Epicardial regulation of cardiomyocyte function via modulation of extracellular signals: toward a model of human muscle pump function

项目摘要 利用多能干细胞的心血管组织工程已经成为一种产生人类 可用于模拟心肌功能和疾病或用于临床植入的心脏组织。然而，在这方面， 现有的组织模型受到低厚度和缺乏结构和功能成熟度的限制， 干细胞衍生的心肌细胞的增殖能力、成熟和胚胎样表型较差 （CM）。在哺乳动物发育过程中，心外膜向心肌提供关键信号， 通过分泌促有丝分裂因子和促进冠状血管平滑肌的心室致密化 细胞（CVSMC）和心脏成纤维细胞（CF）。虽然这些细胞可以用来改善 增殖和成熟的CM在体外，有一个有限的理解的基础细胞 在人类细胞中驱动这些效应的机制。在这个建议中，我们试图阐明中间体 通过开发3D打印的心脏组织模型来驱动人类心外膜-心肌相互作用的信号， 功能性心外膜细胞层，利用来源于人诱导的CM和心外膜祖细胞（EPC）， 多能干细胞（hiPSC）。这种分层的3D组织模型旨在使EPC能够进行 上皮细胞向间充质细胞转化（EMT）并迁移到组织块中，并且还提供了独特的 环境来探测心外膜衍生细胞（EPDC）的ECM重塑，我们假设这一过程是 这些细胞驱动心脏组织成熟的关键机制。利用基因编辑和高- 通过蛋白质组学分析，我们将鉴定EPC分泌的促进hiPSC-CM增殖的生长因子 以及对心脏组织的结构和功能成熟至关重要的EPDC分泌的ECM蛋白。 然后，我们将把心外膜层结合到一个更复杂的几何模型上-一个3D打印的人体模型。 室性心肌泵我们将研究心外膜的影响， 容积压力对泵功能和临床参数如每搏功和射血分数的影响。见解 从这项工作中获得的知识将扩大我们对发展过程的了解，并推动下一代的发展。 工程心脏组织这些研究将首次阐明心外膜-心肌 在人模型中研究EPDC ECM信号传导，并建立EPDC ECM分泌和心脏组织成熟之间的联系。 此外，这些研究将推进临床相关心肌泵模型的结构和功能 它有可能用于药物测试，设备测试和疾病建模-特别是那些 表现为压力-容积动力学的改变

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