Ips mef ng 20d 17

The mouse iPS cell line iPS-MEF-Ng-20D-17 (APS0001; RIKEN Bioresource Center, Japan) used in this study expresses GFP under the control of the Nanog promoter 8) . Undifferentiated cells express Nanog, but its expression disappears with differentiation 8, 9) . Gelatin from porcine skin (Sigma-Aldrich, USA) was added as a 0.1% aqueous solution to a cell culture dish (Becton Dickinson Labware, USA) and incubated at 37°C for 30 minutes, and a gelatin coat was applied to the culture dish. EmbryoMax ® Primary Mouse Embryo Fibroblasts (MEFs; Millipore, USA) treated with mitomycin C (Kyowa Hakko Kogyo Co., Ltd., Japan) were seeded in gelatin-coated culture dishes. The iPS cell line was seeded on mitomycin C-treated MEFs and cultured in ES medium [15% fetal bovine serum (FBS; MP Biomedicals, USA), 1% non-essential amino acids (Millipore, USA), 1% nucleosides (Millipore, USA), and 1% Dulbecco's modified Eagle's medium (Wako Pure Chemical Industries, Ltd., Japan) containing 1% L-glutamine (MP Biomedicals, USA), 500U/mL ESGRO ® Mouse Lukemia Inhibitory Factor (LIF; Millipore, USA), 2(β)-mercaptoethanol (Sigma-Aldrich, USA), and 1% penicillin-streptomycin (Millipore, USA)]. The medium was exchanged every day. iPS cells were passaged once every 2-3 days. For the dispersion of iPS cells, 2.5 g/L trypsin EDTA solutions were used.