Fig. 1
Microglia colonize retinal layers after OPL formation. a Representative brightfield images focusing on the retinal cup at selected 3DRO differentiation time points. Arrow and dashed line: outer plexiform layer formation, visible from WK20 onwards. Scale bar: 100 µm. b, c Images of 3DRO cryostat sections counterstained with the nuclei-dye Hoechst (blue) and immunostained for b photoreceptors with RCVRN (green) and the ribbon synapse marker RIBEYE (magenta, left); presynaptic marker VGLUT1 (magenta, middle); and postsynaptic marker PSD95 (magenta, right) at WK20. White arrow: outer plexiform layer. Scale bar: 10 µm. c IBA1 (magenta) at WK4, WK8, WK12, and WK21. Scale bar: 100 µm. d Experimental schematic to generate iMG-3DRO. e Maximum intensity projection image of entire iMG-3DRO counterstained with the nuclei-dye Hoechst (blue) at different time points of preMG application and collection of iMG-3DRO as outlined in the schematic. Immunostaining for IBA1 (orange), BRN3 (green, for WK16), and OTX2 (green, for WK9 and WK19). White arrowhead: iMG positioned in OPL. White dashed line: OPL. #: retinal cup lumen. Scale bar: 100 µm. Zoom-in: 10 µm. f Images of iMG-3DRO cryostat sections with tdTomato+-iMG (orange) at WK20 immunostained for the presynaptic marker VGLUT1 (magenta, top) or the postsynaptic marker PSD95 (magenta, bottom). Blue: nuclei-dye Hoechst. White arrowhead: iMG located in OPL. Scale bar: 10 µm. g, h Boxplot. g Percent of iMG in the ONL, OPL, INL, GCL, and within the retinal cup lumen (#) at WK20. Each dot: one cryostat section of an independent retinal cup. h Total number of iMG integrated per retinal cup at WK9 and WK20. Each dot represents an entire retinal cup. Students’s t-test. *** p < 0.001. For detailed statistical analysis, see Supplementary Table 4. BRN3 brain-specific homeobox/POU domain protein 3B, GCL ganglion cell layer, hIPSC human induced pluripotent stem cell, IBA1 ionized calcium-binding adapter molecule 1 (alternative name: AIF1), INL inner nuclear layer, iMG microglia-like cell, iMG-3DRO microglia assembled 3D-retinal organoid, ONL outer nuclear layer, OPL outer plexiform layer, OTX2 orthodenticle homeobox 2, preMG microglia precursor cells, PSD95 postsynaptic density protein 95, RCVRN recoverin, 3DRO 3D-retinal organoid, VGLUT1 vesicular glutamate transporter 1, WK week after the start of 3DRO differentiation