Extended Data Fig. 6: Extended data related to Fig.4.

(a) Schematic representation of the microglial differentiation protocol and plating for in vitro experiments, based on Fattorelli et al.³⁵. Created in BioRender. Polanco, P. (2025) https://BioRender.com/83a97ah (b) Representative images of IBA1 immunostaining for control and C9KO iPSC-derived microglia. Scale bar, 20 µm³⁵. (c) Heatmap showing enrichment of microglial-related genes versus macrophages after iPSC differentiation in vitro (n = 7 independent replicates). (d) Example of low-magnification confocal images used for quantification of CTSD clusters in Fig. 4d and f. Upper panel: C9-HRE and C9-ISO; lower panel: C9KO and control microglia. Cells containing at least one CTSD puncta were considered for quantification in ImageJ. The percentage of cells containing CTSD positive puncta was calculated on total DAPI positive cells. (e) Representative low-magnification TEM images for C9KO and control iPSC-derived microglia that were used for quantification of endolysosomes/lysosomes clusters in Fig. 4h. Scale bar, 20 µm. (f) Quantification of percentage of cells containing enlarged endolysosomes/lysosomes. Unpaired T-test (CTRL n = 5 independent replicates, C9KO n = 5 independent replicates). Box plots show the median, interquartile range, and full data range. (g) Microscopy images showing EEA1 (that is, early endosomes) and Lamp1 positive structures in control and C9KO microglial cells. Scale bar, 5 µm. Flow cytometry analysis of uptake of (h, i) amyloid beta and (j, k) pHrodo E. coli particles, showing no difference in the median fluorescence intensity (MFI) between C9KO and controls, at 3 h and 1 h upon treatment, respectively. Ratio paired T-test (amyloid beta, CTRL n = 3 independent replicates, C9KO n = 3 independent replicates; pHrodo E. coli CTRL n = 4 independent replicates, C9KO n = 4 independent replicates).