Fig. 6: LRRK2 regulates the function of human iPSC-derived microglia.

a Overview of microglia generation protocol using iPSC-derived embryoid bodies (EBs). Representative bright-field images of (i) day 3 and (ii) mature EBs and (iii) ramified iPSC-derived microglia. Scale bars, 50, 100, and 20 μm respectively. (iv) Confocal image of iPSC-derived microglia immunostained for IBA1 (red) and nuclear staining DAPI (blue). Scale bar, 20 μm. b–c Confocal images (b) of iPSC-derived microglia immunostained for PU.1 (red, upper), IBA1 (red, lower panels), and nuclear staining DAPI (blue). Scale bar, 20 μm. In (c) corresponding quantification (mean ± SEM, n = 3 independent experiments). d Quantification of migrated iPSC-derived microglia upon ATP stimulation with or without prior IFN-γ treatment, normalized over unstimulated control microglia (mean ± SEM, two-way ANOVA, Bonferroni post hoc; **P = 0.0050, 0.0017 in sequence, *P = 0.0436; n = 5 independent experiments). e Representative confocal images (left) and quantification (right) of microglia phagocytic capacity (green: latex fluorescent beads, red: IBA1). Scale bars, 20 µm; (mean ± SEM, one-way ANOVA, Bonferroni post hoc, ****P < 0.0001, **P = 0.0011; n = 45 control, n = 39 G2019S, n = 67 LRRK2 KO cells examined over three independent experiments). f Concentration of differentially secreted cytokines upon LPS treatment (mean ± SEM, two-way ANOVA, Bonferroni post hoc, treated vs untreated, ^§§§§P < 0.0001; compared to LPS-treated: ****P < 0.0001, ***P = 0.0001, 0.0006 in sequence, **P = 0.0015, 0.0017, 0.0056 in sequence, *P = 0.0432; n = 3 independent experiments). g Metabolic analysis of extracellular acidification rate (ECAR) in LPS-treated microglia with or without prior LRRK2 kinase inhibition (left) or IFN-γ-treated microglia (right) (mean ± SEM, two-way ANOVA, Bonferroni post hoc, treated vs untreated, ^§§§§P < 0.0001, ^§§P = 0.0044 compared to LPS- or IFN-γ-treated^: ****P < 0.0001, ***P = 0.0007; compared to Mli-2-treated: two-tailed t-test, ****P < 0.0001, ***P = 0.0006; n = 8). h Concentration of secreted IL1-β upon poly(I:C) treatment (mean ± SEM, two-way ANOVA, Bonferroni post hoc, treated vs untreated, ^§§§§P < 0.0001, ^§§§P = 0.0002; compared to treated: ***P = 0.0001, *P = 0.0201; ns P = 0.1097; n = 3). i Schematic diagram showing treatment of neurons with either untreated or LPS-treated microglial-conditioned medium (MCM). On the right, quantification of neurite elongation in control and G2019S neurons (mean ± SEM, one-way ANOVA, Bonferroni post hoc, **P = 0.0078, 0.0051 in sequence, compared to UT; n = 5 independent experiments).