and (adult stems cells) are felt not to be able to cross the germ layer lineages as they develop.
と続くんだから三胚葉分化以前の多能性を持っているものではないと信じられていると。
feelしているのは科学者じゃなくて我々ど素人たちという意味なのかなあ。私は三胚葉分化実験を
成功させたわよと言いたいんでしょ?
さて。
Although the existence of adult stem cells has been reported for more than a decade, we have hypothesized the existence of adult stem cells,which are quite small and resistant to external stress, that reside in tissues representative of the three germ layers.
3. Toma, J.G., Akhavan, M., Fernandes, K.J., Barnabe-Heider,F., Sadikot, A., Kaplan, D.R., and Miller, F.D. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol 3, 778, 2001.
4. Kucia, M., Reca, R., Campbell, F.R., Zuba-Surma, E., Majka, M., Ratajczak, J., and Ratajczak, M.Z.A population of very small embryonic-like (VSEL) CXCR4(þ)SSEA-1(þ)Oct-4þ stem cells identified in adult bone marrow. Leukemia 20, 857, 2006.
5.Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D., Ortiz-Gonzalez, X.R., Reyes, M., Lenvik, T.,Lund, T., Blackstad, M., Du, J., Aldrich, S., Lisberg, A., Low, W.C., Largaespada, D.A., and Verfaillie, C.M. Pluripotency
of mesenchymal stem cells derived from adult marrow. Nature 418, 41, 2002.
だね。
we have hypothesized the existence of adult stem cells,which are quite
small and resistant to external stress, that reside in tissues representative
of the three germ layers.というのは、あくまで仮説としてこういうものを想定して
その実在の有無を確かめようとしているという意味だね。
Adult stem cells have generally been felt to be limited to multipotency and unable to cross germ layer lineages as they develop.って
アブストにもあった説明だね。feelという語彙選択はともかくとして、ここまでくると、こう感じでいる人々というのが、あげられた文献を
発表した人々だということらしいのは徐々に明らかになってきたかな。
続きだよ。
Cells derived from many adult tissues, including retina(6),brain(7-10),cornea(11),olfactory neuroepithelium(12、13), pancreas(14),skin(15,16),muscle(17),and bone marrow (BM)(18、19), have now been propagated as nonadherent clusters or spheres, as have ESCs(20).
この論旨の運びだとそうだよ。まだイントロなんだよ。彼女の知ってる
既存論文に書かれていることを知ってるぞと見せるところなんでしょうよ。
続きはこうだ。
In addition, neurospheres have been shown to contain stem cells demonstrating gene express that overlap that of ESCs.
808 ：ダウトファイア：2016/05/09(月) 06:47:03
＞Tissueに投稿しなおした博論の内容で書いた論文だけど、ES細胞がしっかり出てきちゃいますね。
FIG.2のデータでポジコンとしてＥＳ細胞を使っているね。マテメソにも。
"To eliminate the risk of false-positives,RNA was isolated from ES cells and E 6.5 fetal cells as positivecontrols, and RNA was also isolated from heart, muscle, li-ver, and adherent stromal cells as negative controls for em-bryonic gene marker expression in RT-PCR.
Cells derived from many adult tissues, including retina(6),brain(7-10),cornea(11),olfactory neuroepithelium(12、13), pancreas(14),skin(15,16),muscle(17),and bone marrow (BM)(18、19), have now been propagated as nonadherent clusters or spheres, as have ESCs(20).
いや、知識として知らないということは考えにくいんで、普段実際に自分の
実験で持ってこられた姿はnonadherent clusters or spheresではなかったかと。
無論、propagated as nonadherent clusters or spheresで、正しい記述になる
nonadherent の定義も考えられるんでしょ?
ま、その辺はね。後でいくらでも検討できる。進んでみましょうかね。
We believe that adult stem cells described in various reports represent the same adult stem cells at different stages of development, expressing different degrees of potency and that sphere-forming cells are much more immature than previously expected.
we have hypothesized the existence of adult stem cells,which are quite
small and resistant to external stress, that reside in tissues representative
of the three germ layers.
仮説構築はさらに続くぜ。
We hypothesized that adult stem cells procured from any tissue, ectoderm, mesoderm, or endoderm cross germ layers as they develop, when maintained in appropriate environments in vitro and in vivo.
テ、テ、テ。
In this study, we characterized cells isolated from three adult tissues (spinal cord, muscle, and lung) representative of the three different germ layers (ectoderm, mesoderm, and endoderm)and from BM.
はい、続きですわよ。
Cells procured from each source initially expressed many of the markers with ESCs and demonstrated the potential to
differentiate into all three germ layers, in vitro and in vivo.
当時多能性細胞はIPSを除けばESがその代表だったということかな?initially は
次の文にも使われていてこちらは変じゃない。
The isolation initially contained a significant amount of floating debris, or fibers, and other extraneous materials, all of which appeared to participate in the formation of nonadherent nonadherent cells, insoluble proteins spherical clusters that contained the cells.
ちょっとコピペ欲張ったんで文字が化けたな。
The isolation initially contained a significant amount of floating debris, nonadherent cells, insoluble proteins or fibers, and other extraneous materials, all of which appeared to participate in the formation of nonadherent spherical clusters that contained the c ells.
が正しい。
Cells procured from each source initially expressed many of the markers associated with ESCs and demonstrated the potential to differentiate into all three germ layers, in vitro and in vivo.
はい、次です。
The cellular make-up of individual spheres was not identical; that is, spheres were composed of heterogeneous populations of cells, even when the spheres were generated from cells procured from the same tissue at t he same time.
Similarities or differences seen in the cell content of different spheres were believed to be secondary to the environment in whi ch they were cultured.
はい。
BM, spinal cord (ectoderm), muscle (mesoderm), and lung (endoderm) were collected from 3- to 4-week-old and 12-week-old C57BL/6J mice (Jackson Laboratory).
面白くなってきましたわね。うふふ。
The cell suspension obtained was centrifuged at 2000 rpm for 5 min and washed with F12/Dulbecco’s modified Eagle’s medium (DMEM; 1:1, v/v; Gibco).
門外漢は判断しない方がいいでしょうね。疑問をそのまま抱いて忘れなければそれでいいのでは。
The cell suspension was passed through 100 µm nylon filters (BD Falcon) and centrifuged at 2000 rpm for 5 min, washed once more, and centrifuged at 2000 rpm for 5 min.
次は肺よ。
Lung tissues were washed via intratracheal lumen with 10 mL of cold phosphate buffered saline (PBS; Gibco) and were perfused with collagenase type P (Roche).
小保方さんのHPにおける訴訟は
under advisement with relevant people about lawsuits or readmission to other universities.
というアナウンスメントの中に置かれれている言葉だが、relevant people には当然、弁護士グループが
含まれているよ。
Cells from each tissue type were plated at 1x10⁶ cells/cm² in F12/DMEM (1:1, v/v) supplemented with 2% B27 (Invitrogen), 20 ng/mL basic fibroblast growth factor (bFGF; R&D Systems), and 10 ng/mL epidermal growth factor (EGF; R&D Systems).
なぜ、そういう培地なのかは素人には分かりませんわね。次です。
About 50% of the medium was replaced every 2–3 days for the duration of the culture; bFGF and EGF were added every other day.
明日はここからよ。うふふふふ。
Cell size measurement
Marrowspheres from 4-week-old mice, collected after 5 days in the fresh medium, were dissociated into single cells.
感想は全部読んでからにしましょ。
Dissociated cells were stained with propidium iodide filtered through a 40 mm pore filter and measured on a DAKO Galaxy (DAKO) using FloMax software.
あら、失礼。私も小保方さんと性格が似てるのよ。うふふふふ。
Microbeads of predefined sizes (Size Calibration Standards Kit; Bangs Laboratories, Inc.) were re-suspended in isotonic phosphate saline (pH 7.2) and used as a standard for whichto compare size of cells contained in spheres using cytofluorimetric analysis.
そういう時は先を読むとヒントがあったりなかったり、余計分からなくなったり。。。
Both cells and beads were analyzed using the same instrument setting (forward scatter, representing cell and bead size, and side scatter, representing cellular granularity).
ワトソンさん。そのギャグはホームズさんに飛ばしてくださらない。
はい、これでおしまいよ。
Cell size was calculated on a curve employing bead size on the x-axis and forward scatter values on the axis.
ドットプロット解析図なんでしょ? 僕の理解だと
a curve employing bead size on the x-axis ってMicrobeads of predefined sizesの結果なんでしょ。
全部同じものだからカーブにならないんじゃないの。一か所に集中するはずだけど。そこに
本物のスフィア細胞の単離したものを飛ばすんでしょ。どのあたりに分布するかを見て大きさを
比較する。何か間違って理解してるね。
忘却とは忘れ去ることなり。
Each slide was fixed with 4% paraformaldehyde (Wako) for 15 min at room temperature, washed with PBS, incubated with SuperBlock blocking buffer in PBS (P74370; Takara) for 30 min to block nonspecific reactions, and incubated with anti-c-kit rat monoclonal antibody (sc-19619; Santa Cruz Biotechnology, Inc), anti-Sca-1 rat monoclonal antibody (ab25031; Abcam), or anti-E-cadherin rat monoclonal antibody (ab11512; Abcam) overnight at 48C.
�B
専門的な手順を語ってるんでしょ。
After washing with PBS, the cells were incubated with goat anti-rat IgG Texas Red-conjugated antibodies (112-076-062;Jackson ImmunoResearch) and goat anti-rat IgG Fluorescein-conjugated antibodies (112-096-062; Jackson Immuno-Research) for 30 min at room temperature.
ここは彼女の専門ですからね。
Stage-specific embryonic antigen-1 and alkaline phosphatase staining was performed using the ES cell detection Kit (Millipore) in accordance with the manufacturer’s protocol.
少しは、興味が出てきましたか?それとも今日も釣りなの?
In negative controls, the primary antibody was replaced with IgG-negative controls of the same isotype to ensure specificity.
はいはい、詰まんないことやってないで前へ進んでください。
The spheres were collected at 5 days and dissociated into single cells, and placed in DMEM supplemented 20% fetal calf serum (FCS).
The spheres were collected at 5 days and dissociated into single cells, and placed in DMEM supplemented 20% fetal calf serum (FCS).の
single cellsとAfter 7–14 days, muscle cells were stained のmuscle cells とどういう関係にあるのかね?そもそも、The spheres ってどの組織から
得られたものなのかね?
