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

• 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
• 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

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 来自人诱导多能干细胞（iPSC-CMs）的心肌细胞具有研究的潜力 病人特定的心脏病它们的用途从在药物开发中的重要性， 细胞疗法，以及了解人类心脏发育。虽然我们可以产生跳动 培养皿中的心肌细胞，它们代表人类成年亚型（例如，心房、心室和结） 还不成熟为了了解将iPSC-CM调节成各种心肌细胞亚型的因素， 我们将利用单细胞RNA测序的方法来确定关键的转录因子参与， 前体心肌细胞向心房样（视黄酸处理的培养物）或心室样（视黄酸处理的培养物）的转变 （默认分化参数）表型。此外，我们将对我们的iPSC进行基因工程改造， 使用CRISPR/Cas9技术检测核心亚型特异性标志物的表达或标记核心亚型特异性标志物的表达。在使用 这种方法，我们将评估哪些是参与心肌细胞亚型的主要转录因子， 规格以及体外形成的特定心肌细胞亚群是否更好地修复心肌细胞。 受损的心脏在使用小型猪心肌梗死模型中，我们将评估亚群 在缺血的小型猪心脏内整合，以及评估功能恢复参数， 心肌细胞亚群将有助于修复受损的心肌。最后，通过恢复 我们将通过单细胞测序来评估已经整合到心脏中的iPSC-CMs如何与心脏中的iPSC-CMs结合。 这些细胞的转录组在体内发挥功能后发生变化。我们预计，这些研究将摆脱 阐明了如何在体外区分各种亚型，这将进一步加速我们对iPSC-CM的使用 用于研究和治疗应用。