We noticed that layer-specific cortical neurons in hCSs were organized concentrically around a VZ-like zone (Fig. 2k,l). Deep-layer neurons expressing TBR1 and CTIP2 moved immediately outside the proliferative zone, whereas at in vitro day 137, superficial-layer cortical neurons expressing SATB2 and BRN2 had migrated farther away, forming the outside layer of the hCSs. We also examined whether the colocalization of some of these cortical markers was consistent with in vivo studies. Indeed, flow cytometry experiments indicated that the mutually exclusive CTIP2 and SATB2 proteins21, which regulate alternate corticofugal and corticocortical cellular identity programs, were coexpressed by fewer than 3% of the cells (Fig. 2m). Conversely, as expected from a laminated cortical anlage, the cortical-layer markers TBR1 and CTIP2, which are expressed by subtypes of corticofugal neurons in deep layers and a small proportion of neurons in layer II, were colocalized in ~30% of the cells at day 76 in vitro (Fig. 2n).
