Streptomycin p s

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.

Panc1 (American Type Culture Collection), MiaPaCa2, Hs766t (Li et al, 2013 (link)), Kif24-3 Panc1 (this study), and Lenti-X 293T (gift from M Hagiwara) cells were grown in DMEM (Nacalai Tesque) supplemented with 10% FBS (Biosera) and 100 U/ml penicillin and 100 μg/ml streptomycin (P/S) (Nacalai Tesque). CFPAC1 (American Type Culture Collection) cells were grown in IMDM (Nacalai Tesque) supplemented with 10% FBS and P/S.

ESC cell lines (R1, SK7 Pdx1/GFP) [25 (link)] or a mouse Nanog iPS cell line (20D-17) [45 (link)], were maintained on a feeder layer of mouse embryonic fibroblasts (MEFs) in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) supplemented with human recombinant LIF (1:1000, Wako), 10 % fetal bovine serum (FBS, Hyclone), 2 mM l-glutamine (l-Gln, Nacalai Tesque), 100 mM non-essential amino acids (NEAA, Invitrogen), 50 U/mL penicillin, 50 mg/ml streptomycin (PS, Nacalai Tesque), and 100 μM 2-mercaptoethanol (2-ME, Sigma-Aldrich) in 5 % CO₂.

Female ICR mice were treated with pregnant mare serum gonadotropin (PMSG; 7.5 IU) and human chorionic gonadotropin (hCG; 7.5 IU) by intraperitoneal injection. Embryos were rinsed with M2 medium (Sigma) and cultured in KSOM medium until development into blastocysts. WT, Dnmt3a KO, and Dnmt3b KO 2i/L ES cells were trypsinized, and 7–8 cells per embryo were injected into the blastocoels of E3.5 blastocysts. Injected blastocysts were transferred into the uteri of pseudopregnant ICR mice. A Fully Automated Fluorescence Stereo Microscope Leica M205 FA (Leica MICROSYSTEMS) was used to observe GFP-labeled embryos. MEFs were isolated from E14.5 embryos and cultured in DMEM (Nacalai Tesque) with 2 mM L-glutamine (Nacalai Tesque), 100× nonessential amino acids (NEAA) (Nacalai Tesque), 100 U/ml penicillin, 100 μg/ml streptomycin (P/S) (Nacalai Tesque), and 10% fetal bovine serum (FBS) (GIBCO). To remove host cells, GFP-labeled MEFs were collected by fluorescence activated cell sorting (Aria II, BD) after selection with 350 μg/ml G418 disulfate aqueous solution (Nacalai Tesque) for 7 days.