Inducing cord blood and peripheral blood derived EPCs into endothelial cells in vitro

AIM To investigate the feasibility of inducing human umbilical cord blood and peripheral blood derived EPCs into endothelial cells in vitro, and to test their phenotype and function. METHODS Mononuclear cells were isolated from human umbilical cord blood and peripheral blood by density gradient centrifugation, and cultured in M-199 medium. After 3 days, non-adherent cells were removed, the culture was maintained. The EPCs specific surface mark CD34 and endothelial specific surface marker CD31 were assessed by fluorescence activated cell sorter (FACS) analysis. Endothelial specific ecNOS and flk-1/KDR were detected by immunocytochemistry staining, reverse transcription-polymerase chain reaction (RT-PCR), respectively. The endothelial function of the cells was determined by measuring nitric oxide (NO) production in response to different concentrations of VEGF. RESULTS: CD34 positive cells were only (1.1±0.8)% when freshly isolated PBMC, and increased to (16.9±6.2)% after 3 days. When CBMC, PBMC were cultured in medium, a number of cell clusters appeared at 3 d, and spindle-shaped cells were observed and linear cord-like structures were formed at 7 d. Gradually, the attached (AT) cells exhibited cobblestone morphology, which was characteristic of endothelial cells. At 10 d, CD31 positive cells were (76±17)%and (82± 9)%in cultured CBMC and PBMC, respectively. The expression of EC marks of ecNOS and flk-1/KDR was detected by RT-PCR and further confirmed by immunocytochemistry staining. NO production increased with incremental dose of VEGF. CONCLUSION When isolated and cultured in special medium, PBMC and CBMC show EC-like morphology, express the marks of EC lineage and have EC function.