Enhanced Synaptic-Related Transcriptional Programs in Human Forebrain Organoids for Modeling Synaptopathy-Associated Neurodevelopmental Disorders

Human cerebral organoids (COs) are well-recognized in vitro models for recapitulating human fetal brain development. However, spontaneous unguided differentiation of COs leads to severe regional heterogeneity, limiting standardized research and high-throughput drug screening. In this study, we generated optimized human forebrain organoids (FBs) using a triple SMAD/Wnt inhibition strategy modified from classical CO culture systems. Immunofluorescence staining and bulk RNA sequencing validated the physiological authenticity of FBs and elucidated prominent phenotypic differences between FBs and COs. Molecular analyses confirmed robust and specific forebrain specification in FBs, characterized by sustained expression of canonical forebrain markers and dominant enrichment of neocortical and subpallial signatures, while posterior brain lineage programs were markedly suppressed. Furthermore, FBs faithfully recapitulated diverse fetal forebrain cellular types and rudimentary cortical laminar structures. Transcriptomic profiling revealed that FBs exhibited a marked transcriptional shift toward gene sets associated with neuronal differentiation and synaptic-related biological processes. Gene sets linked to synaptic assembly, transmission, and plasticity were transcriptionally up-regulated, whereas cell-cycle- and proliferation-associated transcriptional programs were downregulated as compared to COs. In conclusion, the optimized FBs faithfully recapitulated key human forebrain developmental features and exhibited enhanced synapse-related transcriptional signatures relative to COs. This refined organoid system provides a valuable in vitro platform for studying forebrain circuit ontogeny and dissecting the molecular pathogenesis of synaptopathy-related neurodevelopmental disorders.