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Isolation, cultivation and identification of mesenchymal stem cells and endothelial progenitor cells from murine bone marrow with a modified differential adhesion method
FENG Wen-lei ZHANG Meng YIN Shuang-hong XU Fang-jie WANG Yan-jie CHEN Xue-ling WU Xiang-wei*
Acta Anatomica Sinica ›› 2015, Vol. 46 ›› Issue (2) : 282-288.
Isolation, cultivation and identification of mesenchymal stem cells and endothelial progenitor cells from murine bone marrow with a modified differential adhesion method
Objective To establish a method for simultaneously isolating, culturing and identifing of murine mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) from bone marrow. Methods The cells were isolated by a modified differential adhesion method from murine bone marrow and cultured for 48 hours. The primary adherent cells at 48 hours were cultured in LG-DMEM and the non-adhered cells were collected and induced by EGM-2MV complete medium in human fibronectin-coated dishes. Osteogenic, chondrogenic,and adipogenic induced multi-directional differentiation potentials were performed on the primary adherent cells, their immune phenotypes were detected by flow cytometry (FCM).Tube formation experiment on the matrigelin vitro and the expression of specific surface marker CD31 determined by immunofluoresence cell staining were identified for the subsequent adherent cells, and their immune phenotypes were detected by FCM.
Results The passage 3 of the primary adherent cells were identified by induced differentiation into osteoblasts, adipocytes and chondrocytes after induction. The expression levels of Sca-1, CD29, CD45, and CD11b were (98.30±0.75)%, (97.47±1.32 )% , (1.87±0.15)% and (1.03±0.71)% respectively. The passage 3 of the subsequent adherent cells were cultured on Matrigel, which resulted in the formation of tube-like structures.The expression of the passage 5 of the subsequent adherent cells specific surface marker CD31 was positive. The expression levels of CD34, CD133 and vascular endothelial growth factor receptor(VEGFR)2 were (88.90±1.18 )% , (92.73±2.90)%, and (87.63±1.79 )% respectively. Conclusions The modified differential adhesion is an efficient, stable, and replicable method that can simultaneously isolate and amplify mouse bone marrow MSCs and EPCs.
Mesenchymal stem cells / Endothelial progenitor cells / Bone marrow / Modified differential adhesion method / Cell culture / Flow cytometry / Mouse
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