Cardiac Organoid Models of Hypoplastic Left Heart Syndrome

"Technical Abstract Hypoplastic left heart syndrome (HLHS) is a severe and multigenic congenital heart defect, characterized by underdevelopment of left ventricle and aortic valves. Studies to date, support a genetic etiology for HLHS, however the main genetic component and the molecular mechanism of the disease is unknown. Defects in cardiomyocytes (CM) proliferation and maturation has been reported in patients with HLHS, which has further been proven in our preliminary studies. Moreover, analysis of HLHS hearts suggest that apart from CM, endocardial cells (EC) are also affected. Proper development of the left ventricle depends on the crosstalk between CM and EC. Thus, traditional 2D approaches ability to capture the full spectrum of HLHS is limited. Moreover, due to the genetic heterogeneity of HLHS, small study cohorts are unable to determine the common disease-causing pathways. Here, we intend to utilize cardiac organoids from a large cohort of patient-specific hiPSC to pinpoint factors that can cause HLHS. During the Level I phase, we aim to develop a cardiac organoid model of early ventricular development. We will utilize our stablished CM and EC differentiation approaches to generate multicellular cardiac organoids. Organoids will then be treated with chamber formation factors and be assessed using qRT-PCR, single-cell RNA sequencing, whole organoid staining and 3D imaging (Aim1). Next, we will stablish hiPSC from 10 HLHS patients and 10 age- matched controls. Assessment of gene expression and function of hiPSC-CM, hiPSC-EC and cardiac organoids of HLHS and controls will unveil the disease-casing mechanism (Aim2). In the Level II phase, as an independent investigator, I will embark on studies a) to determine the genes associated with HLHS CM defect using CRISPR/Cas9 genome editing (Aim3), and b) to determine the drugs which can rescue the HLHS phenotype using unbiased high-throughput screening (Aim4). The long-term goal is to explore the molecular mechanism and genes associated with HLHS that will lead to the development of novel genome-informed therapeutic strategies."