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MiR-199a Overexpression Enhances the Potency of Human Induced-Pluripotent Stem-Cell-Derived Cardiomyocytes for Myocardial Repair.

ABSTRACT: Mammalian cardiomyocytes exit the cell cycle during the perinatal period, and although cardiomyocytes differentiated from human induced-pluripotent stem cells (hiPSC-CMs) are phenotypically immature, their intrinsic cell-cycle activity remains limited. Thus, neither endogenous cardiomyocytes nor the small number of transplanted hiPSC-CMs that are engrafted by infarcted hearts can remuscularize the myocardial scar. microRNAs are key regulators of cardiomyocyte proliferation, and when adeno-associated viruses coding for microRNA-199a (miR-199a) expression were injected directly into infarcted pig hearts, measures of cardiac function and fibrosis significantly improved, but the treatment was also associated with lethal arrhythmia. For the studies reported here, the same vector (AAV6-miR-199a) was transduced into hiPSC-CMs, and the cells were subsequently evaluated in a mouse model of myocardial infarction. AAV6-mediated miR-199a overexpression increased proliferation in both cultured and transplanted hiPSC-CMs, and measures of left ventricular ejection fraction, fractional shortening, and scar size were significantly better in mice treated with miR-199a-overexpressing hiPSC-CMs than with hiPSC-CMs that had been transduced with a control vector. Furthermore, although this investigation was not designed to characterize the safety of transplanted AAV6-miR-199a-transduced hiPSC-CMs, there was no evidence of sudden death. Collectively, these results support future investigations of miR-199a-overexpressing hiPSC-CMs in large animals.

SUBMITTER: Bian W

PROVIDER: S-EPMC8209326 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveThe actin-sequestering proteins, thymosin beta-4 (Tβ4) and hypoxia-inducible factor (HIF)-1α, are known to be associated with angiogenesis after myocardial infarction (MI). Herein, we aimed to identify the mechanism of HIF-1α induction by Tβ4 and investigate the effects of bone marrow mesenchymal stromal cells (BMMSCs) transfected with the Tβ4 gene (TMSB4) in a rat model of MI.MethodsRat BMMSCs were isolated, cultured, and transfected with the TMSB4 gene by using the lentivirus-mediated method. Rats with surgically induced MI were randomly divided into three groups (n = 9/group); after 1 week, the rats were injected at the heart infarcted border zone with TMSB4-overexpressed BMMSCs (BMMSC-TMSB4 O E ), wild-type BMMSCs that expressed normal levels of TMSB4 (BMMSC-TMSB4 W T ), or medium (MI). The fourth group of animals (n = 9) underwent all surgical procedures necessary for MI induction except for the ligation step (Sham). Four weeks after the injection, heart function was measured using transthoracic echocardiography. Infarct size was calculated by TTC staining, and collagen volume was measured by Masson staining. Angiogenesis in the infarcted heart area was evaluated by CD31 immunofluorescence histochemistry. In vitro experiments were carried out to observe the effect of exogenous Tβ4 on HIF-1α and explore the various possible mechanism(s).ResultsIn vivo experiments showed that vascular density 4 weeks after treatment was about twofold higher in BMMSC-TMSB4 O E -treated animals than in BMMSC-TMSB4 W T -treated animals (p < 0.05). The cardiac function and infarct size significantly improved in both cell-treatment groups compared to controls. Notably, the cardiac function and infarct size were most prominent in BMMSC-TMSB4 O E -treated animals (both p < 0.05). HIF-1α and phosphorylated HIF-1α (p-HIF-1α) in vitro were significantly enhanced by exogenous Tβ4, which was nonetheless blocked by the factor-inhibiting HIF (FIH) promoter (YC-1). The expression of prolyl hydroxylase domain proteins (PHD) was decreased upon treatment with Tβ4 and further decreased with the combined treatment of Tβ4 and FG-4497 (a specific PHD inhibitor).ConclusionTMSB4-transfected BMMSCs might significantly improve recovery from myocardial ischemia and promote the generation of HIF-1α and p-HIF-1α via the AKT pathway, and inhibit the degradation of HIF-1α via the PHD and FIH pathways.

Project description:AimsThe effectiveness of cell-based treatments for regenerative myocardial therapy is limited by low rates of cell engraftment. Y-27632 inhibits Rho-associated protein kinase (ROCK), which regulates the cytoskeletal changes associated with cell adhesion, and has been used to protect cultured cells during their passaging. Here, we investigated whether preconditioning of cardiomyocytes, derived from human-induced pluripotent stem cells (hiPSC-CM), with Y-27632 improves their survival and engraftment in a murine model of acute myocardial infarction (MI).Methods and resultsAfter MI induction, mice were subjected to intramyocardial injections of phosphate-buffered saline, hiPSC-CM cultured under standard conditions (hiPSC-CM-RI), or Y-27632-preconditioned hiPSC-CM (hiPSC-CM+RI). The resulting engraftment rate calculated 4?weeks after implantation was significantly higher and the abundance of apoptotic transplanted cells was significantly lower in hiPSC-CM+RI recipients than in hiPSC-CM-RI animals. In cultured hiPSC-CM, Y-27632-preconditioning reversibly reduced contractile activity and the expression of troponin genes, while increasing their attachment to an underlying mouse cardiomyocyte (HL1) monolayer. Y-27632 preconditioning also increased the expression of N-cadherin and integrin ß1, the two cell junction proteins. hiPSC-CM+RI were also larger in cell area with greater cytoskeletal alignment and a more rod-like shape than hiPSC-CM-RI, both after transplantation (in vivo) and in culture. The effects of Y-27632 preconditioning on contractile activity and morphology of hiPSC-CMs in culture, as well as on their engraftment rate and apoptotic death in MI mouse grafts, could be recapitulated by hiPSC-CM treatment with the L-type calcium-channel blocker verapamil.ConclusionPreconditioning with the ROCK inhibitor Y-27632 increased the engraftment of transplanted hiPSC-CM in a murine MI model, while reversibly impairing hiPSC-CM contractility and promoting adhesion.

Project description:Rationale: Previous studies have shown that human embryonic stem cell-derived cardiomyocytes improved myocardial recovery when administered to infarcted pig and non-human primate hearts. However, the engraftment of intramyocardially delivered cells is poor and the effectiveness of clinically relevant doses of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in large animal models of myocardial injury remains unknown. Here, we determined whether thymosin β4 (Tb4) could improve the engraftment and reparative potency of transplanted hiPSC-CMs in a porcine model of myocardial infarction (MI). Methods: Tb4 was delivered from injected gelatin microspheres, which extended the duration of Tb4 administration for up to two weeks in vitro. After MI induction, pigs were randomly distributed into 4 treatment groups: the MI Group was injected with basal medium; the Tb4 Group received gelatin microspheres carrying Tb4; the CM Group was treated with 1.2 × 108 hiPSC-CMs; and the Tb4+CM Group received both the Tb4 microspheres and hiPSC-CMs. Myocardial recovery was assessed by cardiac magnetic resonance imaging (MRI), arrhythmogenesis was monitored with implanted loop recorders, and tumorigenesis was evaluated via whole-body MRI. Results: In vitro, 600 ng/mL of Tb4 protected cultured hiPSC-CMs from hypoxic damage by upregulating AKT activity and BcL-XL and promoted hiPSC-CM and hiPSC-EC proliferation. In infarcted pig hearts, hiPSC-CM transplantation alone had a minimal effect on myocardial recovery, but co-treatment with Tb4 significantly enhanced hiPSC-CM engraftment, induced vasculogenesis and the proliferation of cardiomyocytes and endothelial cells, improved left ventricular systolic function, and reduced infarct size. hiPSC-CM implantation did not increase incidence of ventricular arrhythmia and did not induce tumorigenesis in the immunosuppressed pigs. Conclusions: Co-treatment with Tb4-microspheres and hiPSC-CMs was safe and enhanced the reparative potency of hiPSC-CMs for myocardial repair in a large-animal model of MI.

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MiR-199a Overexpression Enhances the Potency of Human Induced-Pluripotent Stem-Cell-Derived Cardiomyocytes for Myocardial Repair.

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