Project Summary Cardiovascular tissue engineering with pluripotent stem cells has emerged as a means to generate human cardiac tissues that can be used to model myocardial function and disease or for clinical implantation. However, existing tissue models are limited by low thickness and a lack of structural and functional maturity, due to the poor proliferative capacity, maturation, and embryonic-like phenotypes of stem cell-derived cardiomyocytes (CMs). During mammalian development, the epicardium provides critical signals to the myocardium, enabling ventricular compaction by secreting pro-mitogenic factors and contributing coronary vascular smooth muscle cells (CVSMCs) and cardiac fibroblasts (CFs) to the heart. While these cells can be harnessed to improve proliferation and maturation of CMs in vitro, there is a limited understanding of the underlying cellular mechanisms that drive these effects in human cells. In this proposal, we seek to elucidate the intermediate signals driving human epicardial-myocardial interactions by developing a 3D printed cardiac tissue model with a functional epicardial cell layer, utilizing CMs and epicardial progenitor cells (EPCs) derived from human induced pluripotent stem cells (hiPSCs). This laminated 3D-tissue model is designed to enable EPCs to undergo epithelial-to-mesenchymal transition (EMT) and migrate into the tissue bulk, and also provides a unique environment to probe ECM remodeling by epicardial derived cells (EPDCs), a process we hypothesize is one of the key mechanisms by which these cells drive maturation of cardiac tissue. Utilizing gene editing and high- throughput proteomic analysis, we will identify EPC-secreted growth factors that promote hiPSC-CM proliferation as well as EPDC-secreted ECM proteins that are critical to structural and functional maturation of cardiac tissue. We will then incorporate an epicardial layer onto a more geometrically complex model - a 3D printed, human chambered myocardial pump. We will investigate the impact of the epicardium with and without imposed volumetric pressure on pump function and clinical parameters like stroke work and ejection fraction. Insights gained from this work will expand our knowledge of developmental processes and propel the next generation of engineered cardiac tissues. These studies will, for the first time, elucidate details of epicardial-myocardial signaling in a human model and establish a link between EPDC ECM secretion and cardiac tissue maturation. Additionally, these studies will advance the structure and function of a clinically relevant myocardial pump model that has the potential to be used for drug testing, device testing, and modeling of diseases – especially those that manifest in altered pressure-volume dynamics