Poly l ornithine laminin

IMR90 NPCs were seeded at a density of 1 × 10⁶ cells per ml in poly-L-ornithine/laminin (Sigma–Aldrich) coated six-well plates and allowed to attach overnight. The following day, cells were treated with 1 µM ketamine or vehicle for 24 h. For all samples, total RNA was isolated using the RNeasy Mini kit from Qiagen, including an on-column DNAase treatment (15 min at room temperature, Qiagen) according to the manufacturer’s instructions. The RNA concentration was determined using the NanoQuant Plate™ (Tecan, Salzburg, Austria). All library preparations were sequenced on an Illumina Hiseq platform with a paired-end read length of 125 bp/150 bp. The library construction and sequencing were performed by Novogene (HK) Company Limited (Wan Chai, Hong Kong).

Fetal brain cells (FBCs) are ReNcells derived form the ventral mesencephalon of human fetal brain (Millipore SCC008). Cells were grown on poly-L-ornithine/laminin (Sigma, St. Louis, MO, USA; P3655-50MG and l2020) coated six-well plates. Cells were maintained in 70% DMEM, 2% B27, 1% Pen/Strep (Life Technologies, Foster City, CA, USA) 30% Ham's F12 (Mediatech, Herndon, VA, USA) and 20 ng ml⁻¹ bFGF (R&D Systems, Minneapolis, MN, USA, 233-FB-025), 20 ng ml⁻¹ EGF (Sigma E9644) and 5 μg ml⁻¹ heparin (Sigma). Differentiation is triggered by removing growth factors from media and letting cells grow for 30 days, with media changes every 3 days; proliferating cells are those maintained in bFGF and EGF.

Cells were plated on poly-l-ornithine/laminin (both Sigma) coated tissue culture plates and cultivated in a stock media (DMEMF12 Glutamax (Gibco) supplemented with 1% penicillin/streptomycin (Gibco), 1:100 N2 supplement (Gibco), 0.8 g D-glucose) with alternating concentrations of growth factors. On day one, cells were plated in stock media supplemented with 10 ng/ml EGF (Gibco) and 10 ng/ml FGF (Gibco). On the second day media was changed by stock media containing 40 ng/ml EGF and 40 ng/ml FGF. For further cultivation of MJD1 lt-NES cells, media was changed every day with alternating concentrations of growth factors (low: 10 ng/ml and high: 40 ng/ml). Accutase (Sigma) was diluted 1:3 with PBS and used for cell detachment (10 min room temperature). Treatments were performed in media with low growth factor concentration.

Human iPSC-derived IMR90 NPCs and Ro-iPSC (Royan iPSC) NPCs were generated by Dr. Tomo Šarić (The University of Cologne, Institute for Neurophysiology, Cologne, Germany) [25 (link)]. Cells were maintained as monolayer cultures on poly-L-ornithine/laminin (Sigma–Aldrich) coated six-well plates in serum-free neural stem cell medium containing dulbecco’s modified eagle medium/ nutrient mixture F-12 (DMEM/F12) glutamax (Gibco), 1× N2 supplement (Gibco), 1.6 mg/L D-glucose (Sigma–Aldrich), 20 µg/mL insulin (Sigma–Aldrich), 1 µL/mL B27 (Gibco), 20 ng/mL fibroblast growth factor-basic (bFGF) (Peptrotech), 20 ng/mL human epidermal growth factor (hEGF) (Sigma–Aldrich) [26 (link)]. The medium was changed every other day. The cells were maintained in a humidified atmosphere at 37 ºC, and 5% CO₂ and subcultured when confluency reached 90%.

NPCs were seeded at a density of 8 × 10⁴ cells per ml in poly-L-ornithine/laminin (Sigma–Aldrich) coated 96-well image-lock plates (Sartorius, Goettingen, Germany) 24 h prior to the experiment. Images were taken every hour for a time frame of three days with the IncuCyte^® Zoom from Sartorius (Goettingen, Germany) using a 10× objective. Confluency of cells was determined as Cell-Body Cluster Area [Phase] (mm²/mm²) using the IncuCyte^® NeuroTrack Software (version 2016B). For protein kinase A (PKA) inhibition studies, cells were pretreated with 1 µM triethylammonium salt (cAMPS-Rp; Tocris Bioscience) for 15 min at 37 °C, and 5% CO₂ before ketamine or DMSO control was added.

For staining of neurofibrils with Bielschowsky (Bio-Optica, Milan, Italy), NPC and cells differentiated over approaches A and B for 28 days were seeded onto poly-L-ornithine/laminin (Sigma-Aldrich, St. Louis, Missouri, USA) precoated Nunc™ Lab-Tek™ II Chamber Slides™ (Thermo Fisher, Waltham, Massachusetts, USA), fixed with 4% w/v PFA in PBS by incubation for 20 min at 4°C and washed twice with PBS. According to the manufacturer's protocol, the slide was washed twice with ultrapure water and was incubated with 10 drops of Reagent A for 15 min at 40°C. After washing two times with ultrapure water, 10 drops of Reagent B were added following incubation for 20 min at 50°C. The supernatant was discarded, and the slide was treated with reduction solution (20 drops Reagent C, 8 drops each of Reagents D, E, and F in 50 mL ultrapure water) for 2 min. Cells were washed twice with ultrapure water and subsequently incubated with 10 drops of Reagent G for 3 min. Before dehydrating the slide with ascending concentration of ethanol and treating twice with Xylene (Sigma-Aldrich, St. Louis, Missouri, USA), it was washed two times with ultrapure water. Finally, the slide was embedded in Entellan Neu (Merck, Darmstadt, Germany) following incubation for 30 min at room temperature to dry. The stained neuronal structures were imaged with Nikon Eclipse TS100 microscope (Nikon, Minato, Japan).

For OL lineage differentiation, a two-step differentiation protocol was utilized. hiPSC-derived NPCs were seeded at a density of 1.5 × 10⁵ cells per well in a 12- well plate coated with poly-L-ornithine/laminin (Sigma‒Aldrich, MO USA). NPCs were transfected with OLIG2^WT or OLIG2^S147A smRNA using ScreenFect transfection reagent (293-75901, Wako, Japan) for 6 days, and the culture medium was changed to glial induction medium (GIM) for 4 days. Then, the medium was replaced with differentiation medium (DM). A detailed description of the GIM and DM is provided in Table S2. The cells were also cryopreserved in medium containing 40% neurobasal medium with B27 supplement, 50% fetal bovine serum (DIB-12B-10X50ML, Data Inventory Biotech, China) and 10% DMSO.