Culture and identification of endothelial progenitor cells from human umbilical cord blood

To elucidate a simple method for isolating endothelial progenitor cells (EPCs) from human umbilical cord blood mononuclear cells and observe the endothelial cell-specific expression profile during proliferation and differentiation in vitro.

Human umbilical cord blood were isolated by Percoll density gradient centrifugation from human cord blood and cultured in vitro. The adherent cells were then identified by immunohistochemical staining and flow cytometric analysis. CD(34), vascular endothelial growth factor receptor-2 (VEGFR-2), EPCs specific antigen CD(133), as well as endothelial cell specific markers CD(31) and vWF were used. The cells were characterized by acetylated LDL (acLDL) up-taking and lectin binding by direct fluorescentstaining.

During culture, the attached cells exhibited spindle-shape in early stage, and gradually display endothelium-like cobblestone morphology with outgrowth. On day 7, flow cytometric analysis showed that the positive staining rate of attached cells for CD(133), CD(34) and VEGFR-2 were 17.8%±3.7%, 22.1%±4.4% and 81.5%±5.0%, respectively. While, immunohistochemical staining showed that the adherent cells were positive to CD(31) and vWF at the rate of 92.7%±2.2% and 73.3%±4.2%, respectively. By direct fluorescentstaining, we observed that 83.0%±4.3% of the attached cells were double positive for DiI-acLDL and FITC-UEA-I.

EPCs can be separated from human cord blood under certain conditions in vitro. This observation may provide a basis for study of relationship between EPCs and retinal neovascularization, as well as further clinical application of EPCs in ischemic retinal lesions.