Abstract
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.
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.
Original language | English |
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DOIs | |
Publication status | Published - 6 Nov 2023 |
Event | Circulation - Duration: 1 Jan 1824 → … http://<Go to ISI>://WOS:000241792804635 |
Conference
Conference | Circulation |
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Period | 1/01/24 → … |
Internet address |