Effects of Apelin13 on efficiency of hESCs differentiating into myocardial cells

CHENG Tao; CHEN Yu; ZHANG Ningkun; GAO Lianru; WANG Ze; ZHANG Yan

doi:10.3969/j.issn.1002-266X.2017.33.001

Abstract：Objective To explore the effects of Apelin13 on the differentiation of human embryonic stems cells (hESCs) into myocardial cells.Methods Feeder-free cultured hESCs were passaged into Matrigel-coated plates after being dissociated by EDTA.The cells were cultured in the differentiation medium with 100 nM Apelin13 (experimental group) or without Apelin13 (control group).On day 2, 4, and 7 of induction, real-time fluorescent quantitative PCR (qRT-PCR)was performed to detect the mRNA levels of the myocardial marker genes, including Brachyury T, Mesp1, Nkx2.5, and APJ.Meanwhile, the protein expression levels of Brachyury T, Mesp1, Nkx2.5, APJ, Nanog, and Sox2 were determined by Western blotting.Furthermore, the expression of TNNT2 and α-actinin at the transcription and protein levels in the differentiated myocardial cells after 7 days of induction was determined by fluorescent quantitative PCR and immunofluorescence, respectively.Results During the differentiation process, the introduction of Apelin13 promoted hESC differentiation, most significantly on day 7.Compared with the control group, the qRT-PCR showed that Apelin13 significantly increased the mRNA levels of Brachury T and Mesp1 after 2 days of induced differentiation, and Nkx2.5 and APJ after 7 days of differentiation.Western blotting showed the similar results with increased expression of Brachury T, Mesp1, Nkx2.5, and APJ after differentiation for 2, 4, and 7 days, and significant decrease in the expression of Nanog and Sox2.The differentiated myocardial cells after 7 days of culture also showed obviously positive expression of TNNT2 and α-actinin.Furthermore, the qRT-PCR demonstrated that Apelin13-treated cells showed much higher levels of TNNT2 and α-actinin after 7 days of induced differentiation (all P<0.05).Conclusion Apelin13 can improve the differentiation efficiency of hESCs into myocardial cells.