Human embryonic stem cell research and protocols were approved by the Ethics Committee of the University of Liège (no. B70720096466); all experiments were conducted according to its guidelines. Human-induced pluripotent stem cell (hIPSC) line GM23446 (Coriell Institute) was maintained on Geltrex-coated dishes (Gibco) in DMEM/F12 supplemented with 20% KO serum replacement (Gibco), 100 μM nonessential amino acids (NEAA) (Gibco), 100 μM 2-mercaptoethanol, and basic fibroblast growth factor (100 ng/ml; PeproTech, London, UK), conditioned on γ-irradiated mouse embryonic fibroblasts. Cells were passaged routinely with collagenase A (1 mg/ml; Roche).

Generation of human cortical neurons was performed as described in (39). Briefly, hiPSCs were dissociated with TrypLE (Gibco) for 4 min at 37°C and cultured on Geltrex-coated dished in Pluripro medium (Cell Guidance Systems) until they reached confluency. Neural induction was triggered with media making use of dual SMAD inhibition and tankyrase inhibitor to enhance forebrain fate: DMEM/F12 containing N2 (Gibco), B27 (Gibco), penicillin/streptomycin (1:100; Gibco), glucose (0.8 mg/ml; Carl Roth HN06.2), NEAA, GlutaMAX (Gibco), cyclic adenosine 3′,5′-monophosphate (0.15 ng/ml; Sigma-Aldrich, A9501-1G), 500 nM A83-01 (Miltenyi Biotec, 130-106-274), 200 nM LDN-193189 (Miltenyi Biotec, 130-103-925), and 2 μM XAV939 (Enzo Life Sciences, BML-WN100-0005). Medium was changed daily, and cells were passaged at day 8 with 0.5 M EDTA. Cells were maintained until day 18 and replated at a density of 12,000 cells/cm2 in neural differentiation media containing 1:1 DMEM/F12/Neurobasal, N2, B27, penicillin/streptomycin, glucose (0.8 mg/ml), NEAA, and GlutaMAX. Neural differentiation media were changed every other day, and Geltrex (1:100) was added 4 days before fixation with 4% paraformaldehyde (PFA) at day 32 to prevent detachment of the neurons.

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