Cultivation and identification of endothelial progenitor cells from human bone marrow

Objective To establish methods of isolation,cultivation,induction and identification for endothelial progenitor cells(EPCs)from human bone marrow in vitro.Methods Mononuclear cells were collected by density gradient centrifugation from the bone marrow of 24 patients with intervertebral disc degeneration.The isolated cells were cultivated in dishes coated with fibronectin and induced by angiogenic growth factors. Immuno-fluorescence staining and flow cytometry were used to indentify EPCs.The differentiation of EPCs to endothelial cells(ECs)was determined by transmission electron microscope(TEM)and in vitro angiogenesis. Results Cell colony-forming units appeared 72 hours after seeding and increased obviously after 5 days.One week later the cells confluenced to 80%.Attached cells formed cobblestone like structure by 14 days.The uptake rate of Dil-ac-LDL and FITC-UEA-I was(95.1±4.0)%.Flow cytometric analysis showed that positive rate of CD133,CD34,KDR,and VE-Cadherin was(18.5±4.4)%,(45.4±7.8)%,(66.7±7.2)%,and(20.5±5.3)%, respectively.Weibel-Palade body was observed in endochylema under TEM.The cells cultivated for 7 days to 12 days formed tube-like structure on ECMatrix.Conclusions EPCs could be efficiently isolated by density gradient centrifugation combined with induction by EGM-2 medium from human bone marrow.EPCs had angiogenic potential between 7 days to 12 days.