Patient-Specific Induced Pluripotent Stem Cells for Modeling Single Ventricle Congenital Heart Disease

项目总结 这一五年计划的目的是为以下人员提供综合和个性化的培训计划 申请人过渡到一个独立的学术职位，成为一名专注于了解 先天性心脏病的根本原因。职业发展计划将在以下方面提供额外培训 诱导多能干细胞来源的心肌细胞(IPSC-CM)表型，CRISPR/Cas9基因组编辑， 和生物信息学。申请者还将在斯坦福接受大量的非正式和说教培训 大学在专业领域的专业发展和赠款写作技能，这将是至关重要的 让申请者获得自主权，并作为一名独立调查员开始富有成效的职业生涯。在.之下 肖恩·吴博士和尤安·阿什利博士的专家指导，以及聚集的咨询委员会(Dr。 Daniel Bernstein、Marlene Rabinovitch博士、James Priest博士和Matthew Porteus博士)，申请者将 获得必要的指导和资源以实现这些目标并高效地过渡到 在K08训练期后独立。 这项建议的研究课题通过鉴定心肌细胞来填补该领域的重大知识空白-- 单心室先天性心脏病的内在因素。通过使用患者特定的IPSCs，内在的 可以研究导致人类先天性心脏病的早期心脏发育异常。 最严重和最致命的先天性心脏病是单心室疾病，它 涉及左或右心室严重发育不良，导致全身或右室发育不全 肺血流量。虽然流行的疾病发病机制理论涉及到脑室发育受损 由于发育中的脑室血流减少，心肌细胞固有的异常也被怀疑，但 很大程度上是未被开发的。在目标1中，心肌细胞生成和功能的固有缺陷将是 评估过了。通过使用优雅的记者系统，特定的异常将在一个房间里被表征- 具体方式并归因于左、右室IPSC-CMS。在目标2中，心肌细胞背后的机制- 将使用单细胞RNA测序分析来研究内在扰动，以识别异常基因 表情模式。此外，这一数据将与来自 儿科心脏基因组联合会(PCGC)验证IPSC-CMS反映疾病的能力 心肌。最后，在AIM 3中，IPSC-CMS将暴露在葡萄糖和缺氧的环境中，以模拟 在发育过程中血流受损的影响，然后评估细胞死亡和增殖。

PROJECT SUMMARY The purpose of this five-year proposal is to provide an integrative and personalized training program for the applicant to transition into an independent academic position as a basic scientist focused on understanding the underlying causes of congenital heart disease. The career development plan will provide additional training in induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) phenotyping, CRISPR/Cas9 genome editing, and bioinformatics. The applicant will also receive a wealth of informal and didactic training at Stanford University in specialized areas such as professional development and grant writing skills, which will be critical for the applicant to gain autonomy and launch a productive career as an independent investigator. Under the expert mentorship of Dr. Sean Wu and Dr. Euan Ashley, along with the assembled advisory committee (Dr. Daniel Bernstein, Dr. Marlene Rabinovitch, Dr. James Priest, and Dr. Matthew Porteus), the applicant will receive the necessary guidance and resources to accomplish these goals and efficiently transition to independence following the K08 training period. The research topic of this proposal fulfills a significant knowledge gap in the field by identifying myocyte- intrinsic contributions to single ventricle congenital heart disease. By using patient-specific iPSCs, intrinsic abnormalities of early cardiac development that contribute to human congenital heart disease can be studied. Among the most severe and lethal forms of congenital heart disease are single ventricle diseases, which involve profound underdevelopment of either the left or right ventricle, with resulting insufficient systemic or pulmonary blood flow. While the prevailing theory of disease pathogenesis involves impaired ventricular growth due to reduced blood flow in the developing ventricle, myocyte-intrinsic abnormalities are also suspected but largely unexplored. In Aim 1, intrinsic deficiencies in ventricular cardiomyocyte generation and function will be assessed. By using an elegant reporter system, specific abnormalities will be characterized in a chamber- specific manner and attributed to left or right ventricular iPSC-CMs. In Aim 2, the mechanisms behind myocyte- intrinsic perturbations will be investigated using single cell RNA sequencing analysis to identify abnormal gene expression patterns. Additionally, this data will be compared to ventricular tissue RNA sequencing data from the Pediatric Cardiac Genomics Consortium (PCGC) to validate the ability of iPSC-CMs to reflect diseased myocardium. Finally, in Aim 3 iPSC-CMs will be exposed to glucose and oxygen deprivation to mimic the effects of impaired blood flow during development, followed by an assessment of cell death and proliferation.