Pathogenic variants in SCN3A, encoding the voltage-gated Na+ channel α-subunit Nav1.3, are associated with SCN3A-related neurodevelopmental disorder (SCN3A-NDD), which includes a clinical spectrum of epilepsy, developmental delay/intellectual disability (DD/ID), and malformation of cortical development (MCD). Affected patients present with some combination of these 3 core features. Interestingly, MCD is atypical of a channelopathy yet is surprisingly a key characteristic phenotype in SCN3A-NDD. Although SCN3A is expressed highly in the brain during embryonic development, the mechanism(s) by which mutations in the Nav1.3 channel result in MCD remain unknown.

In order to address this question, we generated induced pluripotent stem cell (iPSC) lines from a patient with the SCN3A-I875T variant–which is associated with all 3 core phenotypes–and its CRISPR-corrected isogenic control. We perform a 2D directed cortical differentiation of these iPSC lines through a neural progenitor cell (NPC) stage to cortical neurons in order to model human cortical development in vitro.

To specifically examine early-stage neurodevelopment potentially relevant to SCN3A encephalopathy pathogenesis, we employed an S-phase entry assay and developed a neurosphere migration assay to evaluate cell proliferation, migration, and the extension of cell processes active during corticogenesis. In the former assay, we applied EdU, an analogue component of DNA that becomes incorporated during DNA synthesis, for 1 hour at four time points throughout the differentiation ranging from the iPSC to NPC to early neuron stages and quantified for the proportion of EdU positive cells. In the latter assay, we formed neurospheres, or 3D aggregates of NPCs, re-plated them on an adherent substrate, and subsequently fixed, immunostained, imaged, and analyzed them to assess NPC migration and newborn neurite extension. We performed these assays using both cell lines in parallel, finding that patient line has an altered proliferation time course, antibodies NESTIN & DCX can be used to stain for non-overlapping cell processes, and radial migration of neurospheres formed from the NPC to early neuron transition stage is not affected by the patient variant. With the establishment of this hiPSC-derived model system, we can investigate the mechanistic underpinnings of cortical malformation in SCN3A encephalopathy.