Human il 34

Microglia were differentiated according to published methods [42 (link), 43 (link)]. Briefly, embryoid bodies were formed by seeding 10,000 PSCs/well in round-bottomed ultra-low attachment 96-well plates, using Essential 8 medium supplemented with 50 ng/ml human BMP4 (Peprotech), 50 ng /ml human VEGF (Cell Guidance Systems), 20 ng/ml SCF (Cell Guidance Systems) and 10 mM Y27632 ROCK inhibitor (Sigma-Aldrich, Merck). After five days, the embryoid bodies were collected and transferred at one per cm² in flasks containing X-Vivo 15 medium (Lonza) with 2 mM l-glutamine, 100 ng/ml human M-CSF (Cell Guidance Systems), 25 ng/ml IL-3 (Cell Guidance Systems) and 50 μM 2-mercaptoethanol. After three weeks, tissue-resident macrophage precursors were first harvested and at weekly intervals thereafter. These cells were cultured in Corning Primaria plates using DMEM/F12 medium (Gibco, Thermo Fisher Scientific) supplemented with 2 mM l-glutamine, 100 ng/ml GM-CSF (Cell Guidance Systems), 100 ng/ml human IL-34 (R&D Systems, Biotechne), and 50 units/ml penicillin and 50 μg/ml streptomycin, to promote the differentiating cells towards a mature microglial state. They were compared with primary human monocytes and macrophages obtained and cultured as described previously [27 (link)].