Trans isrib

Recovery perfusate was composed from a base of 500 mL Williams’ Medium E (with sodium bicarbonate, without L-glutamine, with phenol red) (Sigma-Aldrich, St. Louis, MO, USA) into which 5 g polyethylene glycol 35,000 (PEG) (Sigma-Aldrich), 12 mg dexamethasone (water-soluble) (Sigma-Aldrich), 5 g bovine serum albumin (Sigma-Aldrich), 400 mg sodium bicarbonate (Sigma-Aldrich),, 5 mL heparin (MGH Pharmacy), and 2 mL penicillin-streptomycin (Thermo Fisher Scientific, Waltham MA, USA) were added. On the day of experiment, 5 mL L-glutamine (Sigma-Aldrich), 50 uL insulin (MGH Pharmacy), 100 uL hydrocortisone (MGH Pharmacy) and 768 mg L-glutathione (Sigma-Aldrich) were added directly to perfusate before beginning perfusion. For the CEPT perfusate, 50 nM chroman 1 (MedChemExpress, Monmouth Junction NJ, USA), 5 uM Emricasan (MedChemExpress), 0.7 uM trans-ISRIB (Tocris Bioscience), 40 ng/mL putrescine (Sigma-Aldrich), 4.5 ng/mL spermidine (Sigma-Aldrich), and 8 ng/mL spermine (Sigma-Aldrich), all suspended in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) were added directly to perfusate before beginning perfusion; the concentrations were identical to those of the original CEPT cocktail. For vehicle perfusate, 800 uL DMSO was added directly to perfusate before beginning perfusion.

All hESCs and hiPSCs (see Table S1) were maintained under feeder-free conditions in Essential 8 (E8) cell culture medium (Thermo Fisher Scientific) and vitronectin (VTN-N; Thermo Fisher Scientific) coated microplates or T175 flasks using the CompacT SelecT (Sartorius). Cells were passaged using 0.5 mM EDTA in phosphate buffered saline (PBS) without calcium or magnesium (Thermo Fisher Scientific) every three days. After passage cells were counted using the automated Vi-cell XR counter (Beckman) on the CTST platform and cells were plated at a density of 1.5 × 10⁵ cells per cm² in E8 cell culture medium supplemented with the CEPT cocktail for the first 24 h (Chen et al., 2019). Manual cell culture was performed in VTN-coated 6-well plates under the conditions mentioned above. All karyotyping analysis were performed by Cell Line Genetics (Madison, WI). Cells were maintained in a humidified 5% CO₂ atmosphere at 37°C. To improve cell survival and provide cytoprotection during cell passaging of pluripotent and differentiated cells, we used the recently developed CEPT cocktail consisting of 50 nM Chroman 1 (#HY-15392; MedChem Express), 5 μM Emricasan (#S7775; Selleckchem), Polyamine supplement (#P8483, 1:1000 dilution; Sigma-Aldrich), and 0.7 μM Trans-ISRIB (#5284; Tocris).

TAK-242 (CAS 243984-11-4 Cat 6587) was purchased from R&D Systems (Minneapolis MN). Lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS) (Catalog # tlrl-rslps) and MCC950 (CAS 210826-40-7, Catalog # inh-mcc) were purchased from InvivoGen (San Diego CA). Trans-ISRIB (CAS 1597403-47-8, Cat 5284) was from Tocris (Ellisville MO). Other chemicals, including glyphosate (CAS 1071-83-6), minocycline (CAS 13614-98-7, Cat# M2280000) and IL1-Ra (Cat# SRP 3084), quercetin (CAS 849061-97-8, PHR1488) and salts were obtained from Millipore Sigma Chemical Company. Roundup, a herbicide containing glyphosate, was purchased from a local store. Drugs were prepared as stock solutions in either ACSF or DMSO and diluted to final concentration at the time of experiment. The concentrations of TAK-242, LPS-RS and minocycline are based on our previous studies using those inhibitors against lipopolysaccharide (LPS) and acrylamide (Izumi, Cashikar, et al., 2021 (link); Izumi et al., 2022 (link)). The concentrations of MCC950 were also based on our previous paper (Izumi et al., 2022 (link)). The dose of TAK-242 in the behavioral study followed a proceeding report by Ono et al. (Ono et al., 2020 (link)).