(英文) Abstract Stimulus-triggered acquisition of pluripotency (STAP) is a cellular reprogramming phenomenon that was recently reported in two papers (Obokata, Nature, 2014a,b). In this reprogramming process, upon strong external stimuli, neonatal somatic cells are converted into cells that express pluripotency-related genes, such as Oct3/4, and acquire the ability to differentiate into derivatives of all three germ layers in vitro and in vivo. These cells, termed STAP cells, can contribute to chimeric fetuses after blastocyst injection. Moreover, in the blastocyst injection assay, injected STAP cells are also found in extra-embryonic tissues, such as placenta.
(英文) STAP cells derived from neonatal somatic cells are thus fully reprogrammed to as state of pluripotency. In the conditions for the establishment of STAP cells, their proliferative capacity is quite limited, distinct from that of embryonic stem cells (ESCs). STAP cells can be further converted into two types of proliferative cell lines: STAP stem cells and FGF4-induced stem cells (FI stem cells). STAP stem cells, which are converted from STAP cells in ACTH-containing medium (see Procedure), lose the ability to contribute to extra-embryonic tissues. FI stem cells, which are generated from STAP cells in FGF4-containing medium, in contrast retain the capacity to contribute to both embryonic and extra-embryonic lineages in blastocyst injection assay, although their embryonic contribution is relatively low.
(英文) The STAP phenomenon induced by external stimuli, thus potentially sheds new light on our understanding of pluripotency and differentiation in mammalian cells. This unforeseen phenomenon can be triggered in neonatal hematopoietic cells, for instance, by transient exposure to low-pH solution. Despite its seeming simplicity, this procedure requires special care in cell handling and culture conditions, as well as in the choice of the starting cell population. The delivery of the optimal level of sublethal stress to cells is essential to the process of STAP cell induction. From our experience, STAP conversion is reproducibly seen with culture conditions in which most cells survive for one day after low-pH treatment, and in which up to 80% of the initial cell number subsequently die at around days 2–3. Control of the pH of the solution is not the only key factor; the delayed onset of sublethal stress is also critically important.
(英文) This biological context can also be affected by many other factors. For example, somatic cell preparation and cell handling before and after the exposure to stress must be done with care, as additional damage to the cells may alter the level of stress, causing excessive cell death or insufficient triggering. The types of cells used for STAP conversion are also critical, and the use of cells from other sources (e.g., the use of cultured fibroblasts after passaging) may also result a failure to achieve STAP conversion. We have reproducibly observed STAP cell conversion when proper procedures are followed in the correct sequence.
(英文) To facilitate the broad testing and use of this technique, we are now preparing a full protocol article with step-by-step instructions. However, as the preparation, submission and publication of a full manuscript takes a significant amount of time, we would like to share a number of technical tips for STAP cell conversion culture (and related experiments) in this Protocol Exchange. We hope that these technical tips may answer many questions frequently asked about the experimental details.
(英文) Procedure Tissue collection and low-pH treatment 1. To isolate CD45+ haematopoietic cells, spleens were excised from 1-week-old Oct4-gfp mice (unless specified otherwise), minced by scissors, and mechanically dissociated using a Pasteur pipette. IMPORTANT (i) Adherent cells should be dissociated into single cells, either mechanically or enzymatically (by trypsin or collagenase). For the tissues described in Fig. 3a (Obokata et al. Nature, 2014a), muscle, adipose tissue and fibroblasts were enzymatically dissociated, whereas others were mechanically dissociated. (ii) Primary cells should be used. We have found that it is difficult to reprogram mouse embryonic fibroblasts (MEF) that have been expanded in vitro, while fresh MEF are competent. (iii) For the experiments reported, we used a Oct-3/4-EGFP transgenic mouse line (Ohbo et al, Dev Biol, 2003; Yoshimizu et al, Dev Growth Differ, 1999), which is maintained by the RIKEN Bioresource Center as GOF18-GFP line11 transgenic mouse (B6;B6D2-Tg(GOF18/EGFP)11/Rbrc). Homozygotes of the transgene were used for the live imaging to obtain the enhanced signal. (iv) Cells from mice older than one week showed very poor reprogramming efficiency under the current protocol. Cells from male animals showed higher efficiency than those from female.
＜手順＞ 組織収集および低pH処理 1. CD45陽性造血細胞を単離するために、1週齢のOct4-GFPマウス（特に指定のない限り）から脾臓が摘出され、ハサミによりミンチされ、そして機械的にパスツールピペットを用いて分離される。 [重要] (i) 接着細胞は機械的または酵素的に（トリプシンまたはコラゲナーゼにより）単一細胞に解離されなければならない。図3ａ (Obokata et al. Nature, 2014a)で説明されている組織のうち、他の細胞が機械的に解離させられたのに対して、筋肉、脂肪組織および線維芽細胞は酵素的に解離させられている。 (ii)一次細胞が使用されるべきである。我々は新鮮なマウス胚線維芽細胞（MEF）なら可能であるが、体外で増殖されたMEFを再プログラムすることは困難であることをすでに知っている。 (iii) 報告されている実験のために、我々は、理化学研究所バイオリソースセンターによってGOF18-GFP株11トランスジェニックマウス(B6;B6D2-Tg(GOF18/EGFP)11/Rbrc)として維持されている、Oct-3/4-EGFPトランスジェニックマウス株を使っている(Ohbo et al, Dev Biol, 2003; Yoshimizu et al, Dev Growth Differ, 1999)。導入遺伝子のホモ接合体は、強化された信号を得るため、ライブイメージング用に使用されている。 (iv)1週間以上経過したマウス由来の細胞は現在のプロトコルの下では非常に貧弱な再プログラミング効率を示した。雄の動物からの細胞は雌からのものよりも高い効率を示している。
(英文) 2. Dissociated spleen cells were suspended with PBS and strained through a cell strainer (BD Biosciences 352340). 3. After centrifuging at 1,000 rpm for 5 min, collected cells were re-suspended in DMEM medium and added to the same volume of lympholyte (Cedarlane), and then centrifuged at 1,000 g for 20 min. IMPORTANT (i) The purity of the starting cells is important for achieving STAP conversion. For lymphocytes, contamination with red blood cells may inhibit the reprogramming event. When using adherent cells, the presence of extracellular matrix may interfere with reprogramming. (ii) Alternatively, red blood cells may be removed by suspension of the cell pellet in 1.8 ml of H2O (Sigma W3500). After 30 seconds, add 0.2 ml of 10× PBS (Gibco 70011-044), followed by 3 ml of 1× PBS (Gibco 10010-023), and strain the cell suspension through a cell strainer.
(英文) 4. The lymphocyte layer was isolated and stained with CD45 antibody (Abcam ab25603). CD45+ cells were sorted by FACS Aria (BD Biosciences). IMPORTANT (i) FACS sorting can be an important step for the confirmation of cell purity, but can affect both cell viability and reprogramming efficiency. Skipping this step may increase reprograming efficiency, although this may result in a reduction in confidence in cell identity. 5. After cell sorting, 1 × 106 CD45+ cells were treated with 500 µl of low-pH HBSS solution (titrated to pH 5.7 by HCl) for 25 min at 37°C, and then centrifuged at 1,000 rpm at room temperature for 5 min. IMPORTANT (i) The buffering action of HBSS is weak, so carry-over of the solution may affect pH. Please adjust pH to 5.7 in cell suspension by the following method. First, suspend the cell pellet with 494 µl of HBSS pre-chilled at 4°C, then add 6 µl of diluted HCl (10 µl of 35% HCl in 590 µl of HBSS) to adjust to a final pH of 5.7. Please confirm the final pH in a pilot experiment, and optimize the volume of HCl added, as necessary. Alternatively, suspend the cell pellet in HBSS-pH 5.4 pre-chilled at 4°C. (ii) The HBSS we used is Ca2+/Mg2+ free (Gibco 14170-112). (iii) Incubate the cells suspended in HBSS in a CO2 incubator. (iv) Cell viability is a critical parameter in this step. Under optimal conditions, massive cell death is observed at two days after plating, as shown in Figure 1d (Obokata et al. Nature, 2014a). (v) If you find massive cell death at one day after plating, it may be ameliorated by shortening the incubation period with low-pH HBSS solution to 15 min.
(英文) 6. After the supernatant (low-pH solution) was removed, precipitated cells were re-suspended and plated onto non-adhesive culture plates (typically, 1×105 cells/ml) in DMEM/F12 medium supplemented with 1,000 U LIF (Sigma) and 2% B27 (Invitrogen). IMPORTANT (i) The use of non-adhesive culture plates is recommended, as the formation of cell clusters is an important step for reprogramming, and the adhesive surface may inhibit cell movement needed to form clusters. (ii) Cell density is critical, and depends on cell viability. Density should be maintained at 1×105~1×106 cells per cm2 of culture surface. (iii) B27 (Invitrogen 17504-044) may show variation between batches. Please check the quality by N2B27-2iLIF culture of ES cells.
(英文) 7. Cell cluster formation was more sensitive to plating cell density than to the percentage of Oct3/4-GFP+ cells. The number of surviving cells was sensitive to the age of donor mice, and was low under the treatment conditions described above when adult spleens were used. IMPORTANT (i) The donor mouse should be 1-week old or younger. Reprogramming efficiency is dramatically reduced using cells from older animals. (ii) STAP cells are derived from clusters of multiple cells; they are not monoclonal. 8. The addition of LIF during days 2–7 was essential for generating Oct3/4-GFP+ STAP cell clusters on day 7, as shown in Extended Data Fig. 1f (Obokata et al. Nature, 2014a). Even in the absence of LIF, Oct3/4-GFP+ cells (most of which showed dim signals) appeared transiently in low-pH-treated CD45+ cells during days 2–5 of culture, but subsequently disappeared, suggesting that there is a LIF-independent early phase, whereas the subsequent phase is LIF-dependent. IMPORTANT (i) Since LIF is essential for the late step of reprogramming, the reprogramming event may depend on the genetic background. We mainly used 129, C57BL6, or their F1 strains, as all of these genetic backgrounds are associated with high responsiveness to LIF. The GFP signal is weaker than that of ES cells carrying the same reporter because of the smaller cell volume of STAP cell than ES cell as found in Figure 1g (Obokata et al. Nature, 2014a).
7. 細胞のクラスター形成はOct3/4-GFP陽性細胞のパーセンテージに対してよりも、プレート移植されている細胞密度に対してより敏感であった。生存細胞数はドナーマウスの年齢に敏感であり、成長したマウス脾臓を用いた場合、上述した処理条件下で低かった。 [重要] (i)ドナーマウスは1週齢もしくはもっと若くあるべきである。高齢の動物からの細胞を用いると再プログラミング効率は劇的に落ちる。 （ii）STAP細胞は複数の細胞のクラスタから誘導される。それらは単一種細胞ではない。 ８. 2から7日目のにおけるのLIFの添加は、拡張データ図1ｆに示すように、7日目のOct3/4-GFP陽性STAP細胞クラスターを生成するために不可欠であった(Obokata et al. Nature, 2014a)。LIFの存在しない場合でも、Oct3/4-GFP陽性細胞（ほとんどが薄暗いシグナルを示したが）は培養2から5日の間に、低pH値で処理したCD45陽性細胞の中に一過的に現れたが、その後に姿を消した。LIF依存の次の段階に対してＬＩＦ非依存の初期段階のあることを示唆している。 [重要] (i)再プログラミングの後期段階ではLIFは不可欠ですので、再プログラミング事象は遺伝的背景に依存しているのかも知れない。我々は主に129、C57BL6、またはそれらのF1株を使用したが、これらの遺伝的背景のすべてがLIFへの高い応答性に関連しているからである。 (ii) GFPシグナルは、同じレポーターを持っているES細胞のそれより弱いが、それは図1ｇに見られるように、STAP細胞の体積がES細胞よりも小さいためである(Obokata et al. Nature, 2014a)。
(英文) STAP stem-cell conversion culture 1. To establish STAP stem-cell lines, STAP cell clusters were transferred to ACTH-containing medium on MEF feeder cells (several clusters, up to a dozen clusters, per well of 96-well plates). IMPORTANT (i) ACTH (1-24) is available from American Peptide and other companies. We used ACTH synthesized by Kurabo on consignment. The composition of this medium is GMEM, 15% Knockout Serum ReplacementTM (KSR, Invitrogen), 1 × non-essential amino acids (NEAA), 1 × Sodium Pyruvate, 10-4M 2-mercaptoethanol, 1000 U/ml LIF, and 10 µM ACTH . The STAP cell cluster was isolated, dissected into small pieces as in the case of injection into blastocysts as shown in Figure 4a (Obokata et al. Nature, 2014a), and seeded on mouse embryonic fibroblast feeder cells in the ACTH medium. (ii) ACTH-containing medium was purchased from DS Pharma Biomedical (Osaka, Japan) as StemMedium@.
(英文) 2. After 4–7 days of culture, the cells were subjected to a first passage using a conventional trypsin method, and the suspended cells were plated in ESC maintenance medium containing 20% FBS. IMPORTANT (i) ESC maintenance medium consists of KnockoutTM DMEM (Life Technologies), 20% FBS, 1 × NEAA, 1 × Glutamine, 1 × Nucleosides, 10-4M 2-mercaptoethanol, and 1000 U/ml LIF. (ii) FBS lots should be confirmed for suitability for use in the culture of mouse ES cells. (iii) We have established multiple STAP stem cell lines from STAP cells derived from CD45+ haematopoietic cells. Of eight clones examined, none contained the rearranged TCR allele, suggesting the possibility of negative cell-type-dependent bias (including maturation of the cell of origin) for STAP cells to give rise to STAP stem cells in the conversion process. This may be relevant to the fact that STAP cell conversion was less efficient when non-neonatal cells were used as somatic cells of origin in the current protocol.
(英文) 3. Subsequent passaging was performed at a split ratio of 1:10 every second day until reaching subconfluency. We tested the following three different genetic backgrounds of mice for STAP stem-cell establishment from STAP cell clusters, and observed reproducible establishment: C57BL/6 carrying Oct4-gfp (29 of 29), 129/Sv carrying Rosa26-gfp (2 of 2), and 129/Sv × C57BL/6 carrying cag-gfp (12 of 16). STAP stem cells with all these genetic backgrounds showed chimaera-forming activity.
(英文) FI stem cell conversion culture 1. STAP cell clusters were transferred to Fgf4-containing trophoblast stem-cell medium (Tanaka et al, Science, 1998) on MEF feeder cells in 96-well plates (Obokata, Nature, 2014b). IMPORTANT (i) TS medium consists of RPMI 1640 with 20% FBS, 1 mM Sodium Pyruvate, 100 µM 2-mercaptoethanol, 2 mM L-glutamine, 25 ng/ml of recombinant FGF4, and 1 µg/ml of heparin. (ii) Different lots of FBS may results in significant differences in the behavior of cultured cells. 2. In most cases (40 of 50 experiments), colonies grew in 10–50% of wells in 96-well plates. In a minority of cases (10 of 50 experiments), no colony growth was observed and/or only fibroblast-like cells appeared. IMPORTANT (i) The cells in proliferative colonies also appear similar to fibroblasts, but gradually change morphology, coming to resemble epithelial cells.
FI幹細胞変換培養 1. STAP細胞クラスターがFGF4を含む96ウェルプレート中のMEFフィーダー細胞上のＴＳ細胞＜栄養芽層幹細胞＞培地 (Tanaka et al, Science, 1998)に移される (Obokata, Nature, 2014b)。 [重要] （i）TS培地は20％のFBS＜ウシ胎児血清＞を含むRPMI1640＜ロズウェルパーク記念研究所培地 Roswell Park Memorial Institute medium＞、1mMのピルビン酸ナトリウム、100μMの2-メルカプトエタノール、2mMのL-グルタミン、組換えFGF4の25 ng / ml及びヘパリンの1μg/mlで構成されている。 (ii)FBSのロットの違いは培養細胞の挙動に重大な差異をもたらしうる。 2. ほとんどの場合（50回のうちの40回の実験）、コロニーは96ウェルプレートのウェルの10から50％までに成長した。少数の例（50のうちの１０回の実験）では、全くコロニー増殖が観察されずに、かつ/または、わずかの線維芽細胞様細胞が出現した。 重要 (i)増殖コロニー内の細胞はまた線維芽細胞のように現れるが、徐々に形態を変更して上皮細胞に似てくる。
(英文) 3. The cells were subjected to the first passage during days 7–10 using a conventional trypsin method. Subsequent passages were performed at a split ratio of 1:4 every third day before they reached subconfluency. IMPORTANT The cells must not be dissociated completely. Partial dissociation is optimal to maintain viability and self-renewal, as seen in the case of embryo-derived trophoblast stem cells.
(英文) References Obokata, H. et al.Obokata. Stimulus-triggered fate conversion of somatic cells into pluripotency, Nature, 505, 641-647 (2014a) Obokata, H. et al. Bidirectional developmental potential in reprogrammed cells with acquired pluripotency. 676–680 (2014b) Ohbo, K. et al. Identification and characterization of stem cells in prepubertal spermatogenesis in mice small star, filled. Dev. Biol. 258, 209–225 (2003) Yoshimizu, T. et al. Germline-specific expression of the Oct4/green fluorescent protein (GFP) transgene in mice. Dev. Growth. Differ. 6, 675-684 (1999) Ogawa, K., Matsui, H., Ohtsuka, S. & Niwa, H. A novel mechanism for regulating clonal propagation of mouse ES cells. Genes Cells 9, 471–477 (2004) Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A. & Rossant, J. Promotion of trophoblast stem cell proliferation by FGF4. Science 282, 2072–2075 (1998)
＜参照＞ Obokata, H. et al.Obokata. 『体細胞の多能性への刺激惹起性運命変換』Nature, 505, 641-647 (2014a) Obokata, H. et al. 『取得多能性を持つ再プログラム細胞における双方向への発生能力』 676–680 (2014b) Ohbo, K. et al. 『マウスの小さな星の中に満たされた思春期前の精子形成における幹細胞の同定と特徴づけ』 Dev. Biol. 258, 209–225 (2003) Yoshimizu, T. et al. 『マウスのOct4/緑色蛍光タンパク質（GFP）導入遺伝子の生殖細胞特異的発現』 Dev. Growth. Differ. 6, 675-684 (1999) Ogawa, K., Matsui, H., Ohtsuka, S. & Niwa, H. 『マウスES細胞のクローン増殖を調節するための新しいメカニズム』 Genes Cells 9, 471–477 (2004) Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A. & Rossant, J. 『FGF4＜線維芽細胞増殖因子＞によるＴＳ細胞増殖の推進』 Science 282, 2072–2075 (1998)
(英文) Associated Publications This protocol is related to the following articles: Stimulus-triggered fate conversion of somatic cells into pluripotency Haruko Obokata, Teruhiko Wakayama, Yoshiki Sasai, Koji Kojima, Martin P. Vacanti, Hitoshi Niwa, Masayuki Yamato, and Charles A. Vacanti Journal title Nature Vol. 505 Issue -7485 Pages 641 - 647 Publication date 29/01/2014 DOI: doi:10.1038/nature12968
＜関連著作物＞ このプロトコルは下記論文に関連している。 『体細胞の多能性への刺激惹起性運命変換』 Haruko Obokata, Teruhiko Wakayama, Yoshiki Sasai, Koji Kojima, Martin P. Vacanti, Hitoshi Niwa, Masayuki Yamato, and Charles A. Vacanti Journal title Nature Vol. 505 Issue -7485 Pages 641 - 647 Publication date 29/01/2014 DOI: doi:10.1038/nature12969
(英文) Bidirectional developmental potential in reprogrammed cells with acquired pluripotency Haruko Obokata, Yoshiki Sasai, Hitoshi Niwa, Mitsutaka Kadota, Munazah Andrabi, Nozomu Takata, Mikiko Tokoro, Yukari Terashita, Shigenobu Yonemura, Charles A. Vacanti, and Teruhiko Wakayama Journal title Nature Vol. 505 Issue -7485 Pages 676 - 680 Publication date 29/01/2014 DOI: doi:10.1038/nature12969
『取得多能性を持つ再プログラム細胞における双方向への発生能力』 Haruko Obokata, Yoshiki Sasai, Hitoshi Niwa, Mitsutaka Kadota, Munazah Andrabi, Nozomu Takata, Mikiko Tokoro, Yukari Terashita, Shigenobu Yonemura, Charles A. Vacanti, and Teruhiko Wakayama Journal title Nature Vol. 506 Issue -7484 Pages 677 - 680 Publication date 29/01/2015 DOI: doi:10.1038/nature12970
(英文) Author Information Affiliations 1. Laboratory for Cellular Reprogramming, RIKEN CDB Haruko Obokata 2. Laboratory for Organogenesis and Neurogenesis, RIKEN CDB Yoshiki Sasai 3. Laboratory for Pluripotent Stem Cell Studies, RIKEN CDB Hitoshi Niwa Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: Hitoshi Niwa (firstname.lastname@example.org)