Effects Of Apelin13 On Efficiency Of Hescs Differentiating Into Myocardial Cells

The First Part The Experimental Study of Human Embryonic Stem Cells Differentiation into Cardiomyocytes in VitroObjective: In this study,embryonic stem cell H9 cell line was used to differ human ESCs into cardiomyocytes,which laid the experimental model and theoretical basis for subsequent experiments.And during the propose,differentiated medium with definite chemical composition was used.Methods: Human ESCs were resuscitated and inoculated onto Matrigel containing growth factors.ESCs were cultured with E8 medium.When the cell confluence reached90%,the medium was changed to CDM3 inducing differentiation medium and the cell was cultured continuously for 7 days until the beating of cardiomyocytes was observed.The cell status was observed during induction and c TNNT2,a marker of cardiomyocyte maturation,was detected by immunofluorescence.Result: The number of beating cells gradually increased on the 7th day and reached the peak on the 14 th day.Immunofluorescence showed that the cardiac myocyte specific marker c TNNT2 of the beating cells was positive,and the sarcomere and intercalated disc structures were observed.Conclusion: The chemically defined CDM3 differentiation medium successfully differed human ESCs into cardiomyocytes.The Second Part Effects of Apelin13 On Efficiency of hESCs Differentiating into Myocardial CellsObjective: 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 with100 n M Apelin13(experimental group)or without Apelin13(control group).On day 2,4,and 7 of induction,real-time fluorescent quantitative PCR(q RT-PCR)was performed to detect the m RNA 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 Blot.Furthermore,the expression of TNNT2 and ?-Actinin at the transcription and protein levels in the differentiated myocardial cells after 7 days of induction was etermined by fluorescent quantitative PCR and immunofluorescence,respectively.Results: During the differentiation process,the introduction of Apelin13 promoted h ESC differentiation,most significantly on day 7.Compared with the control group,the q RT-PCR showed that Apelin13 significantly increased the m RNA levels of Brachury T and Mesp1 after 2 days of induced differentiation,and Nkx2.5 and APJ after 7 days of differentiation.Western Blot 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 c?lture also showed obviously positive expression of TNNT2 and?-Actinin.Furthermore,the q RT-PCR demonstrated that Apelin13-treated cells showed much higher levels of TNNT2 and ?-Actinin after 7 days of induced differentiation(allP <0.05).Conclusion: Apelin13 can improve the differentiation efficiency of hESCs into myocardial cells.