Generation of mature epicardium derived from human-induced pluripotent stem cells via inhibition of mTOR signaling
Reactivating the human epicardium post-cardiac injury holds promise for cardiac tissue regeneration. Despite successful differentiation protocols yielding pure, self-renewing epicardial cells from induced pluripotent stem cells (iPSCs), these cells maintain an embryonic, proliferative state, impeding adult epicardial reactivation investigation. We introduce an optimized method that employs mammalian target of rapamycin (mTOR) signaling inhibition in embryonic epicardium, inducing a quiescent state that enhances multi-step epicardial maturation. This yields functionally mature epicardium, valuable for modeling adult epicardial reactivation. Furthermore, we assess cardiac organoids with cardiomyocytes and mature epicardium, probing molecular mechanisms governing epicardial quiescence during cardiac maturation. Our results highlight iPSC-derived mature epicardium's potential in investigating adult epicardial reactivation, pivotal for effective cardiac regeneration. Additionally, the cardiac organoid model offers insight into intricate cardiomyocyte-epicardium interactions in cardiac development and regeneration. Overall design: Exmination of mTOR inhibition in 3 embryonic epicardial cell types. Epicardium tissue was peeled from adult mouse heart and analyzed using RNA-seq. Human heart organoids were generated from 1390C1 cell line; after a 7-day treatment, untreated or Torin1-treated heart organoids were analyzed using scRAN-seq.