This application is being submitted to PA-18-591 in accordance with NOT-OD-18-194. Parent Award FAIN: R01HL133230 issued by the NHLBI; Principal Investigator: Kunhua Song Project Title: Mechanisms for cell signaling in the control of cardiomyogenesis Project Period: 08/01/2016 ? 06/30/2021 Down syndrome, caused by trisomy of human chromosome 21 (HSA21), affects approximately 1 in 700 newborns in the United States (Parker et al., 2010). Congenital heart defects are very frequent in patients with Down syndrome with a prevalence of 40-50% compared to a risk of < 1% in typical children (Fahed et al., 2013; Antonarakis, 2017). Remarkable advances in health care and cardiac correction surgery have improved the survival rate of children born with Down syndrome. However, congenital heart disease is still a primary and significant risk factor for mortality in people with Down syndrome through age twenty (Kucik et al., 2013; Assenza et al., 2007; Uppal et al., 2015). However, cell and molecular mechanisms by which trisomy 21 causes susceptibility to congenital heart disease are poorly understood. Congenital heart disease can be regarded as a defect of morphogenesis and as dysregulation of expansion and differentiation of subsets of cardiac progenitors that contribute to the heart (Srivastava 2006). We propose a 1-2-year project to test our hypothesis that the heart defects in Down syndrome are caused by dysregulation of a specific population(s) of cardiac cells. Using single-cell RNA Sequencing (scRNA-seq), a cutting-edge single cell genomics approach, we will identify and characterize this cell population(s) and specific signaling pathways responsible for heart defects by analyzing transcriptomics in cardiac progenitors and cardiac lineage cells differentiated from induced pluripotent stem cells (iPSCs) derived from patients with Down syndrome and age-, gender-matched controls. These lines are derived from 6 controls, 6 individuals with both Down syndrome and congenital heart disease, and 1 individual with mosaic Down syndrome and congenital heart disease. Compared to standard RNA-seq, scRNA-seq can reveal individual cell phenotype in heterogeneously developmental processes. Cardiac defects may be caused by a specific cell population that cannot easily be isolated. sc-RNA-seq will allow us to do so and characterize cell-type specific dysregulation in Down syndrome. Animal models are used to investigate disease pathogenesis in vivo. However, many drug candidates based on findings in animal models have failed clinical trials, which is likely due to the physiological differences between animals and humans. Therefore, a combination of animal models and the iPSC-based human model has distinct advantage. Results generated from this project will identify signaling pathways and cell-types that contribute to congenital heart disease in patients with Down syndrome, which would build a strong foundation to discover therapeutics for Down syndrome and congenital heart disease. We believe that the study proposed here meets the criteria of this Administrative Supplement award because of the following reasons. 1. The overall goals of the parent award R01HL133230 are to ?discern the mechanisms underlying cardiomyogenesis and to develop a mechanism-based therapeutic strategy for cardiac regeneration?. In this parent award, we proposed to study mechanisms underlying cardiomyogenesis or formation of the heart using embryoid bodies (EBs) or cardiac lineage cells derived from iPSCs. EBs can spontaneously differentiated into derivatives of the three germ layers (endoderm, mesoderm and ectoderm). RNA-seq was proposed to analyze transcriptome in iPSC-derived EBs. We also proposed to study heart development in embryos using in vivo lineage tracing approach. These studies are proposed in Aim 1 & 2 of the R01HL133230. In this supplement proposal, we will use an optimized differentiation protocol to differentiate iPSCs into cardiac progenitor cells, cardiac neural crest progenitors, and cardiac lineage cells including cardiomyocytes, endothelial cells, endocardial cells and smooth muscle cells. Using the scRNA-seq approach, we will identify molecules and cell types that contribute to cardiac developmental defects in patients with Down syndrome. We also propose to confirm our results from patient-iPSCs using mouse models. Therefore, proposed work using iPSCs derived from individuals susceptible to congenital heart disease provides an additional and powerful model to study heart development to fulfil the overall goals of the parent award R01HL133230. 2. This proposed work aims to address underlying mechanisms that patients with Down syndrome are susceptible to congenital heart disease using the iPSC, scRNA-seq and in vivo mapping approaches. Using scRNA-seq, we will perform analysis of transcriptomic profiling during cardiac differentiation of iPSCs. Our proposed studies address Component 1 of the Down syndrome INCLUDE Project research objective, transcriptomic profiling in iPSCs. According to the NOT-OD-18-194, NHLBI priorities include characterization of differentiation of disease-related tissue types in iPSCs from individuals with Down syndrome and age-, gender-matched controls, and understanding why individuals with Down syndrome are susceptible to congenital heart disease. Therefore, proposed studies address NHLBI priorities as well.
Heart disease and Down syndrome are major health problems and growing economic burden all over the world. Research proposed in this project should provide a foundation for discovery of novel therapeutics to treat patients suffering from heart disease and Down syndrome.
| Riching, Andrew S; Zhao, Yuanbiao; Cao, Yingqiong et al. (2018) Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes. J Vis Exp : |
