Rapid and reproducible in vitro generation of human parvalbumin-expressing cortical interneurons

Summary Parvalbumin-expressing cortical interneurons play a critical role in maintaining the balance between excitatory and inhibitory signalling and are essential for cognition, with dysfunction implicated in numerous brain disorders. Although human pluripotent stem cells have enabled the generation of diverse human neuronal types in vitro , including cortical interneurons, parvalbumin-expressing interneurons - unlike somatostatin-expressing interneurons - remain difficult to generate reliably and consistently. Here, we demonstrate the efficient and reproducible generation of parvalbumin-expressing cortical interneurons in vitro within 50 days of differentiation. Parvalbumin mRNA and protein were detected without forced gene expression, cell sorting, rodent co-culture or intracerebral transplantation, approaches commonly required by previous protocols. Single-cell transcriptomic analyses validated neuronal identity and authenticity, revealing enrichment for gene expression signatures of parvalbumin-expressing cortical interneurons in vivo . Together, these findings establish a robust method that facilitates interneuron research by enabling the reliable generation of authentic human parvalbumin-expressing cortical interneurons within a short time frame. eTOC blurb Azzouni et al . present a rapid and reproducible protocol for generating authentic human parvalbumin-expressing cortical interneurons from pluripotent stem cells in just 50 days, without forced gene expression or co-culture. Single-cell transcriptomics confirm robust acquisition of in vivo -like PVALB interneuron identity, enabling new opportunities for human interneuron research. Highlight Optimising SHH and WNT modulators enables consistent PVALB interneuron generation. 10% of cells express PVALB mRNA within 50 days of 2D differentiation from hPSCs. PVALB expression occurs without gene forcing, sorting, co-culture or grafting. Comparison of gene expression to in vivo interneurons confirms PVALB authenticity. Graphical abstract