Functional human neurospheroids recapitulate key features of cortical complexity

Abstract

BackgroundThree-dimensional in vitro neuronal cultures have emerged as promising platforms for modelling human brain function and disease under controlled conditions. However, their ability to recapitulate in vivo-like complexity and rich dynamics remains underexplored. In this study, we developed and characterized neurospheroids derived from human induced pluripotent stem cells (hiPSCs) to investigate how key features -- three-dimensionality, cellular heterogeneity, and modular organization -- contribute to replicating brain-like network dynamics. MethodsWe engineered neurospheroids with varying excitatory/inhibitory ratios and assembled them into modular constructs (assembloids), evaluating their electrophysiological activity using high-density micro-electrode arrays. We assessed spontaneous and evoked activity through established metrics of dynamical richness and perturbational complexity. ResultsOur findings show that three-dimensionality and modularity significantly enhance the richness and complexity of network activity, approaching levels observed in vivo, while cellular heterogeneity contribute to functional aspects of the network, such as nuanced activity patterns, dynamical variability, and modular coordination. ConclusionsOur work highlights the critical role of spatial organization in reproducing brain-like activity and provide a foundation for future studies using patient-derived neurospheroids to model disease-specific dynamics.