Objectives

Congenital heart diseases (CHDs) are among the most common birth defects. The limited ability to study cardiac organogenesis in utero or maintain embryos ex vivo underscores the need for advanced in vitro models that enable quantitative analysis of normal and pathological heart development.

Methods

We developed a morphogen-free protocol to differentiate murine embryonic stem cells (ESCs) into 3D, functional, and spatially organized cardiac organoids (cardioids). Cardiac development was tracked over time via transcriptional profiling. Cellular heterogeneity, tissue structure, and spontaneous beating were assessed using 3D quantitative imaging. To model CHDs, Greb1l ⁺/tm1a ESCs were used for crisscross heart malformations, and Nodal inhibition was applied to simulate heterotaxy. A perfused droplet-based microfluidic platform was also developed for on-chip cardioid generation to enable scalable and reproducible production.

Results

Transcriptomic analysis revealed sequential activation of first and second heart field markers, followed by expression of cardiomyocyte, epicardial, and endothelial genes - mirroring stages of cardiac organogenesis. Key signaling pathways (Wnt, Nodal, BMP, p38-MAPK) were self-activated in a time-resolved manner, directing lineage commitment toward myocardium-, endocardium-, and epicardium-like tissues. Imaging showed organized tissue structure, cardiac myofibrils, heart-like cavities, and rhythmic beating. Functional heterogeneity across cardioids, reflected by variable beating frequencies, was linked with morphology, calcium transients, and cardiac marker expression. To assess capacity of cardioids to model CHDs, Greb1l-deficient ESCs exhibited disrupted organoid formation, absence of beating, and impaired cardiac progenitor development, consistent with in vivo crisscross phenotypes. Similarly, Nodal inhibition post-progenitor emergence mimicked heterotaxy traits such as reduced differentiation, slower beating, and increased proliferation. Cardioid generation was successfully implemented in a droplet microfluidic platform, supporting high-throughput applications.

Conclusion

Murine cardioids recapitulate key features of normal and pathological heart development. Their integration with microfluidic systems provides a robust, scalable platform for combinatorial drug screening and mechanistic studies of CHDs.