Study on the separation,culturation and identification of human umbilical cord blood progenitor cells

Objective: To study on the separation,culturation and identification of human umbilical cord blood progenitor cells from human cord blood progenitor cells (EPCs),and provide a method to obtain a large number of endothelial progenitor cells.Methods: After cord blood were collected,with methods of 6% hydroxyethyl starch sedimentation and density gradient centrifugation,we obtained cord blood mononuclear cells.And then the cells were seeded in culture flasks laid fibronectin.After the cells were induction culturation,and differentiation in vitro,passage and amplification of the cells were completed;the cells were identified by immunohistochemistry,immunofluorescence,flow cytometry.Results: The cell culture showed colony-like growth at the first five days,and each single cell showed round or spindle-shaped.Two weeks later,the cellscovered the bottom of culture dish,and showed cobblestone-like appearance.Immunohistochemistry got 91.5% positive rate of CD31,72.4% positive rate of vWF;immunofluorescence staining told us that the cell emited red fluorescence,and phagocytose DiI-acLDL(+);but when the cells dyed with FITC marked by UEA-Ⅰ(+) they emited green fluorescence,and when the cells were double stained,they emited the yellow fluorescence.Flow cytometry analysis showed that the rate of CD34-positive cells was 93%,VEGFR-2 positive cells was 88.5%,and CD133 positive cells was 84.8%.Conclusions: CD34⁺、VEGFR-2⁺ and CD133⁺ endothelial progenitor cells can be largly obtained from the umbilical cord blood.