Fig. 4

POLY(I:C)-mediated microglia-dependent consequences on the retinal environment. a, b Release of inflammatory cytokines and chemokines into the supernatant based on the experimental paradigm described in Fig. 3a for control (CTRL, grey) and POLY(I:C) (magenta) after 24 h stimulation. a Heatmap with color-coded mean pixel intensity relative to the reference of three independent differentiations (for individual plots, see Supplementary Fig. 5a). White: n.d. Side-bar: condition with iMG (orange) or without (white) or CTRL versus POLY(I:C). b Bar chart with SEM: Fold change of pixel intensity upon POLY(I:C) stimulation relative to CTRL. Each dot is an independent differentiation (n = 3). Shapiro–Wilk normality test < 0.05, Wilcoxon rank-sum test. Shapiro–Wilk normality test > 0.05, one sample t-test. c–e iMG proliferation rate in iMG-_dissRO for CTRL (left) and POLY(I:C) (right). c Example ROI image of tdTomato⁺-iMG (orange) and immunostained for the proliferation marker KI67 (magenta). White arrow: KI67⁺-expressing iMG. Scale bar: 50 µm. Zoom-in: Scale bar: 10 µm. d Boxplot of KI67⁺/iMG percentage. Wilcoxon rank-sum test. e Boxplot of iMG per area. Students’ t-test. d, e Symbols: single ROI of three biological replicates from five independent differentiations. f–k Proliferation of retinal cells for iMG-_dissRO (f, g), iMG-_3DRO (h, i), _dissRO (j), and _3DRO (k). Example ROI images counterstained for the nuclei-dye Hoechst (blue) and immunostained for the proliferation marker KI67 (green) for CTRL (left) and POLY(I:C) stimulation (right) for iMG-_dissRO (f) and cryostat section with a focus on the retinal cup of iMG-_3DRO (h). Scale bar: 50 µm. Boxplot percent of KI67⁺-cells relative to Hoechst⁺-cells, excluding KI67⁺/iMG, for CTRL and POLY(I:C) in iMG-_dissRO (g), iMG-_3DRO (i), _dissRO (j), and _3DRO (k). Symbols: single ROI of three biological replicates from five independent differentiations. Students’ t-test. For detailed statistical analysis, see Supplementary Table 4. ***p < 0.001. **p < 0.01. *p < 0.05. ^nsp > 0.05, not significant. CTRL untreated control, iMG microglia-like cells, iMG-_dissRO microglia integrated into dissociated retinal organoid culture, iMG-_3DRO microglia assembled 3D-retinal organoids, KI67 marker of proliferation KI-67, n.d. not detectable, POLY(I:C) polyinosinic: polycytidylic acid, _dissRO dissociated retinal organoid cultures without iMG, ROI region of interest, SEM standard error of the mean, WK week after the start of _3DRO differentiation