three commercial astrocyte media (ScienCell, Gibco, and Lonza) for the culture of primary human fetal astrocytes (Table 1). Screening criteria included immunoreactivity for two classical markers of astrocyte identity, S100β and glial fibrillary acidic protein (GFAP) (Ludwin et al., 1976), astrocyte morphology, survival, replicative ability, and cell line variability (Table S1; Figure S1A). When tested on NPCs, most conditions resulted in limited cell proliferation and expression of neuronal markers (Table S1); however, two commercial media, ScienCell and Lonza, yielded S100β- and GFAP-positive astrocyte-like cells (Figures S1B–S1D). These results were confirmed across four representative NPC lines by both flow cytometry and immunocytochemistry by 30 days (Figures 1A and S1E–S1G). Culture of NPCs in both media, when combined with low initial seeding density (nearly single cells: 15,000 cells/cm2) and minimal serum exposure (1%–2%), resulted in astrocyte morphology within 10 days (Figure S1H); star-shaped astrocyte morphologies were evident within 30 days (Figure S1I). Although ScienCell and Lonza astrocyte media showed equivalent efficiencies (Figures S1B–S1D), ScienCell medium was selected owing to its lower cost and relative simplicity.Table 1Screening Conditions for Astrocyte DifferentiationIDReferenceBasal MediumSupplement 1Supplement 2Growth Factors1Brennand and Gage (2011)DMEM/F12N2, B27GlutaMAX + sodium bicarbonate + sodium pyruvateBDNFGDNFcAMPAA2ScienCell 1801ND (AM)AGS2% FBS3Thermo Fisher A1261301DMEMN2GlutaMAX + D-glucose +
