Culture and Surface Markers Identification of Endothelial Progenitor Cells from Mice Bone Morrow

Objective To establish an effective method of separation,culture and surface markers identification of endothelial progenitor cells(EPC) from mice bone marrow.Methods Bone marrow-derived mononuclear cells isolated by density gradient centrifugation were cultured in EGM-2 medium.Cell proliferation was observed in different periods.DIL-AC-LDL and FITC-UEA-1 double fluorescent staining and flow cytometry were performed after 7days′ culture.RT-PCR analysis of the surface markers were also performed on 0,4,7 days.Results After induced culture for 72 hours,bone marrow mononuclear cells almost attached,and the cells could be passed when attached 70% to 80% on 8-9 days.After 2-3 weeks,the morphology showed a typical cobblestone-like appearance.On 7 day,the percentage of DIL-AC-LDL and FITC-UEA-1 double staining positive cells was 90.16±6.77%,and flow cytometry indicated the percentage of Flk-1+CD133+CD34+ cells was 1.73±0.27%.RT-PCR results revealed the expressions of CD34,CD117 and CD133 decreased On 0,4,7 days,while those of endothelium markers such as CD31,FLK-1 and VE-Cad increased gradually.Conclusion The combination of density gradient centrifugation and induced culture can obtain a great number of EPC.The ideal identification method should be based on the flow cytometry,and combined with cell morphology and function assessment.