Abstract Body: We have developed an innovative protocol that produces uniform cardiac organoids, solving the challenges of variable cell composition and morphology in existing iPSC-based models. Our approach combines co-cultured cardiac progenitor cells and fibroblasts derived from human iPSCs with precisely timed VEGF and TGF-β1 administration, resulting in organoids with consistent morphology and cellular makeup.
Developmental analysis shows beating cardiomyocytes concentrated in the core with cavity formation at day 14, followed by increased internal cell density by day 30. Imaging and immunostaining demonstrate uniform distribution of cardiomyocytes, fibroblasts, and endothelial cells, with WT1+ cell alignment in the outer wall suggesting epicardial differentiation capacity.
We investigated BIO (6-Bromoindirubin-3'-oxime), a GSK3 inhibitor with known cardioprotective effects in animal models. BIO treatment enhanced cell proliferation in 2D cultures and expanded cardiomyocyte-concentrated regions with broader WT1+ cell distribution in the organoids. By confirming BIO's myocardial regenerative potential in human cardiomyocytes, we induced the formation of more stable cardiac organoids by incorporating BIO into our protocol. In this study, we developed the reproducible organoids to model human cardiac pathophysiology, enabling disease mechanism studies and therapeutic validation.