Objective: To investigate the Methods for culturing two types of endothelial progenitor cells (EPC) from human umbilical cord blood and study their differentiation traits and the depressant effect of asymmetric dimethylarginine (ADMA) on its proliferation.
Methods: Mononuclear cells were isolated from fresh cord blood by 6% hydroxyethyl starch(HES) and density gradient centrifugation.Isolated cells were cultured in the medium supplemented with vascular endothelial growth factors (VEGF) and basic fibroblast growth factors (bFGF). The growth characteristics and biological features of the cells were observed at different time points and identified by morphology,immunofluorescence staining,reverse transcription polymerase chain reaction (RT-PCR), and flow cytometry.Attached cells were incubated with different concentrations of ADMA (1,5, and 10 micromol/L) for 24,48, and 72 hours. Methylthiazoletetrazolium (MTT) assay and quantified colony forming units (CFUs) were used to assess the proliferation of endothelial progenitor cells.
Results: The attached cells were divided into 2 types:early EPC and late EPC. Early EPC changed from small sized round cells to spindle shaped cells and late EPC formed a typical cobblestone-like cells. Fluorescence microscopy showed that EPC were positive for both Dil-acLDL uptake and FITC-UEA-I binding.RT-PCR and FACS showed the difference of endothelial cell-specific,gene expression and changed AC133,CD34, and KDR among different times.Incubation of EPC with ADMA dose and time-dependently decreased the number and the proliferation of EPC.
Conclusion: There are 2 types of EPC from a source of human umbilical cord blood and ADMA may depress the EPC proliferation, providing a basis for further research.