Identification of Novel Transcriptional Regulators of Stem Cell Derived Cardiomyocyte Maturation

干细胞来源的心肌细胞成熟的新型转录调节因子的鉴定

• 批准号: 9906423
• 负责人: Elaheh Karbassi
• 金额: $ 6.74万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9906423
• 关键词: Action Potentials; Adult; Aging; Animal Model; Animals; Area; Arrhythmia; Binding Sites; Biogenesis; Bypass; CRISPR screen; Calcium; Candidate Disease Gene; Cardiac; Cardiac Myocytes; Cell Therapy; Cells; ChIP-seq; Chromatin; Chromatin Structure; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Culture Media; DNA; Data; Data Set; Development; Disease; Disease model; Electrophysiology (science); Embryo; Energy-Generating Resources; Engraftment; Epigenetic Process; Expression Profiling; Fostering; Future; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Glucose; Goals; Heart; Human; Human body; In Vitro; Infarction; Injections; Injury; Lead; Length; Measures; Membrane Potentials; Mitochondria; Myocardial Infarction; Myofibrils; Natural regeneration; Organ; Pacemakers; Patients; Pattern; Phenotype; Potassium Channel; Property; Protein Isoforms; Protocols documentation; Publishing; Rattus; Regulation; Regulator Genes; Reperfusion Injury; Research; Research Personnel; Research Project Grants; Research Training; Rest; Sarcomeres; Site; Sodium Channel; Structural Genes; Structure; System; Testing; Therapeutic; Time; Tissue-Specific Gene Expression; Tissues; Translations; Transplantation; Up-Regulation; XCL1 gene; base; combinatorial; fatty acid oxidation; fetus cell; follow up assessment; gain of function; genetic testing; heart damage; heart function; human embryonic stem cell; human pluripotent stem cell; human stem cells; in vivo; induced pluripotent stem cell; nonhuman primate; novel; novel strategies; post-transplant; programs; regenerative; screening; single-cell RNA sequencing; stem cells; transcription factor; transcriptome; transcriptome sequencing

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.

项目摘要 该研究项目的目的是使人多能干细胞衍生的心肌细胞（hiPSC-1）成熟。 CM），使得它们显示出类似成人的电生理学特性。hPSC- 由于细胞缺乏相似性，CM在细胞治疗和疾病建模中的应用受到限制 在结构和功能上与成年人相似。心律失常的发展一直是 在将hPSC-CM移植到非人灵长类动物中后，存在一个问题，因此在研究中存在一个主要障碍。 将hPSC-CM翻译到临床。我假设hPSC-CM的不成熟状态是一个主要因素， 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， 增强hPSC-CM的体外成熟状态，用于各种应用。本项目的具体目标 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电成熟和诱导成人样心脏电生理特性，和2）阐明 成熟转录调节因子协调差异基因的染色质结构机制 表情我们通过研究人心肌细胞， 移植后体内成熟。我们将使用CRISPR激活（CRISPRa）来激活这些调节器， 测试它们是否足以产生电成熟的心肌细胞。成熟度将通过单细胞测量 RNA测序分析和电生理特性的后续评估。接下来是染色质 将进行免疫沉淀测序以鉴定候选转录因子靶基因组 结合位点。ATAC测序将用于评估程序性hiPSC中基因组可及性的调节。 CM和表征表观遗传机制，通过该机制，顶级候选因子完全和/或部分编程 成熟基因表达该项目提出了一种新的方法，通过使用高 通量和基于CRISPR的组合筛选，并将提供染色质结构的表征。 特征在于调节心肌细胞成熟。通过产生更成熟的hiPSC-CM，这些编程 心肌细胞可用作细胞疗法，以使术后患者的受损心脏组织肌肉化。 心肌梗死，同时绕过移植物诱导的心律失常。该项目将建立在申请人的 研究培训和技能，并将有助于促进申请人的道路，成为一个独立的 调查员