Single-cell transcriptional analysis of human pluripotent stem cell-derived heart epicardium-myocardium organoids uncover principals of human epicardial-derived cells specification and coronary vascular plexus development

Normal heart function is supported by the integration of functional vasculature and the autonomic nervous system. Therefore, the development of in vitro human heart organoid models that recapitulate not only the myocardium but also the complementary cellular systems that sustain normal cardiac function is crucial for advancing clinical and biomedical applications. Here, we describe a physiologically relevant hiPSC-derived heart organoid model that recreates epicardium-myocardium interactions, exhibiting an in vivo-like structural organization and function. The model also enables compact myocardium development in close crosstalk with a coronary-like, self-organized vascular plexus, as well as epicardial-derived adipose tissue specification within the subepicardial space. We investigated the effect of PDGF-BB and VEGF-A signaling cues on heart organoid development, cellular composition, and spatial organization. Single-cell RNA sequencing revealed that PDGF-BB supplementation enhances subepicardial cell expansion and differentiation of epicardial-derived lineages, whereas VEGF-A promotes the robustness and functionality of the coronary-like vascular network. This system provides a valuable platform for addressing fundamental questions in cardiac development and disease, particularly those related to epicardial-derived cell specification and normal coronary vasculature formation and maturation.