Culture of endothelial progenitor cells from human peripheral blood and real-time identification by flow cytometry

Objective:To establish the method of isolation of endothelial progenitor cells(EPCs)from human peripheral blood and identify the markers of EPCs by FACS to provide basis for angiogenesis therapy.Methods: Total mononuclear cells(MNC) were isolated from peripheral blood by Ficoll density gradient centrifugation,and then the cells were plated on human fibronectin coated culture dishes and cultured in EGM-2 MV,4 days later,the adherent cells were stained with Dil-AC-LDL and FITC-UEA-1 and observed by fluorescence and confocal laser microscope.The cell surface markers(CD31,CD34 and KDR) were detected by FACS analysis at differentiation 4-7 days in cell culture. Results:Adherent cells double-positive for Dil-AC-LDL and FITC-UEA-1 could be observed under fluorescent and confocal laser microscope at 4 days after culture.Flow cytometric analysis revealed that the position rate for CD34,CD31 and KDR was(2.50±1.47)%,(5.72±1.66)% and(36.24±3.24)%,respectively at day 4 of culture.At days 5,6 and 7,CD34+ cells were(8.45±3.97)%,(14.13±2.79)% and(21.14±2.91)% and CD31+ cells were ((22.52±)3.86)%,(42.76±3.67)% and(54.67±3.44)%,respectively.Conclusion:EPCs exist in the peripheral blood and can be differentiated into mature endothelial cells(ECs).FACS can be used to efficiently identify EPCs for angiogenesis study.