Abstract 12887: Cyclin D2 Overexpressing and Hypoimmunogenic Cardiomyocyte Spheroid Transplantation Induced Endogenous Myocardial Regeneration in the Pig Heart After Myocardial Infarction

Abstract

Background: hiPSCs derived cardiomyocytes (hiPSC-CMs) have very low proliferative capacity, while patient hiPSC-CMs may have disease phenotypes which compromise therapeutic efficacy and allogenic hiPSC-CMs are expected to induce an immune response during clinical use. In order to solve these problems, we established a hiPSC line by knocking-out two components of the human leukocyte antigen (HLA) and over-expressing myosin heavy chain driven cyclin D2 (CCND2) systems in hiPSCs ( HLA-KO/CCND2-OE hiPSCs). Aim: To determine the therapeutic efficacy and mechanism of the xeno-transplantation of HLA-KO/CCND2-OE hiPSCs derived cardiomyocytes ( HLA-KO/CCND2-OE hiPSC-CMs) in the pig hearts after myocardial ischemia/reperfusion (IR). Methods and Results: HLA-KO/CCND2-OE hiPSC-CM spheroids were differentiated in shaking flasks. Purified HLA-KO/CCND2-OE hiPSC-CM spheroids were intramyocardially injected into pig hearts after IR (the Spheroid Group), while control animals received basal medium injection (the IR Group). Cardiac magnetic resonance imaging (cMRI) showed that pig heart function improved significantly compared to the IR Group at 4 weeks after IR. This finding was associated with a dramatical reduction in the scar size as determined by late gadolinium enhancement measurement. Immunohistology showed that significantly increased porcine CMs expressed cell proliferation markers, such as Ki67, phospho-histone 3 (pH3), and symmetrical Aurora B, and abundant YAP protein expression in porcine CM nuclei in the porcine hearts of the Spheroid Group compared to the IR Group at 1 week after IR. Western Blot analysis of pig heart tissue showed significantly reduced expression of phospho-YAP in the Spheroid Group. snRNAseq showed that number of cycling porcine CMs was > 5 folds in the Spheroid Groups than that in the IR Group. Angiogenic microarray showed that HLA-KO/CCND2-OE hiPSC-CM spheroids produced abundant follistatin which induced YAP protein expression and cytokinesis in CMs. Conclusions: Transplantation of HLA-KO/CCND2-OE hiPSC-CM spheroids effectively recovered porcine heart function, reduced scar size, and induced porcine CM proliferation through modulating YAP protein expression by follistatin secreted by transplanted cells.