Characterization of inducible astrocytes. (A) Control NPCs (−Dox) and iAstros (+Dox) 6 d after induction from transgenic hPSCs, with the mApple reporter, before cell line purification. Dox treatment induced cell migration and morphological changes. (B) Western immunoblotting confirmed upregulation of Sox9 and NFIA proteins after 2 d of initial transgene induction (dox treatment) compared with the control (left). ICC confirmed expression of Sox9 and NFIA proteins after 4 d (right) in a mixed population. (C) GFAP was evident after 8 d in the presence of, but not without, dox treatment. Expression increased with 2-d treatment of ciliary neurotrophic factor (CNTF), known to stimulate astrocyte GFAP production. (D) Confirmation of transgene induction and astrocyte-restricted gene expression by qPCR in day-8 iAstros, normalized to NPCs and compared to temporally derived human astrocytes (hAstros; n = 1 each). (E) Subset of upregulated mature astrocyte-related transcripts are shown in heatmaps as log₂(FPKM+1) (top) and log₂FoldChange (bottom) of day-12 iAstros compared to NPCs (n = 3 each). (F) Subset of astrocyte-related transcripts plotted as log₂(FPKM+1) for iAstros and NPCs (n = 3 each). (G) Mature iAstros exhibited astrocyte markers including S100 (top) and filamentous GFAP (bottom). Images are replicated from Fig. 1 D with increased magnification of astrocyte morphology. (H) Ki67 expression indicated decreased proliferation with maturation of day-12 iAstros, compared with highly proliferative NPCs (n = 6 images each). (I) GFAP and S100 continued to be highly expressed in day-50+ iAstro spheres. (J) Representative still frames of ATP-induced calcium oscillation in iAstros and NPCs (see Fig. 1 F). (K) Targeting scheme used to generate hM3Dq-expressing iAstros (Astrostim). (L) Confirmation of transgene induction and astrocyte-restricted gene expression by qPCR in Astrostim cells after 2 d of dox treatment, normalized to day-2 no-dox controls (left) and after 15 and 30 d of dox treatment, normalized to day 15 no-dox controls (right; n = 3 each). (M) Live images of Astrostim cells with (bottom) and without (top) CNO treatment, after 1 d (left), 2 d (middle), and 4 d (right). (N) Ki67 expression indicated decreased proliferation with Astrostim cell maturation over a period of 4 wk (n = 8–9 images each). (O) Chronic CNO stimulation up to 14 d did not significantly alter the number of metabolically active Astrostim cells in monolayer culture compared to 2-h CNO treatment, as measured by monolayer viability assay (n = 3 each). Scale bars:I, 20 µm;A right, 500 µm; all others, 100 µm. Results are shown as mean ± SEM where appropriate. For E and F, significance was determined using the DESeq2 R package (see asterisks overlaid on heatmap). For L, significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test, with **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.