Forebrain NPCs were maintained at high density, grown on Matrigel (BD Bioscience) in NPC medium (DMEM/F12, 1× N2, 1× B27-RA [Invitrogen], 1 mg/mL laminin [Invitrogen], and 20 ng/mL FGF2 [Invitrogen]), and split approximately 1:3 to 1:4 every week with Accutase (Millipore). NPCs could be expanded up to 14 passages. Forebrain NPCs were differentiated to astrocytes by seeding dissociated single cells at 15,000 cells/cm2 density on Matrigel-coated plates in astrocyte medium (ScienCell: 1801, astrocyte medium [1801-b], 2% fetal bovine serum [0010], astrocyte growth supplement [1852] and 10 U/mL penicillin/streptomycin solution [0503]). Initial NPC quality, seeding density, and single-cell dissociation are critical, particularly during the first 30 days of differentiation, in order to efficiently generate hiPSC-astrocytes. See detailed protocol for astrocyte differentiation from hiPSC-derived NPCs in the Supplemental Information.
