Direct Coculture of hPSC-derived Cardiac Progenitor Cells with Epicardial cells induces Cardiomyocyte proliferation and ventricular specification

Epicardial cells (EpiCs) form the epicardium, the outer layer of the heart surrounding the myocardium. During development and in response to injury EpiCs undergo an epithelial to mesenchymal transition (EMT) migrating inward to create fibroblasts and smooth muscle cells in the myocardium. EpiCs have been shown to be necessary for proper myocardium formation, yet little is known about crosstalk between EpiCs and cardiomyocyte (CMs) and the potential impact of EpiCs on CM maturation. Here, we differentiated human pluripotent stem cells (hPSCs) to both cardiac progenitor cells (CPCs) and EpiCs, and cocultured EpiCs and CPCs in the presence of A83-01, to inhibit TGFβ signaling and EMT, for two weeks. CPCs were differentiated in the WTC-CAAX-RFP constitutive reporter hiPSC line and EpiCs were differentiated in the WTC-LMNB1-eGFP constitutive reporter hiPSC line. Cells were cocultured for 14 days following the previously described protocol, lifted for flow cytometry with Accutase, and resuspended in PBS (Ca2+/Mg2+ free) supplemented with 0.5% BSA and 5μM DAPI. Dead cells were gated out based on DAPI staining. For single cell sequencing, two samples were prepared of live cells from CMs and EpiCs monocultured or cocultured.