Endothelial Progenitor Cells Derived from Human Umbilical Cord Blood Culture in Vitro with Differential Attachment Method

Objective To establish a practical,stable and high purity endothelial progenitor cells culture meth- od in vitro.Methods Human umbilical cord blood mononuclear cells were isolated by Ficoll density-gradient cen- trifugation,then plated on dishes coated with human fibronectin.After 48 hours,the nonaderent cells were collect- ed and replated onto fibronectin-coated dishes.After 7 days of culture,the cells were identified with the techniques of immunohistochemistry,immunofluorescence and flow cytometer.Results The cultured cells were small and spindle or polygonal in shape.Large numbers of typical endothelial progenitor cell colony-forming units were found,vWF and Flk-1 proteins expression were identified in more than 95% of the attached cells with 98% of them showing positive Dil-ac-LDL and FITC-UEA-1.According to the results from fluorescence-activated cell sorting (FACS),7.0%±1.8% of cells were recognized as CD_(133)~+.Conclusion Differential attachment technique is a practical and stable method for obtaining highly purified endothelial progenitor cells.