-The adult mammalian heart has limited ability to repair itself following injury. Zebrafish, newts and neonatal mice can regenerate cardiac tissue, largely by cardiac myocyte (CM) proliferation. It is unknown if hearts of young large mammals can regenerate. -We examined the regenerative capacity of the pig heart in neonatal animals (ages: 2, 3 or 14 days postnatal) after myocardial infarction (MI) or sham procedure. Myocardial scar and left ventricular function were determined by cardiac magnetic resonance (CMR) imaging and echocardiography. Bromodeoxyuridine pulse-chase labeling, histology, immunohistochemistry and Western blotting were performed to study cell proliferation, sarcomere dynamics and cytokinesis and to quantify myocardial fibrosis. RNA-sequencing was also performed. -After MI, there was early and sustained recovery of cardiac function and wall thickness in the absence of fibrosis in 2-day old pigs. In contrast, older animals developed full-thickness myocardial scarring, thinned walls and did not recover function. Genome wide analyses of the infarct zone revealed a strong transcriptional signature of fibrosis in 14-day old animals that was absent in 2-day old pigs, which instead had enrichment for cytokinesis genes. In regenerating hearts of the younger animals, up to 10% of CMs in the border zone of the MI showed evidence of DNA replication that was associated with markers of myocyte division and sarcomere disassembly. -Hearts of large mammals have regenerative capacity, likely driven by cardiac myocyte division, but this potential is lost immediately after birth.

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 × 10 8 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.

Induced pluripotent stem cells derived cells (iPSCs) not only can be used for personalized cell transfer therapy, but also can be used for modeling diseases for drug screening and discovery in vitro. Although prior studies have characterized the function of rodent iPSCs derived endothelial cells (ECs) in diabetes or metabolic syndrome, feature phenotypes are largely unknown in hiPSC-ECs from patients with diabetes. Here, we used hiPSC lines from patients with type 2 diabetes mellitus (T2DM) and differentiated them into ECs (dia-hiPSC-ECs). We found that dia-hiPSC-ECs had disrupted glycine homeostasis, increased senescence, and impaired mitochondrial function and angiogenic potential as compared with healthy hiPSC-ECs. These signature phenotypes will be helpful to establish dia-hiPSC-ECs as models of endothelial dysfunction for understanding molecular mechanisms of disease and for identifying and testing new targets for the treatment of endothelial dysfunction in diabetes.

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Ischemic heart disease, which is often associated with irreversibly damaged heart muscle, is a major global health burden. Here, we report the potential of stem cell-derived committed cardiac progenitors (CCPs) have in regenerative cardiology. Human pluripotent embryonic stem cells were differentiated to CCPs on a laminin 521 + 221 matrix, characterized with bulk and single-cell RNA sequencing, and transplanted into infarcted pig hearts. CCPs differentiated for eleven days expressed a set of genes showing higher expression than cells differentiated for seven days. Functional heart studies revealed significant improvement in left ventricular ejection fraction at four and twelve weeks following transplantation. We also observed significant improvements in ventricular wall thickness and a reduction in infarction size after CCP transplantation (p-value < 0.05). Immunohistology analyses revealed in vivo maturation of the CCPs into cardiomyocytes (CM). We observed temporary episodes of ventricular tachyarrhythmia (VT) in four pigs and persistent VT in one pig, but the remaining five pigs exhibited normal sinus rhythm. Importantly, all pigs survived without the formation of any tumors or VT-related abnormalities. We conclude that pluripotent stem cell-derived CCPs constitute a promising possibility for myocardial infarction treatment and that they may positively impact regenerative cardiology.