Study On The Maturation Of Induced Pluripotent Stem Cell-derived Cardiomyocytes And Its Mechanism

Background:Human heart failure caused by myocardial infarction(MI)is a major health problem.The scarcity of transplant organs limits treatments for diseased and failing adult hearts.Human induced pluripotent stem cell-derived cardiomyocytes(iPSCCMs)have the potential to provide a long-term,viable seed cell.Although significant progress has been made in the efficient generation of cardiomyocytes from iPSCs.However,it is still immature and has a certain gap with adult cardiomyocytes,which greatly limits its application in cardiac diseases.Therefore,inducing iPSC-CMs to acquire physiological structures,gene expression profiles,and functions similar to mature cardiac tissue is a major challenge at present.In order to break through this technical bottleneck,we plan to combine the small molecule compound BEZ-235 with nano colloidal gelatin(Gel).Through the inhibition of PI3K/AKT/mTOR pathway by BEZ-235 and the structural support provided by Gel for cell growth to promote the functional maturation of iPSC-CMs,and to evaluate the efficacy of iPSC-CMs in myocardial infarction mouse.Objective:Synthesize the Gel required for the experiment,screen the effect of small molecular compounds and combine with the Gel to explore the morphology,microstructure,electrophysiology,organelle and metabolic function of iPSC-CMs.Finally,we used Gel as a carrier to carry mature iPSC-CMs to explore its efficacy in myocardial infarction in mouse.Methods:Gel was synthesized by a two-step method and characterized.The in vitro biocompatibility of different concentrations of Gel was revealed,and BEZ-235 with different concentration gradients and the optimal action time were screened.After combining with Gel,the morphological changes of different groups of cells were displayed by immunofluorescence,and the changes of cell microstructure were revealed.Cell action potentials were recorded by patch clamp,Ca2+ transient dynamics of iPSC-CMs were detected by calcium transients,and synchrony of cell conduction was detected by MEA.The mitochondrial membrane potential changes were detected by JC-1,and the microstructure of mitochondria was observed by TEM.At the same time,the metabolic function changes of iPSC-CMs were revealed.To explore the effect of Gel-loaded mature iPSC-CMs on myocardial infarction.Results:1.The chemical composition of Gel is the chemical composition of typical collagenbased materials,and has good biocompatibility in vitro.2.BEZ-235+Gel significantly improved the sarcomere structure of iPSC-CMs.Promoted electrophysiological maturation of iPSC-CMs,resulting in a more uniform electrical spread.Improved mitochondrial microstructure and enhanced mitochondrial respiratory function.3.Gel enhanced the retention rate of iPSC-CMs in vivo.Gel loaded with mature iPSC-CMs enhanced angiogenesis and gap junction formation at the injection site.Conclusion:Gel has good biocompatibility,and BEZ-235+Gel promotes the maturation of iPSC-CMs.Gel-loaded mature iPSC-CMs enhanced angiogenesis and gap junction formation at the injection site.