Inducing pluripotent stem cells has been considered as a promising treatment for ischemic heart disease. However, an ideal inducing method has not been found yet.OBJECTIVE:
To investigate the role of sodium-calcium exchanger (NCX1) promoter in the differentiation of mouse induced pluriptent stem cells (miPS) into cardiomyocytes.METHODS:
The pLVX-IRES-ZsGreen1 vectors which contain NCX1 promoter constructed by recombinant DNA technology were co-transfected to 293FT cells with ViraPowerTM Lentiviral Packaging Mix. The recombinant lentiviruses infected with miPS were selected and purified by puromycin. miPS were recovered and passaged to form embryoid bodies. The embryoid bodies were induced by differentiation medium containing various concentrations of the virus titer. The number of beating embryoid bodies were calculated. The expression profiles of the myocardial intra-markers were tested to determine the differentiation efficiency of iPSC by RT-PCR and immunofluorescence analysis.RESULTS AND CONCLUSION:
pLVX-IRES-ZsGreen1 vectors which contain NCX1 promoter were constructed and confirmed by PCR. Virus could be packaged, purified and concentrated successfully. The recombinant lentivirus to transduce miPS was sorted by flow cytometry. In contrast to NCX1 -/GFP- cells, NCX1+/GFP+ cells were differentiated and developed prominent beating areas with sustained contractile activity for additional 4 days, and demonstrated positive expression of gap communication marker CX43 and cardiac troponin. The expressions of GATA4, MEF2c and Nkx2.5 in the NCX1 + cells were 4.2, 7.5, and 2.5 times those in NCX1 - cells. Results showed the NCX1 promoter can promote the cardiac differentiation of miPS.Funding:
the National Natural Science Foundation of China, No. 81270189; the Natural Science Foundation of Guangdong Province, No. S2011040000852Funding:
Dai B, Li HT, Liu Z, Xiao DZ, Yu XY, Chen B, He JX, Xiang DC, Qiu J, Wang YG. Sodium-calcium exchanger promoter promotes differentiation of mouse induced pluripotent stem cells into cardiomyocytes in vitro. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(19):3069-3074.