Figure 6: Chimera assay of cell aggregates.
Cell aggregates derived from liver cells prepared from 8-day-old Alb-cre:Rosa-GFP Tg (upper panels) were injected into morula-stage embryos. The manipulated embryos were transferred into the uterus and the embryos were recovered at E10.5. The dissected embryos were observed under fluorescent microscopy for the contribution of GFP-positive cells.
Induced cell aggregates do not give rise to stem cell lines
The original reports stated that two different types of stem cell lines could be established from cell aggregates obtained by the culture of low-pH treated cells: ES-like ‘STAP stem cells’ (Fig. 5 of Obokata et al.1) and trophoblast stem (TS)-like ‘FGF-induced (FI) stem cells’ (Fig. 2 of Obokata et al.2). To reevaluate these claims, we transferred cell aggregates derived from liver cells from various genetic backgrounds into the culture conditions for derivation of either ES-like or TS-like stem cells. In the case of the culture for ES-like stem cells in serum-free culture containing knockout serum replacement (KSR), adrenocorticotropic hormone (ACTH) and LIF19, most of the cell aggregates died without outgrowth, which may attributable to the absence of serum, while a small number aggregates gave rise to colonies containing small cells with large nuclei, resembling the morphology of embryonic stem cells. However, most of these cells ceased proliferation at day 7 and gradually regressed. Marked proliferation after day 7 was observed in only three of 492 cell aggregates and none of these gave rise to cell lines (Fig. 7a). In the case of the culture for TS-like stem cells containing FGF-4 and heparin20, many clumps showed outgrowth of fibroblastic cells, which may be due to the presence of FGF2 in the medium. Few of these (22 of 391 cell aggregates) gave rise to colonies of small stem cell-like cells, and one could be passaged three times (Fig. 7b). However, all ultimately regressed without giving rise to cell lines. These data showed that we are unable to derive stem cell lines from aggregates derived from low-pH treated liver cells.
元の報告は、低pH処理細胞の培養によって得られた細胞凝集体から2つの異なるタイプの幹細胞株を樹立することができたと述べている。即ちES細胞様STAP幹細胞（Obokata et al.1の図5）および栄養膜（TS）様の「FGF誘導（FI）幹細胞」（Obokataらの図2）である。これらの主張を再評価するために、ES様またはTS様幹細胞の誘導のための培養条件に、様々な遺伝的背景の肝細胞由来の細胞凝集体を移した。ノックアウト血清代替物（KSR）、副腎皮質刺激ホルモン（ACTH）およびLIF19を含む無血清培養物におけるES様幹細胞の培養の場合、細胞凝集物の大部分は、おそらく血清の不在に起因して増殖することなく死滅した。一方、少数の凝集物は、ES細胞の形態に似た大きな核を有する小さな細胞を含むコロニーを形成した。しかし、これらの細胞のほとんどは、7日目に増殖を停止し、徐々に退行した。 7日後の顕著な増殖は、492個の細胞凝集体のうちのわずか3つで観察され、いずれも細胞株を生じなかった（図7a）。 FGF-4およびヘパリン20を含有するTS様幹細胞の培養の場合、多くの塊が線維芽細胞の増殖を示したが、これは培地中にFGF2が存在するためである可能性がある。これらのうちの少数（391個の細胞集合体のうち22個）が小幹細胞様細胞のコロニーを生じ、3回継代することができた（図7b）。しかしながら、最終的に細胞株を生じさせることなくすべて退行した。これらのデータは、低pH処理肝細胞由来の凝集塊から幹細胞株を得ることができないことを示している。
Figure 7: Culture of cell aggregates in vitro.
(a) The outgrowth culture of cell aggregate derived from liver cells. Liver cells were prepared from 7-days old of C57BL6 CAG-GFP Tg, treated with ATP and cultured for six days. Single cell aggregates were isolated and cultured on MEF feeder cells with medium containing KSR, ACTH and LIF adapted to the culture of ES cells. The cells continue<→continued> to grow for 15 days but did not give secondary colony after passage. (b) Outgrowth culture of cell aggregates derived from liver cells. Liver cells were prepared from 4-day-old C57BL6/129 mice, treated with ATP, and cultured for six days. The cell aggregates were isolated and cultured on MEF feeder cells with medium containing FGF4 and heparin adapted to the culture of TS cells. The cells continued to grow for 11 days, but did not give rise to secondary colonies after passage.
In the present study, we investigated the properties of cell aggregates obtained by culture of liver cells transiently treated with low-pH stimulus, which was performed by the group directed by the author. We initially followed the protocol described in the original paper with the detail description in protocol exchange where HCl was applied to achieve low-pH condition. However, we merely obtained the cell aggregates expressing the pluripotency makers as described in this report even when it was combined with the culture in medium containing Fgf2, which was not described in the original protocol but subsequently suggested by the authors. However, when we used ATP instead of HCl, also based on a suggestion by the authors, a few cells in a subset of cell aggregates expressed the pluripotency marker Oct3/4 at levels comparable to those in ES cells that were reproducibly detected by QPCR (Fig. 3c) and immunostaining (Fig. 4b). However, the frequency was very low; 5 × 10<to the power of 5> liver cells yielded only ~30 cell aggregates, in which about 20% of the cell aggregates contained 1–2 Oct3/4 positive cells, indicating a frequency per seeded liver cell of 0.0012–0.0024%. Moreover, the pluripotency of such cells was not confirmed by chimera formation assay, and they did not give rise to any stem cell lines. We thus conclude that such cell aggregates do not fulfill the definition for STAP cells proposed in the original studies.
Moreover, since the frequency of Oct3/4-positive cells in the cell aggregates was quite low, it was impossible to determine whether they were selected from the original population or induced in culture, again highlighting the lack of evidence supporting the existence of the reported STAP phenomenon. An independent examination was made on chimeric potency of STAP-like cell aggregates that were generated by Haruko Obokata. Among 1154 embryos injected with the aggregates, 671 developed beyond E8.5; however, none of the aggregates made significant contribution to any tissues, the details of which was reported in a Biorixiv website (bioRxiv doi: ⁑ttp://dx.doi.org/10.1101/028472). These data are consistent to the recent report by De Los Angeles et al..
C57BL/6NJcl (CLEA Japan) and 129X1/SvJJmsSlc (Japan SLC) mice were purchased from suppliers. C57BL/6-Tg(CAG-EGFP)C14-Y01-FM131Osb transgenic mouse (CAG-GFP Tg) line was provided by Research Institute for Microbial Disease, Osaka University. C57BL/6J-Tg(GOFGFP)11Imeg transgenic mouse (GOF-Tg) line was obtained from RIKEN Bio-Resource Center (RBRC00771). B6.Cg-Tg(Alb-cre)21Mgn/J transgenic mouse (Alb-cre Tg) line was supplied by Jackson Laboratory. R26R-H2B-EGFP transgenic mouse (Rosa-GFP) line was generated by Laboratory for Animal Resources and Genetic Engineering (LARGE), RIKEN CDB. All animal experiments were carried out in accordance with our Guidelines for the Care and Use of Laboratory animals and were approved by the Institutional Committee for Laboratory Animal Experimentation (RIKEN Kobe Institute).
4–9-day-old mice were euthanized using carbon dioxide and then sterilized with 70% ethanol. For the isolation of spleen cells, excised spleen was minced with scissors and the tissue fragments were dissociated in phosphate buffered serine (PBS) by pipetting.The cell suspension was strained through a cell strainer followed by the collection of cells by centrifugation at 1,000 rpm for 5 min. The collected cells were re-suspended in 5 ml of Dulbecco’s Modified Eagle medium (DMEM; Life Technologies) and added to the same volume of Lympholyte® (Cedarlane), and then centrifuged at 1,000 g<→rpm > for 20 min. The lymphocyte layer was isolated and washed with PBS to obtain single cell suspension.For the isolation of liver cells, excised liver was minced with scissors and the tissue fragments were dissociated by incubation in Type I collagenase (Worthington Biochemical) solution (0.5 mg/ml in Hanks Balanced Salt Solution (HBSS, no calcium, no magnesium; Life Technologies)). Next, the cell suspension was strained through a cell strainer followed by the collection of cells by centrifugation at 1,000 rpm for 5 min. For the isolation of heart cells, the excised heart was minced with scissors and the tissue fragments were dissociated by incubation in Type II collagenase (Worthington Biochemical) solution (0.5 mg/ml in HBSS). The cell suspension was strained through a cell strainer followed by the collection of cells by centrifugation at 1,000 rpm for 5 min.
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。
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.
How to cite this article: Niwa, H. Investigation of the cellular reprogramming phenomenon referred to as stimulus-triggered acquisition of pluripotency (STAP). Sci. Rep. 6, 28003; doi: 10.1038/srep28003 (2016).
Smith, A. G. et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688–690 (1988).
Okamoto, K., Okazawa, H., Okuda, A., Sakai, M., Muramatsu, M. & Hamada, H. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60, 461–472 (1990).
Bradley, A., Evans, M., Kaufman, M. H. & Robertson, E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255–256 (1984).
Obokata, H. et al. Retraction: Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature 511, 112 (2014).
Obokata, H. et al. Retraction: Bidirectional developmental potential in reprogrammed cells with acquired pluripotency. Nature 511, 112 (2014).
Ohtsuka, S. & Niwa, H. The differential activation of intracellular signaling pathways confers the permissiveness of embryonic stem cell derivation from different mouse strains. Development 142, 431–437 (2015).
Okabe, M., Ikawa, M., Kominami, K., Nakanishi, T. & Nishimune, Y. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 407, 313–319 (1997).
Abe, T. et al. Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging. Genesis 49, 579–590 (2011).
Postic, C. et al. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase. J Biol Chem 274, 305–315 (1999).
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.