Directing GABAergic neuronal identity from human iPSCs through developmental patterning

GABAergic interneurons are implicated in numerous neurodevelopmental and neuropsychiatric disorders. Transcription factor (TF)-mediated induction of human pluripotent stem cells (PSCs) rapidly generates GABAergic neurons, yet their regional identity remains poorly defined. Here, we show that the chromatin state of the starting population constrains the neuronal identities specified by ASCL1 and DLX2. Brief neural patterning via dual-SMAD and WNT inhibition prior to TF induction biases PSCs toward GABAergic neurons with forebrain-associated identities, whereas TF induction of PSCs yields neurons with broader regional signatures, including hypothalamic and thalamic programs. Benchmarking multiple approaches against a human fetal brain atlas reveals distinct regional identity biases and disease gene enrichment profiles across methods, providing a framework for selecting differentiation strategies for disease modeling. Applying this approach to ADNP syndrome, we demonstrate that the recurrent p.Tyr719^∗ mutation disrupts interneuron maturation and subtype-associated transcriptional programs and impairs inhibitory synaptic transmission in human GABAergic neurons.