Human Induced Pluripotent Stem Cells as a Model of Complex Cardiac Disorders

Abstract

Hypoplastic left heart (HLH) is a genetically complex disease, characterized by hypoplasia of the left side of the heart. Although it is one of the most severe forms of congenital heart defects, our current knowledge of the molecular underpinnings of the disease is very limited. Here, we have generated an in vitro model of HLH using human induced pluripotent stem cells (hiPSCs) to uncover disease-causing factors. hiPSCs were generated from 10 unrelated HLH patients and their non-HLH parents (trio design; 3 clones per individual; 87 hiPSC lines in total), providing controls that are as genetically similar to the patients as possible. To investigate differences during early stages of cardiovascular development, hiPSCs were differentiated using both embryoid body and small molecule cardiac-directed differentiation methods. Cellular populations produced upon differentiation and their respective gene expression profiles were studied. Gene expression analysis of the spontaneously differentiated cells showed lower expression of both cardiac and vascular smooth muscle markers in patients compared to controls. Flow cytometry analysis performed on hiPSC cultures after directed cardiac differentiation at 5-day intervals (days 0-30) showed ventricular cardiomyocyte differentiation in HLH-hiPSCs was perturbed. A time-course analysis on 5 HLH families using RNAseq revealed that the greatest differences between patients and parents was at day 20 post-differentiation initiation, with down-regulation of cell cycle related pathways being the main driver. This finding was further validated using a second independent cohort of 5 HLH families. Proliferation assays corroborated our RNAseq data and showed an approximate 50% reduction in the percentage of dividing cardiomyocytes derived from HLH-hiPSCs compared to parents (P < 0.05). Cell phenotyping also indicated that beating cardiomyocytes derived from patients were more immature and their calcium flux properties were significantly different (n > 1000; P < 0.001). In summary, our findings thus far suggest that the progression of cardiogenesis and vasculogenesis in HLH-hiPSCs is perturbed, which may be based on differences in cell cycle properties. Furthermore, the functionality of cardiomyocytes derived from HLH-hiPSCs was altered (with respect to calcium flux properties), suggestive of cardiomyocyte immaturity. Our data suggest a common pathogenic pathway underlying the formation of HLH despite the genetic heterogeneity of disease causation.