Fig. 2

iMG interact with BRN3⁺-ganglion cells in dissociated retinal organoids. a, b Gradual ganglion cell loss with _3DRO maturation. a Images of _3DRO cryostat sections counterstained with the nuclei-dye Hoechst (blue) collected at WK8, 12, 18, 21, 29 and immunostaining for BRN3 (green). Scale bar: 50 µm. b Scatterplot of BRN3⁺-cells relative to Hoechst⁺-cells per cryostat section with SEM and trend curve. Pearson correlation with a significant negative correlation between the differentiation age and the BRN3⁺-cells number. c, d Retinal cell types in _dissRO at WK20. Retinal cell type markers: RCVRN for photoreceptors, OTX2 for photoreceptors and bipolar cells, CALB2 for photoreceptors, bipolar- and amacrine cells, CALB1 for amacrine-, horizontal cells, PRKCA for rod bipolar cells, and BRN3 for ganglion cells. c Immunostaining for retinal markers (green) and nuclei-dye Hoechst (blue). Scale bar: 50 µm. d Bar chart with SEM of retinal cell types relative to Hoechst⁺-cells in _3DROs (black) and _dissRO (blue). Each dot: cryostat section of individual _3DROs (black) or a field of view in _dissRO (blue). Student’s t-test except for CALB1 (Wilcoxon rank-sum test). e Experimental timeline to generate iMG-_dissRO. At WK15, retinal cups dissociated and plated as _dissRO. At WK18.5, independently differentiated tdTomato⁺-preMG were added. iMG-_dissRO was analyzed 10 days later at WK20. f Image of _dissRO (left) and iMG-_dissRO (right) at WK20 counterstained with the nuclei-dye Hoechst (blue) and immunostained for BRN3 (green). Scale bar: 50 µm. Next, boxplot of BRN3⁺-ganglion cells relative to Hoechst⁺-cells in _dissRO (grey) and iMG-_dissRO (orange). Symbols: single ROI of three biological replicates from five independent differentiation. Wilcoxon rank-sum test. g, h Representative images of BRN3⁺-ganglion cells (green), tdTomato⁺-iMG (orange), the nuclei-dye Hoechst (blue, g), and the apoptotic marker CCAS3 (magenta, h) of iMG-_dissRO at WK20. Scale bar: 50 µm, zoom-in: 10 µm. Zoom-in: g 3D-surface rendering of a region of interest. White arrowhead: iMG engulfing BRN3⁺-cell. h White arrowhead: iMG engulfing BRN3⁺/CCAS3⁻-cell. For detailed statistical analysis, see Supplementary Table 4. ***p < 0.001. *p < 0.05. ^nsp > 0.05, not significant. BRN3 brain-specific homeobox/POU domain protein 3B, CALB1 calbindin, CALB2 calretinin, CCAS3 cleaved caspase-3, iMG-_dissRO microglia-assembled dissociated retinal organoids, iMG-_3DRO microglia assembled 3D-retinal organoid, iMG microglia-like cells, preMG microglia precursor, PRKCA protein kinase C alpha, OTX2 orthodenticle homeobox 2, _3DRO 3D-retinal organoid, _dissRO dissociated retinal organoid cultures without iMG, RCVRN recoverin, ROI region of interest, SEM standard error of the mean, WK week after the start of _3DRO differentiation