Isolation, culture and characterization of endothelial progenitor cells from the human peripheral blood

Qiao Wei; Ran Feng; Liu Chang-jian

doi:10.3969/j.issn.2095-4344.2013.36.020

Abstract：BACKGROUND:Endothelial progenitor cel s, known as the precursor cel s of mature endothelial cel s, have the function of neovascularization and neoendothelialization. Therefore, endothelial progenitor cel s have potential applicability in many fields. Endothelial progenitor cel s can be isolated and cultured from different resources with different methods, but the biological properties and identification of endothelial progenitor cel s stil have controversies. <br> OBJECTIVE:To explore the methods of isolation and culture of endothelial progenitor cel s from the human peripheral blood and to identify the biological features of endothelial progenitor cel s. <br> METHODS:Mononuclear cel s were isolated from the human peripheral blood using density gradient centrifugation, and the cel s were resuspended in endothelial basal medium-2 supplemented with the EGM-2-MV-SingleQuots. Then, the cel s were inoculated in human fibronectin-coated culture flasks and cultured in EBM-2MV medium. The morphology of endothelial progenitor cel s was observed. The proliferation potential and surface markers of endothelial progenitor cel s were characterized careful y. Furthermore, the functional properties such as nitric oxide release and tube formation on Matrigel were also evaluated. <br> RESULTS AND CONCLUSION:While adherent cel s maintained, spindle-shaped cel s formed a cel cluster after 6-7 days. Then, adherent cel s developed to endothelial progenitor cel s with a cobblestone appearance after 2-3 weeks. The endothelial progenitor cel s were confluent with an outgrowth appearance. Endothelial progenitor cel s had a higher proliferation potential compared with human aortic endothelial cel s under the same culture condition. Endothelial progenitor cel s expressed CD31, CD34, CD144 and KDR, displaying an obvious endothelial phenotype. Endothelial progenitor cel s were also found to uptake DiL-acLDL and exhibit lectin binding capability. Furthermore, endothelial progenitor cel s were able to form capil ary tubes on Matrigel and had the ability to release nitric oxide. Therefore, endothelial progenitor cel s can be obtained from the human peripheral blood by density gradient centrifugation and adherent culture. A combining method for the identification of endothelial progenitor cel s should be recommended.