Abstract: Cerebral organoids display broad regional heterogeneity and limited capacity to generate cortical cell diversity due to incompetent patterning signals within early developing organoid neural stem cells (NSCs). Here we describe a method to overcome this limitation by applying a short and early dual SMAD/WNT inhibition course, which results in enrichment for cortical NSCs and their progeny while suppressing non-cortical fates. The enhanced cortical cellular diversity achieved with our method is further mirrored by the facilitated emergence of outer radial glial (oRG) NSCs and appearance of molecularly distinct deep and upper layer neurons. In summary, this protocol yields cerebral organoids with considerable cortical regional homogeneity, depicting a short and early pathway Inhibition as a sufficient step for enriching towards cortical NSC identity in organoids. This protocol accompanies Rosebrock D. et al, Nature Cell Biology, 2022.

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