Engineered Mesenchymal Stem Cells And Their Derived Exosomes In The Treatment For Ischemic Heart Disease

BackgroundCardiovascular disease,particularly ischemic heart disease,remains the leading cause of morbidity and mortality worldwide.After myocardial infarction,due to the extremely low regenerative capacity of the adult heart,cardiomyocytes are massively lost and replaced by fibrotic scars.Consequent complications,such as heart failure,create significant medical,social,and economic burdens.At present,regenerative medicine technology(stem cells and their derived exosomes)is in the ascendant for the treatment of ischemic heart disease,and has received extensive attention from clinicians and researchers.At present,an important factor restricting the efficacy of stem cell preparations is the relatively poor cell retention rate in the transplanted area.The same low efficiency of exosome recruitment to the heart is also a bottleneck factor affecting its clinical translation application.Purposes1.To explore the efficacy evaluation of human umbilical cord mesenchymal stem cell sheet constructed by cell sheet engineering technology for chronic ischemic heart failure;2.To construct engineered mesenchymal stem cell exosomes using a biomimetic delivery strategy,and to evaluate their cardiac targeting recruitment ability and cardiac injury repair efficacy.Methods1.Inoculate umbilical cord mesenchymal stem cells by using a culture dish coated with a temperature-sensitive polymer material poly-N-terminal isopropylacrylamide(PIPAAm),by controlling the temperature change(from 37℃ to 32℃),to separate the adherent growing cells from the culture dish to obtain a complete cell sheet.The structure and in vitro function of umbilical cord mesenchymal stem cell sheets were characterized and identified by HE staining,immunofluorescence staining,and cell supernatant factor level detection and analysis.The obtained cell sheets were transplanted into chronic ischemic heart failure model rats,and the benefit of the rat’s cardiac function was evaluated by echocardiography,the level of myocardial fibrosis burden by Masson staining,and the neovascularization ability by CD31 immunofluorescence staining.The changes of myocardial hypertrophy were evaluated by wheat germ agglutinin(WGA)staining,and the changes of PI3K/AKT/mTOR signaling pathway were evaluated by western blotting.2.The mesenchymal stem cell exosomes were isolated and obtained by ultracentrifugation,and the neutrophil-specific antibody Ly-6G antibody was linked to the MMP enzyme-sensitive peptide RVGLP to construct a delivery system.In vitro through transmission electron microscopy,confocal microscopy,nanoparticle tracking analysis and evaluation system was constructed.Evaluation of cardiac targeting properties of engineered exosomes using small animal in vivo imaging.The myocardial protective effect was evaluated in the ischemia-reperfusion injury model.Using mRNA sequencing to explore the molecular mechanism of action of the therapy.TUNEL staining was used to evaluate the level of early cardiomyocyte apoptosis,immunofluorescence staining of macrophage subsets(Argl,CD68)and inflammatory factor ELISA to detect IL-1β,IL-6,TNF-α and IL-10 to evaluate exosome resistance Inflammation treatment effect,cardiac function protection effect was evaluated by echocardiography,fibrosis degree was evaluated by Masson staining,and neovascularization was evaluated by CD31 staining.Results1.Flow cytometry analysis of UC-MSCs was positive for CD73,CD90,and CD105,and negative for CD34,CD45,and HLADR.The thickness of UC-MSC sheet is 80-100 μm,and its supernatant can secrete more HGF,VEGF,IL-10 and endoglin than the same number of monolayer adherent cells,which can reduce myocardial hypoxia/reoxygenation injury in vitro,tubule formation experiments suggest that it has a better role in promoting tube formation.Four weeks after transplantation in the chronic myocardial infarction model,echocardiography showed that the cardiac function of the UC-MSC patch group was significantly better than that of the single-cell suspension group and the MI group(EF 74.2%,35.3%,44.6%,and 52.1%,respectively.%).In addition,histological examination showed that the level of myocardial fibrosis was improved(34.8%,22.1%and 12.8%of fibrosis area,respectively)compared with the single-cell suspension group and MI group.In the UC-MSC sheet group,the neovascular density and cell size in the infarct border area were significantly improved compared to the suspension and MI groups.Furthermore,the UC-MSC sheets inhibited the PI3K/AKT/mTOR signaling pathway in chronic ischemic heart failure.2.The engineered exosomes have the same morphological characteristics as the original exosomes,the potential is reduced,and the particle size has no significant change.Confocal shows that the exosomes co-localize with the Ly-6G antibody.6G antibody binds to exosomes.The engineered exosomes have good freeze-drying and thawing stability,and can be stored at 4℃ for one week.It has the characteristics of MMP enzyme-responsive release in vitro.The results of in vivo imaging in small animals showed that the heart-targeted delivery efficiency of exosomes was about 2.5 times higher than that of traditional intravenous infusion of exosomes.In vivo animal experiments show that exosomes targeting the heart can improve cardiac function,reduce cardiomyocyte apoptosis,promote the polarization of macrophages to M2 type,regulate the immune microenvironment,and promote angiogenesis.Conclusion:1.UC-MSCs can successfully construct cell membranes using temperature-sensitive culture dishes;UC-MSC sheets have a good therapeutic effect on chronic ischemic heart failure,can improve cardiac function,reduce myocardial fibrosis burden and adverse ventricular remodeling,promote angiogenesis,improve myocardial immune microenvironment.2.Mesenchymal stem cell-derived exosomes can be effectively recognized and captured by neutrophils after being engineered,and bionic delivered to the heart,with good cardioprotective efficacy,suitable for early intravenous administration in patients with acute myocardial infarction,which can achieve multiple drug administration interventions,and has great potential for clinical transformation.