Differentiation of iPSCs to Homeostatic Microglia

iPS-microglia were differentiated as detailed in McQuade et al.^{27 (link)}. Briefly, iPSCs are differentiated to hematopoetic progenitor cells using the STEMdiff Hematopoetic kit for 10–12 days before passage into microglia differentiation medium^{27 (link),40 (link)} including DMEM/F12, 2× insulin-transferrin-selenite, 2× B27, 0.5× N2, 1× glutamax, 1× non-essential amino acids, 400 μM monothioglycerol, and 5 μg/mL human insulin. Cultures were maintained in this basal medium supplemented with 100 ng/mL IL-34, 50 ng/mL TGF-β1, and 25 ng/mL M-CSF (Peprotech) for 28 days. For the last 3 days in culture, two additional cytokines were added (100 ng/mL CD200 (Novoprotein) and 100 ng/mL CX3CL1 (Peprotech)) to mature the microglia in a homeostatic brain-like environment^{40 (link)}.