| Background:Heart disease remains the leading cause of morbidity and mortality worldwide.Because of the difficultly of obtaining cardiac myocytes,cell replacement therapy is a significant way for the treatment of cardiovascular diseases in the future.Therefore,the source and quality of cardiac myocytes will be particularly crucial.The emergence of technologies of Induced pluripotent stem cells(iPS)and in vitro cardicmyocytes differentiation(iPS-CM),brings new prospect for the study of cardiovascular disease,cardiac toxicity,cell treatment of myocardial injury and regeneration medicine.Although abudant of researches have been carried out to develop methods of inducing human induced pluripotent stem cells(hiPS)differentiate into cardiomyocytes(CM)in vitro,there are still lots of problems such as unstable,complicated and expensive and so on.At the same time,hiPS-CM obtained by differentiation from hiPS is a mixed population of various subtypes of cardiomyocytes,so it is necessary to establish a single cell culture system of hiPS-CM to facilitate the researches and applications of phenotypic analysis,single cell sequencing,cell therapy and so on.Methods:1.In this study,alkaline phosphatase staining,RT-PCR,immunofluorescence staining and Flow cytometry was performed to identify the pluripotance of human induced pluripotent stem cells(hiPS).On this basis,a monolayer-based directed differentiation platform was used to differentiate hiPS into cardiomyocytes(CM).Optimizing combination of the dose and time of CHIR99021 and IWP2 inhibitors,to achieve a population of mature,autonomic rhythmically and high purity CM.2.Different concentrations of the ROCK kinase inhibitor Y27632 were added into the culture system of hiPS-CM to determine the optimal concentration.The effects of Y27632 on the activity of hiPS-CM were detected by Immunofluorescence,Crystal violet staining and CCK-8 assay.The effect of Y27632 on the proliferation of hiPS-CM was examined by Brdu assay.The effects of Y27632 on the apoptosis of hiPS-CM were determined by Flow cytometry,Real time quantitative PCR and Western blot.3.A hiPS-derived cardiomyocyte injury model was established by doxorubicin(DOX).The effect of different concentrations of doxorubicin on hiPS-CM for 24 h was detected by CCK-8 assay.The expression of γ-H2AX,a DNA damage marker,was detected by immunofluorescence method.The submicroscopic structure of the cells was observed by TEM.The cells were infected with adenovirus of mCherry-GFP-LC3B fusion protein to detect the autophagy level The expression of proteins related by DNA damage,apoptosis and autophagy were detected by Western blotResults:1.Alkaline phosphatase staining of hiPS was positive.The result of RT-PCR and Immunofluorescence chemistry showed that the pluripotent markers were highly expressed in hiPS.After about 8 days in vitro differetation,beating cardiomyocytes were appeared and sheets of monolayer CM were formed about 10 days later.Differentiation for 30 days cells were used for identification.The result of RT-PCR showed that TNNT2,TNNI3 MYL7 and MYL2 were highly expressed in hiPS-CM and the result of Immunofluorescence chemistry showed that cTNT and a-actinin were expressed in hiPS-CM.Flow cytometry showed that the expression of cTNT was 96.79%.In this study,a stable and high-purity population of hiPS-CM was achieved by a monolayer based direct differentiation.2.Different concentrations of the ROCK kinase inhibitor Y27632 significantly increased the cell viability of hiPS-CM.With the increase of Y27632 concentration,the cell viability decreased slightly.To determine the effect of Y27632 on hiPS-CM,the expression of cTNT was detected by immunofluorescence staining.The results showed that the number of cTNT positive cells was significantly higher than that of the control group after treatment with 10 μM Y27632 for 24 h The results of Crystal violet staining and CCK-8 assay showed that the cell viability was significantly increased after Y27632 treatment.On day 3,the means of BrdU+ cells counted in fluorescent images were 4.88±0.07%with Y27632 treatment and 2.12±0.02%without Y27632 treatment(P<0.0001).After 6 days,the proliferation rate of was 5.7±0.12%with Y27632 treatment,compared to 3.34±0.01%without Y27632 treatment(P<0.0001).In a word,the ROCK kinase inhibitor Y27632 promoted the proliferation of hiPS-CM.The apoptosis rate was 10±0.75%with 10 μM Y27632 treatment,compared 22.5±2.6%without Y27632 treatment(P<0.0001).The results showed that ROCK kinase inhibitor Y27632 significantly reduced the apoptosis rate of hiPS-CM.In order to determine underlying mechanism of apoptosis,the apoptosis-related genes and proteins were detected by RT-PCR,qPCR and Western blot.The result of RT-PCR and qPCR showed that the expressions of Caspase-3 and Caspase-8 were significantly down-regulated after Y27632 treatment(P<0.001),while the expressions of Bax and Bcl-2 were not significantly(P>0.05).The result of Western blot showed that the expressions of Cleaved Caspase-3 and Caspase-8 were significantly down-regulated after Y27632 treatment(P<0.001).The result of Caspase-3 activity assay further showed that the activity of Caspase 3 was decreased after Y27632 treatment(P<0.01).Y27632 inhibited the apoptosis of hiPS-CM by reducing the expression level of the apoptotic protein Caspase 3.3.Doxorubicin significantly reduced the cell viability of hiPS-CM,which was positively correlated with the concentration.The cell vibility of hiPS-CM was significantly decreased after treatment with 0.1 μM DOX for 24 h.Low doses of doxorubicin(0.5 μM)treatment for 24 h induced DNA damage in cardiomyocytes and expressed the DNA damage marker y-H2AX.The results of TEM showed that DOX induced the increase of hiPS-CM autophagosome,caused nuclear rupture and shrinkage,and promoted apoptosis.After infection with adenovirus of mCherry-GFP-LC3B,bright yellow fluorescence and red spots were appeared in the DOX group.Western blot results showed that the expression levels of p53 and p21 protein increased with the increase of DOX concentration.The expression of Bax increased,and the anti-apoptotic protein Bcl-2 decreased,and the expression of LC3-II also increased.Therefore,DOX induced DNA damage,autophagy and apoptosis of hiPS-CM.Conclusion:In this study,an optimized hiPS-CM differentiation method was established by optimizing the dose and time of CHIR99021 and IWP2 inhibitors.The culture system of hiPS-CM was improved by the ROCK kinase inhibitor Y27632.Furthermore,DOX was used to establish the injury model of hiPS-CM.In a word,the present study established a foundation for the application of cardiotoxicity research,drug screening and cardiac regenerative medicine. |
