Differentiation of Cortical Neurons from iPSCs

Cortical neurons were differentiated from iPSCs as previously described^{23 (link),69 (link)}. In brief, neuronal differentiation was achieved by adding dual SMAD inhibitors (10 µM SB431542 (Sigma-Aldrich) and 500 nM LDN-193189 (Sigma-Aldrich)) to 3 N medium. At day 10, cells were collected, replated (ratio: 1:3) and further cultivated for 2 days in 3 N medium including 20 ng/ml FGF-2. Cells were further cultivated until day 27 in 3 N medium with medium change every other day. For the specific assays, cells were replated at the desired density (RNA/Protein isolation: 5×10⁵ cells per cm²; Patch clamp: 5 × 10⁴ per cm²) and further differentiated until day 37 to 41. To proof the identity of generated iPSC-derived cortical neurons, cells were immuncytochemically analysed using ß-III-tubulin (TUJ, neuronal marker) and CTIP2 (cortical layer V marker).
Where indicated, 2 mM lithium (Sigma-Aldrich) and/or 10 µM GSK650394 (Sigma-Aldrich) were added for 24 hours.