Fig. 3
iMG respond to POLY(I:C) stimulation but still interact with ganglion cells. a Experimental timeline. At WK18.5, preMG are added to dissRO. After 9 days, the culture medium was replaced with fresh medium either containing POLY(I:C) (magenta) or without as a control (CTRL, grey). Analysis was performed 24 h later on day 10. b RT-qPCR for TLR3 of preMG after CTRL or POLY(I:C) stimulation. Bar chart with SEM: Mean mRNA transcript log2-fold changes compared to CTRL. Symbol: mean of technical triplicate from five independent differentiations. One sample t-test. c iMG-dissRO live imaging for 20 min for CTRL or POLY(I:C) stimulation after 24 h. iMG surveillance index normalized to the mean surveillance of the cells in CTRL with a 95% confidence interval. Four independent differentiations. Wilcoxon rank-sum test. d, e iMG surface area quantification. d iMG surface rendering for CTRL (left) and POLY(I:C) (right) 4 h and 24 h following stimulation, color-coded based on surface area: blue = 1000 µm2 to red = 6000 µm2. Scale bar: 50 µm. e, Boxplot of individual iMG surface areas in iMG-dissRO for CTRL and POLY(I:C). iMG were collected from five independent differentiations. Kruskal–Wallis test with post-hoc Dunn’s test. f, Boxplot quantifying the number of iMG engulfing BRN3+-cells in iMG-dissRO for CTRL and POLY(I:C). Symbols: single ROI of three biological replicates from five independent differentiations. Wilcoxon rank-sum test. g Boxplot determines ganglion cell density based on BRN3+-cells relative to Hoechst+-cells in iMG-dissRO for CTRL and POLY(I:C). Symbols: single ROI of three biological replicates from five independent differentiations. Wilcoxon rank-sum test. h, i Ganglion cell-iMG proximity in iMG-dissRO. h Surface rendering of iMG for CTRL (top) and POLY(I:C) (bottom). BRN3+-spots color-coded based on the proximity to the iMG surface with spots < 5 µm (magenta) and spots > 5 µm (cyan). Scale bar: 10 µm. i Boxplot of percent of magenta BRN3+-spots. Symbols: single ROI of three biological replicates from five independent differentiations. Wilcoxon rank-sum test. 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. CTRL untreated control, iMG microglia-like cells, iMG-dissRO microglia integrated into dissociated retinal organoid culture, POLY(I:C) polyinosinic: polycytidylic acid, preMG microglia precursor cells, dissRO dissociated retinal organoid cultures, RT-qPCR real-time quantitative polymerase chain reaction, ROI region of interest, SEM standard error of the mean, TLR3 toll-like receptor 3, WK week after the start of 3DRO differentiation