One of the central claims in the original reports was that the purported STAP cells had the ability to differentiate into multiple lineages, including germ cells, when placed in a normal developmental environment. The present study focused on assessing pluripotency by chimera production using cell aggregates prepared by Obokata; however, no evidence of pluripotency was observed using this assay. In the original reports, the STAP cells were prepared by Haruko Obokata, while the chimera production and the establishment of ES (embryonic stem)-like STAP-SCs and TS (trophectoderm stem)-like FI-SCs were made by Teruhiko Wakayama.
そうね。はい。
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I encourage readers to recognize a number of limitations in the studies, which were conducted under strict time constraints and in the face of considerable, often adversarial, media scrutiny. Unfortunately, it was not possible to receive technical advice from Teruhiko Wakayama in the chimera production reported here, and it is unclear whether or to what extent the techniques for chimera production in the present study correspond to those used in the previous studies. Previous studies also examined the pluripotency of purported STAP cells by their potency to generate teratomas in immune-deficient mice. However, more than 105 cells are required to form teratoma subcutaneously in the flank of an immune-deficient mouse using ES or EC (embryo carcinoma) cells, and the process takes about one month. No teratoma formation was examined in the present study, since the frequency of green fluorescent cell aggregates was low and time was limited. Teratoma formation under the kidney capsule, which also takes about two months using blastocyst embryos, was also not examined.
はい。
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The more critical question is whether and to what extent the STAP cell aggregates prepared by Obokata in this trial under new experimental conditions recapitulated the STAP cell aggregates reported in the previous study. The frequency of green fluorescent cell aggregates from low pH-treated, Oct-GFP transgenic spleen cells was 10-fold less than that in previous studies. Moreover, green fluorescence due to GFP expression cannot be distinguished from that due to autofluorescence, nor can GFP expression by reprogramming be distinguished from that due to non-specific gene expression in dying cells. The cell aggregates were not characterized in vitro in detail, but the following features were observed:
続きを読んだら。
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(1) Preliminary FACS analysis of low pH-treated, Oct-GFP transgenic spleen cells suggested that the frequency of green fluorescent cells was very low and that the majority of surviving cells were CD45-positive after one week in culture under the conditions used in the present study. In the previous study, CD45+ cells were rare and a significant number of green fluorescent cells were observed (Figure 1c in Obokata et al., 2014a).
そうじゃないと思うよ、 the majority of surviving cells were CD45-positive after one week in culture と
あるじゃないか。残った細胞って、GFPを発していない細胞のことだよ。本物であれ、アーティファクトであれ
今回は以前みたいに出来てないんだから。残ったのはなんでもない細胞じゃないか。
そこはペンディングして全部読みましょうかね。
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(2) Preliminary RT-PCR analysis suggested that the majority of the cell aggregates generated in the present study did not express pluripotency markers, in contrast to the report of pluripotency marker expression in the previous study (Figure 2b in Obokata et al., 2014a), although there were cell aggregates at a low frequency that expressed one or multiple pluripotent markers.
はい。
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(3) Preliminary immunochemical analysis suggested that most of the cell aggregates in the present study did not express pluripotency markers. In contrast to the data shown in Figure 2a of the previous study, they did not express OCT4, SSEA1, NANOG and E-CADHERIN, (Obokata et al., 2014a).
はい、続きよ。
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The possibility cannot be excluded that the experimental conditions used in the present study in some way differed from the previously established optimum conditions for STAP induction. It is my view that it was beyond the scope of this examination to reassign each condition; a definitive answer to the question of whether the previously used conditions for inducing the STAP phenomenon can be indeed established or not must await further study. Nevertheless, I consider it is important to report that Haruko Obokata herself failed to reproduce the reported phenomenon, in that the putative STAP cells described here were unable to contribute to any tissues in a normal developmental environment.
ま、そういうことだね。それが相澤さんの本務だ。科学的事実に関しては
a definitive answer to the question of whether the previously used conditions for inducing the STAP phenomenon can be indeed established or not must await further study.
ということで。
丹羽氏の実験のサンプル量は
Fig.3aは all cells in the wells
Fig.3bは 細胞塊１個
なので、細胞塊１個では、GAPDH値が安定して計れなかったのではないでしょうか？（推測です）
会見でも試行錯誤をしながら実験したと話されています。
*ttps://youtu.be/f9k6tQixpTQ?t=1829
最初の問題をペンディングしたままいわば戦線拡大するのね。ま、いいわ。
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TCR-β chain gene rearrangement analysis
Genomic DNA was extracted from STAP cells and tail tips from chimaeric mice generated with STAP cells derived from CD45+ cells. PCR was performed with 50 ng DNA using the following primers (Dβ2: 5′-GCACCTGTGGGGAAGAAACT-3′ and Jβ2.6: 5′-TGAGAGCTGTCTCCTACTATCGATT-3′) that amplify the regions of the (D)J recombination. The PCR products were subjected to gel electrophoresis in Tris-acetate-EDTA buffer with 1.6% agarose and visualized by staining with ethidium bromide. PCR bands from STAP cells were subjected to sequencing analysis and identified as rearranged genomic fragments of the (D)J recombination.
はい、STAP細胞の方の再構成はあったということでいいのね。学さん達が何か議論されていたのは
キメラの細胞のTCR再構成みたいね。
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Genomic DNA was extracted from STAP cells and tail tips from chimaeric mice generated with STAP cells derived from CD45+ cells.
