アイコン 理研/疑念のSTAP細胞の作成方法を公開 発表原文付

1月28日世界に衝撃を与えた万能細胞STAP細胞の作成発表、しかし、それから写真がほかの学術誌からとったのではないか、いや合成されたのではないか、発表論文も第3者の研究論文を一部コピペしていると指摘されたり、踏んだり蹴ったり。

一 方で、作成した小保方晴子研究ユニットリーダーには理研から緘口令が取られたのか、音沙汰なし、学会までが理研に調査結果を発表しろと迫るなど、一歩間違 えば、捏造データのディオバンどころか、ES細胞によるクローン牛誕生偽証の韓国の黄禹錫教授と同じではと囁かれたりしていた。
 
そうしたなか理化学研究所は5日、小保方晴子研究ユニットリーダーらが開発した新型万能細胞(STAP細胞)の詳しい作製法を公開した。
1月末の発表後、国内外の研究者が論文を基に作製を試みても「うまくできない」との指摘が相次いだことに対応した。
成果の信ぴょう性について疑問視する声も出ており、別のグループが同じ結果を再現できるかが今後の焦点になる。
 一方、論文の一部で別の論文を無断引用していたり、画像を使い回していたりしているとの指摘については「調査結果が出た時点で速やかに公表する」と説明したという。
 科学の世界では、ある成果が出たとき、別の研究者らが同じ条件で実験し、同じ結果を再現した段階で初めて認められる。
京都大学の山中伸弥教授らが開発したiPS細胞は公表後、比較的早イ段階で世界の研究者が同じように作製できたため、世界から注目され、世界中の研究者らが研究に携わっている。
 
  今回の詳細な作製法は、小保方リーダーと理研の2人の研究責任者がまとめ、理研のホームページで公開したほか、論文が掲載された英科学誌ネイチャーの関連サイトにも近く公表するという。
 
 「単純に見えるが、細胞の取り扱いや培養条件は特別の注意を払う必要がある」と指摘。
STAP細胞の元になる体の細胞の選び方から培養法、刺激の与え方まで詳しく説明した。
作製効率を上げるには、生後1週間以内のオスのマウスを使うことなどを明記した。
 
 作製法の公開について、慶応義塾大学の須田年生教授は「ずっと情報が多くなった」と評価する。
一方、横浜市立大学の武部貴則准教授は「多くの研究者が抱いていた当初の印象に比べて難しいとわかった。万能細胞の専門家を除くと、作製できる人は少ないのではないか」とみる。
 
STAP細胞は、紅茶程度の弱い酸性の溶液に細胞を30分漬けるだけで作製できると注目された。しかし、他の研究グループが同様の結果を出したとの報告はなく、論文にはないノウハウの公開を求めていた。
以上、日経ほか参照。
 
英語のできる方は、原文はクリック
 
 
 
 
<原文>
Protocol Exchange
Essential technical tips for STAP cell conversion culture from somatic cells
Haruko Obokata1, Yoshiki Sasai2 and Hitoshi Niwa3*
1Laboratory for Cellular Reprogramming, 2Laboratory for Organogenesis and
Neurogenesis, 3Laboratory for Pluripotent Stem Cell Studies, RIKEN Center for
Developmental Biology, Kobe 650-0047, Japan
*To whom correspondences should be addressed.
Hitoshi Niwa, M.D., Ph.D.
niwa@cdb.riken.jp
 
Introduction
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.
 
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.
 
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. 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.
 
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.
 
issue 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.
 
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.
 
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.
 
6. After the supernatant (low-pH solution) was removed, precipitated cells were e-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.
 
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.
 
(ii) 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).
 
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 (Ogawa et al,Genes Cells, 2004). 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@.
 
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.
 
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.
 
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.
 
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
(i) 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.
 
References
Obokata, H. et al. 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)
 
 
 
 
[ 2014年3月 6日 ]
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