何度でも貼り付けるわ。
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However, as Fgf4-induced stem cells lay between STAP stem cells and trophoblast stem cells in the dendrogram, the possibility of contamination of STAP stem cells in the Fgf4-induced stem-cell population cannot be ruled out. 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). In contrast, the JAK inhibitor treatment had no substantial effect on Oct4-GFP expression in Fgf4-induced stem-cell culture (Extended Data Fig. 5c, d; see Extended Data Fig. 5e, f for control). Expression of neither pluripotency markers (Fig. 2j) nor trophoblast markers (Fig. 2k) was substantially affected, indicating that pluripotency marker expression is unlikely to reflect contaminating STAP stem cells (ICM-type). Consistent with this idea, Fgf4-induced stem cells that were strongly positive for the trophoblast marker Itga7 (a surface marker for trophoblasts but not ES cells) also expressed high levels of Oct4-GFP (Extended Data Fig. 5g).
STAP幹細胞のコンタミと書いてあるよな。ESのコンタミだとは書いてないね。
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However, as Fgf4-induced stem cells lay between STAP stem cells and trophoblast stem cells in the dendrogram, the possibility of contamination of STAP stem cells in the Fgf4-induced stem-cell population cannot be ruled out.
しかしながら、Fgf4誘導幹細胞は、樹状図においてSTAP幹細胞と栄養膜幹細胞との間に位置しているので、Fgf4誘導幹細胞集団におけるSTAP幹細胞の混入の可能性を排除できない。
(Extended Data Fig. 5a, b)はOct4-GFP挿入ESの培地にJAKiを添加した実験だね。
結果は添加後GFP蛍光が消える。つまり多能性を失う。先行する論文の論旨に従えば、
多能性を維持していたJAK(ヤーヌスキナーゼ)が分解されて細胞が分化段階に
入ったということだね。
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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).
先行する研究では、内部細胞塊（ICM)タイプの多能性細胞は、JAK阻害剤で培養を処理することにより培養物から除去することができることを示した（拡張データ図5a,b）。
本文は以下の部分かしらね。
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Expandable pluripotent cell lines from STAP cells•
STAP cells have a limited self-renewal capacity under the conditions used for establishment (Fig. 2g and Extended Data Figs 2e and 5a). However, in the context of the embryonic environment, a small fragment of a STAP cell cluster could grow even into a whole embryo (Fig. 4f). With this in mind, we next examined whether STAP cells have the potential to generate expandable pluripotent cell lines in vitro under certain conditions.
(続き)
STAP cells could not be efficiently maintained for additional passages in conventional LIF+FBS-containing medium or 2i medium20 (most STAP cells died in 2i medium within 7 days; Extended Data Fig. 8a). Notably, an adrenocorticotropic hormone (ACTH)+LIF-containing medium (hereafter called ACTH medium) known to facilitate clonal expansion of ES cells36 supported outgrowth of STAP cell colonies. When cultured in this medium on a MEF feeder or gelatin, a portion of STAP cell clusters started to grow (Fig. 5a, bottom; such outgrowth was typically found in 10–20% of wells in single cluster culture using 96-well plates and in >75% when 12 clusters were plated per well). These growing colonies looked similar to those of mouse ES cells and expressed a high level of Oct4-GFP.
(続き)
After culturing in ACTH medium for 7 days, this growing population of cells, unlike parental STAP cells, could be passaged as single cells (Fig. 5a, bottom, and Fig. 5b), grow in 2i medium (Extended Data Fig. 8a) and expand exponentially, up to at least 120 days of culture (Fig. 5c; no substantial chromosomal abnormality was seen; Extended Data Fig. 8b, c). Hereafter, we refer to the proliferative cells derived from STAP cells as STAP stem cells.
(続き)
STAP stem cells expressed protein and RNA markers for pluripotent cells (Fig. 5d, e), showed low DNA methylation levels at the Oct4 and Nanog loci (Extended Data Fig. 8d), and had a nuclear fine structure similar to that of ES cells (Extended Data Fig. 8e; few electron-dense areas corresponding to heterochromatin). In differentiation culture25, 26, 27, STAP stem cells generated ectodermal, mesodermal and endodermal derivatives in vitro (Fig. 5f–h and Extended Data Fig. 8f, g), including beating cardiac muscles (Supplementary Video 4), and formed teratomas in vivo (Fig.5i and Extended Data Fig. 8h; no teratocarcinomas, n = 40). After blastocyst injection, STAP stem cells efficiently contributed to chimaeric mice (Fig. 5j), in which germline transmission was seen (Extended Data Fig. 8i). Even in tetraploid complementation assays, injected STAP stem cells could generate mice capable of growing to adults and producing offspring (Fig. 5k, l; in all eight independent lines, Extended Data Fig. 8j).
(続き)
In addition to their expandability, we noticed at least two other differences between STAP stem cells and parental STAP cells. First, the expression of the ES cell marker protein Esrrβ, which was undetectable in STAP cells (Extended Data Fig. 5d, e), was clearly seen in STAP stem cells (Fig. 5e). Second, the presence of H3K27me3 foci, which was found in a substantial proportion of female STAP cells, was no longer observed in STAP stem cells (Extended Data Figs 5f and 8k). Thus, STAP cells have the potential to give rise to expandable cell lines that exhibit features similar to those of ES cells.
マテメソは以下だね。
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STAP stem-cell conversion culture
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.
For clonal analysis of STAP stem cells, single STAP stem cells were manually picked by a thin-glass pipette, and plated into 96-well plates at one cell per well. The clonal colonies were cultured in ES medium containing 20% FBS, and expanded for subsequent experiments.
Article Figure 6とExtended Data Figure 8のリジェンドは以下だ。
(Article Figure 6)
a, Growth of STAP stem cells carrying Oct4-gfp. Scale bar, 50 μm. b, Dissociation culture of STAP stem cells to form colonies. Scale bar, 100 μm. c, Robust growth of STAP stem cells in maintenance culture. Similar results were obtained with eight independent lines. In contrast, parental STAP cells decreased in number quickly. d, Immunostaining of STAP stem cells for pluripotency markers (red). Scale bar, 50 μm. e, qPCR analysis of pluripotency marker gene expression. f–h, In vitro differentiation assays into three-germ-layer derivatives.
f, Ectoderm: Rx+/Pax6+ (retinal epithelium; 83%, n = 6). g, Mesoderm: troponin-T+ (cardiac muscle; 50%, n = 6). h, Endoderm: Sox17+/E-cadherin+ (endodermal progenitors; 67%, n = 6). Scale bar, 50 μm. i, Teratoma formation assays. Formation of keratinized epidermis (ectoderm; left), cartilage (mesoderm; middle) and bronchial-like epithelium (endoderm; right) is shown. Scale bar, 100 μm. j, Blastocyst injection assays. These pictures of live animals were taken serially (asterisk indicates the same chimaeric pup). k, l, Tetraploid complementation assay. ‘All-GFP+’ pups were born (k) and germline transmission was observed (l).
(Extended Data Figure 8)
a, Compatibility of 2i conditions with STAP stem-cell derivation from STAP cells and STAP stem-cell maintenance. STAP stem cells could not be established directly from STAP cells in 2i + LIF medium (top). However, once established in ACTH medium, STAP stem cells were able to survive and expand in 2i + LIF medium. Scale bar, 100 μm. b, Q-band analysis (n = 4; all cell lines showed the normal karyotype). c, Multicolour FISH analysis (n = 8; all cell lines showed the normal karyotype) of STAP stem cells. d, Methylation status of the Oct4 and Nanog promoters. e, Electron microscope analysis of STAP stem cells. Scale bar, 1 μm. f, g, Beating cardiac muscle (mesoderm; 38%, n = 8). Red line indicates an analysed region for kymograph (g).
h, Clonability of STAP stem cells. Clonal expansion from single STAP stem cells was performed. Pluripotency of clonal cell lines was confirmed by teratoma formation assay, showing the formation of neuroectoderm (left), muscle tissue (middle) and bronchial-like epithelium (right). Scale bar, 100 μm. i, Production of chimaeric mice from STAP stem-cell lines using diploid embryos. *These STAP stem-cell lines were generated from independent STAP cell clusters. j, Production of mouse chimaeras from STAP stem-cell lines by the tetraploid complementation method. *These STAP stem-cell lines were generated from independent STAP cell clusters. k, No H3K27me3-dense foci are seen in female STAP stem cells (n = 50; the CD45+ cell is a positive control). Scale bar, 10 μm.
これだけのものの移管が行われていて、その後処理として小保方さんが論文同士の整合を取ってるんでしょうね。笹井さんはそこまでは忙しすぎてやってないんじゃないかしら。STAP cells have a limited self-renewal capacity under the conditions used for establishment (Fig. 2g and Extended Data Figs 2e and 5a). のExtended Data Figs 2e ってTCRの説明よね。そこ全部変ね。
(続き)小保方さんは論文では主にリンパ球を使ってSTAP細胞を作っています。TCR再構成の実験をするときだけはCD90でもう一度T細胞だけをFACS選別します。でも厳格な選別ではないから、丹羽さんがヘテロな集団だと言ってるんですね。
マウスの白血球のもともとの構成割合はB細胞:60%、T細胞:30%、マクロファージ:5%、その他、5%です。脾臓の構成細胞全体での細胞の大きさは5から10マイクロメーターです。
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David Aphkhazava
23.19
Size of mouse splenocytes?
Does anyone know what the size of a mouse splenocyte is?
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Joseph michael Cantor
University of California, San Diego
5-10 um on average, with resting B and T cells comprising the lower end of the range and macrophages on the upper end. Adult mouse spleens typically contain 1x10e8 white blood cells: 50-60% B cells, 30% T cells, 5% macrophages, and <5% others.
