相澤論文に対するAustin Smith教授らのコメントに対する相澤氏の回答と論文リバイス

F 1000research.というサイトで、6月1日付で掲載され相澤氏の検証実験論文ですが、Irene de Lazaro氏とAustin Smith教授のコメントに対して、相澤氏が回答し、論文修正をしているそうです（9月27日付）。

※中村公政さんの「白鳥は鳥にあらず」ブログの最新記事での紹介で知りましたが、アクセスを好まれないようにもお書きなので、リンクは貼らないようにします。

　

F 1000research.というサイトでの相澤論文は、次のURLですが、その下のほうの、二人のコメント欄のそれぞれにおいて、相澤氏の回答が掲載されています。

http://f1000research.com/articles/5-1056/v1　　

 

　相澤氏の回答内容や論文リバイス内容の評価については、私にはもちろんわかりませんが、どなたか解説していただけると、皆さんの参考になると思いますし、助かります。

　回答貼り付けておきます。

Author Response 27 9 2016

Shinichi Aizawa,RIKEN Center for Developmental Biology, Japan

Dear Dr. Austin Smith,

I thank you for your comments. The manuscript was revised incorporating your suggestions. My responses are as follows: 

1. The headings in Table 1 have been changed as suggested.
    
2. All oct-gfp cell aggregates exhibited fluorescence to some degrees.
    
3. No cell aggregates were generated from wild-type splenocytes. No direct comparison was made of the intensities of green fluorescence of cell aggregates with those in oct-gfp embryos or ES cells. I cannot state with certainty whether the green and red fluorescence was autofluorescence. RT-PCR analysis for GFP expression showed significant expression in several aggregates, but not in others that had green fluorescence; these data were very preliminary and thus are not shown. This examination focused on the multipotency of cell aggregates generated by Obokata using a chimeric assay, since this was the central feature of the STAP phenomena. Other data were only preliminary given the time constraints under which these experiments were performed, as described in Discussion.
    
4. To make chimeras, cell aggregates were prepared with cag-gfp splenocytes, thus GFP expression or green fluorescence cannot be used as a measure for the selection of cell aggregates. For this reason, they could only be selected by cell cluster morphology. In the present study, the selection was dependent entirely on Obokata’s judgment. If she had succeeded, our plan was next to ask her to describe “cell cluster morphology” precisely.
    
5. Many embryos injected with cag-gfp cell aggregates at 8-cell stage and cultured for one day to the blastocyst stage were examined for the presence of green-fluorescent cells, and such cells were found to be present.
    
6. Chimeric extent was examined at E9.5 or E8.5 in whole mount. The retracted Nature papers show extensive colonization of the cells (Fig. 4 in the Article and Fig. 1 and Extended Data Fig. 1 in the Letter). The article reported eight chimeric embryos, showing more then 50% coat color contribution, of 48 chimeras obtained; these animals yielded “STAP”-derived offspring. This was the central finding in the now-retracted STAP reports. However, in the present study, no chimera equivalent to those in Fig. 4 in the article and Fig. 1 and Extended Data Fig. 1 in the Letter was obtained, nor were any chimeras obtained showing more than 50% contribution to coat pigmentation. Indeed, no chimera showing more than 1% contribution was obtained. I have now revised the text accordingly. We have not examined the limit of detection (minimum number of cells) with the cag-gfp mouse line used, since this was not the point of the present study. However, I believe it to be the case that if dozens of cells had been present together in any tissue, they would have been detectable in whole mount at E9.5 or E8.5.

Best regards,
Shin Aizawa

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Author Response 27 9 2016

Shinichi Aizawa,RIKEN Center for Developmental Biology, Japan

Dear Dr. Irene de Lazaro,

I thank you for your comments. The manuscript was revised incorporating your suggestions. My responses are as follows: 
 

1. There was no FACS cell sorter in the laboratory in which Obokata performed the set of supervised experiments reported here. She had previously obtained “STAP” cells using splenocytes prepared using Lympholyte-M, so we sought to determine whether she was able to repeat this in the present study. If she had succeeded, our plan was next to generate STAP cells using CD45+ cells sorted by FACS.
    
2. The origin of the cag-gfp transgenic mouse line used in the retracted Nature papers is unclear, and was not reported in the papers. Dr. Wakayama informed us that he generated the cag-gfp mouse line himself while at the University of Hawaii, but we did not make a formal investigation into this. The mouse line was no longer maintained in the animal facility of CDB and was not available to us. Alternatively, the cag-gfpmouse line may have been actually an Acr/cag-gfp mouse line (Nakanishi et al., Genomics 80, 564-574 (2002)) as suggested in the report by Konno et al (Konno et al., Nature 525,E4-5 (2015). However, we only became aware of this possibility at the time of that report, which was after the start of Obokata’s replication attempt. In any case, the cag-gfp mouse line reportedly used in the original STAP reports is different from the cag-gfp mouse line (Okabe et al., 1997) we used in the present study. It is nonetheless difficult to conceive how the difference in cag-gfp transgene might affect the efficiency of “STAP cell” production and chimera generation.
    
3. In Fig. 4a of the retracted Nature article, the embryo being injected with “STAP” cells clearly has a zona pellucida. However, E4.5 embryos typically no longer have this structure. In the absence of zona pellucida, injection is practically impossible. We note that E0 is generally defined as 0:00 am of the day when the plug is identified, and suggest that E4.5 may be a typographic error for E3.5. Alternatively, Dr. Wakayama may have artificially delayed the development of the embryo; however, this was not reported in the retracted Nature paper.
    
4. We have now included a statistical analysis (t-test), which indicates that the efficiency of cell aggregate formation is significantly different between ATP treatment and HCl treatment in the C57BL/6 background. However, the difference is slight. We have revised the manuscript accordingly (Table 1 and page 5 in the text).
    
5. This study focused on the multipotency of cell aggregates generated by Obokata using a chimeric assay as this was the central feature of the reported “STAP” phenomena. Given the time constraints of this study, other data were necessarily limited, as noted in the Discussion. As it was not the focus of the present study, I cannot state definitively that the red fluorescence observed was autofluorescence, although I feel that this is highly likely. RT-PCR analysis for GFP expression showed significant expression in several aggregates, but not in others that showed green fluorescence; however, these data were preliminary at best and are not presented.
    
6. The effects on both cell aggregate formation and chimeric potency of the spleens’ genetic background were examined in the C57BL/6 and F1(C57BL6 x 129) background. It is well known that ES culture is strongly influenced by genetic background. Both of these backgrounds were used in the retracted Naturepapers. I have now revised the manuscript (page 4 and page 6) to clarify this point.
    
7. The cell aggregates in Niwa’s report were prepared by Niwa, not by Obokata.
    
8. The two reports are now cited and briefly discussed (page 8–9). These works did not examine multipotency by chimeric assay, and the most important issue of the present report is that cell aggregates prepared by Obokata herself did not exhibit multipotency in chimeric assays.

 
Best regards,
Shin Aizawa