相沢さんが報告しているのは環境の劣悪さ、時間の無さ、スフィア塊はできる。
GFP蛍光は弱い。キメラはできないというものだね。テラトーマに関しては以下だ。
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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.
丹羽さんはイントロでさりげなく書いてるんだよね。無論多義的な読み方をすればだけどね。
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Full reprograming of somatic cells results in the acquisition of the ability to give rise to an entire organism, or totipotency; this can be achieved by somatic cell nuclear transfer.
丹羽さんの報告書のマテメソにある以下は相沢さんの研究だわよね。
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R26R-H2B-EGFP transgenic mouse (Rosa-GFP) line was generated by Laboratory for Animal Resources and Genetic Engineering (LARGE), RIKEN CDB17.
ここもとてもおかしい場所でね。彼らは<Article のメソッド>だけを言ってるんだが
レターにもこのマウスが出ているよね。例のFigure1のaなんだぜ。ES細胞だ。
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Here we have investigated the unique nature of STAP cells, focusing on their differentiation potential into the two major categories (embryonic and placental lineages) of cells in the blastocyst. We became particularly interested in this question after a blastocyst injection assay revealed an unexpected finding. In general, progeny of injected ES cells are found in the embryonic portion of the chimaera, but rarely in the placental portion (Fig. 1a; shown with Rosa26-GFP). Surprisingly, injected STAP cells contributed not only to the embryo but also to the placenta and fetal membranes (Fig. 1b and Extended Data Fig. 1a–c) in 60% of the chimaeric embryos (Fig. 1c).
アーティクルの部分はこれね。
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For establishment of STAP stem-cell lines, STAP cell clusters were transferred to ACTH-containing medium36 on MEF feeder cells (several clusters, up to a dozen clusters, per well of 96-well plates). Four to seven days later, the cells were subjected to the first passage using a conventional trypsin method, and suspended cells were plated in ES maintain medium containing 20% FBS. Subsequent passaging was performed at a split ratio of 1:10 every second day before they reached subconfluency. We tested the following three different genetic backgrounds of mice for STAP stem-cell establishment from STAP cell clusters, and observed reproducible data of 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.
うん、そこはど素人なんでよくわからないな。相沢さんのマウスはどこかでてでくるかな?
肝臓で論文通りに近い数が出たんでそこを中心に調べてるな。
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Induced cell aggregates show poor induction of pluripotency-associated markers
To test the induction of pluripotency markers in cell aggregates obtained from ATP-treated liver cells, we assessed the expression of pluripotency-associated genes. Oct3/4 is a well-defined marker of pluripotent stem cells. Using a primer pair to detect Oct3/4 transcript from the Pou5f1 allele, but not pseudo-genes13, we did not find a detectable level (above 0.1% of the expression level in mouse ES cells, relative to the expression levels of Gapdh) of the transcript by quantitative polymerase chain reaction (Q-PCR) using a total RNA sample prepared from all cells in the culture (Fig. 3a), indicating that extremely few or no cells expressing Oct3/4 were present.
こんなことってあるのね。
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Interestingly, expression of Gfp from the Oct3/4-GFP transgene (GOF18)14 was detected in liver cells cultured for seven days irrespective of ATP treatment, suggesting leaky expression of this transgene.
そうだよ。丹羽さんも同じことをしている。
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We next performed qPCR on individual cell aggregates isolated from culture. Aggregates were selected and RNA samples were prepared separately. These RNAs were reverse-transcribed and qPCR was performed.
小保方さんはスフィア塊２０個に１個Oct4発現があると報告したんだ。
それに対して、丹羽さんは同じ現象を再現確認したうえで、その細胞の数を数えた。
結果は
However, the frequency was very low; 5 × 105 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%.
というものだ。105は10の５乗。
①から⑦までの現象ではこの細胞が多能性を持つということはできない。
小保方さんの論文の題は
<ティシュー誌>
The Potential of Stem Cells in Adult Tissues Representative of the Three Germ Layers
<博論>
Isolation of pluripotent adult stem cells discovered from tissues derived from all three germ layers
えっとね。ディスカッションのところでしょ。
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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).
ここと、
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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.
だろ。
小町ちゃん、 丹羽さんが again highlighting the lack of evidence
supporting the existence of the reported STAP phenomenon. と言ってるのは
キメラができなかったことを理由にしているね。それから別途相沢さんが
この程度の数ではESですらテラトーマはできないと指摘している。違うかね。
<ティシュー誌>
The Potential of Stem Cells in Adult Tissues Representative of the Three Germ Layers
<博論>
Isolation of pluripotent adult stem cells discovered from tissues derived from all three germ layers
