Fig. 7
Cell type-dependent transcriptional differences in PTGS1 and PTGS2 point to iMG impact on calcium dynamics. a–g RT-qPCR. Bar chart with SEM of mean mRNA transcript expression relative to GAPDH for PTGS1 (a, c–g) and PTGS2 (b–g) across untreated (a–c) and CTRL versus POLY(I:C) (magenta) or POLY(I:C) (g) and S(+)-ibuprofen (POLY(I:C) + IBU, blue) (d–f) for 3DRO versus iMG-3DRO (a, b), preMG (c, d), iMG-3DRO (e), 3DRO (f) and hIPSC-derived astrocytes (g). h Experimental timeline similar to with POLY(I:C) and SC-560 exposure for 24 h in iMG-dissRO at WK20. Students t-test (a–c). One-way ANOVA (d–g) except g/PTGS2 Kruskal–Wallis test. i, j Spontaneous calcium dynamics in iMG-dissRO during 5 min recording after 24 h of either fresh medium for control (CTRL, grey), POLY(I:C) (magenta), POLY(I:C) and S(+)-ibuprofen (POLY(I:C) + IBU, blue), or POLY(I:C) and SC-560 (POLY(I:C) + SC-560, green) exposure. Boxplot of mean peak amplitude [F/F0, i] and mean frequency [Hz, j]. Each dot represents an active cell from five independent differentiations. Peak amplitude: reference lines for the median of the control and POLY(I:C) stimulation in dissRO from Fig. 6m. Kruskal–Wallis test with post-hoc Dunn’s test. For detailed statistical analysis, see Supplementary Table 4. ***p < 0.001. *p < 0.05. nsp > 0.05, not significant. CTRL untreated control, GAPDH glyceraldehyde 3-phosphate dehydrogenase, H hour, hIPSC human induced pluripotent stem cell, IBU ibuprofen, iMG microglia-like cells, iMG-dissRO microglia integrated into dissociated retinal organoid culture, iMG-3DRO microglia assembled 3D-retinal organoids, POLY(I:C) polyinosinic: polycytidylic acid, preMG microglia precursor cell, PTGS1 prostaglandin-endoperoxide synthase 1 (COX1), PTGS2 prostaglandin-endoperoxide synthase 2, 3DRO 3D-retinal organoids, RT-qPCR real-time quantitative polymerase chain reaction, SC-560 PTGS1/COX-1 selective inhibitor, SEM standard error of the mean, WK week after the start of 3DRO differentiation