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. 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.心脏损伤后激活人心外膜组织，有望实现心脏组织再生。尽管目前已建立成熟的分化方案，可从诱导多能干细胞（induced pluripotent stem cells, iPSCs）中获得纯净且可自我更新的心外膜细胞，但这类细胞仍维持胚胎期增殖状态，阻碍了成人心外膜激活相关研究的开展。本研究提出一种优化方法：在胚胎心外膜中抑制雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）信号通路，诱导细胞进入静息状态，进而促进多阶段的心外膜成熟，最终获得功能成熟的心外膜组织，可用于成人心外膜激活的相关研究。此外，我们构建了包含心肌细胞与成熟心外膜细胞的心脏类器官模型，以此探究心脏成熟过程中调控心外膜静息状态的分子机制。研究结果表明，由iPSCs诱导获得的成熟心外膜细胞，在成人心外膜激活的相关研究中具有巨大应用潜力，这对实现高效心脏再生至关重要。同时，该心脏类器官模型可为解析心脏发育与再生过程中复杂的心肌细胞-心外膜细胞互作关系提供新的研究视角。本研究针对3种胚胎心外膜细胞类型开展了mTOR抑制相关实验：从成年小鼠心脏中剥离心外膜组织，并通过RNA测序（RNA-seq）进行分析；利用1390C1细胞系构建人类心脏类器官，经7天处理后，分别对未处理组与托林1（Torin1）处理组的心脏类器官采用单细胞RNA测序（scRNA-seq）开展检测分析。