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

doi:10.3969/j.issn.2095-4344.2013.36.020

Abstract

Abstract:

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

Key words: stem cells, endothelial cells, monocytes, cell culture techniques, nitric oxide

CLC Number:

R394.2

Qiao Wei, Ran Feng, Liu Chang-jian. Isolation, culture and characterization of endothelial progenitor cells from the human peripheral blood[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(36): 6508-6514.

Figures/Tables 4

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