During the subsequent 10 days, the organoids increased in size and the number of developed rosettes increased two to three fold (Fig. 1,b1), as compared to 8-day old organoids (Fig. 1, a1). The 18-day organoids formed polarized structures with a distinguishable border that separated a forebrain-like region containing multiple rosettes from a hindbrain-like structure, which typically lacked rosettes (Fig. 1, b1), as reported previously40. In all of the 18-day organoids examined, the cortical rosettes developed three major zones distinguished by DAPI staining (Fig. 1, b2). The VZ contained a large ventricle-like lumen surrounded by compact layers of vertically aligned elongated cells. The area outside the VZ, the IZ, contained uniform, predominantly round cells. The outermost CZ contained horizontally aligned cortical layers (Fig. 1, b2).
