Abstract 16686: A Novel Co-Culture 3D Cardiac Organoid Models Early Heart Development

Introduction: Embryoid body-derived cardiac organoids (CO), incorporating complex chamber-like structures, have been reported since 2021. By contrast, co-culture COs described so far, which have been assembled from primary or hESC derived-cardiomyocytes, endothelial and fibroblast cells, lack complex structures. Cells of the endocardial lineage have a critical role in heart septation and chamber formation in vivo , but their potential role in engineering complex COs is hitherto unreported. We present a co-culture CO method incorporating endocardial lineage cells, showing these cells' critical contribution in forming chambered and vascularised COs suitable to model heart development and congenital heart disease (CHD).

Methods: hESC-cardiomyocytes (CM, Day12), hESC-endothelial cell (EC, Day16), hESC-cardiac fibroblasts (Fb, Day16), and hESC-endocardial like cells (ECC, Day12) are differentiated with 2D protocols. COs are formed by co-culturing cells (1-2x10 ^{^ 4} cells/organoid) in ultra low attachment plates. Mature COs contain CMs, ECs and Fbs (3:1:1). Developing COs contain CMs, ECs, and ECCs (2:1:2). After four weeks, COs are characterised by immunostaining and optical mapping.

Results: Mature and Developing COs form in three days of co-culture, with beating activities restoring during 2-7 days. Fig1A shows that both mature COs and developing COs beat regularly. Mature COs show evenly distributed cell populations (Fig1B). Developing COs form chamber-like cavities with surrounding vascular lumens (Fig1C). The inside chamber is layered by endocardial cells (Fig1C); vascular lumens form networks (Fig1C) within the middle compartment of CMs (Fig1D). Fibroblasts mainly specify the outside layer of developing COs (Fig1D).

Conclusions: Endocardial lineage is critical in forming chamber-like structures in COs. This lineage-controllable developing CO can apply to drug screening and model early heart development and CHDs.