Differentiation of iPSCs to Macrophages and Microglia

iPSCs were differentiated to macrophage precursor cells via embryoid bodies (EBs) using AggreWells (Stem Cell Technologies) using a published methodology with no alterations (45 (link)). Media for embryoid body generation consisted of mTeSR with 50 mg/mL BMP4, 50 mg/mL VEGF and 20 mg/mL SCF. Approx. 300 EBs were split between two T175 flasks in 15 mL of medium per flask. ‘Factory’ medium comprised XVIVO15 (Lonza, Basel, Switzerland) with 100 ng/mL recombinant human M-CSF (Gibco), 25 ng/mL recombinant human IL-3 (Gibco), 2 nM GlutaMAX (Gibco), 50 μM 2-mercaptoethanol and 100 units/mL penicillin with 100 µg/mL streptomycin (Gibco). Precursor cells were harvested and plated into final experimental format for final differentiation to either iPS-Mϕ in macrophage differentiation medium (XVIVO15 with 100ng/mL recombinant human M-CSF (Gibco), 2 mM GlutaMAX (Gibco) and 100 units/mL penicillin with 100 µg/mL streptomycin) for 7 days prior to use, or iPS-microglia-like cells (iPS-MGLs) in microglia differentiation medium (Advanced DMEM/F12 + N2 Supplement (Gibco) with 100 ng/mL recombinant human IL-34 (PeproTech), 10 ng/mL recombinant human GM-CSF (Gibco), 2 mM GlutaMAX, 50 μM 2-mercaptoethanol and 100 units/mL penicillin with 100 µg/mL streptomycin) for 14 days, changing medium twice weekly, prior to use in experiments.