Characterization of Cardiac Progenitors Derived from 22q11-deleted Patients

描述（由申请人提供）：了解先天性心脏病发病机制的一个基本限制是缺乏研究人类心脏发育的模型系统。模式生物的研究已经提供了丰富的信息，但显然存在物种间差异，这对基因突变对心脏发育的影响具有重要影响。近年来的研究进展已经建立了从多能干细胞分化人类心脏的体外模型系统，并允许从先天性心脏病患者的体细胞中培养出多能干细胞。总的来说，这些进展使我们能够首次研究基因突变对人类心脏发育的影响。第二心田（SHF）的多能祖细胞，以转录因子ISL1的表达为标志，有助于右心室和流出道的心肌细胞、平滑肌和内皮细胞谱系。22q11是最常见的染色体微缺失，其缺失导致先天性心脏病，其特征是右心室和流出道发育异常，至少部分原因是TBX1基因剂量减少，TBX1是SHF分化的关键转录调节因子。越来越多的证据表明，22q11缺失通过破坏SHF祖细胞的分化导致先天性心脏病。然而，这一假设尚未得到直接研究，特别是在来自人类患者的SHF祖细胞中。在这项提议中，我们使用来自22q11缺失法洛四联症（TOF）患者的诱导多能（iPS）细胞直接验证了这一假设。首先，我们将生成一个TOF成纤维细胞库和亲代对照。我们将选择22q11缺失的成纤维细胞和未缺失的亲代对照，并生成iPS细胞系。其次，我们将描述22q11缺失与对照SHF祖细胞的分化。第三，我们将使用全转录组测序（RNA-seq）来表征22q11缺失与对照的SHF祖细胞的转录组。这些实验将把确定的基因改变（22q11缺失）与患者特异性细胞表型和基因转录中的分子改变联系起来。该结果将对22q11缺失导致TOF的发病机制产生新的见解，并将为研究其他形式的先天性心脏病的发病机制建立模型。

DESCRIPTION (provided by applicant): A fundamental limitation in understanding the pathogenesis of congenital heart disease has been the lack of model systems to study human heart development. Studies in model organisms have been informative, but clearly interspecies differences exist that importantly influence the effects of gene mutations on heart development. Recent advances have established model in vitro systems of human heart differentiation from pluripotent stem cells, and have permitted development of pluripotent stem cells from somatic cells of congenital heart disease patients. Collectively, these advances position us to study for the first time the effect of gene mutations on human heart development. Multipotent progenitors in the second heart field (SHF), marked by expression of the transcription factor ISL1, contribute to cardiomyocyte, smooth muscle, and endothelial, lineages of the right ventricle and outflow tract. Deletion of 22q11, the most common chromosomal microdeletion, causes congenital heart disease characterized by abnormalities of right ventricle and outflow tract development, at least in part as a result of reduced gene dosage of TBX1, a key transcriptional regulator of SHF differentiation. Growing evidence suggests that 22q11 deletion causes congenital heart disease by disrupting differentiation of SHF progenitors. However, this hypothesis has not been directly studied, particularly in SHF progenitors derived from human patients. In this proposal, we directly test this hypothesis using induced-pluripotent (iPS) cells derived from 22q11-deleted patients with tetralogy of Fallot (TOF). First, we will generate a repository of TOF fibroblasts and parental controls. We will select 22q11-deleted fibroblasts and non-deleted parental controls, and generate iPS cell lines. Second, we will characterize the differentiation of 22q11-deleted versus control SHF progenitors. Third, we will use whole transcriptome sequencing (RNA-seq) to characterize the transcriptome of 22q11-deleted versus control SHF progenitors. These experiments will link a defined genetic alteration (22q11 deletion) with patient-specific cellular phenotypes and molecular alterations in gene transcription. The results will lead to novel insights on the pathogenesis of TOF as a result of 22q11 deletion, and will establish a model for studying the pathogenesis of other forms of congenital heart disease. Public Health Relevance: Congenital heart disease is the leading non-infectious cause of morbidity and mortality among infants. Over the past two decades, studies in model organisms have greatly advanced our understanding of the genetic causes of congenital heart disease. However, it has not been possible to directly study the effect of gene mutations on human heart development. Recent advances in production of pluripotent cells from skin cells of patients allows us for the first time to overcome this hurdle and directly investigate the effect of gene mutations on human heart development. In this proposal, we study the effect of 22q11 deletion, the most common chromosomal microdeletion associated with congenital heart disease, on cardiac differentiation and gene expression in progenitor cells derived from patients with 22q11 deletion and tetralogy of Fallot. These studies will yield unique insights into how 22q11 deletion causes this form of congenital heart disease.