Single Cell Sequencing of Human iPSC-CM Subtype Identity and Function

项目总结 人诱导多能干细胞心肌细胞(IPSC-CMS)具有研究潜力 一盘特定于患者的心脏病。它们的用途从在药物开发中的重要作用， 细胞疗法，以及了解人类心脏发育。尽管我们可以产生击打 培养皿中的心肌细胞，它们代表人类成年亚型(例如，房室和结节)是 仍然是初级的。为了解调节IPSC-CMS分化为不同心肌细胞亚型的因素， 我们将利用单细胞RNA测序的方法来确定参与 祖细胞心肌细胞向心房样(维甲酸处理的培养)或室样的转化 (默认分化参数)表型。此外，我们将通过基因工程使我们的ipscs成为 使用CRISPR/Cas9技术缺乏或标记核心亚型特异标记的表达。在使用中 这种方法，我们将评估哪些主要转录因子参与心肌细胞亚型 在体外形成的特定的心肌细胞亚群是否更适合于修复 心脏受损。在使用小型猪心肌梗死模型时，我们将评估亚群 在缺血的迷你心脏内进行整合，并评估每个 心肌细胞亚群将有助于受损心肌的修复。最后，通过恢复 已经整合到心脏内的IPSC-CMS，我们将通过单细胞测序来评估 这些细胞的转录组在体内发挥功能后会发生变化。我们预计，这些研究将摆脱 阐明不同亚型如何在体外分化，这将进一步加快我们对IPSC-CMS的使用 用于研究和治疗应用。

PROJECT SUMMARY Cardiomyocytes from human induced pluripotent stems cells (iPSC-CMs) possess the potential to study patient-specific heart disease in a dish. Their usefulness ranges from being important in drug development, cellular therapies, as well as understanding human cardiac development. Although we can generate beating cardiomyocytes in a dish, their representation of human adult subtypes (e.g., atrial, ventricular, and nodal) is still rudimentary. To understand the factors which regulate iPSC-CMs into various cardiomyocyte subtypes, we will utilize a single cell RNA sequencing approach to identify critical transcription factors involved in the transition of progenitor cardiomyocytes into an atrial-like (retinoic acid-treated cultures) or ventricular-like (default differentiation parameters) phenotype. Furthermore, we will genetically engineer our iPSCs to be deficient in or mark the expression of core subtype specific markers using CRISPR/Cas9 technology. In using this approach, we will assess what are the master transcription factors involved in cardiomyocyte subtype specification and whether a specific cardiomyocyte subpopulation formed in vitro is better at repairing the damaged heart. In using a miniswine myocardial infarction model, we will assess how well subpopulations integrate within the ischemic miniswine hearts as well as assess the functional recovery parameters that each cardiomyocyte subpopulations will contribute to repairing the damaged myocardium. Finally, by recovering the iPSC-CMs that have integrated within the heart, we will assess by single-cell sequencing how the transcriptome of these cells changes after being functional in vivo. We anticipate that these studies will shed light on how various subtypes can be differentiated in vitro which will further accelerate our use of iPSC-CMs for research and therapeutic applications.