Expanding GABAergic Neuronal Diversity in iPSC-Derived Disease Models

GABAergic interneurons play a critical role in maintaining neural circuit function, and their dysfunction is implicated in various neurodevelopmental and psychiatric disorders. Traditional approaches for differentiating human pluripotent stem cells (PSCs) into neuronal cells often face challenges such as incomplete neural differentiation, prolonged culture periods, and variability across PSC lines. To address these limitations, we developed a new strategy that integrates overexpression of transcription factors ASCL1 and DLX2 with dual-SMAD and Wnt inhibition, efficiently driving the differentiation of human PSCs into diverse, region-specific GABAergic neuronal types. Using single-cell Sequencing, we characterized the cellular heterogeneity of GABAergic induced neurons (iNs) generated with the patterning factors (patterned iNs) and those derived solely with transcription factors (PSC-derived iNs), uncovering the regulatory mechanisms that govern their fate specification. Patterned iNs exhibited gene expression features corresponding to multiple brain regions, particularly ganglionic eminence (GE) and neocortex, while GABAergic PSC-derived iNs predominantly resembled hypothalamic and thalamic neurons. Both iN types were enriched for genes relevant to neurodevelopmental and psychiatric disorders, with patterned iNs more specifically linked to neural lineage genes, highlighting their utility for disease modeling. We further applied this protocol to investigate the impact of an ADNP syndrome-associated mutation (p.Tyr719* variant) on GABAergic neuron differentiation, revealing that this mutation disrupts GABAergic fate specification and synaptic transmission. Overall, this study expands the toolkit for disease modeling by demonstrating the complementary advantages of GABAergic PSC-derived iNs and patterned iNs in representing distinct GABAergic neuron subtypes, brain regions, and disease contexts. These approaches offer a powerful platform for elucidating the molecular mechanisms underlying various neurodevelopmental and psychiatric disorders.