We examined the cytoarchitecture of the hCSs (day 52) in cryosections and observed proliferative zones containing PAX6-expressing progenitors (Fig. 2a). Similarly to what occurs during in vivo cortical development, VZ-like structures inside hCSs were organized around a lumen delimited by N-cadherin (NCAD)-expressing cells (Fig. 2b). Furthermore, the VZ-like zone was surrounded by an intermediate region rich in TBR2+ cells resembling the SVZ15,16 (Fig. 2b). PAX6-expressing cells in the VZ-like zone also contained GFAP+ extensions directed orthogonally to the luminal surface, resembling radial glia (Fig. 2c). When plated in monolayer, these cells had either bipolar or monopolar morphologies (Fig. 2d,e). Both PAX6+ and TBR2+ neural progenitors were actively proliferating, as assessed by the expression of the radial glia–specific mitotic marker phosphovimentin (p-VIM)17 and the G2/M phase marker phosphohistone-3 (PH3) (Fig. 2f,g). In a pattern similar to in vivo cortical development, most of these mitoses were localized close to the luminal side of the proliferative zone rather than being dispersed16. Moreover, live imaging of radial glia fluorescently labeled with a cell-specific reporter (Lenti-GFAP::EGFP) revealed a characteristic division mode reminiscent of interkinetic nuclear migration18 (Fig. 2h, Supplementary Video 1).
