Microglia Differentiation from iPSCs

iPSCs were differentiated into microglia following an established protocol^{75 (link)} with minor changes. In brief, embryoid bodies (EBs) were formed using AggreWell™800 (STEMCELL Technologies), and cultured in mTeSR1 (STEMCELL Technologies) with bone morphogenetic protein 4 (BMP4, 50 ng/mL; ImmunoTools), vascular endothelial growth factor (VEGF, 50 ng/mL; ImmunoTools), and stem cell factor (SCF, 20 ng/mL; ImmunoTools) for four days with 75% medium change daily. On day 4, EBs were collected and transferred into 6-well cell culture plates (12–16 EBs/well) in X-VIVO 15 (Lonza) supplemented with IL-3, (25 ng/mL; ImmunoTools), macrophage colony-stimulating factor (M-CSF, 100 ng/mL; ImmunoTools), 2 mM GlutaMAX (Thermo Fisher), 1% P/S (Merck Millipore), and 0.055 mM β-mercaptoethanol (Sigma-Aldrich), with medium change weekly. After 3–4 weeks, floating cells were collected and seeded at a concentration of 100,000 cells/cm² on Matrigel-coated plates in Advanced DMEM/F-12 (Life Technologies) supplemented with N2 (Thermo Fisher), GlutaMAX, P/S, β-mercaptoethanol, M-CSF (100 ng/mL), IL-34 (100 ng/mL; PeproTech), and GM-CSF (10 ng/mL, ImmunoTools) with medium change twice a week.