Quantitative Imaging Analysis of Internal Structural Complexity in Mouse Heart Organoids; Comparison to Mouse Embryonic Hearts.

We have established mouse heart organoids (mHOs) that are characterized by the presence of atrium- and ventricle-like structures that mimic entire embryonic hearts. However, maturation was not uniform, and little is known about their trabeculation status. Given the essential role of the trabeculae in cardiac morphogenesis, a new method that combines imaging analysis with a machine learning model was developed for quantifying the internal complexity in developing mHOs in a timely manner. We applied this method to screen for modified culture conditions and identified optimal treatment with valproic acid as a Notch activator and both bone morphogenetic protein 10 and Neuregulin 1 as downstream factors of Notch (a trabeculation-regulating signal) to promote mHO maturation. The established method using mouse fetal hearts as tests was suitable for comparing the internal complexity of both mHOs and mouse fetal hearts. The modified culture conditions improved the maturation uniformity as well as the internal structure of mHOs. Thus, this method can be applied to cardiac disorders with trabeculation problems and HOs developed by other methods. In addition, mHOs generated under these modified conditions may be an effective tool for studying the molecular mechanisms of heart development, including the signaling pathway of trabeculation related to these factors.