Characterization of Cardiac Progenitors Derived from 22q11-deleted Patients

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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.