Differentiation of hiPSCs into Telencephalic Neural Progenitors

Previously characterized healthy control hiPSC lines 20b and 18a^{43 (link)} were maintained in mTEsR medium (StemCell Technolgies) on hESC-qualified matrigel (Corning)-coated tissue culture plates and passaged using Dispase (1 mg/ml, Life Technologies). Cell lines were mycoplasma-negative by PCR (LookOut Mycoplasma PCR Detection Kit, Sigma). HiPSCs were differentiated into dorsal telencephalic neural progenitors using a previously published protocol^{44 (link)} without inducing sonic hedgehog signalling (no SHH, see Extended Data Fig. 5). After 18 days, cultures were enzymatically dissociated to single cells using Accutase (StemPro Accutase, Life Technologies) and were replated on Growth Factor Reduced matrigel (1:30, Corning) at 10,000 cells/cm² in human neurobasal (hNB) medium supplemented with 10 μM ROCK inhibitor (Y27632, Tocris). hNB media was replaced 24 h later and supplemented every 2–3 days thereafter. Dissociation and replating (100,000 cells/cm²) was repeated at day 40. Cultures were silenced on day 81 and then KCl-depolarized (see above) and harvested on day 82. hNB medium: neurobasal medium (no glutamine), with 1× penicillin/streptomycin, 1× GlutaMax, 1× MEM non-essential amino acids, 1× B27-supplement without vitamin A (all Life Technologies), 1× N2-B supplement (StemCell Technologies), 1 μM ascorbic acid (Sigma), 20 ng/ml rhBDNF and 10 ng/ml rhGDNF (Peprotech).