Isolation,culture and identification of mesenchymal stem cells and endothelial progenitor cells from murine bone marrow

AIM:To establish an efficient and stable method for simultaneous isolation,culture and differentiation of murine endothelial progenitor cells(EPCs)from bone marrow.METHODS:Mononuclear cells were isolated from murine bone marrow by density gradient(Histopaque-1077,Sigma)after differential centrifugation.The resulting cells were cultured and differentiated to endothelial cells(ECs)in EBM-2.The expressions of specific antigens(CD133,CD34,Flk-1 and CD31)on cell surface were analyzed by immunofluorescence and flow cytometer.Biological functions of endothelial cells were examined by the adsorption of ulex europaeus-agglutinin(UEA)labeled by fluorescein isothiocyanate(FITC)and DiI-ac-LDL internalization.Expression of vWF and CD31 of ECs differentiated from EPCs was also assessed by immunohistochemistry.RESULTS:Cells were obtained from mouse bone marrow by density gradient,and adhesive cells expressed CD133,CD34 and Flk-1 and formed clusters at the 4th day.At the 14th day,UEA adsorption was labeled by FITC and DiI-ac-LDL internalization were positive.Expression of CD133 in adhesive cells decreased,whereas CD31 increased.Positive ratios of CD34+,CD133+,Flk-1+ and CD31+ were(43.55±4.12)%,(18.29±2.56)%,(48.78±3.96)% and(78.89±6.38)%,respectively.After 3 weeks,they differentiated into mature ECs forming cobblestone monolayers.There was no difference in secretion of PGI2 between ECs from EPCs and ECs from mouse aorta.CONCLUSION:An efficient,stable and replicable method for simultaneous isolation and culture of MSCs and EPCs from a single mouse has been established.