Culture Of Endothelial Progenitor Cells In Human Peripheral Blood And Their Characteristics

Objective:To examine whether endothelial progenitor cells(EPCs) could be isolated from peripheral blood and could be cultured and proliferated in vitro.Methods:Total hPBMCs were isolated from peripheral blood of human volunteers and were plated on human fibronectin-coated culture dishes and cultured for 7 days.EPCs were characterized and adherent cells which were positive for DiLDL-up take and lectin binding by direct fluorescent staining were observed under a laser scanning confocal microscope,consistent with endothelial lineage cells.EPCs were further identified by demonstrating the expression of AC133,CD34 and VEGFR-2 with flow cytometry.Results:Culture cells had the ability to endocytose ac-LDL and bind lectin.They also could be shown to have EC specific markers such VEGFR-2,CD34 and AC 133.Conclusions:EPCs can be isolated and cultured from human peripheral blood,in quantities sufficient to permit their harvest and in vitro expansion and provide a cell source for the further investigation of autologous EPCs transplantation contributing to promote angiogenesis inhuman chronic cardiac ischemia.Background:It has been well established that estrogens can increase the number of endothelial progenitor cells(EPCs)by anti-apoptotic effects. Resveratrol,a polyphenolic phytoalexin extracted from grapes and wine, has been reported to act as an estrogen receptor agonist.Therefore,we tested the effects of this putative phytoestrogen on proliferation and survival in vitro.Methods:EPCs were isolated from human peripheral blood and identified immunocytochemically.EPCs were incubated with resveratrol at 1,10,25 and 50μmol·L^-1or vehicle control for indicated time.Cell proliferation,migration and in vitro vasculogenesis were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay,modified Boyden chamber assay and in vitro vasculogenesis detection,respectively.Results:Resveratrol increased the number of EPCs,promoted EPCs proliferation,adhesion and migration in a dose and time dependent manner.Cell number peaked at 50μmol·L^-1after incubation for 24 hours compared with vehicle control(112.8±7.2 vs 62.3±4.5,P＜0.01). Furthermore,cell cycle analysis showed that 50μmol·L^-1resveratrol significantly increased the S phase and decreased the G0-G1 phase of EPCs.In addition,resveratrol increased vascular endothelial growth factor production and further induced vasculogenesis in vitro effectively.Cnclusions:Resveratrol significantly induces EPCs proliferation, migration and further promotes angiogenesis in vitro.Resveratrol reduces human endothelial progenitor cell senescence through augmentation of telomerase activityBackground:Estrogens can reduce endothelial progenitor cell senescence through augmentation of telomerase activity.Resveratrol,a polyphenolic phytoalexin found in grapes and wine,has been reported to act as an agonist at the estrogen receptor and to extend saccharomyces cerevisiae lifespan.Therefore,we tested the effect of this putative phytoestrogen on senescence in human endothelial progenitor cells. Objective:To investigate whether resveratrols are able to prevent senescence of EPCs.Method:Human EPCs were isolated from peripheral blood and characterized.After in vitro cultivation,the cells became senescent as determined by acidicβ-galactosidase staining.Because cellular senescence is critically influenced by telomerase,which elongates telomeres,we measured telomerase activity using a polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay(ELISA)technique.We also measured the expression of hTERT by Western blotting and real-time polymerase chain reaction.Results:Resveratrol dose-dependently inhibited the onset of EPC senescence in culture.Resveratrol significantly increased telomerase activity.Conclusions:The inhibition of EPC senescence by resveratrol in vitro may improve the functional activity of EPCs in a way that is important for potential cell therapy.