R37 - Cardiac Organoids Model EndoMT Dysregulation in Trisomy 21-Linked Congenital Heart Defects

Introduction: : Congenital heart defects (CHDs) occur in ~50% of individuals with Down syndrome (DS), contributing significantly to early mortality. These defects are linked to dysregulation of the endocardial-mesenchymal transition (EndoMT), a process critical for cardiac valve and septal development. Trisomy 21 disrupts genes such as COL6A1 and RCAN1, which are implicated in EndoMT, though mechanistic insights remain limited. Current cardiac models often lack endocardial cells, hindering study of this transition. To address this, we developed a human induced pluripotent stem cell (hiPSC)-based cardiac organoid model incorporating cardiomyocytes, fibroblasts, and endocardial cells from DS and control lines. This platform enables direct investigation of how trisomy 21 alters EndoMT dynamics, offering a path to unravel CHD pathogenesis. Our study bridges a critical gap by integrating endocardial cells into 3D organoids, providing a spatially organized system to model developmental defects.

Materials and

Methods: : Cardiac organoids were generated from DS and control hiPSCs differentiated into cardiomyocytes, fibroblasts, and endocardial cells. A tdTomato reporter was knocked into the AAVS1 locus via CRISPR-Cas9 to lineage-trace endocardial cells. Knock-in validation included Sanger sequencing and droplet digital PCR. Organoids were assembled in AggreWell™ plates (60% cardiomyocytes, 20% fibroblasts, 20% endocardial cells) and cultured for 8 days. CD31+ endocardial cells were sorted using magnetic-activated cell sorting. Organoids were fixed, stained for CD31 (endothelial) and αSMA (mesenchymal), and imaged via confocal microscopy. Imaris software quantified marker overlap, cell counts, and tdTomato+ cell distribution.

Results, Conclusions, and Discussions:: Cardiac organoids exhibited chamber-like structures and spatial stratification of cell types. The tdTomato+ endocardial cells localized to peripheral layers, with migration to organoid interiors by Day 7, suggesting EndoMT (Figure). Quantitative analysis revealed increased αSMA (mesenchymal) volume in DS organoids, though CD31/αSMA overlap showed no significant difference. Control organoids demonstrated higher mesenchymal surface area (p=0.0872), hinting at enhanced transition dynamics. Additionally, DS organoids showed earlier compaction and structural durability. This model addresses a critical gap by incorporating endocardial cells, enabling mechanistic insights into CHD pathogenesis linked to trisomy 21. While trends suggested altered EndoMT in DS, statistical power was limited by sample heterogeneity. Future work will expand sample sizes, extend culture durations, and incorporate additional markers to refine EndoMT assessment. This platform offers promise for studying heart structural development defects in patient-specific genetic backgrounds.

Acknowledgements and/or References (Optional):: We thank members of the Linda Crnic Institute for Down Syndrome Research Human Trisome Project Biobank involved in recruitment and consent of research participants; biospecimen collection, processing, and storage; maintenance of iPSC lines; and funding. We thank members of the Gates Institute for Regenerative Medicine who completed iPSC reprogramming.