ObjectiveTo compare the biological features of early and late endothelial progenitor cells (EPCs) by isolating and culturing early and late EPCs from the human peripheral blood so as to find some unique properties of EPCs and to propose a suitable strategy for EPCs identification. MethodsMononuclear cells were isolated from the human peripheral blood using density gradient centrifugation. Then, the cells were inoculated in human fibronectin-coated culture flasks and cultured in endothelial cell basal medium 2. After 4-7 days and 2-3 weeks culture, early and late EPCs were obtained respectively. The morphology, proliferation potential, surface markers, cytokine secretion, angiogenic ability, and nitric oxide (NO) release were compared between 2 types of EPCs. Meanwhile, the human aortic endothelial cells (HAECs) were used as positive control. ResultsThe morphology of early and late EPCs was different:early EPCs formed a cell cluster with a spindle shape after 4-7 days of culture, and late EPCs showed a cobblestone appearance. Late EPCs were characterized by high proliferation potential and were able to form capillary tubes on Matrigel, but early EPCs did not have this feature. Both types EPCs could ingest acetylated low density lipoprotein and combine with ulex europaeus Ⅰ. Flow cytometry analysis showed that early EPCs did not express CD34 and CD133, but expressed the CD14 and CD45 of the hematopoietic stem cell markers;however, late EPCs expressed CD31 and CD34 of the endothelial cell markers, but did not express CD14, CD45, and CD133. By RT-PCR analysis, the expressions of vascular endothelial growth receptor 2 and vascular endothelial cadherin in early EPCs were significantly lower than those in the late EPCs and HAECs (P<0.05), but no significant difference was found in the expression of von Willebrand factor and endothelial nitric oxide synthase (eNOS) between 2 type EPCs (P>0.05). The concentrations of vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8 were significantly higher in the supernatant of early EPCs than late EPCs (P<0.05). Western blot assay indicated eNOS expressed in both types EPCs, while the expression of eNOS in late EPCs was significantly higher than early EPCs at 5 weeks (P<0.05). Both cell types could produce similar amount of NO (P>0.05). ConclusionThe expression of eNOS and the production of NO could be used as common biological features to identify EPCs, and the strategy of a combination of multiple methods for EPCs identification is more feasible.