Enhanced cortical neural stem cell identity through short SMAD/WNT inhibition in human cerebral organoids facilitates emergence of outer radial glial cells.

Cerebral organoids exhibit broad regional heterogeneity accompanied by limited cortical cellular diversity under a variety of derivation methods, suggesting inadequate patterning of early neural stem cell (NSC) starting populations. Here we show that a short combined Dual SMAD/WNT inhibition course during early organoid establishment is sufficient for yielding robust and lasting cortical identity with efficient suppression of non-cortical fates in organoid NSCs. In contrast, other widely used methods are inconsistent in their cortical specification capacity. Furthermore, combined inhibition selectively enriches for outer radial glia (oRG) cells in organoids that demarcate well-defined outer sub-ventricular (oSVZ)-like regions. Finally, combined inhibition enables superior NSC radial organization, further facilitates the generation of molecularly distinct deep and upper cortical layer neurons, and uncovers cortex-specific microcephaly defects. Thus, combined inhibition is critical for establishing a rich cortical cell repertoire, for enabling fundamental cytoarchitectural features of cortical development, and for meaningful disease modeling. Overall design: Single cell RNA-sequencing of brain organoids from 4 iPSC lines across 3 derivation protocols after 50 days and 80 days in culture. Bulk RNA-Seq of H9-derived microcephaly and wild-type organoids. Please note that raw data is not provided as it requires controlled access.