Identification of Novel Transcriptional Regulators of Stem Cell Derived Cardiomyocyte Maturation

项目摘要 本研究的目的是成熟人多能干细胞来源的心肌细胞(hiPSC- CMS)通过转录编程，使它们表现出成体一样的电生理特性。HPSC- 由于细胞缺乏相似性，CM在细胞治疗和疾病建模方面的应用受到限制 在结构上和功能上都与成年对应物相同。心律失常的发展一直是一个主要的 HPSC-CMS移植到非人类灵长类动物后的问题，因此是 将hPSC-CMS移植到临床。我推测hPSC-CMS的不成熟状态是一个主要因素 对于移植物诱导的心律失常，这些细胞的转录编程将是一个有效的系统 提高hPSC-CMS的体外成熟状态，以适应各种应用。这个项目的具体目标是 是：1)确定一组核心的心脏转录调节因子，可用于组合编程 HiPSC-CM电成熟和诱导成人型心脏电生理特性；2)阐明 成熟转录调控因子协调差异基因的染色质结构机制 表情。我们通过对人类心肌细胞的研究确定了候选转录调控因子 移植后体内成熟。我们将使用CRISPR激活(CRISPRa)激活这些调节器以 检测其在产生电成熟心肌细胞方面的充分性。成熟度将由单个细胞来衡量 RNA测序分析和电生理特性的后续评估。接下来，染色质 将进行免疫沉淀-测序以确定候选转录因子目标基因组 结合部位。ATAC测序将用于评估程序化HiPSC中基因组可及性的调节。 并表征表观遗传机制，通过这些机制，顶级候选因子完全和/或部分编程 成熟基因的表达。本项目提出了一种成熟的HiPSC-CMS的新途径 产量和基于CRISPR的组合筛选，并将提供染色质结构的特征 具有调节心肌细胞成熟的特点。通过产生更成熟的HiPSC-CMS，这些编程 心肌细胞可用于细胞治疗，以重建受损的心肌组织 心肌梗死，同时绕过移植物引起的心律失常。这个项目将建立在申请人的基础上 研究、培训和技能，将有助于促进申请者成为独立的 调查员。

PROJECT ABSTRACT The purpose of this research project is to mature human pluripotent stem cell-derived cardiomyocytes (hiPSC- CMs) via transcriptional programming, such that they display adult-like electrophysiological properties. hPSC- CM applications for cell therapy and disease modeling are limited due to the cells’ lack of resemblance structurally and functionally to their adult counterparts. The development of arrhythmias has been a major problem after transplantation of hPSC-CMs into non-human primates and thus presents a major obstacle in the translation of hPSC-CMs to the clinic. I hypothesize that the immature state of hPSC-CMs is a major contributor to the graft-induced arrhythmias, and transcriptional programming of these cells will be an effective system to enhance the maturation state of hPSC-CMs in vitro for various applications. The specific aims for this project are: 1) to identify a core set of cardiac transcriptional regulators that can be used to combinatorially program hiPSC-CM electrical maturation and induce adult-like cardiac electrophysiological properties, and 2) to elucidate the chromatin structural mechanisms by which maturation transcriptional regulators coordinate differential gene expression. We have identified candidate transcriptional regulators by studying human cardiomyocyte maturation in vivo after transplantation. We will activate these regulators using CRISPR activation (CRISPRa) to test their sufficiency in generating electrically mature cardiomyocytes. Maturation will be measured by single cell RNA-sequencing analyses and follow-up assessment of electrophysiological properties. Next, chromatin immunoprecipitation-sequencing will be performed to identify candidate transcription factor target genomic binding sites. ATAC-sequencing will be used to assess regulation of genome accessibility in programmed hiPSC- CMs and to characterize the epigenetic mechanisms by which top candidate factors fully and/or partially program maturation gene expression. This project presents a new approach to mature hiPSC-CMs by using high throughput and combinatorial CRISPR-based screening and will provide characterization of chromatin structural features regulating cardiomyocyte maturation. Through generating more mature hiPSC-CMs, these programmed cardiomyocytes can be applied as cell therapy to remuscularize damaged heart tissue in patients after myocardial infarction, while bypassing graft-induced arrhythmias. This project will build on the applicant’s research training and skillsets and will serve to foster the applicant’s path towards becoming an independent investigator.