A DEFINED MONOLAYER CULTURE SYSTEM FOR PSC-DERIVED BRG CELLS FACILITATES MECHANISTIC INSIGHTS INTO HUMAN NEURODEVELOPMENT

Modeling human corticogenesis has been hindered by limited access to primary fetal tissue. While human induced pluripotent stem cell (hiPSC)-derived cerebral organoids have advanced our understanding of cortical development, they underrepresent certain progenitor populations, including basal (outer) radial glia (bRG). Predominantly localized in the outer subventricular zone (oSVZ), bRG are key neural progenitors that drive the expansion of the human neocortex and whose dysfunction have been implicated in neurodevelopmental disorders (NDD). They are defined by characteristic molecular markers, specific signaling pathways and hallmark behaviors including mitotic and interphasic somal translocation (MST and IST). To date, however, bRG could only be studied in small numbers, either in primary tissue or within cerebral organoids. While acute isolation into 2D cultures is possible, this yields very limited numbers of cells that cannot be expanded, posing major technical barriers to systematic and scalable analyses. Here, we present a defined 2D-culture system enabling generation, expansion and differentiation of bRG-like cells from human iPSCs. These cultures closely resemble primary bRG, retain canonical markers and exhibit niche-like behaviors including cell-cell crosstalk and dispersive movement. Network-based transcriptomic screen identified PAK2 as regulator of MST, a defining feature of bRG dynamics. This simplified and reproducible 2D system provides an accessible experimental platform for dissecting bRG biology, enabling controlled interrogation of human cortical progenitor dynamics and signaling mechanisms. By bridging the gap between primary tissue and complex organoid models, it advances our capacity to model human neocortical development and associated NDD in vitro.