Establishing Area-Specific Brain Organoids Through Transcription Factor-Mediated Patterning

The human cerebral cortex is organized into distinct area-specific regions along the rostral-caudal axis, yet current human brain organoid models incompletely recapitulate this regional diversity. Here, we establish an area-specific brain organoid platform by leveraging transcription factors (TFs) identified through re-analysis of in vivo human cortical transcriptomic datasets. Publicly available single-cell RNA sequencing datasets from human developing cortex were re-analyzed to identify differentially expressed genes associated with rostral and caudal cortical identities. From this analysis, we identified SP9 (rostral-enriched) and DMRTA2 (caudal-enriched) as candidate TFs governing regional specification. To model cortical area identity, these TFs were overexpressed in an inducible manner during human cerebral organoid (hCO) generation. Overexpression of SP9 resulted in hCOs exhibiting rostral cortical characteristics, whereas DMRTA2 overexpression promoted caudal cortical features. The resulting hCOs showed distinct regional identities, reflected by differential expression of area-specific markers. In addition, these regional identities were accompanied by distinct functional phenotypes, as calcium imaging revealed divergent patterns of spontaneous neural activity between rostral and caudal hCOs. Altogether, our findings demonstrate that overexpression of TFs enables the controlled generation of area-specific hCOs. This approach provides a scalable and reproducible platform for studying human cortical regionalization and offers a framework for investigating region-specific mechanisms underlying neurodevelopmental and neurological disorders.