Single rosette-based generation of uniform cortical assembloids recapitulating cellular interactions between neurons and glial cells

Abstract Despite recent advances, current brain organoid technologies face ongoing challenges in managing heterogeneity and representing the diverse structure and cell types of the human brain. Here, we develop a module-based cellular reconstitution technology to sequentially build uniform cortical assembloids with mature cortical structures and functional connectivity. The uniformity and maturity of the cortical assembloids are achieved by creating single-rosette-based organoids at the early stage, whose sizes were big and consistent with the treatment of Wnt and Hedgehog agonists, followed by spatial reconstitution with the Reelin-expressing neuronal layer and non-neuronal glial cells. The resulting single-rosette-based cortical assembloids are highly uniform and reproducible without significant batch effects, solving major heterogeneity issues caused by difficulties in controlling the number and size of rosettes in conventional multi-rosette organoids. Furthermore, these cortical assembloids structurally and functionally recapitulate the physiology of the human brain, including the six-layered cortical structure, functional connectivity, and dynamic cellular interplay between neurons and glial cells. Our study thus provides an innovative preclinical model to study a range of neurological disorders, understanding the pathogenesis of which requires an organoid system capable of representing the dynamic cellular interactions and the maturity of the human brain.