Summary Human induced pluripotent stem cells (hiPSCs) hold promise for myocardial repair following injury, but preclinical studies in large animal models are required to determine optimal cell preparation and delivery strategies to maximize functional benefits and to evaluate safety. Here, we utilized a porcine model of acute myocardial infarction (MI) to investigate the functional impact of intramyocardial transplantation of hiPSC-derived cardiomyocytes, endothelial cells, and smooth muscle cells, in combination with a 3D fibrin patch loaded with insulin growth factor (IGF)-encapsulated microspheres. hiPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomeric structures, and endothelial and smooth muscle cells contributed to host vasculature. Tri-lineage cell transplantation significantly improved left ventricular function, myocardial metabolism, and arteriole density, while reducing infarct size, ventricular wall stress and apoptosis without inducing ventricular arrhythmias. These findings in a large animal MI model highlight the potential of utilizing hiPSC-derived cells for cardiac repair.

Current EC differentiation protocols are inefficient, and the phenotypes of the differentiated ECs are only briefly stable, which significantly inhibits their utility for basic science research. Here, a remarkably more efficient hiPSC-EC differentiation protocol that incorporates a three-dimensional (3D) fibrin scaffold is presented. With this protocol, up to 45% of the differentiated hiPSCs assumed an EC phenotype, and after purification, greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks in vitro. Gene and protein expression levels of CD31, CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. Collectively, these data demonstrate that a 3D differentiation protocol can efficiently generate ECs from hiPSCs and, furthermore, the differentiated hiPSC-ECs are functional and can maintain EC fate up to 4 weeks in vitro.

Background-We investigated the feasibility and efficacy of polyethylenimine (PEI) based human vascular endothelial growth factor-165 (hVEGF 165 ) gene transfer into human skeletal myoblasts (HSM) for cell based delivery to the infarcted myocardium. Methods and Results-Based on optimized transfection procedure using enhanced green fluorescent protein (pEGFP), HSM were transfected with plasmid-hVEGF 165 (phVEGF 165 ) carried by PEI (PEI-phVEGF 165 ) nanoparticles. The transfected HSM were characterized for transfection and expression of hVEGF 165 in vitro and transplanted into rat heart model of acute myocardial infarction (AMI): group-1ϭDMEM injection, group-2ϭ HSM transplantation, group-3ϭ PEI-phVEGF 165 -transfected HSM (PEI-phVEGF 165 myoblast) transplantation. A total of 48 rats received cyclosporine injection from 3 days before and until 4 weeks after cell transplantation. Echocardiography was performed to assess the heart function. Animals were sacrificed for molecular and histological studies on the heart tissue at 4 weeks after treatment. Based on optimized transfection conditions, transfected HSM expressed hVEGF 165 for 18 days with Ͼ90% cell viability in vitro. Apoptotic index was reduced in group-2 and group-3 as compared with group-1. Blood vessel density (ϫ400) by immunostaining for PECAM-1 in group-3 was significantly higher (Pϭ0.043 for both) as compared with group-1 and group-2 at 4 weeks. Regional blood flow (ml/min/g) in the left ventricular anterior wall was higher in group-3 (Pϭ0.043 for both) as compared with group-1 and group-2. Improved ejection fraction was achieved in group-3 (58.44Ϯ4.92%) as compared with group-1 (Pϭ0.004). Conclusion-PEI

Background Thymosin β4 (Tβ4) has been shown to enhance the survival of cultured cardiomyocytes. Here, we investigated whether the cytoprotective effects of Tβ4 can increase the effectiveness of transplanted swine mesenchymal stem cells (sMSCs) for cardiac repair in a rat model of myocardial infarction (MI). Methods and Results Under hypoxic conditions, cellular damage (lactate dehydrogenase leakage), apoptosis (TUNEL+ cells), and caspase-8 activity were significantly lower, while Bcl-XL protein expression was significantly higher, in sMSCs cultured with Tβ4 (1 µg/mL) than in sMSCs cultured without Tβ4, and Tβ4 also increased sMSC proliferation. For in-vivo experiments, animals were treated with basal medium (MI: n=6), a fibrin patch (Patch: n=6), a patch containing sMSCs (sMSC: n=9), or a patch containing sMSCs and Tβ4 (sMSC/Tβ4: n=11); Tβ4 was encapsulated in gelatin microspheres to extend Tβ4 delivery. Four weeks after treatment, echocardiographic assessments of left-ventricular ejection fraction (LVEF) and fractional shortening (FS) were significantly better (p<0.05) in sMSC/Tβ4 animals (LVEF=51.7±1.1%; FS=26.7±0.7%) than in animals from MI (39±3%; 19.5±1.7%) and Patch (43±1.4%; 21.6±0.9%) groups. Histological assessment of infarct wall thickness (TH) was significantly higher (p<0.05) in sMSC/Tβ4 animals (50%, (45%, 80%)) than in animals from MI (25%, (20%, 25%)) group. Measurements in sMSC (LVEF=45±2.6%; FS=22.9±1.6%; TH=43% (25%, 45%)), Patch, and MI animals were similar. Tβ4 administration also significantly increased vascular growth, the retention/survival of the transplanted sMSCs, and the recruitment of endogenous c-Kit+ progenitor cells to the infarcted region. Conclusions Extended-release Tβ4 administration improves the retention, survival, and regenerative potency of transplanted sMSCs after myocardial injury.

Transforming growth factor beta-1 (TGFβ1) is a major driver of vascular smooth muscle cell (VSMC) phenotypic switching, an important pathobiology in arterial disease. We performed RNA-sequencing of TGFβ1-stimulated human aortic or arterial VSMCs which revealed large and consistent upregulation of Interleukin 11 (IL11). IL11 has an unknown function in VSMCs, which highly express the IL11 receptor alpha, suggestive of an autocrine loop. In vitro, IL11 activated ERK signaling, but inhibited STAT3 activity, and caused VSMC phenotypic switching to a similar extent as TGFβ1 or angiotensin II (ANGII) stimulation. Genetic or therapeutic inhibition of IL11 signaling reduced TGFβ1- or ANGII-induced VSMC phenotypic switching, placing IL11 activity downstream of these factors. Aortas of mice with Myh11-driven IL11 expression were remodeled and had reduced contractile but increased matrix and inflammatory genes expression. In two models of arterial pressure loading, IL11 was upregulated in the aorta and neutralizing IL11 antibodies reduced remodeling along with matrix and pro-inflammatory gene expression. These data show that IL11 plays an important role in VSMC phenotype switching, vascular inflammation and aortic pathobiology.