Developmental cues from epicardial cells simultaneously promote cardiomyocyte proliferation and electrochemical maturation
Accumulating evidence indicates that maturation limits cardiomyocyte proliferation and conversely, that proliferation occurs rarely in cardiomyocytes of the adult myocardium. Here we  show results that expand on that theory when we co-culture hiPSC-CM with epicardial cells and epicardial-derived cells in two-dimensional (2D) direct co-culture and 3D engineered heart tissues (EHT). In 2D direct co-cultures the percentage of proliferating CM increased at the same time as stark electrophysiologic improvements in calcium handling. Single-cell transcriptomics revealed a significant shift in the bulk CM population in epicardial-CM co-cultures as characterized by more fetal-like myofilament isoforms but with enhanced pathways associated with electrochemical maturation and ventricular development. Co-culture of epicardial cells in the 3D-EHT model showed more limited proliferation but a similar improvement in CM electrophysiologic function. These results indicate that certain aspects of CM maturation are enhanced, especially electrophysiologic properties, by epicardial cells while an embryonic phenotype is maintained (i.e., proliferation and myofilament isoforms). Next, consistent with developmental progression and the initiation of epicardial-to-mesenchymal transition (EMT), epicardial-derived fibroblasts were added to the EHTs containing epicardial cells. Interestingly, we observed significant myofilament maturation and increased force generation by more than an order of magnitude. Taken together, our results suggest that some aspects of CM maturation (i.e., electrochemical) occur during embryonic development when proliferation rates are relatively high and that sarcomere-associated mechanical maturation occurs at later developmental stages when proliferation has largely ceased. Overall design: Cardiomyocytes and epicardial cells derived from human induced pluripotent stem cells were co-cultured with or without TGF-Beta inhibitors that were used to inhibit epicardial epithelial-to-mesenchymal transition.