Pathogenic variation in SCN3A, a gene encoding the voltage-gated Na+ channel α-subunit Nav1.3, is associated with SCN3A-related neurodevelopmental disorders, which include three core clinical features: epilepsy, developmental delay/intellectual disability (DD/ID), and/or malformation of cortical development (MCD). Patients present all three or some overlapping combination of these core phenotypes; however, the prominence of MCD among the patient cohort is surprising considering it is an atypical feature of a channelopathy. SCN3A is expressed highly in the embryonic forebrain, yet the mechanism by which variants in SCN3A cause MCD remain unknown.

Here, we generated induced pluripotent stem cell (iPSC) lines of the patient SCN3A-I875T variant and its CRISPR/Cas9-corrected control. Using these isogenic cell lines, we performed a 2D cortical differentiation of iPSCs to neural progenitor cells (NPCs) to cortical neurons. Cells of the SCN3A-I875T variant and its corresponding CRISPR/Cas9-corrected control demonstrate a healthy propensity to be differentiated and both lines express proliferative markers at the same time points, overall showing no obvious qualitative differences when compared. In order to study early-neurodevelopment–the period when SCN3A gene peaks in expression–we created 3D aggregates of NPCs, or neurospheres, to evaluate cell migration from both patient and corrected lines by measuring NPC migration and neurite outgrowth. Total cell migration was quantified utilizing a trace analysis of fixed and immunostained spheroids. The development of this assay using an hiPSC-derived model system mimicking human cortical development will allow for investigation of the mechanism(s) of MCD in SCN3A encephalopathy.