・Mouse embryonic fibroblast cells (Prepared from ICR mouse; see Procedure).
・E14 mouse ES cells (American Type Culture Collection; Manassas, VA).
・Dulbecco’s Phosphate Buffered Saline (PBS; Sigma-Aldrich, cat.no. D5652-10L)
・DMEM containing high glucose (4500.00mg/L), L-gultamine (584.00mg/L), Sodium pyruvate (110.00mg/L). (Wako; Osaka, Japan, cat.no. 043-30085)
・DMEM containing low glucose (1000.00mg/L), L-gultamine (584.00mg/L), Sodium pyruvate(110.00mg/L). (Wako, cat.no. 041-29775)
・0.25% Tripsin-EDTA solution (Wako, cat.no. 201-16945)
・Fatal Bovine Serum (FBS; Nichirei Bioscience; Tokyo, Japan, cat.no. 171012 lot.no. 7G0031)
・Knock-out Serum Replacement (KSR; Life Technologies; Carlsbad, CA, USA, cat.no. 10828-028)
・Nonessential amino acid solution (NEAA; Sigma-Aldrich, cat.no. M7145-100ML)
・β-Mercaptoethanol (Sigma-Aldrich, cat.no. M6250-100ML)
・Penicillin/streptomycin (Wako, cat.no. 168-23191)
・Milli-Q water (Merck; Darmstadt, Germany, cat.no. TANKPE060)
・N2 supplement (Wako, cat.no. 17502048)
・B27 supplement (Invitrogen, cat.no. 17504044)
・Cell Banker 1 (Takara Bio; Shiga, Japan, cat.no. CB011)
・Stem Cell Banker (Takara Bio, cat.no. CB041)
・Mitomycin C (Kyowa Kirin; Toyko, Japan, cat.no. 4231400D2038; see Reagent set up)
・Human recombinant FGF10 (R&D Systems; Minneapolis, MN, cat.no. 345-FG-025)
・Human recombinant WNT3A (R&D Systems, cat.no. 5036-WN-010)
・Human recombinant NOGGIN (R&D Systems, cat.no. 6057-NG-025)
・Human recombinant R-SPONDIN1 (R&D Systems, cat.no. 4645-RS-025/CF)
・Human recombinant SHH (R&D Systems, cat.no. 1314-SH-025/CF)
・Human recombinant DKK (R&D Systems, cat.no. 5439-DK-010/CF)
・Human recombinant EGF (Peprotech; Rocky Hill, NJ, cat.no. AF-100-15)
・Matrigel Growth Factor Reduced (BD Biosciences; San Jose, CA, cat.no. 354230; see Reagent set up)
・Dispase (BD Biosciences, cat.no. 354235)
・Gelatin (Sigma-Aldrich) (see Reagent set up)
・ES medium (see Reagent set up )
・MEF medium (see Reagent set up)
・EB medium (see Reagent set up )
・KSR medium (See Reagent set up)
・DS medium (See Reagent set up)
・FGF-based Medium (See Reagent set up)
Reagent set up
・Mitomycin C (MMC) solution stock Dissolve 2 mg MMC in 1 mL PBS, and mix gently to dissolve MMC completely. Store at -80°C for 6 months with protection from light.
・Matrigel Thaw the whole Matrigel solution in a vial at 4°C overnight. After thawing, dispense 100 μL Matrigel to each autoclaved 1.5 mL tube on ice. Store at - 30°C as a frozen stock for a month. When you use Matrigel, thaw each Matrigel solution in 1.5 mL tube at 4°C overnight.
・Gelatin Prepare 0.1w/v% Geraitn solution with Milli-Q water, and sterilize by autoclave. Store at room temperature for a month.
・ MEF Medium DMEM low glucose (Wako) containing 15% FBS (Nichirei Bioscience), and 0.1mg/mL penicillin/streptomycin (Wako). To prepare 500 mL of the medium, mix 50 mL of FBS, and 5 mL penicillin/streptomycin from x 100 concentrated stock. Store at 4 °C for a month.
・ES medium DMEM high glucose (Wako) containing 15% FBS (Nichirei Bioscience), 0.1 mM NEAA, 0.1 mM β-mercaptoethanol (Sigma Aldrich), and 0.1 mg/mL penicillin/streptomycin (Wako), supplemented with 1000 U/mL human recombinant LIF (Wako). To prepare 500 mL of the medium, mix 75 mL of FBS, 5 mL penicillin/streptomycin from x 100 concentrated stock, 5 mL NEAA from x 100 concentrated stock, 3.6 µL β-mercaptoethanol, and 500 µL of human recombinant LIF. Store at 4 °C for a month.
・EB medium DMEM high glucose (Wako) containing 15% FBS (Nichirei Bioscience), 0.1 mM NEAA, 0.1 mM β-mercaptoethanol (Sigma Aldrich), and 0.1mg/mL penicillin/streptomycin (Wako). Do not add any human recombinant LIF. To prepare 500 mL of the medium, mix 75 mL of FBS, 5 mL penicillin/streptomycin from x100 concentrated stock, 5 mL NEAA from x 100 concentrated stock, 3.6 µL β-mercaptoethanol. Store at 4 °C for a month.
・KSR medium DMEM high glucose (Wako) containing 15% KSR (Life Technologies), 0.1 mM NEAA, and 0.1mg/mL penicillin/streptomycin (Wako). To prepare 500 mL of the medium, mix 75 mL of KSR, 5 mL penicillin/streptomycin from x 100 concentrated stock, and 5 mL NEAA from x 100 concentrated stock. Store at 4 °C for a month.
・ DS medium KSR medium containing 500 ng/mL recombinant human SHH, 500 ng/mL DKK1 (All from R&D Systems). You can store DS medium containing growth factors at 4 °C for a week, however, we recommend to add all growth factors just before use.
・FGF-based medium DMEM/F12 (Wako) containing 1% KSR (Life Technologies), 0.1 mM NEAA , 100 ng/mL recombinant human FGF10, 100 ng/mL WNT3A, 100 ng/mL NOGGIN, 250 ng/mL R-SPONDIN1 (All from R&D Systems), and 50 ng/mL EGF (Peprotech) supplemented with N2 (Wako)/ B27 (Invitrogen). To prepare 500 mL of the medium, mix 5 mL of KSR, 5 mL NEAA from x 100 concentrated stock, 5 mL of N2 supplement, 10 mL of B27 supplement, and each growth factors. You can store FGF-based medium containing growth factors at 4 °C for a week, however, we recommend to add all growth factors just before use.