Self-assembled chamber-like cardiac organoids for modeling cardiac chamber formation and cardiotoxicity assessment

The human heart remains a central focus of biomedical research, yet capturing early chamber morphogenesis in vitro with structural and functional fidelity remains challenging. Here we show the generation of chamber-like cardiac organoids (CCOs) from human pluripotent stem cells that reproduce key features of heart chamber development. CCO formation is orchestrated by coordinated FGF, WNT, BMP, RA, and AA signaling, and regulated by transcription factors including HAND1, TFAP2B and NR2F2. These CCOs exhibit stable self-organized architectures with myocardial, endothelial layers and inner chamber, displaying spontaneous contraction and calcium transients. Single-cell transcriptomics revealed endothelium-to-cardiomyocyte interactions mediated by WNT2B, suggesting coordinated tissue patterning. We further validated the essential roles of RA and AA in modulating chamber enlargement and stability. Furthermore, CCOs enable efficient evaluation of cardiotoxicity and teratogenicity through direct visualization of drug-induced changes. Together, this platform provides a robust model for studying cardiac development, mechanistic research, and pharmacological screening.