hPSC-derived microglia shape neuronal morphology and enhance network activity in vitro 171 5 Microglia differentiation Human microglia-like cells were generated using an adapted version of a previously published protocol (Haenseler et al., 2017). First, hPSCs were detached in small fragments using 0.5 mM EDTA. EDTA was inactivated by dilution in PBS. hPSC fragments were resuspended in TeSR1 medium containing 1x P/S supplemented with 50 ng/ml Bone Morphogenetic Protein-4 (BMP4, PeproTech, 120-05ET), 50 ng/ml Vascular Endothelial Growth Factor (VEGF, PeproTech, 100-20), 20 ng/ml Stem cell factor (SCF, R&D systems, 255SC), and 10 µM ROCK inhibitor Y-27632 (Selleckchem, S1049) and transferred to low attachment 6 well plates for the formation of embryoid bodies (EBs). The following three days, the medium was replaced daily using the same formulation but without supplementation of ROCK inhibitor. After, differentiation was started by plating 15-20 EBs onto 6 well plates in X-vivo 15 medium (Lonza, 02-060F) containing 1x P/S and supplemented with 100 ng/ml M-CSF + 25 ng/ml hIL3 + 1x GlutaMAXTM + 55 µM BetaMercaptoethanol, this is considered day 0 of differentiation. From this point on, the medium was replaced entirely weekly. Microglia-like cells were collected between day 55-70 by collecting the medium and passing it through a 40 µm cell strainer. Microglia mono-culture To address conversion into microglia-like cells they were plated on poly-l-ornithine and fibronectin-coated plates with Advanced DMEM/F-12 + N2 supplement, Glutamax, penicillin, streptomycin, and β-mercaptoethanol supplemented with IL-34 (100 ng/ml), GMCSF (10 ng/ml), M-CSF (25 ng/ml) and TGFB-1 (50 ng/ml) and Rock-Inhibitor Y-27632. Media refreshments were performed every 6 days using the same media but excluding RockInhibitor. For immunocytochemistry, cells were plated on glass coverslips at a density of 25k cells/cm2 and PFA fixed after 14 days, immunostainings were performed according to the protocol described below. For RNA isolation microglia were plated at a density of 50k cells/cm2 and collected in Trizol Reagent (Thermo Fisher Scientific) after 14 days. Total RNA was extracted according to manufacturer's protocol. cDNA synthesis was performed using SuperScript™ IV First-Strand Synthesis System (Invitrogen, 18091050) with 1 μg of RNA and random hexamer primers, according to the manufacturer’s protocol. 1x Phire Reaction Buffer (Thermo Scientific, F524L) with dNTPs (0.2 mM) and Phire Hot Start II DNA polymerase (Thermo Scientific, F122L) and primers (0.5 µM, TMEM119 FW: ACTCT CTCTT CCAGC CCAGG, RV: CCAGG ACCAG TTCCT TGGCG TA, IBA1, FW: CCCTC CAAAC TGGAA GGCTT C, RV: CTTTA GCTCT AGGTG AGTCT TGG, P2RY12 FW: TCCAG GGTCA GATTA CAAGA GCAC, RV: ACTGT TGATT CTGGA GGGTT TGA) were combined and PCR reaction was started with 1
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