Engineered Cardiac Microtissue Biomanufacturing Using Human Induced Pluripotent Stem Cell Derived Epicardial Cells

Epicardial cells are a crucial component in constructing in vitro 3D tissue models of the human heart, contributing to the ECM environment and the resident mesenchymal cell population. Studying the human epicardium and its development from the proepicardial organ is difficult, but induced pluripotent stem cells can provide a source of human epicardial cells for developmental modeling and for biomanufacturing heterotypic cardiac tissues. This study shows that a robust population of epicardial cells (approx. 87.7% WT1 ⁺ ) can be obtained by small molecule modulation of the Wnt signaling pathway. The population maintains WT1 expression and characteristic epithelial morphology over successive passaging, but increases in size and decreases in cell number, suggesting a limit to their expandability in vitro. Further, low passage number epicardial cells formed into more robust 3D microtissues compared to their higher passage counterparts, suggesting that the ideal time frame for use of these epicardial cells for tissue engineering and modeling purposes is early on in their differentiated state. Additionally, the differentiated epicardial cells displayed two distinct morphologic sub populations with a subset of larger, more migratory cells which led expansion of the epicardial cells across various extracellular matrix environments. When incorporated into a mixed 3D co-culture with cardiomyocytes, epicardial cells promoted greater remodeling and migration without impairing cardiomyocyte function. This study provides an important characterization of stem cell-derived epicardial cells, identifying key characteristics that influence their ability to fabricate consistent engineered cardiac tissues.