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Cardiac Progenitors Induced from Human Induced Pluripotent Stem Cells with Cardiogenic Small Molecule Effectively Regenerate Infarcted Hearts and Attenuate Fibrosis.

ABSTRACT: Cardiac progenitor cells (CPCs) being multipotent offer a promising source for cardiac repair due to their ability to proliferate and multiply into cardiac lineage cells. Here, we explored a novel strategy for human CPCs generation from human induced pluripotent stem cells (hiPSCs) using a cardiogenic small molecule, isoxazole (ISX-9) and their ability to grow in the scar tissue for functional improvement in the infarcted myocardium. CPCs were induced from hiPSCs with ISX-9. CPCs were characterized by immunocytochemistry and RT-PCR. The CPC survival and differentiation in the infarcted hearts were determined by in vivo transplantation in immunodeficient mice following left anterior descending artery ligation and their effects were determined on fibrosis and functional improvement. ISX-9 simultaneously induced expression of cardiac transcription factors, NK2 homeobox 5, islet-1, GATA binding protein 4, myocyte enhancer factor-2 in hiPSCs within 3 days of treatment and successfully differentiated into three cardiac lineages in vitro. Messenger RNA and microRNA-sequencing results showed that ISX-9 targeted multiple cardiac differentiation, proliferation signaling pathways and upregulated myogenesis and cardiac hypertrophy related-microRNA. ISX-9 activated multiple pathways including transforming growth factor ? induced epithelial-mesenchymal transition signaling, canonical, and non-canonical Wnt signaling at different stages of cardiac differentiation. CPCs transplantation promoted myoangiogenesis, attenuated fibrosis, and led to functional improvement in treated mice.

SUBMITTER: Xuan W

PROVIDER: S-EPMC6109623 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Project description:IntroductionThe adult heart lacks the regenerative capacity to self-repair. Serum response factor (SRF) is essential for heart organogenesis, sarcomerogenesis, and contractility. SRF interacts with co-factors, such as NKX2.5 and GATA4, required for cardiac specified gene activity. ETS factors such as ELK1 interact with SRF and drive cell replication. To weaken SRF interactions with NKX2.5 and GATA4, one mutant, SRF153(A3) named STEMIN, did not bind CArG boxes, yet induced stem cell factors such as NANOG and OCT4, cardiomyocyte dedifferentiation, and cell cycle reentry. The mutant YAP5SA of the Hippo pathway also promotes cardiomyocyte proliferation and growth.AimInfarcted adult mouse hearts were injected with translatable STEMIN and YAP5SA mmRNA to evaluate their clinical potential.Methods and resultsMice were pulsed one day later with alpha-EDU and then heart sections were DAPI stained. Replicating cells were identified by immuno-staining against members of the DNA replisome pathway that mark entry to S phase of the cell cycle. Echocardiography was used to determine cardiac function following infarcts and mRNA treatment. To monitor cardiac wall repair, microscopic analysis was performed, and the extent of myocardial fibrosis was analyzed for immune cell infiltration. Injections of STEMIN and YAP5SA mmRNA into the left ventricles of infarcted adult mice promoted a greater than 17-fold increase in the DAPI stained and alpha-EDU marked cardiomyocyte nuclei, within a day. We observed de novo expression of phospho-histone H3, ORC2, MCM2, and CLASPIN. Cardiac function was significantly improved by four weeks post-infarct, and fibrosis and immune cell infiltration were diminished in hearts treated with STEMIN and YAP5SA mmRNA than each alone.ConclusionSTEMIN and YAP5SA mmRNA improved cardiac function and myocardial fibrosis in left ventricles of infarcted adult mice. The combinatorial use of mmRNA encoding STEMIN and YAP5SA has the potential to become a powerful clinical strategy to treat human heart disease.

Project description:Тhe most pressing issue in generating induced pluripotent stem cells (iPSCs) in clinical practice is the cell source. Compared to human dermal fibroblasts (HDFs), which have been widely used, human peripheral blood could be a more easily obtainable alternative. However, iPSCs generated from fresh peripheral blood require inconvenient specific methods including isolation. Recently, we succeeded in isolating and culturing human heart-derived circulating cells called circulating multipotent stem (CiMS) cells. Here, we investigated the generation efficiency of CiMS-derived iPSCs (CiMS-iPSCs) and tested their differentiation potential into mesodermal lineages and cardiovascular cells. We isolated and cultured CiMS cells from peripheral mononuclear cells with a high efficiency. Moreover, our method succeeded in reprogramming the CiMS cells and generating iPSCs with higher efficiency compared to when HDFs were used. Compared to HDF-iPSCs or human embryonic stem cells (hESCs), CiMS-iPSCs showed high differentiation potential into mesodermal lineage cells and subsequently into endothelial cells, vascular smooth muscle cells, and cardiomyocytes. Further, we checked the epigenetic status of each cell type. While methylation of the CpG site of the brachyury T promoter did not differ between cell types, the histone H3 lysine 4 trimethylation level in the brachyury T promoter region was enhanced in CiMS-iPSCs, compared to that in other cell types. In contrast, histone H3 lysine 9 acetylation was downregulated during the differentiation process of the CiMS-iPSCs. In the myocardial infarction model, the CiMS-iPSCs group showed more therapeutic potential in regenerating the myocardium than other cell types. Our study showed a new method to isolate human heart-derived stem cells from human peripheral blood and to generate iPSCs efficiently. Due to epigenetic memory, these CiMS-iPSCs easily differentiated into cardiovascular lineage cells, resulting in improved efficiency in vivo. These results suggest that our new method using CiMS cells has therapeutic potential in regenerative medicine using cell therapy.

Project description:BackgroundHuman-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have shed great light on cardiac regenerative medicine and specifically myocardial repair in heart failure patients. However, the treatment efficacy and the survival of iPSC-CMs in vivo after transplantation have yielded inconsistent results.ObjectivesThe objective of this study was to evaluate the ability of human iPSC-CMs to improve myocardial function in a rat postinfarction heart failure model.MethodsEight-week-old male Sprague-Dawley rats were randomly selected to receive an intramyocardial injection of 5% albumin solution with or without 1 × 107 human iPSC-CMs 10 days after undergoing left anterior descending (LAD) coronary artery ligation. Cyclosporine A and methylprednisolone were administered before iPSC-CM injection and until the rats were killed to prevent graft rejection. Cardiac function was evaluated by echocardiography. The survival of grafted cardiomyocytes was confirmed by observing the fluorescent cell tracer Vybrant™ CM-DiI or expression of the enhanced green fluorescent protein (eGFP) in transplanted cells, or survival was demonstrated by polymerase chain reaction (PCR)-based detection of human mitochondrial DNA. Sirius red stain was used to evaluate the fibrosis ratio. Hematoxylin-eosin staining was used to observe the formation of teratomas.ResultsFour weeks after intramyocardial injection of iPSC-CMs, animals undergoing iPSC-CM transplantation had lower mortality than the control group. Animals injected with cell-free solution (control group) demonstrated significant left ventricular (LV) functional deterioration, whereas grafting of iPSC-CMs attenuated this remodeling process. In the control group, the ejection fraction deteriorated by 10.11% (from 46.36 to 41.67%), and fractional shortening deteriorated by 9.23% (from 24.37 to 22.12%) by 4 weeks. In the iPSC-CM injection group, the ejection fraction improved by 18.86% (from 44.09 to 52.41%), and fractional shortening improved by 23.69% (from 23.08 to 28.54%). Cell labeling, tracking, and molecular biology techniques indicated that the grafted cardiomyocytes survived in the rat heart 1 month after iPSC-CM transplantation. Myocardial fibrosis was also attenuated in the iPSC-CM treatment group.ConclusionsHuman iPSC-CM grafts survived in infarcted rat hearts and restored myocardial function 4 weeks after transplantation. Cell replacement therapy also reversed ventricular remodeling, indicating the potential of iPSC-CMs for cardiac repair strategies.

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Cardiac Progenitors Induced from Human Induced Pluripotent Stem Cells with Cardiogenic Small Molecule Effectively Regenerate Infarcted Hearts and Attenuate Fibrosis.

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