Fig. 5
oMG form functional interactions with neurons and respond to inflammatory stimuli in situ. a Microglia (IBA-1)–neuron (TUJ1) interaction at an early (day 31) and late (day 52) timepoint was visualized by immunostainings. Representative pictures of cerebral organoids from iPSC 1 are shown. Scale bars 40 μm. b gSTED microscopy showing synaptic content inside microglial processes visualized by immunohistochemistry for IBA-1 and PSD-95 on day 66 organoids. Maximum intensity projection of the entire cell (scale bar 10 μm), and close-up of region of interest (box in dashed line) with maximum intensity projection (scale bar 1 μm) and orthogonal view (voxel size 0.18 μm). c Pilot experiment in which the IL6 inflammatory response was measured in organoids in situ. The cytokine response was significantly increased after 24 h as determined with a Friedman’s ANOVA test (p = 0.03; Dunn’s test t = 6 vs. baseline: -3 with p = 0.44; Dunn’s test t = 24 vs. baseline: -6 with p = 0.03). N = 3 iPSC 1 organoids per group. Error bars indicate standard deviation. d Inflammatory response in situ was measured in whole organoids upon 24 and 72 h of LPS stimulation by ELISA for cytokine release (IL6, TNF-α, and IL10; both timepoints) and transcript fold change by qRT-PCR (after 72 h) for mRNA levels (IL6, TNF, IL10) in day 52 organoids. Secretion of both IL6 and TNF-α, but not IL10, was significantly increased after 24 and 72 h as analyzed with Wilcoxon matched-pairs signed rank test (IL6 and TNF-α: W = 21, p = 0.03). Similarly, mRNA levels of IL6 and TNF, but not IL10, were increased after 72 h stimulation (IL6: W = 21, p = 0.03; TNF: W = 19, p = 0.06). mRNA levels were determined by qRT-PCR and normalized to the geomean of the reference genes SDHA2 and ACTB. Two batches of cerebral organoids from iPSC 1, 3, and 5 were used (n = 6). IL10 levels were below detection level for the second batch of organoids. (*p < 0.05)
