Inducement In Vitro And Culture Of Endothelial Progenitor Cells From Human Peripheral Blood

During embryogenesis , endothelial progenitor cells ( EPCs ) participate in the initial processes of primitive blood vessel formation (vasculogenesis) .It has become evident that progenitors to vascular endothelial cells (ECs) also exist in the adult. EPCs normally reside in the adult bone marrow but may become mobilized into circulation by cytokine or angiogenic growth factor signals, migrate to the sites of physiological or pathological and differentiate into endothelial cells in situ, and contribute to neovascularization (adult vasculogenesis) .At present, post-natal vascularization is a hotspot of clinical investigation. Autologous endothelial progenitors, mobilized in situ or transplanted, has become a major target of therapeutic revascularization approaches. Moreover, endothelial progenitors represent a potential target of strategies to block tumor growth. To find out the best condition for isolation, culture and identification of EPCs, thus becomesthe major problem before any practical clinical application of this technique.The present aim is to study the isolation, inducement , differentiation, culture and identification of endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells (PMC), and to probe into its biological activity in vitro for the angiogenesis therapy.Two methods are used to obtain the PMC, the first is from the whole blood and the second is the enriched MNC. After isolation, freshly isolated PMCs were plated at 1.0×10~6 cells per wells on 24 well culture dishes in EGM-2, Which contains VEGF, bFGF , IGF, EGF and in EBM which don't containthe above. After 3 days, the adherent cells were removed and fresh culture medium was applied. At the same time, the adherent cells were incubated with Dil-AOLDL and UEA-1. After staining, the samples were abserved using fluorescent microscope and further demonstrated by laser scaning confocal microscope. On the seventh day, surface markers were assessed by FACS analysis.The second method decreased the interference from erythrocyts. Double positive staining for Dil-AC-LDL and UEA-1 cab be observed. After culture for 7 days , CD34+, CD31+, KDR+ cells were 2. 79%, 6. 27%, 31. 24% respectively, while the CD45 were not expressed.In conclusion: EPCs exist in the peripheral blood, and can be differentiated into mature ECs by the stimulation of VEGF, bFGF, IGF and EGF.