Abstract 13459: Human iPS-Derived Pre-Epicardial Cells Direct Cardiomyocyte Aggregation Expansion and Organization in vitro

Abstract

During heart maturation, epicardial-derived cells emanating from the pro-epicardial organ envelope the developing heart, then integrate within the myocardium and undergo epithelial-mesenchymal transition (EMT) to become fibroblasts, smooth muscle cells, and endothelial cells that enable several processes of healthy heart morphogenesis (i.e., ventricular thickening, compaction, and angiogenesis). Directed cardiac differentiation of human induced-pluripotent stem cells (hiPSCs) holds great potential for generating several cardiovascular cell types, which can theoretically be combined to model cardiac development more completely and engineer sophisticated myocardial grafts. Here, we show that differentiating hiPSC-derived lateral plate mesoderm (LPM) with BMP4, RA and VEGF (BVR) for 96 hours can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) with high efficiency (86.8 ± 4.1% WT1 + by FACS), that also show significant increases in WT1, TBX18, SEMA3D , TBX5, BNC, and SCX transcript expression within 7 days. This protocol was successfully reproduced in three commercial lines of hiPSCs. BVR stimulation after Wnt inhibition of LPM demonstrated efficient co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidated CMs into dense aggregates, which then formed a connected beating syncytium with significantly enhanced contractility (0.029 ± 0.002 mN/mm 2 ; p<0.05 vs. CMs-alone and co-culture controls), improved calcium handling (i.e. amplitude, upstroke, and decay), sarcomere length (1.82 ± 0.02 μm vs. 1.72 ± 0.03 μm, p=0.00264) and mitochondrial density (79.4 ± 3% vs. 54.9 ± 6%, p=0.0029). Co-cultured PECs demonstrated enhanced maturity to epicardial cells with significant upregulation of UPK1B, ITGA4, and ALDH1A2 transcript expressions. Our study also demonstrated that PECs secrete IGF2 and stimulate CM proliferation in co-culture (37.2 ± 2.1% vs. 28.1 ± 1%, p=0.016). These characteristics suggest PECs could play a key role in enhancing cell-cell signaling and tissue organization, which may help to recapitulate important aspects of cardiac morphogenesis in vitro and advance the maturation of bioengineered cardiac constructs.