The Effect Of T3 On The Maturation Of Human Induced Pluripotent Stem Cell- Cardiomyocytes

Background Cardiac death caused by acute myocardial infarction has become one of the leading causes of death worldwide.Due to limited ability of self-regeneration,the necrotic cardiomyocytes are replaced by fibrous scar tissue which results in cardiac remodeling and ventricular mechanical dysfunction,and often finally culminates in irreversible congestive heart failure.Therefore,how to increase the cardiomyocyte numbers and improve function of the infarcted area is the key problem in the treatment of ischemic heart disease.In recent years,hi PSCs(human induced pluripotent stem cells)have successfully been differentiated into cardiomyocytes,which provides a potential cell source for stem cells transplantation therapy of ischemic heart disease.However,the function of cardiomyocytes derived from induced pluripotent stem cells(induced pluripotent stem cell-Cardiomyocytes,i PSC-CMs)is often not perfect.Fully mature i PSC-CMs can better reflected cardiomyocyte physiology,establish more effective disease model and drug screening system,obtain better therapeutic effect of cell transplantation and reduce the risk of arrhythmia.The research about thyroid hormone T3 used to promote i PSC-CMs mature is still only a few now,and there is no related research report about T3 hi PSC-CMs mature in the period of inducing differentiation from day o to day 7.At present,there are few studies on the effect of thyroid hormone T3 on hi PSC-CMs maturation,especially the effect of thyroid hormone T3 on hi PSC-CMs differentiation.ObjectivesThis study was aimed to explore the feasibility of thyroid hormone T3 used to promote hi PSC-CMs maturation in the period of inducing differentiation.Methods 1.Draw venous blood from a healthy adult,then isolate and culture peripheral blood mononuclear cells by gradient centrifugation.The Cyto Tune?-i PS Sendai Reprogramming Kit was used to derive i PSCs by transfecting exogenous transcription factors into PBMCs.IPSCs were analyzed and identified by the following identification method: alkaline phosphatase living cells staining,immunofluorescence staining,embryoid bodies forming experiment.2.Inducing hi PSCs differentiate into cardiomyocytes by chemically defined methods and hi PSC-CMs were analyzed and identified by the following identification method: morphological observation,the expressions of Nkx2.5,c Tn T and MLC2 v by immunofluorescence staining.3.Experiment groups were designed into control group and thyroid hormone T3 treatment in the period of inducing differentiation.On the 7th day,the following indicators such as the express of Nkx2.5,c Tn T,MLC2 v,cell cycle,mitochondria volumes and ultrastructures of cells were analyzed.Results1.Typical clones were obtained at 14 days after transfecting exogenous transcription factors into PBMCs.Positive expressions of alkaline phosphatase living cells staining and Nkx2.5,c Tn T and MLC2 v by immunofluorescence staining were observed.Embryoid bodies were formed from hi PSCs by suspension culture.2.Hi PSCs were differentiated successfully into cardiomyocytes with the morphological changes from clones into Polygon or fusiform.The indicators by immunofluorescence staining such as Nkx2.5,c Tn T and MLC2 v were positive.3.There was no significant difference in the expression levels of Nkx2.5,c Tn T and MLC2 v by immunofluorescence staining between T3 treatment group and control group.Flow cytometry detected G1/G0 was increased in T3 treatment group which meant the decrease of cell proliferation ability,and mitochondrial volumes of T3treatment group were increased significantly.Transmission electron microscopy showed that hi PSCs-CMs of T3 treatment group had more glycogen and mitochondrion,the early myofibrils appeared in cells,some cells appeared disc structure and tight junction,the ultrastructure of T3 treatment group were closer to the mature cardiomyocytes.Conclusion The study indicated that the Sendai virus system can be used to obtain a stable proliferating hi PSCs from PBMCs and hi PSCs can be induced differentiated to sufficient for cardiomyocytes further research.In addition,thyroid hormone T3 could be used to promote hi PSC-CMs maturation in the period of inducing differentiation.