Induced cell aggregates show poor induction of pluripotency-associated markers
To test the induction of pluripotency markers in cell aggregates obtained from ATP-treated liver cells, we assessed the expression of pluripotency-associated genes. Oct3/4 is a well-defined marker of pluripotent stem cells. Using a primer pair to detect Oct3/4 transcript from the Pou5f1 allele, but not pseudo-genes[13], we did not find a detectable level (above 0.1% of the expression level in mouse ES cells, relative to the expression levels of Gapdh) of the transcript by quantitative polymerase chain reaction (Q-PCR) using a total RNA sample prepared from all cells in the culture (Fig. 3a), indicating that extremely few or no cells expressing Oct3/4 were present. Interestingly, expression of Gfp from the Oct3/4-GFP transgene (GOF18)「14 」was detected in liver cells cultured for seven days irrespective of ATP treatment, suggesting leaky expression of this transgene.
(英文)
Figure 3: Q-PCR analysis for the expression of pluripotency markers in induced cell aggregates.
Figure 3
(a) Q-PCR analysis of the low-pH treated liver cells cultured for 7 days. Liver cells were prepared from 7-day old GOF mice and treated with either ATP or HCl, or without stressor. RNA samples were prepared from all cells in the wells at day 7 of culture and the relative expression levels of Gfp (derived from GOF Tg) and Oct3/4 (derived from the endogenous Pou5f1 allele) to Gapdh were indicated with standard deviation. The expression levels in control ES cells carrying CAG-GFP Tg were set at 1.0. (b) Q-PCR analysis of the single cell aggregates derived from the ATP-treated or non-treated liver cells cultured for seven days. The liver cells were prepared from 4-days old of C57BL6/129 mice and the single cell aggregates were separately treated for quantification of gene expression. The relative expression levels of pluripotency-associated genes to Gnb2l1 were indicated with standard deviation. The expression levels in 10 control ES cells were set at 1.0. (c) Frequency of cell aggregates showing the levels of Oct3/4 expression comparable to ES cells. The relative expression levels of Oct3/4 in single cell aggregates derived from liver cells were measured as b and the frequency of the cell aggregates with the levels of Oct3/4 expression over 0.001 of relative expression to ES cells is indicated.
で、問題は仮に小保方さんの発見したOct4蛋白発現細胞が多能性細胞であったとしたら、
それはES細胞と同じなのですから１個でも入るとキメラになる確率が高い。となると
３万個に１２個でもいいからどれだけ数をこなしたらそれにあたるかということになりますね。
そこで３万個に12個というのは2500個に１個だねという計算ができますよね。ナイフ切り分けで
できたと書かれているのでその数を仮に写真でみらけめように２０個程度としたら125個の
キメラ胚を作ったらそのうちの１個が当たるという計算になる。それを丹羽さん達は1154個の
キメラ胚を作って確かめたんです。８．５日胚まで発生したのは671個でしたが、どこにもキメラ貢献
事実は無かったと報告した。確率的にはうまくいけば125回に一回当たるものを671回やってみたが
一つも当たらなかったということです。６回振れば１回１の目が出る確率のサイコロを３０回振ってみたが
１の目が出なかったということです。１００回振ったらその後１６回続けて１の目が出たかもしれない。
そもそも１の目の無いダイスだったのか。そこは分かりませんよね。
however, none of the aggregates made significant contribution to any tissues,
関連個所は貼っときましょう。
>>
Figure 5: アnalyses of fluorescent signals from GOF transgene.
Figure 5
(a) Fluorescent microscopic アnalysis of cell aggregates derived from GOF Tg mice. The cell aggregates (top) were derived from liver cells of 6-days old of GOF Tg mice. Fluorescent images with the filter sets for detection of GFP and RFP signals are shown. Images of ES cells carrying CAG-GFP (middle) captured with the same conditions are shown as a control to confirm no leaky signal of GFP in RFP channel. Images of wild-type ES cells (bottom) are shown as a control to confirm spesific signal of GFP in GFP channel. (b) FACS analysis of the low-pH treated spleen cells derived from GOF Tg mice. The spleen cells were isolated from 7-day-old GOF Tg mice and prepared with Lympholyte followed by treatment with the indicated stressors. After the culture for seven days, the cells were dissociated, stained with anti-E-cadherin with PE and anti-CD45 with APC, and analyzed by FACS. Wild-type ES cells were used as a positive control for E-cadherin staining and a negative control for CD45-staining as well as GFP fluorescence.
