Inducing bone marrow mesenchymal stem cells to differentiate into cardiomyocyte-like cells via Wnt-11 in vitro
10.7652/jdyxb201702009
- VernacularTitle:不同浓度Wnt-11诱导骨髓间充质干细胞向心肌样细胞定向分化的实验研究
- Author:
Yuan LIU
;
Haiping WANG
;
Yang Lü
;
Haoyu WANG
;
Yanli HUO
- Keywords:
bone marrow mesenchymal stem cell;
Wnt-11;
cardiomyocyte-like cell;
cell differentiation
- From:
Journal of Xi'an Jiaotong University(Medical Sciences)
2017;38(2):199-205
- CountryChina
- Language:Chinese
-
Abstract:
Objective To probe into the optimal concentration of Wnt-11 to induce the differentiation of bone marrow mesenchymal stem cells (BMMSCs)into cardiomyocyte-like cells in vitro.Methods BMMSCs were isolated from the bone marrow of SD rats using whole bone marrow culture method.After cultured for 48 h, BMMSCs of the second generation were utilized for directed induction.Based on the final concentration of Wnt-11 , BMMSCs were divided up into Group A (100 ng/mL),Group B (200 ng/mL),Group C (400 ng/mL)and Group D (blank control).After 72-hour induction,the cells were cultured in complete medium for 4 weeks while cells in Group D were cultured only in the complete medium.The morphological changes were observed under the phase contrast microscope.Surface antigen expression of BMMSCs was identified by flow cytometry.When cells were cultured for 4 weeks,the expressions of Desmin,Connexin43 and cTnI were detected by immunocytochemistry. Meanwhile, the ultrastructural changes were observed using transmission electron microscope. The mRNA expressions of cardiac transcription factors GATA-4,Nkx2.5 andα-MHC in BMMSCs were detected by RT-qPCR at 1,2 and 4 weeks after induction.Results Primary BMMSCs formed cell colonies at 2 weeks;the cells were mainly fusiform or star-shape,and a few irregularly-shaped ones were also visible.The passaged cells were larger than those of primary culture.After induction,the cells exhibited long shuttle-shape and were aligned in parallel. Flow cytometery displayed that the positive rate of the surface antigens of BMMSCs CD29,CD45,and CD90 was 97.9%,0.4% and 99.5%,respectively.When BMMSCs-induced via Wnt-11 were cultured for 4 weeks,Desmin, cTnI and Connexin43 were all positively expressed in induction groups.Whereas in the blank control group they were slightly positive or negative;the positive rate in Group B was the highest (P<0 .05 ).Transmission electron microscopy exhibited that organelles such as rough endoplasmic reticulum,mitochondria,as well as some ribosomes were visible in the cytoplasm of these cells in each induction group.In addition,myofilaments were arranged in parallel in the cytoplasm.The cells in induction groups could express GATA-4 and Nkx2 .5 in the first week,and then the expression of them decreased in the second week,but then increased in the fourth week;gene expression in induction Group B was significantly higher than in the other two induction groups (P<0 .05 ).The expression of GATA-4 and Nkx2 .5 in Group D was 1 ,α-MHC was not expressed in the four groups during the culture period. Conclusion Wnt-11 can induce the differentiation of BMMSCs into cardiomyocyte-like cells in vitro,and the optimal concentration of Wnt-11 is 200 ng/mL.
