The human iPSC line WTSIi015-A (EBiSC through Sigma-Aldrich) was maintained on Matrigel (Corning) in mTeSR1 medium (Stemcell Technologies). IPSC colonies were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) and 4 × 106 iPSCs seeded per Aggrewell 800 (Stemcell Technologies) in a 24-well plate in embryonic body medium (EBM). EBM consisted of mTeSR1 medium supplemented with 10 μM ROCK inhibitor, 50 ng/mL BMP-4, 20 ng/mL SCF, and 50 ng/mL VEGF-121 (all from Peprotech). Cells were cultured for 4 days in Aggrewells to form embryonic bodies (EBs) with half media change every day. EBs were harvested using an inverted cell strainer (40 μm), and around 15 EBs were plated per 6 wells in hematopoietic medium (HM) consisting of X-VIVO 15 medium (Lonza) supplemented with 2 mM Glutamax, 100 U/mL penicillin, 100 μg/mL streptomycin, 55 μM β-mercaptoethanol, 100 ng/mL human M-CSF (Peprotech), and 25 ng/mL human IL-3 (Cell Guidance Systems). Two milliliters medium was replaced every 7 days by fresh HM. After around 30 days, primitive macrophage precursors could be harvested during the media change and plated in microglia medium (MiM) into 48 wells at a density of 105 cells/cm2. MiM consists of contained X-VIVO 15 medium (Lonza) supplemented with 2 mM Glutamax, 100 U/mL penicillin, 100 μg/mL streptomycin, 55 μM β-mercaptoethanol, 100 ng/mL human IL-34 (Peprotech), and 10 ng/mL human GM-CSF (Peprotech). Finally, cells were differentiated in MiM for subsequent 7–10 days with full MiM change every second day.

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