Additional file 1 of Microglia determine an immune-challenged environment and facilitate ibuprofen action in human retinal organoids
Creators: name: Schmied, Verena
name: Korkut-Demirbaş, Medina
name: Venturino, Alessandro
name: Maya-Arteaga, Juan Pablo
name: Siegert, Sandra
Publisher: name: figshare
Year: 2025
Subjects: subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Neuroscience
subject: Pharmacology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Mental Health
subject: Infectious Diseases
subject: FOS: Health sciences

description: Supplementary Material 1. Figure 1. Characterization of 3D retinal organoids. a-b, Immunostaining of 3DRO cryostat sections counterstained with the nuclei-dye Hoechst (blue) and immunostained for a, the presynaptic marker VGLUT1 (magenta) and b, the postsynaptic marker PSD95 (magenta) at WK13, WK17 and WK20. White arrow: outer plexiform layer forming between the outer- and inner nuclear layer (ONL, INL, respectively). Scale bar: 10 µm. c, Representative cryostat section images of 3D-retinal organoid counterstained with the nuclei-dye Hoechst (blue) and immunostained for retinal cell type-specific markers (green) and at week 20: RCVRN (recoverin; photoreceptors). OTX2 (orthodenticle homeobox 2; photoreceptors, bipolar cells). CALB2 (calretinin; photoreceptors, bipolar-, amacrine cells). CALB1 (calbindin; amacrine-, horizontal cells). CHAT (choline acetyltransferase; amacrine cells). BRN3 (brain-specific homeobox/POU domain protein 3B; ganglion cells). RLBP1 (cellular retinaldehyde-binding protein; Müller glia). ONL: outer nuclear layer. INL: inner nuclear layer. White dashed line: outer plexiform layer. #: retinal cup lumen. White arrow: BRN3+-cells close to lumen. Scale bar: 50 µm. d, Expression of microglia transcript markers in USCS Cell Browser of Cowan et al. [10]: Dataset ID: ‘Developed human retinal organoid.’ Uniform manifold approximation and projection (UMAP) of transcript expression for AIF (also known as IBA1, ionized calcium-binding adapter molecule 1), CX3CR1 (C-X3-C motif chemokine receptor 1), SPI1 (also known as PU.1, Spi-1 proto-oncogene) and P2RY12 (purinergic receptor P2Y12) of 3D-retinal organoid at week 32 and 38. AC: amacrine cell. BC: bipolar cell. Cone: cone photoreceptors. HC: horizontal cell. MC: Müller glia. RPE: retinal pigment epithelium. Rod: rod photoreceptors. Blue dot: not detected. Figure 2. Generation of tdTomato+-hIPSC cell line and characterization of differentiated tdTomato+-microglia precursor cells (preMG). a Integration strategy into the adeno-associated virus integration site 1 (AAVS1) locus. DSB: double-strand break. CAG: CMV immediate enhancer/β-actin promoter. HA-L: homologous arm left. HA-R: homologous arm right. HR: homologous recombination. Puro: puromycin selection side. tdTom: tdTomato. b, c, Validation strategy. Reaction 1: wildtype allele: PCR product 1.4 kbp. Reaction 2: tdTomato allele: PCR product 1.2 kbp. PCR: polymerase chain reaction. c PCR product size. Top: Reaction 1-wildtype AAVS1 locus (1.4 kbp). Bottom: Reaction 2 - construct integrated into AAVS1 (1.2 kbp). Orange: wildtype clone. Red: clone with homozygous integration of the construct. NTC: non-template control. Kbp: kilobase pair. d, Validating pluripotency for the wildtype human induced pluripotent stem cell (hIPSC) line SC102A (top) and the tdTomato+-hiPSC line SC102A (bottom). Immunostaining of hIPSC colonies for NANOG (nanog homeobox, green), OCT3/4 (octamer-binding protein 3, cyan), and counterstaining for the nuclei-dye Hoechst (blue). Intrinsic tdTomato expression (orange). Scale bar: 100 µm. e, Bar chart of tdTomato+/ IBA1+-preMG with standard error of the mean. f-k, Representative images of tdTomato-expressing microglia precursor cells (preMG, orange) harvested from the supernatant and plated on a new dish. Cells counterstained for the nuclei-dye Hoechst (blue, merged image), immunostained for IBA1 (ionized calcium-binding adapter molecule 1, green) and the microglia/macrophage markers in magenta for f, PU.1 (hematopoietic transcription factor PU.1); g, RUNX1 (runt-related transcription factor 1); h, ITGAM (integrin subunit alpha m); i, CD45 (cluster of differentiation 45/ protein tyrosine phosphatase receptor); j, CX3CR1 (chemokine (C-X3-C) receptor 1); k, MYB (MYB proto-oncogene). Scale bar: 20µm. Figure 3. tdTomato+-microglia precursor cells (preMG) integration patterns into 3D retinal organoids. Representative images of 3D-retinal organoids. Left: fluorescence image, right: brightfield image. 4x magnification (top) and 10x magnification (bottom). Scale bar: 20µm. a, tdTomato+-microglia precursor cells (preMG, white arrow) attach on week 17 at the surface of 3D-retinal organoids (3DRO). b, tdTomato+- microglia-like cells (iMG) integrate into the 3D-retinal organoids at week 20, showing a bipolar shape (white arrow). c, Images of iMG-3DRO cryostat sections with tdTomato+-iMG (orange) counterstained with the nuclei-dye Hoechst (blue) and at WK20. White arrowhead: iMG located in the outer plexiform layer forming between the outer- and inner nuclear layers (ONL, INL, respectively). #: lumen. Scale bar: 50 µm. Figure 4. Validating iMG signature. a, Impact of brain-derived neurotrophic factor (BDNF) on ganglion cell survival. Bar chart of percentage of BRN3+-cells relative to Hoechst+-cells with standard error of the mean in 3D-retinal organoids (3DRO, left) and dissociated retinal organoid culture (dissRO, right) cultured either in standard retinal organoid differentiation media without (-BDNF, grey) or supplemented with BDNF (+BDNF, green) from week 15 to 20. 3DRO: Each dot is one cryostat section of independent retinal cups. Welch's t-test. dissRO: Each dot is one region of interest. One-sample Wilcoxon signed rank test. b, Image of iMG distribution within iMG-dissRO (orange) at WK20, counterstained with the nuclei-dye Hoechst (blue). Scale bar: 100 µm. c, Schematic timeline of microglia marker expression during development. d, RT-qPCR for the microglia marker C1QA (complement component C1q), CX3CR1 (C-X3-C motif chemokine receptor 1), P2RY12 (purinergic receptor P2Y G-protein-coupled 12) and TMEM119 (transmembrane protein 119) in iMG-dissRO after 1 and 10 days of coculture. Bar chart with SEM of mean mRNA transcript expression relative to GAPDH. Each dot is one biological replicate. Student’s t-test. e-g, Representative images of iMG-dissRO with tdTomato+-iMG (orange), counterstained for the nuclei-dye Hoechst (blue) and immunostained in magenta for e, early transcription factors PU.1 (hematopoietic transcription factor PU.1), RUNX1 (runt-related transcription factor 1) and MYB (MYB Proto-Oncogene); f, ‘early’ microglia marker IBA1 (ionized calcium-binding adapter molecule 1) and CD45 (cluster of differentiation 45/ protein tyrosine phosphatase receptor); and g, ‘mature’ microglia marker P2Y12 and TREM2 (Triggering Receptor Expressed On Myeloid Cells 2). Scale bar: 10 µm. For detailed statistical analysis, see Supplementary Table 4. ***p &lt; 0.001. **p &lt; 0.01. *p &lt; 0.05. nsp &gt; 0.05, not significant. Figure 5. iMG mediated consequences in the dissociated retinal organoid model. a-c, Boxplot of percent of a, CCAS3+-cells per area; b, Hoechst+-cells per area and c, TX2+-photoreceptor- and bipolar cells relative to Hoechst+-cells in dissRO (grey) and iMG-dissRO (orange). a-b, Students’s t-test. c, Welch's t-test. d, Representative images of iMG-dissRO for tdTomato expression (orange), counterstained for the nuclei-dye Hoechst (blue) and immunostained for the apoptotic marker CCAS3 (cleaved caspase3, green). White arrowhead: iMG engulfing CCAS3+-fragment. Scale bar: 50µm. e, Hoechst+-fragments (white) in dissRO (left) and iMG-dissRO (right). Scale bar: 50 µm. f, Boxplot of percent of Hoechst+-fragments per area in dissRO (grey) and iMG-dissRO (orange). Welch's t-test. g, Representative images of iMG-3DRO cryostat sections counterstained with the nuclei-dye Hoechst (blue) and tdTomato+-iMG (orange) at WK20. Arrow: iMG engulfing Hoechst+-fragment. Scale bar: 50µm. Zoom in: Scale bar: 10µm. Symbols: single ROI of three biological replicates from five independent differentiations. For detailed statistical analysis, see Supplementary Table 4. ***p &lt; 0.001. nsp &gt; 0.05, not significant. Figure 6. Individual inflammatory proteome profiler results. a, Release of inflammatory cytokines and chemokines into the supernatant based on the experimental paradigm described in Fig. 3a for control (CTRL, grey) and 24h- POLY(I:C) (magenta) stimulation. Individual heatmap plots with color-coded mean pixel intensity relative to the reference of three independent differentiations White: n.d. (not detectable). Side-bar: condition with iMG (orange) versus without (white) or CTRL versus POLY(I:C). b, Representative membranes for each condition. Numbers refer to the legend below. c, Example images of dissRO counterstained with the nuclei-dye Hoechst (blue) and immunostained for the glial marker GFAP (glial fibrillary acidic protein, green). Scale bar: 20 µm. d, List of proteins assayed on the membrane but not detected in the supernatant of any condition. Figure 7. Timeline POLY(I:C)-mediated response. a, Same assay as for in Supplementary Figure 6a with additional measurement of cytokine and chemokine release after two and four hours compared to 24h in iMG-dissRO with annotated example membranes iMG-dissRO. b, Boxplot of the total branch length (left) and the number of endpoints (right) per iMG for CTRL (grey) and POLY(I:C) (magenta) following 4h and 24h stimulation in iMG-dissRO. iMG were collected from five independent differentiations. Kruskal-Wallis test with post-hoc Dunn’s test. Figure 8. The POLY(I:C)-mediated proliferation rate increase cannot be replicated with CCL2 alone. a, Experimental timeline. At WK18.5, preMG are added to dissRO. After nine days, cultures received fresh medium for control (CTRL, grey) or CCL2 stimulation iMG-dissRO for 24 hours. b, Effect of CCL2 on retinal cell proliferation excluding iMG. Boxplot of percent KI67+-cells relative to Hoechst+-cells in iMG-dissRO for CTRL and CCL2 stimulation at a final concentration of 10 ng/mL (yellow), 20 ng/mL (orange), and 50 ng/mL (red). Magenta line: Median proliferation rate in POLY(I:C) stimulation of iMG-dissRO (Fig. 4g). Symbols: three biological replicates from five independent differentiations. One-way ANOVA. c, Effect of CCL2 on iMG proliferation. Boxplot of percent KI67+/iMG for CTRL and CCL2 stimulation at a final concentration of 10 ng/mL (yellow), 20 ng/mL (orange), and 50 ng/mL (red). Magenta line: Median iMG-proliferation rate in POLY(I:C) stimulation of iMG-dissRO (Fig. 4d). Symbols: three biological replicates from five independent differentiations. Kruskal-Wallis test. For detailed statistical analysis, see Supplementary Table 4. *p &lt; 0.05. nsp &gt; 0.05, not significant. Figure 9. Comparison of individual secreted inflammatory mediators after ibuprofen exposure. a, Release of inflammatory cytokines and chemokines into the supernatant based on the experimental paradigm described in Fig. 3a for control (CTRL, grey), POLY(I:C) (magenta), and POLY(I:C) and S(+)-ibuprofen (POLY(I:C)+IBU, blue) stimulation. Release of different inflammatory mediators into the supernatant of iMG-dissRO. Bar chart of pixel intensity normalized to reference with standard error of the mean for CTRL, POLY(I:C), and POLY(I:C)+IBU. Each symbol: an independent differentiation (n=3). One-way ANOVA with post-hoc Tukey’s test, except IL13, IL18, IL21 Kruskal-Wallis test. For detailed statistical analysis, see Supplementary Table 4. ***p &lt; 0.001. **p &lt; 0.01. *p &lt; 0.05. nsp &gt; 0.05, not significant. Figure 10. GCAMP6s expression across retinal cell types. a-e, Example ROI images of dissRO infected with AAV2-GCAMP6s at WK17, analyzed at WK20, counterstained for the nuclei-dye Hoechst (blue) and the calcium sensor GCAMP6s (green), and immunostaining for retinal cell types (magenta): a, RCVRN (recoverin; photoreceptors). b, OTX2 (orthodenticle homeobox 2; photoreceptors, bipolar cells). c, CALB2 (calretinin; photoreceptors, bipolar-, amacrine cells). d, CALB1 (calbindin; amacrine-, horizontal cells). e, BRN3 (brain-specific homeobox/POU domain protein 3B; ganglion cells). Arrow: Co-expression of calcium sensor and retinal marker. Scale bar: 50µm. f, Spontaneous calcium dynamics in iMG-dissRO (orange) and dissRO (grey) for control (CTRL, grey), POLY(I:C) (magenta), and POLY(I:C) and S(+)-ibuprofen (POLY(I:C)+IBU, blue) stimulation. Boxplot of the mean frequency [Hz] during five minutes of recording. Each dot represents an active cell. Recordings from five biological replicates from independent differentiations. Kruskal-Wallis test. For detailed statistical analysis, see Supplementary Table 4. nsp &gt; 0.05, not significant. Figure 11. PTGS1, PTGS2, and TLR3 mRNA expression profile. a-b, Expression of (a) PTGS1 and PTGS2 (prostaglandin-endoperoxide synthase 1 and 2) as well as (b) TLR3 (toll-like receptor 3) in USCS Cell Browser of Cowan et al. [10].