Fig. 2. Functional characterization of iTF-Microglia.

a, Phagocytosis of pHrodo-Red-labeled rat brain-derived synaptosomes by iTF-Microglia. Representative images at 0 h and 12 h after synaptosome addition are shown. Treatment with 5 μM actin polymerization inhibitor Cytochalasin D decreases phagocytosis. Scale bar, 100 μm. b, Phagocytosis of pHrodo-labeled rat brain-derived synaptosomes with or without Cytochalasin D treatment was quantified by flow cytometry at 0.5 h, 1.5 h and 2.5 h after synaptosome addition (mean ± s.d., n = 3 biological replicates; P values from two-tailed Student’s t-test). c, Morphological changes of iTF-Microglia after LPS treatment are visualized by fluorescence microscopy. Samples were treated for 24 h with 100 ng ml⁻¹ LPS or buffer control and fixed samples were stained with Alexa Fluor 488-phalloidin for F-actin (green) and with Hoechst 33342 for nuclei (blue). Scale bar, 100 μm. d, Transcriptomic changes caused by 50 ng ml⁻¹ LPS treatment in day 15 iTF-Microglia (n = 3 biological replicates). DEGs (P_adj < 0.05, two-tailed Student’s t-test) are labeled in black (increase). Other colors label genes associated with specific pathways that are discussed in the main text. e, Cytokines secreted by iTF-Microglia. Analysis of cytokine array signal (integrated density of dot blots) from supernatants of cultures treated with LPS or buffer control (mean ± s.d., n = 6 biological replicates; P values from two-tailed Student’s t-test). *GM-CSF is a component of the culture medium. f, Coculture with iPSC-derived excitatory neurons promotes ramified morphology of iTF-Microglia. Representative fluorescence micrographs at low and high magnification of day 9 iTF-Microglia after 24 h in coculture. iTF- Microglia express membrane-localized Lck-mNeonGreen (green). Neurons are stained for the pre-synaptic marker synaptophysin (magenta). Nuclei are stained with Hoechst 33342 (blue). Scale bars, 100 µm. AU, arbitrary units; NS, not significant.