Fig. 8: MS-relevant inflammatory stimuli increased the number of senescent-like cells in a hiPSC-derived neural glia-enriched organoid model.

a Immunostaining of NeuN⁺ and MAP2⁺ neurons, MBP⁺ oligodendrocytes, SOX10-eGFP⁺ OPCs, GFAP⁺ astrocytes, and MHCII⁺ microglia out of DAPI in whole cryosections from 8 weeks-old submillimetric glia-enriched organoids (30×). Scale bar: 100 μm. b Trasmission electron microscopy showing myelinated axons (red arrows) within the glia-enriched organoid. Scale bar: 500 nm. c Representative images and quantification of SA-β-galactosidase activity along the organoid differentiation (n = 3 MS hiPSC-lines; at least 25 glia-enriched organoids per condition; one-way ANOVA p < 0.0001, Tukey’s post-hoc multiple comparison analysis * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). The violin plots show the median (red line), quartiles (dotted lines), as well as the minima and maxima; each dot corresponds to one glia-enriched organoid. d Representative images of untreated organoids (left) vs. organoids exposed to 10% CSF (right) for 24 h (top, > 100 glia-enriched organoids analyzed per experimental condition) and 6 days (bottom, > 40 analyzed organoids per experimental condition) and relative quantification of SA-β-gal staining area (n = 3 MS hiPSC-lines, t-test *** p = 0.001; **** p < 0.0001). The violin plots show the median (black line), quartiles (dotted lines), as well as the minima and maxima; each dot corresponds to one glia-enriched organoid. e Single-cell RNA-seq clustering of 118,706 cells, visualized as UMAP plot, from 8-week-old glia-enriched organoids (from 3 MS hiPSC-lines). Each dot corresponds to a single cell and each colour to a cell cluster. f Averaged percentage of each cell population by experimental condition. g Dot plot of averaged z-transformed gene expression of marker genes for each cluster. h Box plot showing the percentage of SIT-defined senescent cells (mean ± SEM; n = 3 MS hiPSC-lines; ANOVA p = 0.028, Kruskal–Wallis post-hoc multiple comparison analysis *p = 0.011). Increased number of senescent-like cells is seen after stimulation with MS CSF or inflammatory cytokines. i Heatmap showing the averaged percentage of SIT-defined senescent cells in the different cell-types by experimental condition (two-way ANOVA p = 0.05, row effect p = 0.012, column effect p = 0.015, post-hoc multiple comparison analysis *p < 0.05). j Volcano plot reports gene expression changes in SIT-defined senescent vs. non-senescent microglia from glia-enriched organoids. W8 week 8, MS multiple sclerosis, SEM standard error of the mean, CSF cerebrospinal fluid, NEU neurons, ASTRO astrocytes, OPC oligodendrocyte precursor cells, OL oligodendrocytes, GLIA_IMM immature glia, PROG cycling progenitors, MG hiPSC-derived microglia, SA-β-gal senescence-associated β-galactosidase activity, MBP myelin basic protein, SIT senescence index tool.