hiPSCs were differentiated to astrocytes based on our recent protocol [10 (link)]. Fetal human cortical astrocytes were obtained from ScienCell (#1800). Cells were cultured according to suppliers’ instructions and used at passage <8 post-thawing. One million cells were thawed in complete astrocyte media (AM; ScienCell 1801). For assays, 500,000 cells/well were seeded in 6-well cell culture plates coated with 1x attachment factor (AF; Thermo S-006-100). On the next day of subculture (day 1), the media were changed to a 1:1 mixture of AM and “defined media,” and on day 2 to defined media only. Our defined media were composed of DMEM without glucose and glutamine (Thermo A14430-01) supplemented with 1% G-5 (Thermo 17503012; serum-free supplement for growth and expression of glial cells), 1% Penicillin-Streptomycin (P/S; ScienCell 0503), 10mM GlutaMax (Sigma 35050-38), 25 mM HEPES (EMD 391340), and 5.55 mM glucose (Sigma G7528) to match the AM glucose content. We used these defined media to have better control over the initial concentration of glucose within the media. Exemplary bright-field images of the cells on day 2 are shown in Figure 1b,c.
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