It is recommended to conduct cryosections or lightsheet imaging to inspect the cortical structure forming within the organoids. In day 25 organoids, the rosette structures and ventricular zones are clearly visible in DAPI (Figure 4A) and phospho-vimentin (Figure 4B) staining. The presence of TBR2 (Figure 4C) indicates that intermediate progenitors are forming, with a morphology that suggests outward migration. MAP2+ (Figure 4D) neurons populate the outer regions of each rosette. From these proteins, one can visualize the ventricular zone (VZ), subventricular zone (SVZ), intermediate zone (IZ), and cortical plate (CP) within each rosette (Figure 4E,4E'). One can continue to culture these organoids in order to observe later stages of development. While cortical layering is not as prominent in this type of organoid, the natural switch to gliogenesis does occur much like it does in vivo. This can be observed in day 108 organoids, in which a large portion of the cells express either MAP2 or GFAP (Figure 4F-I).
