(英文)
We recently discovered an intriguing phenomenon of cellular fate conversion: somatic cells regain pluripotency after experiencing sublethal stimuli such as a low-pH exposure. When splenic CD45+ lymphocytes are exposed to pH 5.7 for 30 min and subsequently cultured in the presence of LIF, a substantial portion of surviving cells start to express the pluripotent cell marker Oct4 (also called Pou5f1) at day 2. By day 7, pluripotent cell clusters form with a bona fide pluripotency marker profile and acquire the competence for three-germ-layer differentiation as shown by teratoma formation. These STAP cells can also efficiently contribute to chimaeric mice and undergo germline transmission using a blastocyst injection assay.
Although these characteristics resemble those of ES cells, STAP cells seem to differ from ES cells in their limited capacity for self-renewal (typically, for only a few passages) and in their vulnerability to dissociation. However, when cultured in the presence of ACTH and LIF for 7 days, STAP cells, at a moderate frequency, further convert into pluripotent ‘stem’ cells that robustly proliferate (STAP stem cells).
(英文)
We recently discovered an intriguing phenomenon of cellular fate conversion: somatic cells regain pluripotency after experiencing sublethal stimuli such as a low-pH exposure. When splenic CD45+ lymphocytes are exposed to pH 5.7 for 30 min and subsequently cultured in the presence of LIF, a substantial portion of surviving cells start to express the pluripotent cell marker Oct4 (also called Pou5f1) at day 2. By day 7, pluripotent cell clusters form with a bona fide pluripotency marker profile and acquire the competence for three-germ-layer differentiation as shown by teratoma formation. These STAP cells can also efficiently contribute to chimaeric mice and undergo germline transmission using a blastocyst injection assay.
Although these characteristics resemble those of ES cells, STAP cells seem to differ from ES cells in their limited capacity for self-renewal (typically, for only a few passages) and in their vulnerability to dissociation. However, when cultured in the presence of ACTH and LIF for 7 days, STAP cells, at a moderate frequency, further convert into pluripotent ‘stem’ cells that robustly proliferate (STAP stem cells).
(英文)
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).
(英文)
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).
このEpiSCはアーティクルのExtended Data Figure 5-d〜hの遺伝子発現解析と免染のみに使われていて、レター論文には使われていない。このエピブラスト幹細胞がなぜ木星リストにも若山さんの持ち出しリストにも無いのかは調査が必要だろうね。これがあったらこの129/Svがcarrying Rosa26-gfpであったか否か、つまりAC129が「僕のマウス」ESであったのとは別のマウスであった可能性を調べることができたろうね。
胎盤の光っていると言われるSTAP由来キメラ写真は
①Extended Data Figure 1-aとb,cの証拠写真(小保方氏B6CAG-GFP x 129/Sv***若山氏2011/11/28 撮影129/Sv x B6GFPの4Nキメラ)
②Figure 1-a,bの写真(小保方氏aはESキメラでbはSTAPキメラ***若山氏aもbもSTAPキメラ、長時間露光は嘘でデジタル増感だと言うが桂調査は否定)
の二つなのね。
(英文)
In quantitative polymerase chain reaction (qPCR) analysis, STAP cells (sorted for strong Oct4-GFP signals) expressed not only pluripotency marker genes but also trophoblast marker genes such as Cdx2 (Fig. 1d and Supplementary Table 1 for primers), unlike ES cells. Therefore, the blastocyst injection result is not easily explained by the idea that STAP cells are composed of a simple mixture of pluripotent cells (Oct4+Cdx2－) and trophoblast-stem-like cells (Oct4－Cdx2+).
In contrast to STAP cells, STAP stem cells did not show the ability to contribute to placental tissues (Fig. 1e, lanes 2–4), indicating that the derivation of STAP stem cells from STAP cells involves the loss of competence to differentiate into placental lineages. Consistent with this idea, STAP stem cells show little expression of trophoblast marker genes (Fig. 1f).
a bは若山氏が違うといった？胎児胎盤の写真
c STAP細胞はキメラの胎盤・卵黄のう形成に寄与するとの棒グラフ（キメラ胎児の６割に貢献）
d STAP細胞は胎児形成能を持つOct4,Nalog,Rex1を発現するが、ESとの比較で示す
およびTSマーカー（Cdx2、Eomes, Elf5）の発現についてTSとの比較で示す
e STAP 幹細胞3種は、胎盤形成に寄与しない。 TSとのGFP細胞の割合の比較
f STAP 幹細胞、ES 細胞は Cdx2、Eomes,、Elf5 マーカーを発現しない。TSとの比較
(英文)
Figure 1: STAP cells contribute to both embryonic and placental tissues in vivo.
a, b, E12.5 embryos from blastocysts injected with ES cells (a) and STAP cells (b). Both cells are genetically labelled with GFP driven by a constitutive promoter. Progeny of STAP cells also contributed to placental tissues and fetal membranes (b), whereas ES-cell-derived cells were not found in these tissues (a). Scale bar, 5.0 mm. c, Percentages of fetuses in which injected cells contributed only to the embryonic portion (red) or also to placental and yolk sac tissues (blue). ***P < 0.001 with Fisher’s exact test. d, qPCR analysis of FACS-sorted Oct4-GFP-strong STAP cells for pluripotent marker genes (left) and trophoblast marker genes (right). Values are shown as ratio to the expression level in ES cells. Error bars represent s.d.
e, Contribution to placental tissues. Unlike parental STAP cells and trophoblast stem (TS) cells, STAP stem cells (STAP-SCs) did not retain the ability for placental contributions. Three independent lines were tested and all showed substantial contributions to the embryonic portions. f, qPCR analysis of trophoblast marker gene expression in STAP stem cells. Error bars represent s.d.
STAP細胞は増殖しないと論文に書かれている。ところがSTAP幹細胞はSTAP細胞を培地誘導してできたと書かれている。それは通常は酸浴細胞製作時に記載されているSTAP細胞が自己増殖しないという記載が間違いだということを意味するはずだ。
>>アーティクル論文
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.
>>レター論文
Although these characteristics resemble those of ES cells, STAP cells seem to differ from ES cells in their limited capacity for self-renewal (typically, for only a few passages) and in their vulnerability to dissociation. However, when cultured in the presence of ACTH and LIF for 7 days, STAP cells, at a moderate frequency, further convert into pluripotent ‘stem’ cells that robustly proliferate (STAP stem cells).
僕はあれは暗喩だと思う。彼はどこかの時点でかあの暗喩を論文の中に滑り込ませたんだ。それは彼がこれはいけないと内心思っていたことを証明していると思うよ。彼は12/28にアーティクル論文を書き上げた時はまだ気づいていなかったと思う。3/10に小保方さんがメール投稿する前までのどこかではないかな。培養によってSTAP細胞の性質が変わったからSTAP幹細胞由来キメラの胎盤は光らないという論旨を作らないで、方や自己増殖しない、方や自己増殖するというあの両論文を矛盾なく結びつけることはできない。ところが、その変わったという証明はキメラでの実際の確認ではなく遺伝子発現解析で代替されている。笹井さんほどの論理家が、そんな矛盾に気づかなかったはずはないよ。
>>
Thus, our findings indicate that epigenetic fate determination of mammalian cells can be markedly converted in a context-dependent manner by strong environmental cues.
まともな論文なら何を置いてもまずSTAP幹細胞由来キメラの胎盤は光らないというコントロールを示さないといけないのよね。遺伝子解析なんて、そもそもキメラの本物があったらどうしてそんな補完証明が必要なのよってことね。元が無いから補完にもなってない。元は言葉だけなのよね。in a context-dependent manner だわね。
