Human umbilical cord blood-derived endothelial progenitor cells (EPCs) have been proven to contribute to postnatal angiogenesis, and have been applied in various models of ischemia. However, to date, to the best of our knowledge, there is no available data on the angiogenic properties of EPCs during the process of in vitro expansion. In this study, we expanded EPCs to obtain cells at different passages, and analyzed their cellular properties and angiogenic ability. In the process of expansion, no changes were observed in cell cobblestone-like morphology, apoptotic rate and telomere length. However, the cell proliferative ability was significantly decreased. Additionally, the expression of CD144, CD90 and KDR was significantly downregulated in the later-passage cells. Vascular formation assay in vitro revealed that EPCs at passage 4 and 6 formed more integrated and organized capillary-like networks. In a murine model of hind limb ischemia, the transplantation of EPCs at passage 4 and 6 more effectively promoted perfusion recovery in the limbs on days 7 and 14, and promoted limb salvage and histological recovery. Furthermore, the phosphorylation levels of platelet-derived growth factor receptor-beta (PDGFR-beta) were found to be significantly decreased with the in vitro expansion process, accompanied by the decreased activation of the PI3K/Akt signaling pathway. When PDGFR inhibitor was used to treat the EPCs, the differences in the angiogenic potential and migratory ability among the EPCs at different passages were no longer observed; no significant differences were also observed in the levels of phosphorylated PI3K/Akt between the EPCs at different passages following treatment with the inhibitor. On the whole, our findings indicate that the levels of phosphorylated PDGFR-beta are decreased in EPCs with the in vitro expansion process, which impairs their angiogenic potential by inhibiting PI3K/Akt signaling. Our findings may aid in the more effective selection of EPCs of different passages for the clinical therapy of ischemic disease.
