Details of PSC lines used in this study can be found in Supplemental Experimental Procedures . TREM2 mutant lines were generated from fibroblasts derived from skin punch biopsies obtained following informed consent, after approval from the ethics committee of the Istanbul Faculty of Medicine, Istanbul University, or the joint research ethics committee of the National Hospital for Neurology and Neurosurgery and the Institute of Neurology, UCL. Microglia were differentiated from PSCs via embryoid bodies and PMPs (Karlsson et al., 2008 (link), van Wilgenburg et al., 2013 (link)). In brief, at least 2 days after last passaging, PSCs (cultured feeder-free in E8) were passaged to single cells with TrypLE Express (Gibco), and plated at 10,000 per well in 96-well ultra-low attachment plates (Corning) in 100 μL embryoid body medium (10 μM ROCK inhibitor, 50 ng/mL BMP-4, 20 ng/mL SCF, and 50 ng/mL VEGF-121 in E8), before centrifugation at 300 × g for 3 min. Embryoid bodies were cultured for 4 days, with half medium change after 2 days. Ten to 16 embryoid bodies were plated per well of tissue culture-treated 6-well plates and cultured in 3 mL hematopoetic medium (2 mM GlutaMax, 100 U/mL penicillin, 100 μg/mL streptomycin, 55 μM β-mercaptoethanol, 100 ng/mL M-CSF, and 25 ng/mL IL-3 in X-VIVO 15 [Lonza, LZBE02-060F]). From this point on, 2 mL medium was exchanged every 4–7 days.
PMPs were harvested from suspension during medium exchange and plated in RPMI 1640 at 180,000 cells/cm2 in 6-, 12-, or 96-well plates. In the absence of serum, PMPs adhered to uncoated tissue culture-treated plastic within 1 hr, at which point medium was switched to complete microglia medium (10% FBS [Gibco, 16000044 or Sigma, [F2442], 2 mM GlutaMax, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 ng/mL IL-34, and 10 ng/mL GM-CSF in RPMI1640).
Final differentiation of PMPs into microglia occurred over 6–10 days, with full medium change every 2–3 days. All cytokines and growth factors obtained from PeproTech, except for IL-3 (Cell Guidance Systems).
PMPs were harvested from suspension during medium exchange and plated in RPMI 1640 at 180,000 cells/cm2 in 6-, 12-, or 96-well plates. In the absence of serum, PMPs adhered to uncoated tissue culture-treated plastic within 1 hr, at which point medium was switched to complete microglia medium (10% FBS [Gibco, 16000044 or Sigma, [F2442], 2 mM GlutaMax, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 ng/mL IL-34, and 10 ng/mL GM-CSF in RPMI1640).
Final differentiation of PMPs into microglia occurred over 6–10 days, with full medium change every 2–3 days. All cytokines and growth factors obtained from PeproTech, except for IL-3 (Cell Guidance Systems).
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