REGULATORY GENOMIC STUDIES IN A COHORT OF IPS CELL DERIVED CARDIOMYOCYTES

描述(由申请人提供)： 我们项目的首要目标是使用来自基因分型个体的IPSC来源的心肌细胞作为细胞模型，以研究人类遗传变异如何影响心脏生物学和疾病涉及的基因调控网络。尽管目前的治疗方案，心血管疾病仍然是美国和发达国家发病率和死亡率的主要原因。全基因组关联研究已经确定了一些与心血管疾病易感性相关的基因座。我们的研究将通过将细胞重编程策略与集成的分子图谱和细胞分析相结合来阐明这些发现的功能意义。我们已经组建了一支在干细胞生物学、心脏细胞生物学、基因组学、分子遗传学/表观遗传学、生物统计学和临床医学方面成就卓著的研究团队，并已做好准备在五年内实现项目目标。在收集了加州大学圣迭戈分校TSP队列中个体的成纤维细胞和角质形成细胞后，该项目将分三个阶段进行。在第一阶段，我们将建立产生IPSC的标准化试剂和程序。此外，我们将利用我们正在进行的研究努力，将心肌细胞分化的效率提高到80%，比目前的方案(~20%)有很大的提高。在第二阶段，我们将开发尖端技术，用于生产高吞吐量的IPSC，这将使我们能够在大约24个月内生产600条IPSC生产线(每条生产线3条，每条200人)。我们还将扩大我们从IPSC系中分离心肌细胞的优化方案。在第三阶段，我们将首先进行验证实验，以测量同基因(来自同一个体)心肌细胞之间的基因组图谱变异性。然后，我们将使用衍生的心肌细胞来1)识别和表征具有心电特征的强Gwas信号背后的因果DNA变异；以及2)识别IPSC衍生的心肌细胞在基线(未处理)和刺激后的队列中的表达数量性状基因座(EQTL)。(摘要结束)

DESCRIPTION (provided by applicant): The overarching goal of our project is to use IPSC derived cardiomyocytes from genotyped individuals as cellular models to investigate how human genetic variation influences the gene regulatory networks Involved In cardiac biology and disease. Despite current treatment regimens, cardiovascular diseases remain the leading cause of morbidity and mortality in the United States and developed countries. Genome-wide association studies have identified a number of loci associated with cardiovascular disease susceptibility. Our Study will clarify the functional significance of these findings by combining cellular reprogramming Strategies with integrated molecular profiling and cellular assays. We have assembled a team of highly accomplished researchers in stem cell biology, cardiac cell biology, genomics, molecular genetics/epigenetics, biostatistics, and clinical medicine, and are well positioned to achieve the project goals within five years. After collecting fibroblasts and keratinocytes from individuals in the UCSD TSP cohort, the project will be carried out in three phases. In PHASE I, we will establish standardized reagents and procedures for the generation of iPSCs. Additionally, we will take advantage of our ongoing research efforts to increase the efficiency of cardiomyocyte differentiation to 80%, a substantial increase over current protocols (~20%). In PHASE II, we will develop cutting-edge technologies for high throughput generation of IPSCs, which will enable us to generate 600 iPSC lines (3 lines each from 200 individuals) in ~ 24 months. We will also scale our optimized protocols for deriving cardiomyocytes from the IPSC lines. In PHASE III we will initially perform validation experiments to measure the genomic profile variability between isogenic (derived from the same individual) cardiomyocytes. We will then use the derived cardiomyocytes to 1) Identify and characterize the causal DNA variants underlying strong GWAS signals with electrocardiographic traits; and 2) Identify expressed quantitative traits loci (eQTL) in the cohort of IPSC derived cardiomyocytes at baseline (untreated) and after stimulation. (End of Abstract)