Therapeutic Effect Of Induced Pluripotent Stem Cell-derived Exosomes On Myocardial Injury Repair

[Background]Coronary artery disease is one of the major public health problem in China,which seriously threatens the health and economy of the country.Due to the lack of regenerative capacity of adult cardiomyocytes,scar tissue replaces myocardial tissue after infarction,resulting in decreased left ventricular function and heavy heart structure.Although medical advances in pharmacological,intracardiac coronary intervention,and cardiac surgery have reduced acute mortality from cardiovascular disease,current treatments have not yet provided the ideal cardiomyocyte regeneration strategy.Induced pluripotent stem cells（iPSCs）have similar pluripotency with embryonic stem cells（ESCs）,but iPSCs do not involve in ethics problem and have a wide range of sources,providing scientists a new model to explore the occurrence and development of disease and study related pathophysiological mechanisms.In recent years,there have been many studies on ESC-derived exosomes and their applications for myocardial repair.The one of possible mechanisms is that ESC play its role through paracrine.As a carrier of cell contact,exosomes contain unique biologically active substances.As an intercellular messenger and epigenetic regulator,it exerts biological functions.It is widely used as a means of myocardial repair due to its low immunogenicity,the multi-differentiation capabilities and improved technologies of induction and isolation.The pluripotency of iPSC enables the production of exosomes from any human tissue cell and applies them to treat degenerative diseases extensivly,which could accomplish transforming precision medicine through the cell-free clinical implementation of personalized iPSC biology.The present study hypothesized whether iPSC-derived exosomes also have anti-apoptosis,necrosis,remodeling,fibrosis,pro-angiogenesis,and promotion of myocardial repair in damaged cardiomyocytes.[Research purposes]1.Extraction of exosomes from iPSC serum-free culture supernatant and identification of them;2.To establish an in vitro experimental model of oxidative stress injury in rat cardiomyocytes H9C2 cells,and evaluate the efficacy of iPSC-exosomes on damaged H9C2 cells.3.To construct a model of acute myocardial infarction in SD rats,establish a completed analysis and evaluation system,and provide an animal model for exploring the efficacy of iPSC-exosomes in the treatment of acute myocardial infarction;[Experimental method]1.Ultra-high-speed centrifugation method was used to extract exosomes from iPSC serum-free medium supernatant.The morphological characteristics of exosomes was obseved by transmission electron microscope.Nanoparticle tracking technology was used to measure the size of exosomes.The expressed markers CD9,CD63,CD81 and TSG101 on the surface of exosomes was detected by western blot.2.H9C2 was treated with 200μg/ml of hydrogen peroxide solution for 12 h,and the cell culture supernatant was collected to detect the increase of lactate dehydrogenase,indicating that the model was successfully prepared.The cell viabilities of damaged H9C2 without or with iPSC-exosomes were detected by CCK8 assay,which was used for the evaluation of the protected efficiency of iPSC-exosomes.The expression of apoptosis-related proteins including Caspase3,Bax and Bcl-2 after iPSC-exosomes intervention in injured H9C2 cells were also detected by western blot.3.SD rats were emplyed for our in vivo study,that randomly divided into three groups.After tracheotomy and intubation,the fourth intercostal transverse incision was performed on the left side of the rat.The left anterior wall myocardium was caused by ligation of the left anterior descending coronary artery under direct vision.After infarction was observed for several minutes,iPSC-exosomes were intramyocardially injected.The judgement for successful establishment of myocardial infarction model was included:electrocardiogram,ST-segment elevation within a few hours after surgery,elevated troponin 3 hours after postoperation;ultrasound examination before operation,and after 1 day,1 week,4 weeks of postoperation seperately.After 4 weeks,the rats were sacrificed,and the heart was collected for further analysis.H&E staining and Masson staining were performed to observe myocardial fibrosis.The efficacy of the iPSC-exosomes through anti-apoptosis and/or neovascularization was evaluated by immunofluorescence.4.Statistical methods:Unpaired t-test was performed to compare the difference between two groups.Statistical analyses were conducted using SPSS（22.0）and GraphPad Prism 6.0.Differences were considered to be statistically significant when p<0.05[Results]1.Extraction and identification of iPSC-exosomes1.1 The iPSC-exosomes could be stably isolated by polyethylene glycol-8000（PEG-8000）precipitation method,and then purified by sucrose density gradient centrifugation to obtain iPSC-exosomes.The physical and chemical properties of exosomes was identifed and consistent with previous studies,which was indicated that they could be used in subsequent experiments..2.Establishment of an in vitro injury model of H9C2 cardiomyocytes and analysis of the efficacy of iPSC-exosomes2.1H₂O₂-induced H9C2 cardiomyocytes injury could be used as an ideal in vitro experimental model of myocardial infarction;2.2iPSC-exosomes enhanced the resistance of H9C2 cardiomyocytes to H₂O₂ exposure;2.3iPSC-exosomes treatment reduced the H₂O₂-induced H9C2 cardiomyocyte apoptosis;2.4iPSC-exosomes reduced H9C2 cardiomyocyte apoptosis might be related to mitochondrial pathway-mediated apoptosis;3.Establishment of SD rat AMI model and evaluation of therapeutic effect of iPSC-exosomes3.1 The electrocardiogram,echocardiography,histological examination and other methods were confirmed that the successful establishment of the AMI model by ligating the anterior descending coronary artery in SD rats.3.2 iPSC-exosomes improved cardiac function in rats after AMI;3.3 iPSC-exosomes reduced the apoptosis of myocardial tissue cells in AMI rats;3.4 iPSC-exosomes increased neovascularization in myocardial tissue of AMI rats.[Conclusion]1.iPSC-exosomes could be separated stably by Polyethylene glycol-8000 precipitation method,and then purified by sucrose density gradient centrifugation,which were further identified for their the physical and chemical properties of exosomes.2.H₂O₂-induced H9C2 cardiomyocytes injury could be used as an ideal model for myocardial infarction in vitro.iPSC-exosomes enhanced the resistance of H9C2 cardiomyocytes to H₂O₂,enhanced cell viability and reduced H9C2 cardiomyocyte apoptosis.3.The AMI model could be successfully established by ligation of the anterior descending coronary artery in SD rats.iPSC-exosomes improved the heart function of rats after AMI,reduced the apoptosis of myocardial cells and increased the neovascularization in myocardial tissue.4.In this study,group t-test was used to analyze the difference between the intervention group and the control group.SPSS（22.0）and GraphPad Prism 6.0 were used for data statistics.P<0.05 indicated statistical difference.[Analysis conclusion]Ⅰ.Polyethylene glycol-8000 precipitation method can stably separate iPSC-exosomes,and then purified by sucrose density gradient centrifugation to obtain iPSC-exosomes,which is qualified to meet the physical and chemical properties of exosomes.Ⅱ.The AMI model can be successfully established by ligation of the anterior descending co’ronary artery in SD rats.iPSC-exosomes can improve the heart function of rats after AMI,reduce the apoptosis of myocardial cells and increase the neovascularization in myocardial tissue.Ⅲ.H₂O₂ intervention H9C2 cardiomyocytes can be used as an ideal model formyocardial infarction in vitro;iPSC-exosomes can enhance the resistance of H9C2 cardiomyocytes to H₂O₂,enhance cell viability and reduce H9C2 cardiomyocyte apoptosis;Ⅳ.iPSC-exosomes can be used as a potential treatment for myocardial injury repair.