Three-dimensional co-culturing reveals human stem cell-derived somatostatin interneurons with subclass expression

Cortical interneuron deficiencies, particularly involving the somatostatin (SST) subtypes, contribute to neurological and neuropsychiatric disorders. These interneurons are difficult to derive in vitro from human embryonic stem cells (hESCs) due to their late embryonic development and dependence on glial interaction. To this end, we developed a three-dimensional co-culture model of hESC-derived neurons, enabling long-term development, functional maturity, and neuron-glial interaction. Under these conditions, hESCs successfully differentiated into functional GABAergic interneurons expressing the SST gene and protein within 50 days. Single-nuclei RNA sequencing revealed transcripts for SST subclasses, including Martinotti, non-Martinotti, and long-projecting neurons, that have not yet been described for hESC cultures. Upon injection into forebrain organoids, the interneuron progenitors spread and functionally matured while retaining their SST subclass identities, suggesting cell-intrinsic fate specification. Our in vitro model provides a robust platform for studying human SST interneurons, offering new avenues for investigating their role in health and disease.