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

人类 iPSC-CM 亚型身份和功能的单细胞测序

基本信息

批准号：
9763916
负责人：
THOMAS QUERTERMOUS
金额：
$ 70.11万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9763916
关键词：
Adult Area CRISPR/Cas technology Cardiac Cardiac Myocytes Cardiac development Cardiovascular Diseases Cells ChIP-seq Characteristics Collaborations Complex Data Disease model Electrocardiogram Exogenous Factors Female Fostering Genetic Engineering Heart Heart Atrium Heart Diseases Histology Human In Vitro Injections Injury Knock-out Light Link Magnetic Resonance Imaging Miniature Swine Modeling Molecular Morbidity - disease rate Myocardial Infarction Myocardium Nodal Patients Phenotype Population Protocols documentation Recovery of Function Reporter Research Saline Source Stem cells Telemetry Therapeutic Time Tissue-Specific Gene Expression Transplantation Tretinoin Ventricular apoAI regulatory protein-1 drug development drug testing genetic signature heart damage heart function improved in vivo induced pluripotent stem cell male monolayer mortality novel overexpression patch clamp progenitor regenerative therapy repaired single cell analysis single cell sequencing single-cell RNA sequencing tool transcription factor transcriptome transcriptome sequencing transcriptomics treatment strategy

项目摘要

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.

项目摘要来自人类诱导多能茎细胞（IPSC-CM）的心肌细胞具有研究的潜力菜肴中特定于患者的心脏病。它们的实用性范围从在药物开发中很重要，细胞疗法以及了解人类心脏发展。虽然我们可以产生殴打菜肴中的心肌细胞，它们对人类成人亚型（例如心房，心室和淋巴结）的表示仍然是基本的。要了解将IPSC-CMS调节为各种心肌细胞亚型的因素，我们将利用单个细胞RNA测序方法来识别涉及的关键转录因子祖细胞心肌细胞转变为心房样（视黄酸治疗的培养物）或室状样（默认分化参数）表型。此外，我们将在基因上设计我们的IPSC 使用CRISPR/CAS9技术缺乏或标记核心亚型特定标记的表达。在使用中这种方法，我们将评估心肌细胞亚型涉及的主要转录因子是什么规格以及体外形成的特定心肌细胞亚群是否更好地修复受损的心。在使用Miniswine心肌梗死模型时，我们将评估亚群的良好状态整合在缺血性迷你葡萄酒中，并评估每个功能恢复参数心肌细胞亚群将有助于修复受损的心肌。最后，通过恢复已经整合在心脏中的IPSC-CMS，我们将通过单细胞测序评估这些细胞的转录组在体内发挥作用后会发生变化。我们预计这些研究将丢弃阐明如何在体外区分各种亚型，这将进一步加速我们对IPSC-CMS的使用用于研究和治疗应用。