Glutamax l glutamine supplement

Cortical spheroids were produced following an adaption to Pasca methods, Nature Methods 2015^{41 (link)}. Briefly, enzymatically lifted hIPSCs were transferred to ultra-low attachment plates and cultured in DMEM supplemented with Knockout Serum Replacement (Gibco) supplemented with 5 μM Dorsomorphin (BioVision), 10 μM SB431542 (Miltenyi Biotec), 10 μM Y27632 (Selleck Chemicals) for 6 days. The resulting spheroids were then maintained in neurobasal media until day 90: Neurobasal A medium, 2% B-27 supplement without vitamin A, GlutaMAX L-glutamine supplement (Gibco) penicillin/ streptomycin (Gibco). Spheroids were next supplemented with 20 ng/mL of bFGF and EGF (PeproTech) from day 6 to 25, and 20 ng/mL of BDNF and NT3 (Shenandoah Biotechnology) from day 26 to 42. Spheroids were harvested beginning at day 90 for analysis (refer to Fig. 3 for experimental details).

Cortical spheroids were produced following the methods described by Pasca et al. [38 (link)]. Briefly, enzymatically lifted hIPSCs were transferred to ultra-low attachment plates and cultured in DMEM supplemented with Knockout Serum Replacement (Gibco) supplemented with 5 μM Dorsomorphin (BioVision, Milpitas, CA, USA), 10 μM SB431542 (Miltenyi Biotec, Bergisch Gladbach, Germany), 10 μM Y27632 (Selleck Chemicals) for 6 days. The resulting spheroids were then maintained in neurobasal media until day 90: Neurobasal A medium, 2% B-27 supplement without vitamin A, GlutaMAX L-glutamine supplement (Gibco) penicillin/streptomycin (Gibco). Spheroids were next supplemented with 20 ng/mL of bFGF and EGF (PeproTech, Cranbury, NJ, USA) from day 6 to 25, and 20 ng/mL of BDNF and NT3 (Shenandoah Biotechnology, Warminster, PA, USA) from day 26 to 42. Spheroids were harvested beginning at day 90 for analysis.