HyClone Dulbecco's Modified Eagle Medium (DMEM)/ F12 1:1: Liquid

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Pluripotent stem cell culture and organoid differentiation: hPSC experiments were conducted with prior approval from the UCLA Embryonic Stem Cell Research Oversight (ESCRO) Committee. hPSC lines H9, UCLA1, hIPS2 and XFiPS were obtained from the UCLA Broad Stem Cell Research Center Core and maintained on 0.1% gelatin-coated plates with irradiated mouse embryonic feeders in DMEM/F12 (Hyclone) with 20% Knockout Serum Replacement (KSR: Invitrogen), MEM Non-Essential Amino Acids (NEAA: Invitrogen), GlutaMAX (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 100 μg/ml Primocin (InvivoGen), and 10 ng/ml of FGF2 (Invitrogen). hPSC were maintained at 5% CO2 at 37 °C with daily media change, and were passaged every 6 days at 1:3-1:6 using StemPro EZ Passage tool (Invitrogen). Experiments were performed on cells between passages 40-80 for H9, UCLA1, and hIPS2, and between passages 10-20 for XFiPS. Cortical organoid differentiation was performed as described (Kadoshima et al., 2013) with several modifications. Briefly, hPSC were dissociated to single cells and plated into low attachment V-bottom 96-well plates (Sumitomo Bakelite, #MS-9096V) to form aggregates in GMEM (Invitrogen), 20% KSR, NEAA, 100 μg/ml Primocin, 0.1 mM β-mercaptoethanol, sodium pyruvate (Invitrogen), and 20 μM ROCK inhibitor (BioPioneer) without Wnt and TGFβ inhibitors. Half of the media was changed every 2-3 days. ROCK inhibitor was removed after 6 days. Aggregates were then transferred to a hyperoxygenated incubator at 5% CO2 and 40% O2 and maintained in DMEM/F12 with N2 (Invitrogen), GlutaMAX, Chemically Defined Lipid Concentrate (CDLC: Invitrogen), and 0.4% methylcellulose (Sigma). Culture media was completely changed every 2-3 days thereafter. On day 35, organoids were cut in half using Vannas spring scissors (Fine Science Tools) and media changed to N2B27 media containing DMEM/F12 supplemented with N2, GlutaMAX, CDLC, 0.4% methylcellulose, B27 without vitamin A (Invitrogen), 1% Growth Factor Reduced Matrigel (Fisher Scientific, #CB-40230), and 5 μg/ml heparin (Sigma). For the FBS method, 10% FBS (Invitrogen) was used in place of B27 without vitamin A. On day 56, organoids were cut in half and transferred to oxygen permeable dishes (Lumox, Sarstedt) containing N2B27 media. Organoids were subsequently cut in half every 2 weeks and routinely sustained for up to 150 days. For STAT3 activation, Leukemia Inhibitory Factor (LIF, Millipore) was added at 2,000 U/mL from D35 onward. At the end of the experiments, organoids were processed for immunohistochemical and RNA analyses as described in the Supplemental Experimental Procedures. See Table S6 for key resources and Table S7 for antibody information.

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