どうもな。
CD45+ cells were sorted by fluorescence-activated cell sorting (FACS) from the lymphocyte fraction of postnatal spleens (1-week old) of C57BL/6 mice carrying an Oct4-gfp transgene, and were exposed to various types of strong, transient, physical and chemical stimuli (described below). We examined these cells for activation of the Oct4 promoter after culture for several days in suspension using DMEM/F12 medium supplemented with leukaemia inhibitory factor (LIF) and B27 (hereafter called LIF+B27 medium).
まあ、それは別問題として
Among the various perturbations, we were particularly interested in low-pH perturbations for two reasons. First, as shown below, low-pH treatment turned out to be most effective for the induction of Oct4. Second, classical experimental embryology has shown that a transient low-pH treatment under ‘sublethal’ conditions can ヘンリー・フォンダ
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alter the differentiation status of tissues. Spontaneous neural conversion from salamander animal caps by soaking the tissues in citrate-based acidic medium below pH 6.0 has been demonstrated previously.
そのへんもな。何しろ塩酸でもＡＴＰでもできなかった。
Without exposure to the stimuli, none of the cells sorted with CD45 expressed Oct4-GFP regardless of the culture period in LIF+B27 medium. In contrast, a 30-min treatment with low-pH medium (25-min incubation followed by 5-min centrifugation; Fig. 1a; the most effective range was pH 5.4–5.8; Extended Data Fig. 1a) caused the emergence of substantial numbers of spherical clusters that expressed Oct4-GFP in day-7 culture (Fig. 1b). Substantial numbers of GFP+ cells appeared in all cases performed with neonatal splenic cells (n = 30 experiments).
ま、先に進もうかな。
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).
このまま放置は出来ないと思うよ。
Among maintenance media for pluripotent cells, the appearance of Oct4-GFP+ cells was most efficient in LIF+B27 medium, and did not occur in mouse epiblast-derived stem-cell (EpiSC) medium (Extended Data Fig. 1e). The presence or absence of LIF during days 0–2 did not substantially affect the frequency of Oct4-GFP+ cell generation on day 7 (Extended Data Fig. 1f), whereas the addition of LIF during days 4–7 was not sufficient, indicating that LIF dependency started during days 2–4.
＜and did not occur in mouse epiblast-derived stem-cell (EpiSC) medium＞って
丹羽さんの研究論文関連なんだけど何のためにここに書き込んでるのかと疑わせるよね。
LIF+B27 でいいんでしょ。後は飾りの知識だよね。なんかここに引っ張り出して、批判使用と
いうわけでもない。ただ知ってるぞと。
何度も解説された場所だね。
Most of the surviving cells on day 1 were still CD45+ and Oct4-GFP−. On day 3, the total cell numbers were reduced to between one-third to one-half of the day 0 population (Fig. 1d; see Extended Data Fig. 1g, h for apoptosis アナリシス), and a substantial number of total surviving cells became Oct4-GFP+ (Fig. 1d), albeit with relatively weak signal intensity. On day 7, a significant number of Oct4-GFP+CD45− cells (one-half to two-thirds of total surviving cells) constituted a distinct population from the Oct4-GFP−CD45− cells (Fig. 1c, top, day 7, and Fig. 1d). No obvious generation of Oct4-GFP+CD45− populations was seen in non-treated CD45+ cells cultured similarly but without low-pH treatment (Fig. 1c, bottom).
そうだな。次がこの実験が笹井さんの追試だということを証明してる場所だね。
Low-pH-treated CD45+ cells, but not untreated cells, gradually turned on GFP signals over the first few days (Fig. 1e, Supplementary Videos 1 and 2 and Extended Data Fig. 2a), whereas CD45 immunoreactivity became gradually reduced in the cells that demonstrated Oct4-GFP expression (Fig. 1f and Extended Data Fig. 2b). By day 5, the Oct4-GFP+ cells attached together and formed clusters by accretion. These GFP+ clusters (but not GFP− cells) were quite mobile and often showed cell processes on moving (Supplementary Video 1).
＜These GFP+ clusters (but not GFP− cells) were quite mobile and often showed cell processes on moving (Supplementary Video 1).＞ってとこ
我々素人でも変だと思ったよね。動いてますって、直ぐに次に浮かぶ疑問は
どうして動くのってことでしょ。物理学の力って4つの力とその応用以外にないって
誰でも知ってる。
The Oct4-GFP+ cells demonstrated a characteristic small cell size with little cytoplasm and also showed a distinct fine structure of the nucleus compared with that of parental CD45+ lymphocytes (Fig. 1g). The Oct4-GFP+ cells on day 7 were smaller than non-treated CD45+ cells (Fig. 1g, h and Extended Data Fig. 2c) and embryonic stem (ES) cells (Fig. 1h), both of which are generally considered to be small in size. The diameter of low-pH-treated CD45+ cells became reduced during the first 2 days, even before they started Oct4-GFP expression (Fig. 1f), whereas the onset of GFP expression was not accompanied by cell divisions. Consistent with this, no substantial 5-ethynyl-2′-deoxyuridine (EdU) uptake was observed in the Oct4-GFP+ cells after the stressor (Extended Data Fig. 2d).
このあたりは笹井自らが確認したしじつなんだね。それが本当はなんであったかは別として。
それは相沢検証で行なわれたこととの矛盾点の検討課題を生み出すことになるね。
The lack of substantial proliferation argues against the possibility that CD45− cells, contaminating as a very minor population in the FACS-sorted CD45+ cells, quickly grew and formed a substantial Oct4-GFP+ population over the first few days after the low-pH treatment. In addition, genomic rearrangements of Tcrb (T-cell receptor gene) were observed in Oct4-GFP+ cells derived from FACS-purified CD45+ cells and CD90+CD45+ T cells (Fig. 1i, lanes 4, 5, and Extended Data Fig. 2e–g), indicating at least some contribution from lineage-committed T cells. Thus, Oct4-GFP+ cells were generated de novo from low-pH-treated CD45+ haematopoietic cells by reprogramming, rather than by simple selection of stress-enduring cells.
＜possibility that CD45− cells, contaminating as a very minor population in the FACS-sorted CD45+ cells,＞って
なんか生物系の実験って幅ありすぎだよね。これじゃ確率的に何回以上と条件つけないと
そこから結論なんてひきだせないでしょうね。
