Isolation, cultivation and identification of endothelial progenitor cells from rat bone marrow

Objective To investigate the methods for isolating, identifying and culturing endothelial progenitor ceUs (EPCs) from rat bone marrow, as well as the differentiation of EPCs into endothelial cells.Methods The mononuclear cells were isolated from rat femoral and tibial bone marrow using percoll density gradient eentrifugafion, then plated on dishes coated with fibronectin, and induced with vascular endothlial growth factor (VEGF), basic fibroblast growth factor(bFGF) and epidermal growth factor (EGF) for two weeks.The expression of cell markers was assessed by immunocytochemistry, and the attached cells were stained with Dil-ac-LDL and FITC-UEA-1.Results The attached EPCs were able to line up in the typical cord-like structure and formed clusters and cobblestone.Adherent cells showed CD34, Flk-1, CD133, vWF positive in different time and took up Dil-acLDL, bound FITC-UEA-1 double positive fluorescence by laser confocal microscopy.On day 2 of induced differentiation, CD133 positive rate was (79.4±4.5)%.Flk and vWF showed high level of expression in (74.2±3.5)% and (72.2±4.3)% of the cells respectively from day 6.Conclusions The mononuclear cells can be isolated from rat bone marrow by percoll density gradient eentrifugation.After induction of VEGF, bFGF and EGF, highly purified EPCs are obtained with the same characteristics of endothelial cells.EPCs can be differentiated into endothelial cells in vitro. Key words: Bone marrow cells;  Endothelial calls;  Stem cells;  Differentiation;  Induction