Stem cell-based therapy has been used to treat ischaemic heart diseases for two decades. However, optimal cell types and transplantation methods remain unclear. This study evaluated the therapeutic effects of human umbilical cord mesenchymal stem cell (hUCMSC) sheet on myocardial infarction (MI). Methods hUCMSCs expressing luciferase were generated by lentiviral transduction for in vivo bio-luminescent imaging tracking of cells. We applied a temperature-responsive cell culture surface-based method to form the hUCMSC sheet. Cell retention was evaluated using an in vivo bio-luminescent imaging tracking system. Unbiased transcriptional profiling of infarcted hearts and further immunohistochemical assessment of monocyte and macrophage subtypes were used to determine the mechanisms underlying the therapeutic effects of the hUCMSC sheet. Echocardiography and pathological analyses of heart sections were performed to evaluate cardiac function, angiogenesis and left ventricular remodelling. Results When transplanted to the infarcted mouse hearts, hUCMSC sheet significantly improved the retention and survival compared with cell suspension. At the early stage of MI, hUCMSC sheet modulated inflammation by decreasing Mcp1-positive monocytes and CD68-positive macrophages and increasing Cx3cr1-positive non-classical macrophages, preserving the cardiomyocytes from acute injury. Moreover, the extracellular matrix produced by hUCMSC sheet then served as bioactive scaffold for the host cells to graft and generate new epicardial tissue, providing mechanical support and routes for revascularsation. These effects of hUCMSC sheet treatment significantly improved the cardiac function at days 7 and 28 post-MI. Conclusions hUCMSC sheet formation dramatically improved the biological functions of hUCMSCs, mitigating adverse post-MI remodelling by modulating the inflammatory response and providing bioactive scaffold upon transplantation into the heart. Translational perspective Due to its excellent availability as well as superior local cellular retention and survival, allogenic transplantation of hUCMSC sheets can more effectively acquire the biological functions of hUCMSCs, such as modulating inflammation and enhancing angiogenesis. Moreover, the hUCMSC sheet method allows the transfer of an intact extracellular matrix without introducing exogenous or synthetic biomaterial, further improving its clinical applicability.

Muscle LIM protein (MLP, CSRP3 ) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca 2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca 2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca 2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca 2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.

Rationale: Genetic editing has shown great potential for the treatment of human hereditary disorders via the elimination of mutations in embryos. However, the efficiency and safety of germline gene editing are not well understood. Objective: We aimed to examine the preclinical efficacy/safety of embryonic base editing in a mouse model of hypertrophic cardiomyopathy (HCM) using a novel adenine base editor (ABE) platform. Methods and Results: Here, we described the use of an ABEmax-NG to directly correct the pathogenic R404Q/+ mutation (Myh6 c.1211C>T) in embryos for a mouse model of HCM, increasing the number of wild-type embryos for in vitro fertilization. Delivery of the ABEmax-NG mRNA to embryos from R404Q/+ HCM mice resulted in 62.5-70.8% correction of the Myh6 c.1211C>T, reducing the level of mutant RNA and eliminating HCM in the post-natal mice as well as their offspring. In addition, the same sgRNA was also used to target an intronic locus (TGG PAM) with an overall editing rate of 86.7%, thus confirming that ABEmax-NG can efficiently edit target loci with different PAMs (NG) and genomic distribution in vivo. Compared with CRISPR/ssODN-mediated correction, ABEmax-NG displayed a much higher correction rate without introducing indels. DNA and RNA off-target analysis did not detect off-target editing in treated embryos and founder mice. In utero injection of adeno-associated virus 9 (AAV9) encoding the ABEmax-NG also resulted in around 25.3% correction of the pathogenic mutation and reduced of mutant RNA, thereby indicating ABEmax-NG has the potential to correct the HCM mutation in vivo. Conclusions: We developed an ABEmax-NG system, which efficiently corrected a pathogenic Myh6 HCM mutation in mouse embryos without off target lesions, thus safely eliminating HCM in derived mice and their progeny.