トコトンやりましょう。
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Retraction: Stimulus-triggered fate conversion of somatic cells into pluripotency
Haruko Obokata, Teruhiko Wakayama, Yoshiki Sasai, Koji Kojima, Martin P. Vacanti, Hitoshi Niwa, Masayuki Yamato & Charles A. Vacanti
Several critical errors have been found in our Article and Letter (dx.doi.org/10.1038/nature12969), which led to an in-depth investigation by the RIKEN Institute. The RIKEN investigation committee has categorized some of the errors as misconduct (see Supplementary Data 1 and Supplementary Data 2). Additional errors identified by the authors that are not discussed in RIKEN’s report are listed below.
(1) Figure 1a and b in the Letter both show embryos generated from STAP cells, not a comparison of ES- and STAP-derived chimaeric embryos, as indicated in the legend.
(2) Extended Data Fig. 7d in the Article and Extended Data Fig. 1a in the Letter are different images of the same embryo and not, as indicated in the legends, a diploid chimaera embryo and tetraploid chimaera embryo.
(3) There is an erroneous description in Fig. 1a in the Letter. The right panel of Fig. 1a is not a ‘long exposure’ image at the camera level but a digitally enhanced one.
(4) In Fig. 4b of the Letter, STAP cell and ES cell are wrongly labelled in a reverse manner.
(5) In the Article, one group of STAP stem cells (STAP-SCs) was reported as being derived from STAP cells induced from spleens of F1 hybrids from the cross of mouse lines carrying identical cag-gfp insertions in chromosome 18 in the background of 129/Sv and B6, respectively, and that they were maintained in the Wakayama laboratory. However, further analysis of the eight STAP-SC lines indicates that, while sharing the same 129×B6 F1 genetic background, they have a different GFP insertion site. Furthermore, while the mice used for STAP cell induction are homozygous for the GFP transgene, the STAP-SCs are heterozygous. The GFP transgene insertion site matches that of the mice and ES cells kept in the Wakayama laboratory. Thus, there are inexplicable discrepancies in genetic background and transgene insertion sites between the donor mice and the reported STAP-SCs.
We apologize for the mistakes included in the Article and Letter. These multiple errors impair the credibility of the study as a whole and we are unable to say without doubt whether the STAP-SC phenomenon is real. Ongoing studies are investigating this phenomenon afresh, but given the extensive nature of the errors currently found, we consider it appropriate to retract both papers.
(1) Figure 1a and b in the Letter both show embryos generated from STAP cells, not a comparison of ES- and STAP-derived chimaeric embryos, as indicated in the legend.
(3) There is an erroneous description in Fig. 1a in the Letter. The right panel of Fig. 1a is not a ‘long exposure’ image at the camera level but a digitally enhanced one.
桂報告じゃなくてリトラクト理由書だね。
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(2) Extended Data Fig. 7d in the Article and Extended Data Fig. 1a in the Letter are different images of the same embryo and not, as indicated in the legends, a diploid chimaera embryo and tetraploid chimaera embryo.
ここね。
>>
Figure 2. SNPs detected in FI-SC mRNAs indicating contamination. (A) Allele distributions obtained from ESC and FI-SC RNA-seq experiments used in the STAP paper. Both ESCs (blue) and FI-SCs (red) are annotated as having a 129B6F1 genetic background. The number of applied SNPs for each experiment is shown in parentheses in the boxes. (B) SNPs detected in Sall4 and Klf4, which are highly expressed in ESCs. B6-type alleles are shown in blue and 129-type alleles (i.e., non-B6) are in yellow. (C) SNPs detected in the TSC-specific genes Elf5 and Sox21. (D) The number of homozygous/heterozygous SNPs observed in the stem cells used in the original paper. Only the composition observed in FI-SCs would be predicted to affect gene expression. P-values were calculated using Fisher's exact test of genotype distribution between TSC-specific genes and ESC-specific genes. Rep1 and rep2 denote two replicated experiments. (E) Heatmap of representative cytokine and extracellular matrix genes that are highly expressed in MEFs. Normalized log ratios of fragments per kilobase of exon per million reads (FPKM) against the medians of all samples were shown.
隣に論文貼ってくれた人がいるね。
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J Biol Chem. 2015 Dec 11;290(50):30152-62. doi: 10.1074/jbc.M115.659094. Epub 2015 Oct 21.
Gene Expression Profiling Reveals a Novel Regulatory Role for Sox21 Protein in Mouse Trophoblast Stem Cell Differentiation.
アブストだけだけど。
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Abstract
Appropriate self-renewal and differentiation of trophoblast stem cells (TSCs) are key factors for proper placental development and function and, in turn, for appropriate in utero fetal growth. To identify novel TSC-specific genes, we performed genome-wide expression profiling of TSCs, embryonic stem cells, epiblast stem cells, and mouse embryo fibroblasts, derived from mice of the same genetic background. Our analysis revealed a high expression of Sox21 in TSCs compared with other cell types. Sox21 levels were high in undifferentiated TSCs and were dramatically reduced upon differentiation. In addition, modulation of Sox21 expression in TSCs affected lineage-specific differentiation, based on both marker analysis and functional assessment. Our results implicate Sox21 specifically in the promotion of spongiotrophoblast and giant cell differentiation and establish a new mechanism through which trophoblast sublineages are specified.
あ、そうね。頼んどくわ。
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J Biol Chem. 2015 Dec 11;290(50):30152-62. doi: 10.1074/jbc.M115.659094. Epub 2015 Oct 21.
Gene Expression Profiling Reveals a Novel Regulatory Role for Sox21 Protein in Mouse Trophoblast Stem Cell Differentiation.
Moretto Zita M1, Soncin F1, Natale D2, Pizzo D1, Parast M1.
Author information
Abstract
Appropriate self-renewal and differentiation of trophoblast stem cells (TSCs) are key factors for proper placental development and function and, in turn, for appropriate in utero fetal growth. To identify novel TSC-specific genes, we performed genome-wide expression profiling of TSCs, embryonic stem cells, epiblast stem cells, and mouse embryo fibroblasts, derived from mice of the same genetic background. Our analysis revealed a high expression of Sox21 in TSCs compared with other cell types. Sox21 levels were high in undifferentiated TSCs and were dramatically reduced upon differentiation. In addition, modulation of Sox21 expression in TSCs affected lineage-specific differentiation, based on both marker analysis and functional assessment. Our results implicate Sox21 specifically in the promotion of spongiotrophoblast and giant cell differentiation and establish a new mechanism through which trophoblast sublineages are specified.
和モガさんは理系だとは言え素人だからね。自分で勉強されてブログに書いているだけだからね。
遠藤さんとは違うよね。正式に論文になって採用されている。その論文に<(C) SNPs detected
in the TSC-specific genes Elf5 and Sox21. >と書いたからには、これは公共データベースを
見たまんまというわけには行かないんじゃないのかな。
学さんはMariなる人物がノフラーブログに自分のブログを紹介したために
彼が覗くであろうことを想定して英語でブログを書いたようだね。
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She wrote in her book that she used the mice just she was given from Dr Wakayama without knowing its strain, therefore, she did not check its strain at all.
その前に、この問題はややこしい経緯があるよな。若山さんはこのLetterの表の
ESとSTAPが逆になってると取り下げ理由の一つに挙げている。
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(4) In Fig. 4b of the Letter, STAP cell and ES cell are wrongly labelled in a reverse manner.
注意深く追ってもらったら分かるが、11次元がティシュー論文で捏造操作指摘しているのは
先ほどから言ってるGIG.2と3の逆転写PCRの結果表だけだ。二つとも左端にES細胞が
コントロールとしておかれている。それに対して、2は骨髄からのスフイアの三つのサンプル比較が
おかれていて、3の方は脊髄、筋肉、肺の組織からのスフイアが置かれているんだ。
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FIG. 2. Gene expression profiles of cell contained in bone marrow spheres. Gene expression of each bone marrow sphere was analyzed by reverse transcription-polymerase chain reaction. Gene expression of cells in each bone marrow sphere was compared to those of embryonic stem cells.
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FIG. 3. Gene expression profiles of oct4-expressing cells in spheres from each tissue. Spinal spheres (SS), myospheres (MS), and pneumospheres (PS) at day 5 were compared to those of embryonic stem cells.
①Increasing the Cell Number of Host Tetraploid Embryos Can Improve the Production of Mice Derived from Embryonic Stem Cells
Hiroshi Ohta Yuko Sakaide Kazuo Yamagata Teruhiko Wakayama
Published: 01 September 2008
②Generation of Normal Progeny by Intracytoplasmic Sperm Injection Following Grafting of Testicular Tissue from Cloned Mice That Died Postnatally
Hiroshi Ohta Teruhiko Wakayama
Published: 01 September 2005
③Establishment of Male and Female Nuclear Transfer Embryonic Stem Cell Lines from Different Mouse Strains and Tissues
Sayaka Wakayama Hiroshi Ohta Satoshi Kishigami Nguyen Van Thuan Takafusa Hikichi Eiji Mizutani Masashi Miyake Teruhiko Wakayama
Published: 01 April 2005
the Molecular Biology Society of Japan=日本分子生物学会
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Genes to Cells is a peer-reviewed scientific journal that publishes original research on the molecular mechanisms of biological events. The journal has been published since 1996 by Wiley-Blackwell on behalf of the Molecular Biology Society of Japan.[1][2]
了解。私の早とちりよ。あなたのコメントは以下ね。
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現京都大学研究者の神戸理研CDB時代の若山研での2008年論文2本のタイトル
Generation of mice derived from embryonic stem cells using blastocysts different developmental ages
もう一本は
Increasing the Cell Nunber of Host Tetraploid Embryos Can Improve the Production of mice Derived from Embryonic stem Cells
です。確認下さいませ。
2005年論文もあります。
2018/4/8(日) 午前 3:08[ Ooboe ]返信する
我々の知っている論文は①②③です。今④が加わった。これが知りたかった論文です。
①Increasing the Cell Number of Host Tetraploid Embryos Can Improve the Production of Mice Derived from Embryonic Stem Cells
Hiroshi Ohta Yuko Sakaide Kazuo Yamagata Teruhiko Wakayama
Published: 01 September 2008
②Generation of Normal Progeny by Intracytoplasmic Sperm Injection Following Grafting of Testicular Tissue from Cloned Mice That Died Postnatally
Hiroshi Ohta Teruhiko Wakayama
Published: 01 September 2005
③Establishment of Male and Female Nuclear Transfer Embryonic Stem Cell Lines from Different Mouse Strains and Tissues
Sayaka Wakayama Hiroshi Ohta Satoshi Kishigami Nguyen Van Thuan Takafusa Hikichi Eiji Mizutani Masashi Miyake Teruhiko Wakayama
Published: 01 April 2005
④Generation of mice derived from embryonic stem cells using blastocysts of different developmental ages.
Ohta H1, Sakaide Y, Wakayama T.
Reproduction. 2008 Nov;136(5):581-7. doi: 10.1530/REP-08-0184. Epub 2008 Aug 29.
えっとねえ、このことはずいぶん後になって寂聴さんとの対談の中に
小保方さんの言葉として出てくるね。
>>
O 先生は、この本の中に恋愛がないとおっしゃいましたが、私の恋愛対象が研究だったとは思われませんでしたか?
S ええ、思います。
O 『あの日』は失恋の物語です。何よりも愛していたものを失った、失恋の物語として私は書きました。
S 相手が物も言ってくれないからね。でも、失恋は必ずするんですよ、みんな。また恋愛は生まれます。
O 愛した相手が、あまりにも美しく、大きく・・・・・。
S ちらっと見たのよね。
O でも、心を許してくれなかった。閉じられてしまいましたね。まさに失恋です。
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 50mL tube. Hank’s balanced salt solution (20mL) was added to each tube and subsequently centrifuged at 800rpm for 3min. The supernatant was discarded and the pellet was resuspended in 50mL of DMEM with 10% fetal bovine serum. This solution was seeded onto a sheet 3x3x1mm, composed of a nonwoven mesh of polyglycolic acid fibers, 200mm in diameter, and implanted subcutaneously into the dorsal flanks of a 4-week-old NOD/SCID mice (Jackson Laboratory).
つづき
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 4mm 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 with IgG-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.
つづき
Differentiation potential of cells in vivo
When implanted subcutaneously into NOD/SCID mice, spheres generated from cells procured from each of the three germ layers demonstrated the potential to form tissue-like teratoma containing cells representative of all three germ layers. The tissues generated were encapsulated and easily resected. Each explant was *25mm3 (Fig. 6A). Individual explants contained cells representative of all three germ layers. Tissue generated from spinal spheres contained nerve (ectoderm; Fig. 6Bi, Bii), muscle (mesoderm; Fig. 6Biii, Biv), and duct-like tissue (endoderm; Fig. 6Bv, Bvi). Tissue generated from myospheres contained epithelium (ectoderm; Fig. 6Ci, Cii), muscle (mesoderm; Fig. 6Ciii, Civ), and ductlike tissue (endoderm; Fig. 6Cv, Cvi). Tissue generated from pneumospheres contained epithelium (ectoderm; Fig. 6Di, Dii), cartilage (mesoderm; Fig. 6Diii, Div), and gland (ectoderm; Fig. 6Dv, Dvi). Specific tissues were identified using immunohistochemical techniques. Nerves were identified using beta III-tubulin, epithelium identified using pancytokeratin, and muscle identified using desmin and myosin. Duct-like structures and gland were identified using FOXA2.
The article also describes teratomas derived from Oct4-GFP STAPcells as evidence for pluripotency (Fig. 2 and Extended Data Fig. 4 inref. 1).
We found a glass slide specimen from which all these teratomaimages were taken, and its corresponding paraffin block.
Quantitative PCR of genomic DNA extracted from this paraffin block reproducibly indicated that these teratoma tissues formed from FES1-derived cells(Fig. 1d, e).
Immunostaining revealed that intestinal epithelium tissue(Fig. 2e, right in ref. 1) and pancreatic tissue (Extended Data Fig. 4c inref. 1),
shown as teratomas from STAP cells1, were GFP-negative and,thus, of host mouse origin (Fig. 1f–h).
The article also describes teratomas derived from Oct4-GFP STAPcells as evidence for pluripotency (Fig. 2 and Extended Data Fig. 4 inref. 1).
We found a glass slide specimen from which all these teratomaimages were taken, and its corresponding paraffin block.
Quantitative PCR of genomic DNA extracted from this paraffin block reproducibly indicated that these teratoma tissues formed from FES1-derived cells(Fig. 1d, e).
Immunostaining revealed that intestinal epithelium tissue(Fig. 2e, right in ref. 1) and pancreatic tissue (Extended Data Fig. 4c inref. 1),
shown as teratomas from STAP cells1, were GFP-negative and,thus, of host mouse origin (Fig. 1f–h).
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 50mL tube. Hank’s balanced salt solution (20mL) was added to each tube and subsequently centrifuged at 800rpm for 3min. The supernatant was discarded and the pellet was resuspended in 50mL of DMEM with 10% fetal bovine serum. This solution was seeded onto a sheet 3x3x1mm, composed of a nonwoven mesh of polyglycolic acid fibers, 200mm in diameter, and implanted subcutaneously into the dorsal flanks of a 4-week-old NOD/SCID mice (Jackson Laboratory).
明日は釣りだ。
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 4mm 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 with IgG-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.
明日は釣りだ。
Differentiation potential of cells in vivo
When implanted subcutaneously into NOD/SCID mice, spheres generated from cells procured from each of the three germ layers demonstrated the potential to form tissue-like teratoma containing cells representative of all three germ layers. The tissues generated were encapsulated and easily resected. Each explant was *25mm3 (Fig. 6A). Individual explants contained cells representative of all three germ layers. Tissue generated from spinal spheres contained nerve (ectoderm; Fig. 6Bi, Bii), muscle (mesoderm; Fig. 6Biii, Biv), and duct-like tissue (endoderm; Fig. 6Bv, Bvi). Tissue generated from myospheres contained epithelium (ectoderm; Fig. 6Ci, Cii), muscle (mesoderm; Fig. 6Ciii, Civ), and ductlike tissue (endoderm; Fig. 6Cv, Cvi). Tissue generated from pneumospheres contained epithelium (ectoderm; Fig. 6Di, Dii), cartilage (mesoderm; Fig. 6Diii, Div), and gland (ectoderm; Fig. 6Dv, Dvi). Specific tissues were identified using immunohistochemical techniques. Nerves were identified using beta III-tubulin, epithelium identified using pancytokeratin, and muscle identified using desmin and myosin. Duct-like structures and gland were identified using FOXA2.
明日は釣りだ。
Discussion
・・・
Although the spheres in this study described were capable of generating teratoma-like tissue, the transplanted cells did not form large teratomas as did ES cells, nor they did express Eras in vitro as ESCs,31 which suggests that the teratoma-like tissue they generate may be very different from true teratomas generated from ESCs. In addition, the cells studied did not express the trophectoderm marker Cdx232 or also associated with ESCs. These differences of gene expression pattern may explain the differences of the biological function between ESCs and adult stem cells in this study.
・・・
翻訳機君、頼んどくぜ。
明日は我々は早くに釣りだ。小保方さんはESのテラトーマも作ったことがあってその大きさの違いを知ってる。
12/27Harukoを切り出した時はおどろいたろうな。くひひひひひ。
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the transplanted cells did not form large teratomas as did ES cells
はい、はい、機械の癖に大口叩いていないで、とっとと仕事おし。
>>
Materials and Methods
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 50mL tube. Hank’s balanced salt solution (20mL) was added to each tube and subsequently centrifuged at 800rpm for 3min. The supernatant was discarded and the pellet was resuspended in 50mL of DMEM with 10% fetal bovine serum. This solution was seeded onto a sheet 3x3x1mm, composed of a nonwoven mesh of polyglycolic acid fibers, 200mm in diameter, and implanted subcutaneously into the dorsal flanks of a 4-week-old NOD/SCID mice (Jackson Laboratory).
はい、翻訳機君。
>>
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 4mm 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 with IgG-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.
細かいことは後にして、続きだわ。
>>
Differentiation potential of cells in vivo
When implanted subcutaneously into NOD/SCID mice, spheres generated from cells procured from each of the three germ layers demonstrated the potential to form tissue-like teratoma containing cells representative of all three germ layers. The tissues generated were encapsulated and easily resected. Each explant was *25mm3 (Fig. 6A). Individual explants contained cells representative of all three germ layers. Tissue generated from spinal spheres contained nerve (ectoderm; Fig. 6Bi, Bii), muscle (mesoderm; Fig. 6Biii, Biv), and duct-like tissue (endoderm; Fig. 6Bv, Bvi). Tissue generated from myospheres contained epithelium (ectoderm; Fig. 6Ci, Cii), muscle (mesoderm; Fig. 6Ciii, Civ), and ductlike tissue (endoderm; Fig. 6Cv, Cvi). Tissue generated from pneumospheres contained epithelium (ectoderm; Fig. 6Di, Dii), cartilage (mesoderm; Fig. 6Diii, Div), and gland (ectoderm; Fig. 6Dv, Dvi). Specific tissues were identified using immunohistochemical techniques. Nerves were identified using beta III-tubulin, epithelium identified using pancytokeratin, and muscle identified using desmin and myosin. Duct-like structures and gland were identified using FOXA2.
Although the spheres in this study described were capable of generating teratoma-like tissue, the transplanted cells did not form large teratomas as did ES cells, nor they did express Eras in vitro as ESCs,[31] which suggests that the teratoma-like tissue they generate may be very different from true teratomas generated from ESCs. In addition, the cells studied did not express the trophectoderm marker Cdx2[32 ]or also associated with ESCs. These differences of gene expression pattern may explain the differences of the biological function between ESCs and adult stem cells in this study.
参考文献の31と32は以下よ。
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31. Takahashi, K., Mitsui, K., and Yamanaka, S. Role of ERas in promoting tumour-like properties in mouse embryonic stem cells. Nature 423, 541, 2003.
32. Ralston, A., and Rossant, J. Genetic regulation of stem cell origins in the mouse embryo. Clin Genet 68, 106, 2005.
