and showed to be spindle-shaped with a tendency to form colonies which weakly potential,and were positive for CD14 at 7 days,those cells can express CD34KDR.In addition,cells took up Ac-LDL and bound lectin UEA-1.The adherent cells during 48 hours demonstrated colony forming cells with typical cobblestone morphology during the first 9～17 days showed robust proliferative potential and give rise to secondary colonies.During the 8 and 15 days,cells continued to be positively expressed CD34CD133 and CD34KDR.In addition,cells took up Ac-LDL and bound lectin UEA-1.Those cells became positive for vWF and CD31 after 40 days of culture,those cells are late outgrowth EPCs.Conclusion:These data showed that two types EPCs can be isolated and cultured from umbilical cord blood.During the culture conditions containing growth factors,EPCs gradually lost progenitor characterization and differentiated towards mature endothelial cells.