石井報告でPCRのバンドの件は錯綜してしまうので今は論じないけど、
テラトーマの件に関係する場所はみておきましょうかね。
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ウ なお、審査の過程で、以下の事実が認められた。
（ア）不服申立て者は、2012 年 4 月に Nature 誌に投稿した 2012 年論文について、同誌か ら掲載を拒否された後の同年 7 月、2012 年論文にＴ細胞受容体再構成を示すための電気泳 動写真（Supplemental Figure 6）などを加えた上、類似した内容の論文を Science 誌に投 稿したところ、同誌査読者から、「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.」と指摘 されている。ここでいうレーン３は、論文１の Figure 1i で見られたレーン３と同一のもの と推測することも可能であるが、そうでないとしても、2012 年 8 月の段階で、すでにレー ン３の両側に線を加える等して、異なるゲルに由来するレーン３を区別しなければならないことなど真正なデータの提示が求められていたことは認識していたと認めるのが相当である。
我々は若山さんが結果的に捏造犯であることを既に知っている。にもかかわらず、
我々は小保方さんの石井調査に対するテラトーマに関する問いに対する答えには
嘘が含まれていると睨んでいる。アーティクルを振り返ってみたいね。
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In vivo differentiation assay
1 × 10(power of 7) STAP cells were seeded onto a sheet composed of a non-woven mesh of polyglycolic acid fibres (3 × 3 × 1 mm; 200 μm in pore diameter), cultured for 24 h in DMEM + 10% FBS, and implanted subcutaneously into the dorsal flanks of 4-week-old mice. In this experiment, to better support tumour formation from slow growing STAP cells by keeping cells in a locally dense manner, we implanted STAP cells with artificial scaffold made of polyglycolic acid fibres. Given the artificial nature of the material, we used NOD/SCID mice as hosts, to avoid possible enhancement of post-graft inflammation caused by this scaffold even in syngenic mice. STAP stem cells were dissociated into single cells and cell suspension containing 1 × 107 cells was injected into the testis. Six weeks later, the implants were analysed using histochemical techniques.
(続き)
The implants were fixed with 10% formaldehyde, embedded in paraffin, and routinely processed into 4-µm-thick sections. Sections were stained with haematoxylin and eosin. Endoderm tissues were identified with expression of anti-α-fetoprotein (mouse monoclonal antibody; MAB1368, R&D Systems). Ectodermal tissues were identified with expression of anti-βIII tubulin (mouse monoclonal antibody; G7121, Promega). Mesodermal tissues were identified with expression of anti-α-smooth muscle actin (rabbit polyclonal; DAKO). In negative controls, the primary antibody was replaced with IgG-negative controls of the same isotype to ensure specificity.
もう一つある107も10 to the power of 7 ね。
博論の草稿にあるテラトーマ記述は以下だね。
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3.3.2 Differentiation potential in vivo.
Bone marrow spheres and ES cells were transplanted subcutaneously into immune deficient mice to examine their tumor-initiating capacity. As a result, after 6 weeks ES cells formed a tumor. Spheres did not form tumor as big as ES cells did. We concluded that the proliferative potential of sphere cells was much weaker than that of ES cells (Fig. 13).
Next, we investigated if transplanted cells differentiated in vivo after transplantation. Transplanted cells were harvested after 6 weeks, and processed for immunohistochemical analyses. According to results of immunohistochemical analyses, spheres differentiated into tissues derived from three germ layers in vivo (Fig. 14).
あ、ごめん。張り付け順を間違えた。先にここね。
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3.2.2 In Vivo Differentiation.
Spheres were seeded onto biodegradable scaffolds and implanted into subcutaneous of NOD/SCID mice (Charles River laboratories). After 6 weeks, the implants were harvested and fixed with 10% formaldehyde, then examined by immunocytochemistry.
僕の張り付けまだ終わってないよ。ティシューのテラトーマだ。
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In vivo differentiation assay
Sphere cultures from representative tissues were washed twice with Hank’s balanced salt solution (Gibco). Under a microscope, 2000 spheres, each containing *1000 cells, were collected using a glass pipette and placed in a 50 mL tube. Hank’s balanced salt solution (20 mL) was added to each tube and subsequently centrifuged at 800 rpm for 3min. The supernatant was discarded and the pellet was resuspended in 50 mL of DMEM with 10% fetal bovine serum. This solution was seeded onto a sheet 3�B3�B1 mm, composed of a nonwoven mesh of polyglycolic acid fibers, 200 mm in diameter, and implanted subcutaneously into the dorsal flanks of a 4-week-old NOD/SCID mice ( Jackson Laboratory). Four weeks later the implants were harvested, and analyzed using immunohistochemical techniques. The implants were fixed with 10% formaldehyde, embedded in paraffin, and routinely processed into 4 mm thickness. Sections were stained with hematoxylin and eosin. Cartilage was confirmed using Safranin-O (Fisher; S670-25). Duct and gland-like tissues were identified using the endoderm marker, rabbit polyclonal FOXA2 (Abcam; ab40874). Epithelium-like structures were identified using mouse monoclonal [PCK-26] Pan cytokeratin antibody (Abcam; ab6401). Muscle-like structures were identified using mouse monoclonal desmin antibody (Sigma;D1033) and anti-a-smooth muscle actin antibody (N1584;Dako). Nerve-like structures were identified using mouse monoclonal beta III tubulin antibody (Promega; G7121). In negative controls, the primary antibody was replaced withIgG-negative controls of the same isotype to ensure specificity. All sections were then peroxidase stained using the LSAB 2 kit (DakoCytomation) according to the manufacturer’s protocol. The experiments were reviewed and approved by Harvard Medical Area Standing Committee in Animals.
博論草稿にもESより小さいテラトーマしかできないことが書かれているわね。
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As a result, after 6 weeks ES cells formed a tumor. Spheres did not form tumor as big as ES cells did. We concluded that the proliferative potential of sphere cells was much weaker than that of ES cells (Fig. 13).
