Fig 2: iAs efficiently express classical phenotypic markers in vitro irrespective of Matrigel coating. RUCDR-iAs and ATCC-iAs were cultured on glass or tissue culture plates with and without Matrigel coating for 72 h and subjected to IF or flow cytometry. (A) IF staining of RUCDR-iAs and ATCC-iAs showing expression of GFAP, S100β, and EAAT1 when plated with and without Matrigel coating (200× magnification). Scale, 50 μΜ. (B) Proportion of cells positive for GFAP, S100β, and EAAT-1 (relative to DAPI staining) in RUCDR-iAs (n = 4). (C) Proportion of cells positive for GFAP, S100β, and EAAT-1 (relative to DAPI staining) in ATCC-iAs (n = 4). (D) Representative flow histograms showing GFAP, S100β, and MAP-2 expression in RUCDR-iAs. (E–G). Quantitative flow for GFAP, S100β, and MAP-2 expression in RUDCR-iAs, ATCC-iAs, and Axol-iAs with and without Matrigel, respectively (n = 4). p-values determined by unpaired two-tailed t-test. Abbreviations: GFAP: glial fibrillary acidic protein, iA: iPSC-derived astrocytes, MAP-2: microtubule-associated protein 2.

Fig 3: Necroptosis induction in activated EGCs during Th1/Th17-driven intestinal inflammation. (A) Uniform Manifold Approximation and Projection (UMAP) plots reanalyzed from Progatzky et al 7 showing EGC clusters with elevated cell death signature and reduced Plp1 expression. Ridge plots display Ifngr1 and Tnfrsf1a expression in Glia_2 cluster with higher expression of the cell death pathway signature and lower Plp1 expression. (B and C) Flow cytometry density plots of tdTomato+ EGCs from aCD3- or Iso-treated mice, showing activation markers and cell death dye. (D and E) Whole-mount images of submucosal plexus: (D) GFAP and tdTomato colocalization, (E) pMLKL staining in tdTomato+ EGCs from aCD3 vs Iso groups.

Fig 4: Glia-specific Casp8 deficiency increases susceptibility to T cell–driven enteritis. (A) Whole mount immunostaining for CASP8 and GFAP from the colon tissues of Casp8flox and Casp8iΔGlia mice on day 10 post 3 cycles of tamoxifen administration, showing the glial specificity of the knockout. Insets show magnified views of selected areas. (B) Representative photomicrographs of immunostained plexanoids cultured from intestines of C57BL/6J mice displaying S100B-positive EGCs surrounding TUBB3-positive neurons, displaying morphologies reminiscent of plexuses in culture. (C) Heatmap visualizing normalized counts of selected cell type marker genes from bulk RNA-seq delineating the composition of plexanoid cultures from 3 independent biological replicates (M, mouse: M1 to M3) showing dominance of EGCs in the culture. (D and E) Mean with standard deviation of disease activity scores (D), muscle thickness and H&E bleeding scores (E) from littermate Casp8flox controls and Casp8 glia-deficient Casp8iΔGlia mice, treated with 75 mg per kg body weight of tamoxifen for 5 days, followed by the induction of intestinal inflammation via the aCD3 model. Data are derived from 4 independent biological replicates. Asterisks in (E) indicate P < .05 in Welch’s 2 tailed t test.

Fig 5: Induction of cell death in activated EGCs in vivo. (A) Plots showing gating strategy for tdTomato+ single EGC from digested muscularis samples for further analysis. (B and C) Activated (tdTomato+, GFAP+) live (B) and dead (fixable dead cell dye+) (C) EGC and their proportions in aCD3 and Iso groups. (D and E) Analysis of glia subsets from 2 independent scRNA-seq datasets29,38 showing the expression of CASP8 and MLKL in the indicated patient groups. Fist.Inv, fistulating involved; Fist.N.Inv, fistulating noninvolved; Inf.Inv, inflammatory involved; Inf.N.Inv, inflammatory noninvolved; Stric.N.Inv, stricturing noninvolved; Stric.Inv, stricturing involved.38