L glutamine l gln

We thank Dr. S. Yamanaka (Kyoto University, Kyoto, Japan) for providing the human iPS cell (hiPSC) line, 201B7. We thank Drs. M. Toyoda, N. Kiyokawa, H. Okita, Y. Miyagawa, H. Akutsu, and A. Umezawa (National Institute for Child Health and Development, Tokyo46 (link)) for providing the Toe hiPS cell line. Undifferentiated hiPS cells were maintained on a feeder layer of mouse embryonic fibroblasts (MEFs) in Knockout DMEM/F12 (Invitrogen) supplemented with 5 ng/ml FGF2 (Peprotech), 20% Knockout Serum Replacement (KSR, Invitrogen), 2 mM L-glutamine (L-Gln, Nacalai Tesque), 100 mM non-essential amino acids (NEAA, Invitrogen), 50 units/mL penicillin, 50 mg/ml streptomycin (PS, Nacalai Tesque), and 100 μM 2-mercaptoethanol (2-ME, Sigma-Aldrich) in 5% CO₂. To passage hiPSCs, colonies were detached from the feeder layer by treating with 0.25% trypsin (Invitrogen) and 0.1 mg/ml collagenase IV (Gibco) in phosphate buffered saline (PBS) containing 20% KSR and 1 mM CaCl₂ at 37 °C for 6 min, followed by adding culture medium and disaggregating ES cell clumps into smaller pieces (5–20 cells) by gently pipetting several times. HepG2 and Caco2 cells were maintained in DMEM supplemented with 10% FBS (Hyclone) and PS.