In vitro differentiation into endothelial-like cells from bone marrow mesenchymal stem calls in diabetic rats

Abstract

Objective To investigate the methods for separation and culture of bone marrow mesenchymal stem cells (BMSCs) in vitro in diabetic rats and the capability of differentiation into endotheliallike cells.Methods Rat diabetic model (DM) was induced by injection of stretopzin (STZ) in SD rats.After 12 weeks of DM duration,BMSCs were separated from bone marrow with density gradient centrifugation,wall sticking screening and amplified in vitro.The surface markers of BMSCs were evaluated with fluorescence-activated cell sorter (FACS).BMSCs of the third passage were obtained and divided into two groups.In the induction group,the cells were induced to differentiate in M199 medium containing 20％ fatal bovine serun ( FBS),vascular endothelial growth factor ( VEGF,10 μg/L) and basic fibroblast growth factor (bFGF,2 μg/L).No cytokine was added in the blank control group.Two weeks later,the cell morphology was observed under the phase contrast microscopy.The expression levels of VEGFR-2 were detected by using immunohistochemistry.EPC-specific marker CD34 was evaluated with FACS,and release of nitrous oxide (NO) from culture cells was also detected by NO detection reagent.Weibel-Palade (W-P) bodies in the cytoplasm were observed under the transmission electron microscopy.Results Rat diabetic model in SD rats that were injected with STZ intraperitoneally was established.Cultured BMSCs at the third passage were positive for CD44 and CD90 [ (97.8 ± 0.9 ) ％ and (96.8 ± 1.4) ％,respectively ],but negative for CD11 b/c and CD34 [ ( 13.2 ± 0.6 ) ％ and ( 1.2 ± 0.5 ) ％,respectively ].On the 14 day in the induction group,the cell morphology observed under the phase contrast microscopy was not distinctly different.Differentiation antigens VEGFR2 and CD34 in the induction group were higher [ (97.1 ± 1.0) ％,(65.0 ± 3.9) ％ ] than in the blank control group [ (7.0 ± 1.0 ) ％,( 0.9 ± 0.3 ) ％ ] ( P ＜ 0.05 ).The content of nitrous oxide in the induction group was also obviously increased as compared with the blank control group [ (94.14 ± 3.25 ) vs.( 70.37 ± 2.1 0) μmol/L,P ＜ 0.05 ).W-P bodies in the cytoplasm of the cultured cells were not found in the two groups.Conclusion BMSCs in diabetic rats can be isolated by density gradient centrifugation and adherent culture.VEGF and bFGF can induce differentiation of BMSCs in diabetic rats into endothelial-like cells in vitro.It is suggested that BMSCs may be used as a new type of seed cells for the diabetic lower limb ischemia disease. Key words: Diabetic; Bone marrow mesenchymal cell; Induction differentiation; Endothelial-like cell