N2 supplement

To generate neural progenitors, iPSCs were disassociated into single cells using ACCUTASE™ Cell detachment solution as described above, and seeded at high density (1 × 10⁶ cells per well of a 12-well plate) in SMAD inhibition medium as previously described^{27 (link)}. Cell counting was performed using the Countess 3 Automated Cell Counter (ThermoFisher Scientific) with trypan blue (Gibco, Cat. 15250061) staining to identify dead cells, as per manufactures instructions. Neural induction of iPSC was performed using the STEMdiff™ SMADi Neural Induction Kit (StemCellTechnologies, Cat. 08582) and protocol per manufacturer was followed^{28 (link)}. Briefly, iPSCs were cultured for 8–12 days on BME matrix (as before) and passaged using 1 mg/mL Collagenase type IV solution (Gibco, Cat. 17104019) upon the appearance of neuroepithelial cells onto BME matrix coated 6-well plates in STEMdiff™ SMADi Neural Induction medium. The next day, medium was switched to neural maintenance medium (NMM: 48.4 mL KnockOut DMEM/F12, Gibco, Cat. 12660-102; 48.5 mL Neurobasal, Gibco, Cat. 12348-017; 1 mL B-27 Supplement, Gibco, Cat. 17504-044; 500 µL N-2 Supplement, Gibco, Cat. 17502-048; 500 µL Non-essential amino acids, Gibco, Cat. 11140-050; 500 µL GlutaMAX, Gibco, Cat. 35050-061; 500 µL Penicillin-Streptomycin, Gibco, Cat. 15140-122; 90 µL 2-Mercaptoethanol, Gibco, Cat. 31350010; 25 µL Insulin solution, Sigma, Cat. I9278-5ML; as previously described^{27 (link)}) and henceforth medium was refreshed daily. Upon formation of rosettes (as described in refs. ^{27 (link),67} ), cultures were treated with 5 μL/mL Recombinant Human Basic Fibroblast Growth Factor (bFGF, 4 μg/mL, Gibco, Cat. PHG0024). After four consecutive days of bFGF treatment, cells were passaged with 1 mg/mL Collagenase type IV solution and transferred to BME matrix (Cultrex®) coated 6-plates. Once morphologically apparent neuronal cells (polarised with leading axon and trailing dendrite) could be seen migrating from the apical border of the rosette, cultures were passaged with ACCUTASE™ Cell detachment solution and plated onto 10 μg/mL laminin (Sigma-Aldrich, Cat. L2020) and poly-L-Lysine (Sigma-Aldrich, Cat. P6282-5MG). Cells were passaged between day 21 and 26, and either continued as neural cultures or frozen for storage. For freezing, cells were detached using ACCUTASE™ Cell detachment solution, transferred to neural freezing medium (90% NMM, 10% DMSO (Sigma, Cat. D2438), 20 ng/mL bFGF), and placed in a Mr. Frosty (Thermo Scientific, Cat. 5100-0001) at -80°C and stored long-term in liquid nitrogen, as described^{27 (link)}.
Neural cultures were fed daily with NMM until day 45, after which 48-hour cycles were implemented whereby every second day two thirds of the medium was removed and 2 mL NMM plus maturation supplements GDNF and BDNF (both at 20 ng/mL, StemCellTechnologies, Cat. 78058 and 78005) were supplied to neuronal cultures. In this way, neuronal cultures were maintained up to a maximum of day 120.

V6.5 mESCs were taken from a male individual derived from a cross of C57BL/6(F) × 129/sv(M) and were kindly provided by the Jaenisch laboratory of the Whitehead Institute.
mESCs were cultured at 37°C, 5% CO₂ in a humidified incubator on tissue-culture plates covered by 0.2% gelatin (Sigma, G1890) in 2i medium with LIF, made by 960 ml Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Life Technologies, 11320082), 5 ml N2 supplement (Life Technologies, 17502048; stock 100×), 10 ml B27 supplement (Life Technologies, 17504044; stock 50×), 5 ml additional l-glutamine (Gibco, 25030-081; stock 200 mM), 10 ml Minimum Essential Medium (MEM) non-essential amino acids (Gibco, 11140076; stock 100×), 10 ml penicillin–streptomycin (Life Technologies, 15140163; stock 10⁴ U/ml), 333 μl bovine serum albumin (BSA) fraction V (Gibco, 15260037; stock 7.50%), 7 μl β-mercaptoethanol (Sigma, M6250; stock 14.3 M), 100 μl leukemia inhibitory factor (LIF - Chemico, ESG1107; stock 10⁷ U/ml), 100 μl PD0325901 (Stemgent, 04-0006-10; stock 10 mM), and 300 μl CHIR99021 (Stemgent, 04-0004-10; stock 10 mM).
When cells were passaged, TrypLE inhibition occurred using stem cell media (SCM) made by 500 ml DMEM KO (Gibco, 10829-018), MEM non-essential amino acids (Gibco, 11140076; stock 100×), 5 ml penicillin–streptomycin (Life Technologies, 15140163; stock 10⁴ U/ml), 5 ml l-glutamine (Gibco, 25030-081; stock 100×), 4 μl β-mercaptoethanol (Sigma, M6250; stock 14.3 M), 50 μl LIF (Chemico, ESG1107; stock 10⁷ U/ml), and 75 ml of fetal bovine serum (Sigma, F4135). Cells were then pelleted by centrifugation at 1000 rpm for 3 min and resuspended in 2i medium containing LIF.
Freezing of cells was performed in SCM, supplemented with a 1:1 ratio of 2× freezing media: 60 ml DMEM (Gibco, 11965-052), 20 ml dimethyl sulfoxide (Sigma, D2650), and 20 ml fetal bovine serum (Sigma, F4135).
Human embryonic stem cell (hESC) H1, purchased from WiCell, was cultured and fixed in 4% paraformaldehyde (PFA) on coverslips by Ido Sagi.