DMEM/F-12

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Cerebral organoids were generated from WIBR3 hESCs using a previously reported protocol (Lancaster et al., 2013), with some modifications. Briefly, hESCs were dissociated from MEFs using collagenase type IV, and further separated from residual MEFs by gravity separation, before trypsinization of hESC colonies to generate single cells. A total of 9000 cells were then plated into each well of an ultra-low-attachment 96-well plate (Corning) to form single EBs, in medium containing DMEM/F12, 20% KSR, 3% fetal bovine serum, 2mM GlutaMAX, 1% non-essential amino acids, 50nM bmercaptoethanol and 4ng/ml bFGF. ROCK inhibitor Y27632 (50uM) was included in the first 24 hours. EBs were maintained in 96-well plates for 6 days, then transferred to ultra-low-attachment 24-well plates (Corning), in neural induction medium containing DMEM/F12, 1X N2 supplement, 1% non-essential amino acids, 2mM GlutaMAX and 1ug/ml heparin (Stem Cell Technologies). On days 10–12, EBs were embedded in droplets of matrigel, and were allowed to gel at 37C. Embedded EBs were subsequently cultured in neural maturation medium containing 50% DMEM/F12, 50% Neurobasal, 0.5X N2 supplement, 0.5X B27 supplement, 2mM GlutaMAX, 2.5ng/ml human insulin, 0.5% non-essential amino acids, and 25nM b-mercaptoethanol. Droplets were cultured in stationary condition in 6cm suspension dishes for 4 days, followed by transfer to an orbital shaker (Unimax-1010, Heidolph Brinkmann) rotating continuously at 80rpm. Mouse cerebral organoids were generated as previously described(Lancaster et al., 2013). Multiple independent experiments were performed to generate cerebral organoids from WIBR1 (n=3), WIBR2 (n=3), WIBR3 (n>15) and V6.5 (n=3). Each experiment generates 24–96 individual organoids of each condition (genotype and treatment). Various concentrations of AKT inhibitors GDC-0068 (Selleckchem) or MK-2206 (Selleckchem) were added to growth medium for EBs on day 1. To pre-pattern organois towards the dorsal forebrain fate, 2.5uM dorsomorphin was added to the culture on day 0 for 7 or 14 days. To stimulate phospho-AKT in 12-week organoids, BDNF (100ng/ml, Peprotech) was added for 30 minutes. All human cerebral organoid experiments were performed on WIBR3-derived control and mutant lines unless otherwise specified.

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