4から9日齢のマウスを二酸化炭素を用いて安楽死させ、次いで70％エタノールで滅菌した。脾臓細胞の単離のために、摘出した脾臓をはさみで細かく刻み、組織断片をピペットでリン酸緩衝セリン（PBS）中で解離させた。細胞懸濁液を細胞濾過器に通し、続いて1,000rpmで5分間遠心分離することによって細胞を回収した。回収した細胞を5mlのダルベッコ改変イーグル培地（DMEM; Life Technologies）に再懸濁し、同量の Lympholyte® (Cedarlane)に加え、次いで1,000rpmで20分間遠心分離した。リンパ球層を単離し、PBSで洗浄して単細胞懸濁液を得た。肝臓細胞の単離のために、切除した肝臓をハサミで細かく刻み、I型コラゲナーゼ（Worthington Biochemical）溶液(0.5 mg/ml in Hanks Balanced Salt Solution (HBSS, no calcium, no magnesium; Life Technologies))の中で組織断片を保温維持によって単離した。次に、細胞懸濁液を細胞濾過器に通し、続いて1000rpmで5分間の遠心分離によって細胞を回収した。心臓細胞の単離のために、切除した心臓をハサミで細かく刻み、II型コラゲナーゼ（Worthington Biochemical）溶液（HBSS中0.5mg / ml）中での保温維持により組織断片を解離させた。細胞懸濁液を細胞濾過器に通し、続いて1,000rpmで5分間遠心分離することによって細胞を回収した。
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Low-pH treatment and culture of cell aggregates
Diluted HCl solution was prepared with 10 μl of 35% HCl (Nakarai) in 590 μl HBSS. Diluted ATP solution was prepared with ATP (Sigma) in distilled water at 200 mM. Titration of pH with various amount of diluted HCl or ATP was performed with 500 μl of HBSS containing 7 × 10<to the power of 5> liver cells. As a routine method, 10 μl of either diluted HCl or ATP solution was added into 500 μl of cell suspension containing 5 × 10<to the power of 5> cells in HBSS followed by incubation for 25 min at 37 °C, and then centrifuged at 1,000 rpm at room temperature for 5 min. After the supernatant was removed, precipitated cells were re-suspended and plated onto either adhesive or non-adhesive plates at cell density of 1–5 × 10<to the power of 5> cells per well in 1 ml of the culture medium. The culture medium consists of DMEM/HamF12 (Life Technologies) supplemented with 1,000 U/ml of mouse LIF (home-made) and 2% of B27® Supplement (Life Technologies). Optionally, recombinant human Fgf2 (Wako) was added at final concentration of 10 ng/ml.
To quantify the levels of mRNA transcripts, total RNA was prepared by TRIzol® (Life Technologies). cDNA were synthesized from 1 μg of total RNA using SuperScript® III (Life Technologies), and quantified by real-time PCR using a CFX384 system (BioRad). All samples were tested in triplicate, and the mean relative amounts of each transcript were calculated by normalization to an endogenous control Gapdh.
Each cell aggregate was washed with PBS and transferred in 2 μl of PBS into 8 μl RealTime ready Cell Lysis Buffer (Roche) supplied with NP-40, RNAsin and RNase inhibitor. Then 3 μl of cell lysis solution was mixed with 1.5 μl of DNaseI solution (0.2 U/μl) to degradate genomic DNA followed by addition of 1.5 μl of 8 mM EDTA solution to stop the reaction. For reverse transcription of RNA, 3 μl of pre-mixture of SuperScript® VILO reverse transcriptase (Life Technologies) was added into 6 μl of DNaseI-treated cell lysate and incubated at 42 °C for 1 hour. The reverse-transcribed product was pre-amplified with Plutinum multiplex PCR master mix using pooled primer mixture using the reaction cycle (95 °C for 30 sec; 60 °C for 90 sec; 72 °C for 60 sec) for 14 cycles. The mixture was treated with Exonuclease I to remove the primers for pre-amplification, and quantitative PCR was performed with the primer pairs specific for each gene using Quantitest SYBR Green PCR mix (Qiagen) in BioRad CFX384 Real-Time System (Bio-Rad). All samples were tested in triplicate, and the mean relative amounts of each transcript were calculated by normalization to an endogenous control Gapdh or Gnb2l1.
Cells were fixed by 4% paraformaldehyde in PBS for 30 min at 4 °C and then permeabilized by 0.1% Triton X-100 in PBS for 15 minutes at room temperature (RT). After brief washing with PBS followed by blocking with PBS containing 2% FCS, the cells were incubated with the following primary antibodies: anti-Oct3/4 rabbit antiserum15 and anti-Nanog rat monoclonal antibody (R&D) for overnight at 4 °C.After washing with PBS, the cells were incubated with Alexa Fluor 488- or 633-conjugated donkey antibodies (Invitrogen) <which>were used in a proper combination of species specificity as indicated in Figure legends.Fluorescent images were captured with an IX51 microscope with DP70 digital camera (Olympus) or a Leica SP8 confocal microscope (Leica).
For flow cytometric analyses, cell aggregates were harvested, washed by PBS, and incubated with TrypLETM Select (Life Technologies) for 5 min. After dilution with culture medium, aggregates were dissociated into single cells by gentle pipetting. Cells adhered to the culture substrate were also harvested following a standard method. These cells were mixed and collected as pellets by a centrifugation. For quantification of GFP-positive population, dissociated cells were re-suspended in 500 μl HBSS containing 1 μl of DRAQ7, a cell-nonpermeable DNA dye (for the detection of dead cells; Cell Signaling). When combined with a staining for CD45 antigen, cell pellets were suspended with 50 μl HBSS containing 10 μl of APC-conjugated rat anti-CD45 antibody (BD Pharmingen), and incubated for 30 min on ice. For co-staining with CD45/E-cadherin antibodies, cell pellets were suspended in culture medium, and then incubated for 30 min in CO2 incubator. The cells were harvested and suspended with 50 μl HBSS containing 5 μl biotin-labeled rat anti-E-cadherin antibody (ECCD2). After incubation for 30 min on ice, the stained cells were once washed by HBSS, re-suspended with 50 μl HBSS containing 1 μl PE-conjugated streptavidin (Life Technologies) and 10 μl APC-conjugated anti-CD45 antibody, and further incubated for 30 min on ice. These stained cells were once washed by HBSS and suspended with 500 μl HBSS.
After the cell suspension was passed through a filter mesh, the cells were analyzed using a FACSAria IIIu cell sorter (Becton Dickinson).
Cell aggregates obtained by the ATP treatment of liver cells were cut into smaller pieces using a laser (XYClone, Nikko Hansen & Co., Ltd) or shaped glass capillaries, and single piece was then microinjected into each 8-cell or blastocyst stage embryo from ICR mice (Charles River Laboratories Japan, Inc.). Injected embryos were transferred to the uterus of 2.5 dpc<Transration machine note:dpc=days post copulation> pseudopregnant ICR females (Charles River Laboratories Japan, Inc.) on <Transration machine note:Maybe the omission of the word "the day">or the next day of injection.
肝細胞のATP処理により得られた細胞凝集体を、レーザー（XYClone、Nikko Hansen＆Co.、Ltd）または成形ガラス毛細管を用いてより小さい断片に切断し、次いで単片をそれぞれICRマウス（Charles River Laboratories Japan、Inc.)の8細胞期胚または胚盤胞期胚に注射注入した。 挿入胚は移植当日もしくは翌日、交尾後<訳注:不妊オスを使う>2.5日のメスの偽妊娠ICRマウス(Charles River Laboratories Japan、Inc.）の子宮に移入された。
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.
