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Objective To obtain myoclonic epilepsy with ragged-red fibers (MERRF)-specific cardiomyocytes by the differentiation in vitro of inducing pluripotent stem cells (iPSC) derived from a MERRF patient and evaluate the application of the prepared cardiomyocytes in construction of MERRF syndrome model.Methods The patient-derived iPSCs and H9 embryonic stem (ES) cells,the control cell line,were unidirectionally differentiated into cardiomyocytes n in vitro.The obtained cardiomyocytes were identified and validated by detecting the presence of cardiomyocyte-specific markers using immunofluorescence staining and RT-PCR.The beating frequencies were recorded to compare the functional evaluation for the two groups of cardiomyocyte.Results Both the patient-derived iPSC and H9 ES cells were differentiated into cardiomyocytes successfully.The average beating frequencies of MERRF-induced cardiomyocytes (iCMs) were 13,24,15 and 18 times/min on the day 10,13,15 and 16 during the cell differentiation process.The average beating frequencies of H9-iCMs were 80,96,120 and 120 times/min,respectively.The beating ability of iPSC-differentiated cardiomyocytes was significantly lower than that of corresponding control (all P ＜ 0.05).Conclusion The patient-derived iPSCs may differentiated into cardiomyocytes.Based on the functional evaluation for these cardiomyocytes,the model for MERRF syndrome with mitochondrial mutations was generated and characterized in vitro.
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Objective To identify the rale of NKX2-5 gene in cardiomyocyte differentiation and its mechanism.Methods P19 cells were divided into transfected and non-transfected groups.In the transfected group,P19 cells were with stable expression of NKX2-5 gene.The P19 cells were cultured in suspension for 4 days,and the formed aggregates were transferred to Petri dish for adherent culture.On days 4,8,12,and 16 of the adherent culture,the expressions of ct-saicomeric actin(α-SA)and cardiac troponin T(cTnT)were detected with double-labeling immunofluorescence and Western blot.The ultrastruetural changes were observed on day 16.Results In the transfected group,no expression of α-SA and cTnT was found on day 4,and the expression of these 2 proteins or co-expression existed on days 8,12,and 16.There were early cell junction and myofilament-like structure in the cytoplasm of some cells in the transfected group.In the non-transfected group,these 2 proteins were negative,and no differentiated cell was found.Conclusion Stable expression of NKX2-5 gene can induce cardiomyocyte differentiation from P19 cells,but the P19 cells with stable expression of JVKX2-5 gene is not suitable to be an in vitro model of cardiac development.
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@# ObjectiveTo study the differentiation of adipose tissue-derived mesenchymal stem cells (ADMSCs) into cardiomyocytes in vitro. MethodsADMSCs were isolated and purified by the method of digesting and adhering to the culture plastis. The third generation of cells were determined with immunocytochemistry method and induced by 5-aza. On the 7th, 14th, 21st, 28th day after being induced, the surface antigen of myoblast cell and cardiac myocyte were determined, the expression of gene of GATA4 and Nkx2.5 were tested, and the content of atrial natriuretic polypeptide (ANP) assayed. ResultsImmunocytochemical staining showed CD44, CD13, CD105 positive, CD45, CD34, HLA-DR, factor Ⅷ negative. After being induced by 5-aza, the direction of cell arraying was gradually similar. The cells were stained positively for Desmin, α-Sarcomeric Actin, myosin heavy chain and Troponin T. The cells express GATA4 and Nkx2.5 and secrete ANP. ConclusionThere was mesenchymal stem cells in human adipose tissue. 5-aza may induce ADMSCs to differentiate into cardiomyocytes in vitro.
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BACKGROUND AND OBJECTIVE: AT-1 cells have been derived from the left atrial tissue in which the ANF promoter targeted SV40 large T antigen expression. When cultured, clusters of spontaneously contracting cells were observed after 4-5 days and contiguous sheets of synchronously beating cardiomyocytes were formed after 10 days. In this study, expression of several cell cycle regulatory genes were monitored through Northern blot analyses in AT-1 cells during beating and after formation of beating sheets (BS). MATERIALS AND METHOD: AT-1 RNAs were obtained in 3 days after plating, during beating and after formation of BS, and used for Northern blot analyses. RESULTS: alpha-Cardiac myosin heavy chain expression was prominent in beating cells, as would be expected for this contractile protein isoform but ANF was decreased after beating. Gax was not expressed in cultured AT-1 cells but in AT-1 tumor and murine heart. p53 and p21 were decreased after beating which indicate transcription level of p53 and p21 correlated well in AT-1 cells. In contrast, pRB and p107 were increased after beating but p68 (2.4 kb) which arose by alternative splicing of p107 and lacks the pocket domain B was decreased in beating cells. pTCS2, murine tuberous sclerosis gene, represented similar levels during beating but a little was decreased after formation of BS. mRAD50, the murine homologue of yeast DNA recombinational repair gene RAD50, was increased in beating cells, a similar pattern to p107 and pRB. But the p50 arose by alternative splicing of mRAD50 and has 3' half of mRAD50 had unexpectedly appeared and maintained after beating. CONCLUSION: The expression of cell cycle regulatory genes after beating and formation of BS in AT-1 cells showed gene-specific pattern and the p50 which has homology to the mRAD50 may participate in differentiation of cardiomyocytes.