The advent of 3D cultures started with the pioneering work of Y. Sasai and colleagues in 2008, in which they derived 3D-organized aggregates of neuronal cells from mouse and human ES cells 46. In this study the authors also showed that the regional identity of specific cortical neurons could be specified and manipulated by the addition of patterning factors into the medium. Similar organoid protocols were refined and optimized for the 3D differentiation of human iPS cells 47,48. More recently Lancaster and colleagues developed an organoid method designed to mimic the organization of the entire brain and used this method to model intrinsic neuronal differentiation defects in individuals with microcephaly 49. Organoids obtained with this method are heterogeneous in the sense that they contain several different brain regions (such as cerebral cortex, brainstem, retina and choroid plexus) within each organoid. We have used 3D organoid cultures to study of ASD50, and used a method that differed in the initial enrichment of neural progenitor cells through the manual selection of neural rosettes, which allows a more homogeneous differentiation of each organoid in dorsal and ventral neurons mainly representing the telencephalon.
