iPSC-Derived Cortical Neuron Differentiation

iPSCs were differentiated into cortical neurons using a two-step approach as previously described (Karch et al., 2019 (link)) (https://dx.doi.org/10.17504/protocols.io.p9kdr4w). iPSCs were plated at a density of 65,000 cells per well in neural induction media (StemCell Technologies) in a 96-well v-bottom plate to form neural aggregates and after 5 days, transferred into culture plates. The resulting neural rosettes were then isolated by enzymatic selection (Neural Rosette Selection Reagent; StemCell Technologies) and cultured as neural progenitor cells (NPCs). NPCs were differentiated in planar culture in neuronal maturation medium (neurobasal medium supplemented with B27, GDNF, BDNF, cAMP). Neurons typically arose within 1 week after plating, identified using immunocytochemistry for β-tubulin III (Tuj1). The cells continue to mature and were analyzed at 6 weeks.