PROJECT SUMMARY
Hypoplastic left heart syndrome (HLHS) is one of the most fatal congenital heart diseases (CHD) that occurs in
1 of every 4,344 newborns. The HLHS patients’ hearts show the severe underdevelopment of the left ventricle,
heart valves, and ascending aorta, and specifically lower capillary density in the myocardium. Moreover, the
genetic variant of NOTCH1 is implicated in HLHS patients showing the relevance to endocardial defects,
endothelial dysfunction, and NOTCH1 signaling pathways. However, how the NOTCH1 signaling determines
the underdevelopment of cardiac vasculature in the HLHS patients is still inconclusive. There is still a missing
link between the pathogenesis in the cardiovascular underdevelopment and dysfunction found in the HLHS
hearts and the NOTCH1 mutation. In this proposal, the genome-edited NOTCH1+/- hiPSCs will be co-
differentiated in an already optimized protocol into the vascularized cardiac organoid (VCO) with
cardiomyocytes, endothelial cells, smooth muscle cells, and other cardiac cells to recapitulate the
underdevelopment and dysfunction of the cardiovascular system in the early development of HLHS heart with
NOTCH1 mutation and to further delineate the pathogenesis of HLHS in the dynamic multicellular crosstalk of
cardiovascular cells via NOTCH-DLL-JAG receptor-ligand interactions. In the preliminary results, we observed
a reduced vascular formation with a smaller myocardial-like ring in VCO when NOTCH signaling was inhibited
by g-secretase inhibitor, and smaller organoid size was noticed in the NOTCH1+/- VCO compared to the
isogenic wildtype VCO. We will further explore the NOTCH1 signaling on vascular and cardiac formation in
VCOs by measuring the cardiovascular structure, proliferation, function, and corresponding gene expressions
(Aim 1). Moreover, our preliminary study also shows upregulation of NOTCH1 signaling via the NOTCH-DLL-
JAG ligand-receptor interactions in the VCO compared to the non-vascularized cardiac organoid by the single-
cell RNA-seq and ligand-receptor analysis. We will further investigate the ligand-receptor interactions and
pseudotime trajectories of NOTCH1 signaling in the process of NOTCH1+/- VCO formation. The proteome
profile over the HLHS VCO development will be uncovered by the proteomic analysis in correlation and
validation with the single-cell RNA-seq data (Aim 2). The completion of the proposed project will be for better
determining the NOTCH1 signaling-mediated multicellular crosstalk and downstream pathways of
underdeveloped cardiac vascularization in HLHS.
