Characterization of Cardiac Progenitors Derived from 22q11-deleted Patients

描述（由申请人提供）：理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。在模式生物中的研究已经提供了信息，但很明显，种间差异的存在，重要地影响基因突变对心脏发育的影响。最近的进展已经建立了从多能干细胞分化人类心脏的体外系统模型，并且已经允许从先天性心脏病患者的体细胞发育多能干细胞。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。第二心脏区域（SHF）中的多能祖细胞以转录因子ISL 1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。22 q11缺失是最常见的染色体微缺失，导致以右心室和流出道发育异常为特征的先天性心脏病，至少部分是由于TBX 1基因剂量减少，TBX 1是SHF分化的关键转录调节因子。越来越多的证据表明，22 q11缺失通过破坏SHF祖细胞的分化而导致先天性心脏病。然而，这一假设尚未得到直接研究，特别是在来源于人类患者的SHF祖细胞中。在这个建议中，我们直接测试这一假设使用诱导多能（iPS）细胞来自22 q11缺失的法洛四联症（TOF）患者。首先，我们将生成TOF成纤维细胞和亲本对照的储存库。我们将选择22 q11缺失的成纤维细胞和非缺失的亲本对照，并产生iPS细胞系。其次，我们将描述22 q11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22 q11缺失与对照SHF祖细胞的转录组。这些实验将确定的遗传改变（22 q11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。该结果将为22 q11缺失导致的TOF发病机制提供新的见解，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。 公共卫生相关性：先天性心脏病是婴儿发病率和死亡率的主要非感染性原因。在过去的二十年里，对模式生物的研究极大地促进了我们对先天性心脏病遗传原因的理解。然而，目前还无法直接研究基因突变对人类心脏发育的影响。最近从患者皮肤细胞中生产多能细胞的进展使我们首次克服了这一障碍，并直接研究基因突变对人类心脏发育的影响。在这个建议中，我们研究22 q11缺失，最常见的染色体微缺失与先天性心脏病，心脏分化和基因表达的祖细胞来自22 q11缺失和法洛四联症患者的影响。这些研究将对22 q11缺失如何导致这种形式的先天性心脏病产生独特的见解。

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.