We screened 11 methods to differentiate hiPSC-derived NPCs into astrocytes, identifying a medium with low FBS (1%–2%) that produces populations of hiPSC-astrocytes within 30 days. Although this is a commercial medium sold for the culture of primary astrocytes, it has not previously been demonstrated to support differentiation of hiPSC to astrocytes. Our method is fast and robust: unlike previous reports, it does not require prolonged culture (∼6 months) or a serial sorting process (Krencik et al., 2011, Shaltouki et al., 2013, Yuan et al., 2011). This protocol was evaluated across 42 NPC lines from 30 individuals (16 males and 14 females) generated from three independent hiPSC cohorts (Table S2). We caution that the quality of the starting NPC population is a critical predictor of success and note that for a few particularly intransient NPC lines, starting from very low-passage stocks proved critical to the ultimate successful differentiation of hiPSC-astrocytes. By both fluorescence-activated cell sorting and qPCR, GFAP seems to be a more variable marker of astrocyte fate (Figures 1A and 1B) and we recommend instead using S100β when evaluating hiPSC-astrocyte
