One of the central claims in the original reports was that the purported STAP cells had the ability to differentiate into multiple lineages, including germ cells, when placed in a normal developmental environment. The present study focused on assessing pluripotency by chimera production using cell aggregates prepared by Obokata; however, no evidence of pluripotency was observed using this assay. In the original reports, the STAP cells were prepared by Haruko Obokata, while the chimera production and the establishment of ES (embryonic stem)-like STAP-SCs and TS (trophectoderm stem)-like FI-SCs were made by Teruhiko Wakayama.
そうね。はい。
>>
I encourage readers to recognize a number of limitations in the studies, which were conducted under strict time constraints and in the face of considerable, often adversarial, media scrutiny. Unfortunately, it was not possible to receive technical advice from Teruhiko Wakayama in the chimera production reported here, and it is unclear whether or to what extent the techniques for chimera production in the present study correspond to those used in the previous studies. Previous studies also examined the pluripotency of purported STAP cells by their potency to generate teratomas in immune-deficient mice. However, more than 105 cells are required to form teratoma subcutaneously in the flank of an immune-deficient mouse using ES or EC (embryo carcinoma) cells, and the process takes about one month. No teratoma formation was examined in the present study, since the frequency of green fluorescent cell aggregates was low and time was limited. Teratoma formation under the kidney capsule, which also takes about two months using blastocyst embryos, was also not examined.
はい。
>>
The more critical question is whether and to what extent the STAP cell aggregates prepared by Obokata in this trial under new experimental conditions recapitulated the STAP cell aggregates reported in the previous study. The frequency of green fluorescent cell aggregates from low pH-treated, Oct-GFP transgenic spleen cells was 10-fold less than that in previous studies. Moreover, green fluorescence due to GFP expression cannot be distinguished from that due to autofluorescence, nor can GFP expression by reprogramming be distinguished from that due to non-specific gene expression in dying cells. The cell aggregates were not characterized in vitro in detail, but the following features were observed:
続きを読んだら。
>>
(1) Preliminary FACS analysis of low pH-treated, Oct-GFP transgenic spleen cells suggested that the frequency of green fluorescent cells was very low and that the majority of surviving cells were CD45-positive after one week in culture under the conditions used in the present study. In the previous study, CD45+ cells were rare and a significant number of green fluorescent cells were observed (Figure 1c in Obokata et al., 2014a).
そうじゃないと思うよ、 the majority of surviving cells were CD45-positive after one week in culture と
あるじゃないか。残った細胞って、GFPを発していない細胞のことだよ。本物であれ、アーティファクトであれ
今回は以前みたいに出来てないんだから。残ったのはなんでもない細胞じゃないか。
そこはペンディングして全部読みましょうかね。
>>
(2) Preliminary RT-PCR analysis suggested that the majority of the cell aggregates generated in the present study did not express pluripotency markers, in contrast to the report of pluripotency marker expression in the previous study (Figure 2b in Obokata et al., 2014a), although there were cell aggregates at a low frequency that expressed one or multiple pluripotent markers.
はい。
>>
(3) Preliminary immunochemical analysis suggested that most of the cell aggregates in the present study did not express pluripotency markers. In contrast to the data shown in Figure 2a of the previous study, they did not express OCT4, SSEA1, NANOG and E-CADHERIN, (Obokata et al., 2014a).
はい、続きよ。
>>
The possibility cannot be excluded that the experimental conditions used in the present study in some way differed from the previously established optimum conditions for STAP induction. It is my view that it was beyond the scope of this examination to reassign each condition; a definitive answer to the question of whether the previously used conditions for inducing the STAP phenomenon can be indeed established or not must await further study. Nevertheless, I consider it is important to report that Haruko Obokata herself failed to reproduce the reported phenomenon, in that the putative STAP cells described here were unable to contribute to any tissues in a normal developmental environment.
ま、そういうことだね。それが相澤さんの本務だ。科学的事実に関しては
a definitive answer to the question of whether the previously used conditions for inducing the STAP phenomenon can be indeed established or not must await further study.
ということで。
丹羽氏の実験のサンプル量は
Fig.3aは all cells in the wells
Fig.3bは 細胞塊１個
なので、細胞塊１個では、GAPDH値が安定して計れなかったのではないでしょうか？（推測です）
会見でも試行錯誤をしながら実験したと話されています。
*ttps://youtu.be/f9k6tQixpTQ?t=1829
最初の問題をペンディングしたままいわば戦線拡大するのね。ま、いいわ。
>>
TCR-β chain gene rearrangement analysis
Genomic DNA was extracted from STAP cells and tail tips from chimaeric mice generated with STAP cells derived from CD45+ cells. PCR was performed with 50 ng DNA using the following primers (Dβ2: 5′-GCACCTGTGGGGAAGAAACT-3′ and Jβ2.6: 5′-TGAGAGCTGTCTCCTACTATCGATT-3′) that amplify the regions of the (D)J recombination. The PCR products were subjected to gel electrophoresis in Tris-acetate-EDTA buffer with 1.6% agarose and visualized by staining with ethidium bromide. PCR bands from STAP cells were subjected to sequencing analysis and identified as rearranged genomic fragments of the (D)J recombination.
はい、STAP細胞の方の再構成はあったということでいいのね。学さん達が何か議論されていたのは
キメラの細胞のTCR再構成みたいね。
>>
Genomic DNA was extracted from STAP cells and tail tips from chimaeric mice generated with STAP cells derived from CD45+ cells.
でも、ESであったら絶対に無かったはずよね。で、小保方さんはアーティクルに
キメラの尻尾の検査の結果を書いてない。
>>
PCR bands from STAP cells were subjected to sequencing analysis and identified as rearranged genomic fragments of the (D)J recombination.
キメラの尻尾の確認は分からないけど、キメラを作った時の幹細胞とされている
FLSにTCR再構成が後から調べた限りでは無かったことは事実ね。
>>
(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.
ちと、中断。サイエンス査読部分は以下だよね。
>>
The DNA analysis of the chimeric mice is the only piece of data that does not fit with the contamination theory. But the DNA fragments in the chimeras don’t look the same as those in the lymphocytes. This assay is not properly explained. If it is just an agarose gel then the small bands could be anything. Moreover this figure has been reconstructed. It is normal practice to insert thin white lines between lanes taken from different gels (lanes 3 and 6 are spliced in). Also I find the leading edge of the GL band suspiciously sharp in #2-#5.
<the DNA fragments in the chimeras don’t look the same as those in the lymphocytes. >ということは
何らかのゲル写真があったということなのかな。血球の分は今アーティクルに
ってるような奴だよな。同じ画像にキメラの尻尾の分が並べられていたのか?
小保方さんは12/11ヴァージョンにそのままキメラの尻尾もつけてたのね。
でも先生たちにそれじゃ証明にはならないと言われて外したのね。でも
キメラも調べたという意味合いで言葉は残しておいたということね。実際には
あったのよね。小保方さんがESコンタミしてたら無いと知ってる筈よね。
でも2Nで調べてあったから喜んで使ってたら、サイエンスでThe DNA fragments
in the chimeras don’t look the same as those in the lymphocytes.と指摘され、
笹井さんや、丹羽さんにもリシピエントの血球のTCR再構成をひろったんじゃないのと
言われて、写真は削除したのね。
まあ、捏造するならそんな試料はどこからでも取って来れるわね。まさに、査読者みたいに
The DNA fragments in the chimeras don’t look the same as those in the lymphocytes.
と意地悪く疑うことはできるのね。丹羽さん達は信じているから捏造は疑って無くて、ただ2Nじゃ
証明にならないでしょとアドヴァイスしただけね。
ではどうして若山さんはアドヴァイスしなかったのかしら。
>>
プロトコル公開されました．
論文と矛盾する点が多いので，苦し紛れに見えます．
ゲノム再構成についてはこの部分でちゃんと述べています．
(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.
８つのサンプルのうちゲノム再構成がみられたものはなかった，だそうです．
これは私が提出した資料を見た可能性があります．そうだとすればこの数日のうちに書いたものでしょう．
だとすれば分化した細胞から作成されたという証拠はなくなるし，論文の図も誤っていることになります．数枚の図の訂正ではすまない「修正」が必要になるでしょうね．
次の日記で書こうと思っていましたが元々CD45はここで述べられているようにT細胞のマーカーではなく血球系の広い細胞のマーカーです．論文中でT細胞だと書いた部分は全て間違っており，ゲノム再構成についての記述も全て削除しなければならないでしょう．
最初はCD45+で細胞を選択した理由は単なる無知か，わざと（幹細胞を含む）雑多な細胞を混ぜるためだと思っていました．その中にSTAPとなりうる細胞が含まれているかもしれないからと．しかし恐らくそうではありません．他の研究者が理解しづらくするための煙幕だと，今は思っています．
--
kaho
相澤報告に戻るのね。ペンディングされている問題だわよね。
>>
411： デラ・ストリート ：2018/06/26(火) 17:18:32
続きを読んだら。
>>
(1) Preliminary FACS analysis of low pH-treated, Oct-GFP transgenic spleen cells suggested that the frequency of green fluorescent cells was very low and that the majority of surviving cells were CD45-positive after one week in culture under the conditions used in the present study. In the previous study, CD45+ cells were rare and a significant number of green fluorescent cells were observed (Figure 1c in Obokata et al., 2014a).
<元々CD45はここで述べられているようにT細胞のマーカーではなく>って、誰も
そんなこと言ってないわよね。アーティクルもちゃんと読んでないのよね。
>>
The emergence of Oct4-GFP+ cells at the expense of CD45+ cells was also observed by flow cytometry (Fig. 1c, top, and Extended Data Fig. 1b, c). We next fractionated CD45+ cells into populations positive and negative for CD90 (T cells), CD19 (B cells) and CD34 (haematopoietic progenitors), and subjected them to low-pH treatment. Cells of these fractions, including T and B cells, generated Oct4-GFP+ cells at an efficacy comparable to unfractionated CD45+ cells (25–50% of surviving cells on day 7), except for CD34+ haematopoietic progenitors, which rarely produced Oct4-GFP+ cells (<2%; Extended Data Fig. 1d).
(英文)
a, Immunostaining for pluripotent cell markers (red) in day 7 Oct4-GFP+ (green) clusters. DAPI, white. Scale bar, 50 μm. b, qPCR analysis of pluripotency marker genes. From left to right, mouse ES cells; parental CD45+ cells; low-pH-induced Oct4-GFP+ cells on day 3; low-pH-induced Oct4-GFP+ cells on day 7. n = 3; error bars show average ± s.d. c, DNA methylation study by bisulphite sequencing. Filled and open circles indicate methylated and non-methlylated CpG, respectively. d, Immunostaining analysis of in vitro differentiation capacity of day 7 Oct4-GFP+ cells. Ectoderm: the neural markers Sox1/Tuj1 (100%, n = 8) and N-cadherin (100%, n = 5). Mesoderm: smooth muscle actin (50%, n = 6) and brachyury (40%, n = 5). Endoderm: Sox17/E-cadherin (67%, n = 6) and Foxa2/Pdgfrα (67%, n = 6). Scale bar, 50 μm.
つぎはbね。
>>
b, qPCR analysis of pluripotency marker genes. From left to right, mouse ES cells; parental CD45+ cells; low-pH-induced Oct4-GFP+ cells on day 3; low-pH-induced Oct4-GFP+ cells on day 7. n = 3; error bars show average ± s.d.
参考
調査報告
９）Article Fig.2b、3d、3g、Extended Data Fig.1a、Extended Data Fig.6dについて
エラーバーが不自然である点
（調査結果）
本件に関しては、Extended Data Fig.1aについて過去に投稿された論文原稿に遡りグラフが投稿毎に一致しないこと（特にエラーバーのサイズ、有無など）を確認した。小保方氏本人対する聞き取り調査で、図を描画ソフト等で修正したことはないかを含め原因の心当たりを確認したところ、修正したことはないが、本人としてもエラーバーが不自然であること、ただ表計算ソフトの問題でこのようなことはよく起きると考えていたとの回答を得た。パソコンに入っていると思われるオリジナルデータの提出を小保方氏に求めたが、提出されなかった。
Science査読
Reviewer 1
In Fig.2B and the other RT-PCR studies, it is not stated whether the Y-axis is linear or logarithmic. If it is linear, which seems more likely, then I am very surprised that all of the pluripotency genes measured in the ESC control have virtually the same RNA abundance, which exceeds that of GAPDH.
Reviewer 2
Regarding the existing molecular data on the identity of the cell lines, the embryonic gene expression qPCRs (Figure 2B, S3C) show unusually high values for expression levels relative to GAPDH levels. Even though the figure has an ESC control, and it may be a primer-specific phenomenon, mRNA levels of genes such as Nanog and Rex1 are more like 0.05 or 0.15 of GAPDH levels, whereas the authors observed levels as high as 12 -14 times GAPDH.
まだ最初のあなたの昨日の指摘まで行ってないよ。あなたの昨日の指摘はdでしょ。
そんなにどんどん先走らないでください。
>>
d, Immunostaining analysis of in vitro differentiation capacity of day 7 Oct4-GFP+ cells. Ectoderm: the neural markers Sox1/Tuj1 (100%, n = 8) and N-cadherin (100%, n = 5). Mesoderm: smooth muscle actin (50%, n = 6) and brachyury (40%, n = 5). Endoderm: Sox17/E-cadherin (67%, n = 6) and Foxa2/Pdgfrα (67%, n = 6). Scale bar, 50 μm.
<Article論文上では、インビトロ分化誘導は、外胚葉で再現性100％になっている>という
部分に対応する部分が以下で、ここに至るために順番に検討してきたんだな。
>>
d, Immunostaining analysis of in vitro differentiation capacity of day 7 Oct4-GFP+ cells. Ectoderm: the neural markers Sox1/Tuj1 (100%, n = 8) and N-cadherin (100%, n = 5). Mesoderm: smooth muscle actin (50%, n = 6) and brachyury (40%, n = 5). Endoderm: Sox17/E-cadherin (67%, n = 6) and Foxa2/Pdgfrα (67%, n = 6). Scale bar, 50 μm.
Science査読
Reviewer 1
In Fig.2B and the other RT-PCR studies, it is not stated whether the Y-axis is linear or logarithmic. If it is linear, which seems more likely, then I am very surprised that all of the pluripotency genes measured in the ESC control have virtually the same RNA abundance, which exceeds that of GAPDH.
Reviewer 2
Regarding the existing molecular data on the identity of the cell lines, the embryonic gene expression qPCRs (Figure 2B, S3C) show unusually high values for expression levels relative to GAPDH levels. Even though the figure has an ESC control, and it may be a primer-specific phenomenon, mRNA levels of genes such as Nanog and Rex1 are more like 0.05 or 0.15 of GAPDH levels, whereas the authors observed levels as high as 12 -14 times GAPDH.
まあ、遊び無いと飽きるわね。そもそも問題自体がどうでもいいようなことだものね。
>>
Science査読
Reviewer 1
In Fig.2B and the other RT-PCR studies, it is not stated whether the Y-axis is linear or logarithmic. If it is linear, which seems more likely, then I am very surprised that all of the pluripotency genes measured in the ESC control have virtually the same RNA abundance, which exceeds that of GAPDH.
そうね。ところが小保方さんのHPには対数表示で対数コントロールESの1に対して
100のOct4-GFP発現と10のOct4蛋白製造遺伝子の発現があって、これは明らかに
GAPDH製造遺伝子の数を超えていることになるのね。つまり一つの細胞に2つ以上の
同じ遺伝子が存在していることになる。これが、レフェリーがIf it is linear,
which seems more likely, then I am very surprised that all of the pluripotency
genes measured in the ESC control have virtually the same RNA abundance,
which exceeds that of GAPDH.と書いている理由で、私たちの戸惑いと同じだわね。
>>747
査読にある
Even though the figure has an ESC control, and it may be a primer-specific phenomenon, mRNA levels of genes such as Nanog and Rex1 are more like 0.05 or 0.15 of GAPDH levels, whereas the authors observed levels as high as 12 -14 times GAPDH.
の部分から判断して特許の図Fig.2Aが対応しているのではないかと思っています。
この図は、2012.4.24の仮出願にも載せられている図で、これを並び替えたものが、Article Fig.2bと考えています。
Article Fig.2bにはRex1が載っていませんが、6つの遺伝子はほぼ対応していますし、コメントの内容から同類の図が示されていたと考えられます。
これらは、2014年5月に疑義をまとめたものの中にあった指摘です。
桂報告書P.23
「９）Article Fig.2b、3d、3g、Extended Data Fig.1a、Extended Data Fig.6dについてエラーバーが不自然である点」
と若干の違いはありますが、上記の指摘があって調査に含めたのではないかと思います。
調査報告書のExtended Data Fig.6dは肉眼では不自然な部分がみつけられなかったので、2chまとめ13）のext. fig5dの方ではないかと思いますが、専門的な部分で不自然な点があったのかもしれないのでこの部分はよく分かりません。
「本件に関しては、Extended Data Fig.1aについて過去に投稿された論文原稿に遡りグラフが投稿毎に一致しないこと（特にエラーバーのサイズ、有無など）を確認した。」
については、外部の指摘に、過去の投稿論文との比較が含まれていたとは考えにくく、調査委員会が入手した資料から確認できた内容を記述したものと思われます。
