三誌論文には幹細胞化の実験のことは書かれていないからね。まさに8月に提出する以前の
作業に関して語っているんだね。ここはどうなの?
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Letter Fig.2iは2012年にTruseqによるRNA-seqを行い、そのデータに基づいて作成し た系統樹と考えられるが、
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Letter Extended Data Fig.6dは、2012年にSMARTERキットによるRNA-seqを行い、その データをもとに作成した系統樹と考えられるが、
リジェンドは以下だね。
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3. Extended Data Figure 3: Transcriptome analyses of STAP cells shown by heat maps. (494 KB)
a, Heat maps of expression profiles of top-ranked up- and downregulated genes in STAP cells (Oct4-GFP+ clusters converted from CD45+ cells) compared to ES cells. Their respective expression levels in STAP stem cells, trophoblast stem cells and Fgf4-induced stem cells are shown. Absolute expression values are scaled by log2. The genes expressed differentially between ES cells and STAP cells tended to show more similar expression profiles to ES cells in STAP stem cells and Fgf4-induced stem cells than in trophoblast stem cells. Expression of some early endodermal lineage genes such as Gata4 and Sox17 was moderately elevated in STAP cells as compared to ES cells, whereas its biological significance remains elusive (these genes are shown to be strongly expressed in Oct4-GFP-dim cells1). b, Heat maps of expression profiles of top-ranked up- and downregulated genes in ES cells compared to CD45+ cells and their respective expression levels in STAP cells.
The genes expressed differentially between CD45+ and ES cells tended to show similar expression profiles in ES cells and STAP cells. c, Heat maps of expression profiles of representative genes implicated in haematopoietic lineage development in CD45+, ES and STAP cells. No strong correlation was seen between CD45+ cells and STAP cells in their expression profiles (a similar tendency of no correlation was seen for the data in b).
分かりにくいのはaとbとで遺伝子の並びが違うことだね。もう一度本文を見てみよう。
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To investigate the relationship among STAP cells, STAP stem cells, Fgf4-induced stem cells, ES cells and trophoblast stem cells, we performed genome-wide RNA-sequencing analysis (Fig. 2i for dendrogram; Extended Data Figs 3 and 4 for expression analyses of representative genes ; Supplementary Tables 2 and 3 for analysis conditions). Whereas STAP cells formed a cluster with STAP stem cells, Fgf4-induced stem cells, ES cells and trophoblast stem cells and not with the parental CD45+ cells, STAP cells were an outlier to the rest of the cell types in the cluster. In contrast, STAP stem cells were closely clustered with ES cells. Fgf4-induced stem cells formed a cluster with a sub-cluster of ES cells and STAP stem cells, whereas trophoblast stem cells comprised an outlier to this cluster, indicating a close relationship of Fgf4-induced stem cells with these pluripotent cells.
学さんの説明は端折りすぎだね。リジェンドは以下だ。
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j, qPCR analysis of Fgf4-induced cells (cultured under feeder-free conditions) with or without JAK inhibitor (JAKi) treatment for pluripotent marker genes.
k, qPCR analysis of FI-SCs with or without JAK inhibitor (JAKi) treatment for trophoblast marker genes. Values are shown as ratio to the expression level in ES cells (j) or trophoblast stem cells (k). ***P < 0.001; NS, not significant; t-test for each gene between groups with and without JAK inhibitor treatment. n = 3. Statistical significance was all the same with three pluripotency markers. None of the trophoblast marker genes showed statistical significance. Error bars represent s.d.
j,多能性マーカー遺伝子のためのJAK阻害剤(JAKi)処置の有無で分けられた（フィーダー無し条件下で培養された）Fgf4誘導細胞のqPCR分析。
k,栄養膜幹細胞マーカー遺伝子へのJAK阻害剤(JAKi)処置の有無で分けられたFI-SCsのqPCR分析。値はES細胞（j）または栄養膜幹細胞（k）の中の発現レベルに対する比として示されている。 *** P <0.001; NS、重要でない;JAK阻害剤処理の有無によるグループ間の各遺伝子についてのt検定。 N = 3。三つの多能性マーカーの統計的有意性はまったく同じであった。栄養膜幹細胞のマーカー遺伝子のいずれも統計的有意性を示さなかった。エラーバーは±標準偏差を表す。
親分、ここでしょ。
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Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues.
面白くなってきたわね。その件に関する本文は以下ね。
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Previous studies have indicated that inner cell mass (ICM)-type pluripotent cells can be removed from culture by treating the culture with a JAK inhibitor <16>(Extended Data Fig. 5a, b).
Previous studiesの一つが参考文献に挙げられている16番の以下だね。ペンディングしていた奴だ。
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16. Yang, J. et al. Stat3 activation is limiting for reprogramming to ground state pluripotency. Cell Stem Cell 7, 319–328 (2010)
<16. Yang, J. et al. Stat3活性化は、基底多能性への再プログラミングを制限している。 Cell Stem Cell 7, 319–328 (2010)>
その論理が先に示した以下よね。
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Previous studies have indicated that inner cell mass (ICM)-type pluripotent cells can be removed from culture by treating the culture with a JAK inhibitor <16>(Extended Data Fig. 5a, b).
inner cell mass (ICM)-type pluripotent cells can be removed from culture by treating the culture with a JAK inhibitor <16>(Extended Data Fig. 5a, b).って表現は
笹井さんが書いたはずね。
この論文は多能性段階を維持している細胞にJAKiを入れるとその機能が壊れて分化を開始するから
多能性マーカー遺伝子の発現が減少してくるというものね。
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16. Yang, J. et al. Stat3 activation is limiting for reprogramming to ground state pluripotency. Cell Stem Cell 7, 319–328 (2010)
その意味ではレター論文の記述も変だね。原論文では普通のES細胞やEpiSCの他にiPS細胞も使われている。
inner cell mass (ICM)-type pluripotent cellsとは限らない。多能性細胞に関しての研究だ。
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Previous studies have indicated that inner cell mass (ICM)-type pluripotent cells can be removed from culture by treating the culture with a JAK inhibitor <16>(Extended Data Fig. 5a, b).
Fig3
FI細胞をLIF+FBS培地で培養するとOct4、Nanog, SSEA-1を発現してきてES様細胞（STAP幹細胞）となり、このES様細胞は,TSマーカ（インテグリンα７、Eomes９）の発現を失う。
b ES様細胞はES並みの遺伝子発現（Oct4,Nalog,Rex1Kelf24,Sox2,Esrrb・・・）を保つが
TS並みの遺伝子発現（Cdx2、Eomes,、Elf5）を失う。
e f FI細胞はMEKインヒビターを入れて培養するとバラバラになって死んでしまうが、ES細胞は生き残る （TSとFIはFGF-MEKシグナル系に依存しているが、ESは依存していない）。
リジェンドは以下だね。
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a, Culture of Oct4-GFP Fgf4-induced cells in LIF + 20% FBS medium.
b, qPCR analysis of ES-like cells derived from Fgf4-induced cells for pluripotent marker genes (left) and trophoblast marker genes (right). Values are shown as ratio to the expression level in ES cells (left) or trophoblast stem (TS) cells (right).
c, d, Culture of Oct4-GFP Fgf4-induced cells sorted by FACS for strong integrin α7 (Itga7) expression in LIF + 20% FBS medium.
d, Formation frequency (shown by percentage) of Oct4-GFP+ colonies from cells plated on gelatin-coated dishes at a clonal density. **P < 0.01; t-test; n = 3.
e, f, Culture of Oct4-GFP Fgf4-induced cells (dissociated) in LIF + 20% FBS medium with MEK inhibitor. **P < 0.01; NS, not significant; Tukey’s test; n = 3.
e, No substantial formation of Oct4-GFP+ colonies was seen from Fgf4-induced cells in the presence of MEK inhibitor (left), whereas colonies frequently formed when cells were co-plated with Oct4-GFP ES cells (right; plated cells were 1/20 of Fgf4-induced cells).
f, Quantification of colony formation per plated cells (1 × 103 Fgf4-induced cells and/or 1 × 103 ES cells). Unlike Fgf4-induced cells, ES cells formed colonies (regardless of co-plating with FI-SCs) in the presence of MEK inhibitor. Bars and error bars represent mean values and s.d., respectively (b, d, f). Scale bars: 100 μm (a, c, e).
キメラができて初めてその細胞が多能性細胞であったということになるんで、何が
キメラになったのかが分からないのに分かってないものを分からないままにいくら
分析しても意味ないよね。T細胞がキメラになったとは証明されていない。T細胞は
実際FACSで分画されて酸浴細胞になってるんだけどね。
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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).
本文にあるね。
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To confirm further that Fgf4-induced stem cells with a trophoblast-like nature were converted into ES-like cells, rather than just selecting ES-like cells pre-existing in the Fgf4-induced stem cell culture, we examined the effect of the MEK inhibitor PD0325901 on the ES-like cell generation from Fgf4-induced stem cells. Like trophoblast stem cells, Fgf4-induced stem-cell survival is dependent on FGF–MEK signals, and the inhibition of MEK activity caused massive cell death (Extended Data Fig. 6c). However, PD0325901 is also known to be a main effector in 2i medium[17] and to promote ES cell maintenance. Addition of PD0325901 to LIF+FBS-containing medium strongly inhibited the formation of ES-like colonies from Fgf4-induced stem cells (Fig. 3e, left, and Fig. 3f). This inhibition was unlikely to be due to secondary toxic effects from massive cell death of Fgf4-induced stem cells, as colonies formed in the presence of PD0325901 when ES cells were co-plated in the same culture with Fgf4-induced stem cells (Fig. 3e, right, and Fig. 3f).
リジェンドには以下のようにあるわね。
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g, FACS analysis of integrin α7 expression in Fgf4-induced stem cells. Over 40% of Fgf4-induced stem cells strongly expressed both the pluripotency marker Oct4-GFP and the trophoblast marker integrin α7. The bottom panel shows an isotype control for integrin α7 antibody. In ES cells, integrin-α7-expressing cells were less than 0.1% (data not shown; three independent ES cell lines were examined).
