Rapid and Robust Generation of Human Cortical Interneurons from Induced Neural Stem Cells

Current protocols for generating cortical interneurons from human pluripotent stem cells are hindered by slow differentiation kinetics and poor reproducibility across cell lines. Here, we present a defined small-molecule-based strategy that efficiently directs human-induced neural stem cells (hiNSCs) toward cortical GABAergic interneurons within 14–18 days, which is substantially faster than conventional methods. Short-term dual-SMAD and WNT inhibition rapidly commits hiNSCs to an interneuron progenitor fate, reaching transcriptional states equivalent to those obtained with prolonged protocols. Prolonged activation of Sonic Hedgehog (via SAG) further enhances lineage specification, markedly upregulating NKX2.1, FOXG1, GABA, somatostatin (SST), and parvalbumin (PV) expression, and enriching pathways associated with early functional maturation. Importantly, RNA-sequencing reveals that under identical induction conditions, hiNSCs differentiate more rapidly and homogeneously than human-induced pluripotent stem cells (hiPSCs), which exhibit broader, less lineage-focused transcriptional trajectories. This differentiation strategy is highly reproducible across four genetically distinct hiNSC lines, with minimal off-target populations. Functionally, hiNSC-derived cortical interneurons display robust migratory behavior, produce abundant GABA, and survive transplantation into the adult mouse hippocampus, where they extend processes and form synapse-like structures. These findings establish a rapid, scalable, and robust approach for generating human cortical interneurons, supporting their safety and integration potential as a foundation for future cell replacement strategies in neurological disorders.