The culture medium for derivation of ES-like stem cells consists of Glasgow-modified eagles medium (GMEM, Sigma), 15% KnockOut Serum Replacement® (KSR, Life Technologies), 1 × non-essential amino acids (NEAA, Nakarai), 1 × Sodium Pyruvate (Nakarai), 10−4 M 2-mercaptoethanol (Nakarai), 1,000 U/ml of LIF and 10 μM ACTH (Kurabo on consignment). We confirmed the medium is optimal for the culture of conventional ES cells. The culture medium for derivation of TS-like stem cells consists of GMEM, 20% FCS, 1 × NEAA, 1 × Sodium Pyruvate, 10−4 M 2-mercaptoethanol, 25 ng/ml of recombinant mouse Fgf4 (Wako) and 1 μg/ml of heparin (Wako).We confirmed the medium is optimal for the culture of conventional TS cells. To derive stem cells, cell aggregates were isolated under a microscope and transferred into a well of 96-well plate with 100 μl of the culture medium and 1,000 feeder cells. Feeder cells were prepared by treatment of mouse embryonic fibroblasts prepared from day 14 C57BL6 embryos with Mitomycin C (Wako) for 3 hours.
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We would like to thank the assistance of the members of the Scientific Validity Examination Team, Dr. Hiroshi Kiyonari and Mr. Kenichi Inoue for chimera production and animal breeding, and Laboratory of Animal Resources and Genetic Engineering for animal housing. We also thank Mr. Douglas Sipp for critical discussion of this report. This examination was supported by the grant for Scientific Validity Examination by RIKEN President.