s********3
发帖数: 231 | 1 《科学》对STAP手稿的专家评审意见曝光
《科学》对传说中的STAP细胞论文的三名评审专家意见，过去一直被雪藏，今天终于
被曝光。专家对这一研究的评价不高，认为非常全面的描述性研究，如果结果确定，可
能导致发育生物学大厦的颠覆，其实是不相信这种研究结论。因为没有当时的投稿件，
所以没有办法对这些评价进行更深入分析。不过这些资料显然都在某个地方，只是拥有
的人不愿意拿出来。
Retraction Watch readers are of course familiarwith the STAP stem cell saga,
which was punctuated by tragedy last month whenone of the authors of the
two now-retracted papers in Nature committed suicide.
In June, Science‘s news section reported:
Sources in the scientific community confirm that early versions of theSTAP
work were rejected by Science, Cell, and Nature.
Parts of those reviews reviews havesurfaced, notably in a RIKEN report.
Science‘s news section reported:
For the Cell submission, there were concerns about methodology and thelack
of supporting evidence for the extraordinary claims, says [stem
cellscientist Hans] Schöler, who reviewed the paper and, as is standard
practice atCell, saw the comments of other reviewers for the journal. At
Science,according to the 8 May RIKEN investigative committee’s report, one
reviewerspotted the problem with lanes being improperly spliced into gel
images. “Thisfigure has been reconstructed,” the RIKEN report quotes from
the feedbackprovided by a Science reviewer. The committee writes that the “
lane 3”mentioned by the Science reviewer is probably the lane 3 shown in
Figure 1i inthe Nature article. The investigative committee report says [co-
author Haruko]Obokata told the committee that she did not carefully consider
the comments ofthe Science reviewer.
The entire reports, however, have not beenmade available. Retraction Watch
has obtained the full text of the editor’scover letter and reviews of the
rejected Science paper. The reviews are full ofsignificant questions and
doubts about the work, as would be expected in arejection. We present them
here, to fill in some of the gaps and help readersconsider how the research
eventually made it through peer review:
21 August 2012
Dr. Haruko Obokata
Anesthesiology
[ROOM NUMBER REDACTED]
Brigham and Womens Hospital/Harvard MedicalSchool
75 Francis Street
Boston MA 02115
USA
Dear Dr. Obokata:
Manuscript number: [REDACTED]
Thank you for submitting your manuscript“Stress altered somatic cells
capable of forming an embryo” to Science. We havenow received the detailed
reviews of your paper. Unfortunately they are notpositive enough to support
publication of the paper in Science. Although werecognize that you could
likely address many of these specific criticisms in arevised manuscript, the
overall nature of the reviews is such that the paperwould not be able to
compete for our limited space. We hope that you find thecomments helpful in
preparing the manuscript for submission to another journal.
We are grateful that you gave Science theopportunity to consider your work.
Sincerely,
NAME REDACTED, Ph.D.
Senior Editor
REVIEWS
Reviewer 1
This paper claims that cells from anysomatic tissue can be reprogrammed to a
fully pluripotent state by treatmentfor a few days with weak acid.
This is such an extraordinary claim that avery high level of proof is
required to sustain it and I do not think thislevel has been reached. I
suspect that the results are artifacts derived fromthe following processes:
(1) the tendency of cells with GFP reporters to gogreen as they are dying. (
2) the ease of cross contamination of cell lines keptin the same lab.
I believe that the green transformation isindeed due to stress as reporters
are often upregulated in stressed or dyingcells. But the cells that go green
may not be the ones in the later greencolonies. I think these are more
likely to be ES cells acquired by crosscontamination and selected for growth
by the B27-LlF medium. This would explainthe results on marker expression,
promoter demethylation, differentiation, andchimera formation. In Fig.2B and
the other RT-PCR studies, it is not statedwhether the Y-axis is linear or
logarithmic. If it is linear, which seems morelikely, then I am very
surprised that all of the pluripotency genes measured inthe ESC control have
virtually the same RNA abundance, which exceeds that ofGAPDH.
The claim about all the other tissues beingsimilarly reprogrammed by low pH
treatment is truly extraordinary. Much moredetail needs to be provided about
the nature of the cells and the cultureconditions. Otherwise this is simply
not credible, since the principal celltype of several of these tissues is
postmitotic.
The DNA analysis of the chimeric mice isthe only piece of data that does not
fit with the contamination theory. But theDNA fragments in the chimeras don
’t look the same as those in the lymphocytes.This assay is not properly
explained. If it is just an agarose gel then thesmall bands could be
anything. Moreover this figure has been reconstructed. Itis normal practice
to insert thin white lines between lanes taken fromdifferent gels (lanes 3
and 6 are spliced in). Also I find the leading edge ofthe GL band
suspiciously sharp in #2-#5.
Minor points:
1. It is by no means clear that newt cellscan revert to “stem cells” (
presumably this means pluripotent stem cells).Recent work on newt
regeneration has indicated conservation of tissue type inmost cases. The
Wolf (1895) reference is out of date.
2. p.8 heading: “exposure” not “expose”.
3. The sentence on p.12 line 6 up “mixture…. analyzed” is very confusing.
4. In the Fig. 4 legend it should be madeclear which experiments are done
with 2N and which with 4N hosts.
On the positive side, I do agree with theauthors that the many claims of
pluripotent stem cells from adult mammalsprobably arise from partial
reprogramming due to stress followed by selectionin culture. But I don’t
think such cells often match ESC in pluripotentbehavior, especially the
ability to form chimeras in 4N hosts.
Reviewer 2
Obokata et aI. describe a novel method forreprogramming somatic cells to a
state that possesses many features ofpluripotent stem cells. By subjecting
C045+ hematopoietic cells to low pH,mechanical trituration, and culture in
LlF-B27 medium, ESC-like properties canbe induced, including ESG-like levels
of Oct4, Sox2, and Nanog mRNA andcritically, the potential for germline
transmission and tetraploidcomplementation — two of the most stringent
functional assays for there-acquisition of pluripotency. If these data are
indeed robust then theobservation is highly significant.
Unfortunately, the paper presents only asuperficial description of many
critical aspects of the work. A detaileddescription of the methods used to
induce and maintain SACs was not provided,and the mechanisms and
explanations are either not compelling or poorly defined(Figure 3). Given
the novelty of the claims, a thorough characterization of theSACs is
warranted. as is some probing of the mechanisms. This would necessitatea
more sophisticated genomic analysis of SACs, through microarray or RNA-seq,
and genome-wide DNA methylation analysis — analyses that other pluripotent
stemcell lines have been routinely subjected to and for which methods for
smallercell numbers have been developed.
Issues to be addressed:
1) From the experiments performed by theauthors, it cannot be ruled out that
rare multipotent progenitors are beingselected for and expanded under
stress conditions. While this in itself wouldbe extremely interesting, it
suggests a very concept [sic] than what is beingclaimed about reprogramming
of “mature” adult somatic populations. It isunclear whether cells are
harvested from any other stages than young (7-day old)mice. Might the cells
in these young mice be errant migratory germ cells orsome other stem cell-
like progenitors? CD45+ cells are harvested from thespleens and these are
called lymphocytes, but hematopoietic stem cells (HSCs)express CD45, and the
spleen contains HSCs at this young age. Thus stressconditions may be acting
on HSCs, rather than fully differentiated somaticcells, which would imply a
very different main conclusion of this work. Shouldthe authors wish to
maintain their conclusion, they should rule out thepotential germ cell or
HSC origin of SACs. This could involve perhaps theexamination of genomic
imprints, or expression of c-Kit.
2) The analysis of TCRb gene rearrangementin fig S6 purporting to show
derivation from fully mature T cells with TCRbrearrangement is flawed. If
mice are clonally derived, they should have asingle gene rearrangement, not
a population of polyclonal rearrangements asappears in at least some of
these animals. This analysis should be done usingSouthern Blots to avoid the
problems of PCR contamination; see Hochedlinger andJaenisch, Nature 2002.
3) The evaluations of stress-mediatedresponse pathways and analysis of
mitochondria are purely correlative and haveno demonstrated mechanistic link
to the observed reprogramming.
4) The ability to permanently maintainthese cell lines is not well-described
. The authors claim that “sphericalcolonies grew to approximately 70 um in
diameter … and spherical colonies couldbe maintained for another 7 days in
that culture condition.” Figure 2C–itappears that the expression of mESC-
associated pluripotency genes is decreasingsignificantly over time in bulk
SAC populations. If these cells are trulypluripotent. they should not only
exhibit the developmental potential of ESCs/iPSCsbut also the ability to
indefinitely self•renew. For ESCs/iPSCs, past groupsprovided evidence
of telomerase activity to indicate the capture of animmortalized cell line.
In the case that the cells cannot self-renewindefinitely, a description of
what happens (differentiation, death, etc.), andan explicit statement on
unlimited or limited proliferative potential of theSACs should be provided.
5) Reprogramming efficiency alter stressexposure should be monitored for
each cell population. Presumably many cellsdie after low pH treatment, and
the 40% of surviving cells expressing Oct4-GFPafter 7 days represents a
selected and subsequently expanded population, butthis is not clear. This
would help understand the proportion and give clues tothe nature of the
cells undergoing reprogramming. For example, more refinedcell isolation
followed by analysis of conversion efficiency could be veryinformative.
6) The nature of the B27 medium was notdescribed. Is it serum-free or serum-
containing? What is the base medium used?These are critical details because
“B27 medium” is not a conventional conditionfor mouse ESC/iPSC propagation
. but rather for primitive neural stem cellderivation/propagation. For serum
-free culture of mouse ESCs/iPSCs in LlF andB27 supplement, they require N2
supplement and BMP4 or N2 supplement and 3i/2i(Ying et al. Cell. 2003; Ying
et al. Cell. 2008). The original work that firstdescribes the use of LlF-B27
medium was not cited (Tropepe et al. Neuron.2001). In that study, they
critically observed that LlF-B27 ESC-derived neuralcolonies are competent to
colonize both neural and non-neural when exposed toan appropriate
environment Given the claim of acquired pluripotency, theauthors need to
rule out that they have generated primitive neural stem cellsby genomic
characterization of the SACs they generated, and more preciselycapture the
nature of the “reprogramming” that is claimed (microarray, DNAmethylation
analysis, etc.). A systematic genome-wide characterization of SACswill
establish the molecular identity of SACs in relation to other
mousepluripotent stem cell lines.
7) From the FACS analysis of Oct4expression in day 1 CD45+ cells, it is not
clear if there is a small populationof weakly-expressing Oct4-GFP cells. How
can the expansion of a pre-existingQct4-GFP expression population be ruled
out, rather than de novo expressionfrom mature cells? Because the authors
claim a significant increase inefficiency and pace of Oct4 locus
reactivation, they should compare theirmethod with the predominantly
established method by which hematopoietic cellsare reprogrammed: by defined
factor induction. A head-to-head comparison of SACinduction and iPSC
generation is needed (a la Figure 1B).
8) The authors surveyed changes in the mRNAexpression levels of an array of
stress-response genes, but did not assesstheir functional relevance by shRNA
knockdown or overexpression during conversion.
9) Considering that mouse strains known tobe either recalcitrant (e.g. Bl6)
or permissive (e.g. 129) to ESC derivationwere used in this study, was there
any correlation between SAC derivation andstrain?
10) It is stated that there was no differentiationtendency of SACs derived
from any tissues when incorporated into chimeras. Thisdoes not appear to be
true as liver-derived SACs exhibit a low contribution,and are skewed to
liver differentiation. Similarly, skin-derived SACs appear todemonstrate a
tendency to contribute to the skin.
11) Embryos generated by tetraploidcomplementation should be taken to term.
In figure S5E, the example of theBDF1/GFP embryo presented does not look
normal.
12) Regarding the existing molecular dataon the identity of the cell lines,
the embryonic gene expression qPCRs (Figure2B, S3C) show unusually high
values for expression levels relative to GAPDHlevels. Even though the figure
has an ESC control, and it may be aprimer-specific phenomenon, mRNA levels
of genes such as Nanog and Rex1 aremore like 0.05 or 0.15 of GAPDH levels,
whereas the authors observed levels ashigh as 12 -14 times GAPDH.
13) The authors did not describe “the lowpH solution (pH5.5)” treatment in
detail (Methods). The authors need to providea detailed description of what
the composition of the pH solution was, how longthe treatment was, and how
the cells were handled.
14) The authors did not describe thesubstrate used during conversion. Did
they use feeders or gelatin? These are theconventional substrates on which
pluripotent stem cells are derived andmaintained.
15) Because the authors claimed acombination of pH decrease and mechanical
stress caused Oct4 reactivation, theauthors should show data indicating how
the two procedures additively orsynergistically promote Oct4-GFP
reactivation.
16) Figure S1C — the authors quantified thenumber of spherical colonies,
but they did not provide a normalization. Thisfigure would additionally be
significantly enhanced if the authors showed themorphologies of the
spherical colonies they are obtaining in the differentculture conditions
17) A more detailed description on thecomposition of the different media
tested was not provided (Figure S1C).Additionally, ACTH is not
conventionally used for pluripotent stem cellculture. The manuscript would
be enhanced if the authors provided anexplanation for the use of ACTH.
18) Decreased cellular size is a feature ofpluripotent stem cells, but the
authors did not include data or a discussion onthe ESCs/iPSCs cell size in
their examination of cell size (Figure 1D).
19) Figure 2B — the nuclear localization ofthe Nanog signal in SACs should
be very clear, however the staining appears tobe non-specific.
20) The authors should refer to the 2007Cell Stem Cell paper from the
Jaenisch lab when discussing reports of theexistence of Oct4-expressing
pluripotent adult stem cells. Here it was shownthat Oct4 gene ablation in
adult tissues did not affect regenerative capacity.
21) Figures are often not referred to inorder, making the manuscript
slightly difficult to follow at times.
Reviewer 3
The finding described in this manuscript isvery unusual and unexpected.
Under certain circumstances, it appears that anon-physiological non-specific
stress can trigger reprogramming of terminallydifferentiated cells, such
that the cells enter a pluripotent stem cell-likestate. If these results are
repeatable, a paradigm of developmental biologywould be changed. Currently,
that paradigm is that terminally differentiatedstates are set and cannot be
reset. Although Yamanaka broke this biologicalrule by overexpressing
pluripotency-associated factors, that system is highlyartificial. The
authors of the present manuscript propose that cells have anintrinsic
capacity to reprogram. I found the manuscript to be clearly writtenand
concise, although sometimes mildly unorthodox in terms of literature cited.
However, the methods and cell protocolsused must be described in far more
detail. For example, the section on Oct4should state how many cells were
sorted and describe the appearance of thecells. Is it possible that rare
populations of cells pre-exist or are alreadyapparent on day 1 (thus, what
are the “dots” of Fig. 1?). The authors willargue that, indeed, under
certain circumstances, they were able to reprogramterminally differentiated
cells, and that this was attributable to TCRrecombination. I think, ideally,
that the cells should be experimentally taggedand traced. This would
unequivocally clarify the source of the cells and,further, would exclude the
possibility that some cells pre-existed in apluripotent state.
Critically, it is necessary to determinewhether SAC cells can propagate
stably in culture and whether such cells can bepassaged. CD45.2 cells from
the spleen are differentiated and, unless activatedby an antigen, are
supposed to be in G0. Do these cells re-enter the cellcycle? The cells
should be further characterized.
Some negative controls are missing. SeeFigs. 2A, S3B, S3C, S5A, and S5B.
Unstressed cultured cells should be used asnegative controls.
In Figs. 3C and D, it would be interestingto see the ATP and ROS levels in
both ES and SAC cells.
In Figs. 3C and D, it is apparent that mEScells show rises in ATP and ROS
levels, and in mtDNA copy number. These resultsshould be compared to other
publications.
In Fig. 2, Nanog is not located in thenucleus. Also, do the authors have
data on staining of Oct4 in thisexperimental context?
Update, 6:30 p.m. Eastern: Here’s PaulKnoepfler’s take on these reviews.
http://blogs.nature.com/news/2014/09/new-details-emerge-on-retr | s******r
发帖数: 1245 | 2 science不厚道，爆这些是为了打nature脸？
saga,
【在 s********3 的大作中提到】
: 《科学》对STAP手稿的专家评审意见曝光
: 《科学》对传说中的STAP细胞论文的三名评审专家意见，过去一直被雪藏，今天终于
: 被曝光。专家对这一研究的评价不高，认为非常全面的描述性研究，如果结果确定，可
: 能导致发育生物学大厦的颠覆，其实是不相信这种研究结论。因为没有当时的投稿件，
: 所以没有办法对这些评价进行更深入分析。不过这些资料显然都在某个地方，只是拥有
: 的人不愿意拿出来。
: Retraction Watch readers are of course familiarwith the STAP stem cell saga,
: which was punctuated by tragedy last month whenone of the authors of the
: two now-retracted papers in Nature committed suicide.
: In June, Science‘s news section reported:
| s********3
发帖数: 231 | 3 Science一直在影响因子上吃了Nature激进主义的亏。这会好不容易站在道德制高点上
，肯定对Nature狂羞辱啊。
【在 s******r 的大作中提到】
: science不厚道，爆这些是为了打nature脸？
:
: saga,
| F*Q
发帖数: 3259 | 4 打得好！Nature应该也有一堆打Science脸的机会。它们是需要互相打。看看谁被打出
更多的shxt来。
【在 s******r 的大作中提到】
: science不厚道，爆这些是为了打nature脸？
:
: saga,
| z*t
发帖数: 863 | 5 reiewer的意见是confidential的吧？虽然nature这件事上有编辑的责任，但是这么搞
还是坏了规矩
【在 s******r 的大作中提到】
: science不厚道，爆这些是为了打nature脸？
:
: saga,
| s******y
发帖数: 28562 | 6 哈哈哈。挺搞笑的。
不过，Science 爆的是他们自己这个杂志对这个文章的审稿意见吧，应该也征求了那些
审稿人的同意，所以不好说是违背professional guideline。而且好几个杂志其实是公
开把审稿意见和文章一起发表的。
从Science 的说法看来，这个文章先投到Cell, 被据了，然后投到Science, 也被据了
，而且Science 的reviewer 还详细的指出某个图片貌似有作假的嫌疑。然后这个文章
投到了Nature, 居然很快就被接受了。可见Nature的审稿比另外两家都更不严谨，感觉
是只要看到题材火热就立刻给发了。
当然，Science 自己也发过一些被领域广泛质疑的报道（大家还记得砷细菌么?)
saga,
【在 s********3 的大作中提到】
: 《科学》对STAP手稿的专家评审意见曝光
: 《科学》对传说中的STAP细胞论文的三名评审专家意见，过去一直被雪藏，今天终于
: 被曝光。专家对这一研究的评价不高，认为非常全面的描述性研究，如果结果确定，可
: 能导致发育生物学大厦的颠覆，其实是不相信这种研究结论。因为没有当时的投稿件，
: 所以没有办法对这些评价进行更深入分析。不过这些资料显然都在某个地方，只是拥有
: 的人不愿意拿出来。
: Retraction Watch readers are of course familiarwith the STAP stem cell saga,
: which was punctuated by tragedy last month whenone of the authors of the
: two now-retracted papers in Nature committed suicide.
: In June, Science‘s news section reported:
| w*****r
发帖数: 2061 | 7 支持互爆
【在 s******y 的大作中提到】
: 哈哈哈。挺搞笑的。
: 不过，Science 爆的是他们自己这个杂志对这个文章的审稿意见吧，应该也征求了那些
: 审稿人的同意，所以不好说是违背professional guideline。而且好几个杂志其实是公
: 开把审稿意见和文章一起发表的。
: 从Science 的说法看来，这个文章先投到Cell, 被据了，然后投到Science, 也被据了
: ，而且Science 的reviewer 还详细的指出某个图片貌似有作假的嫌疑。然后这个文章
: 投到了Nature, 居然很快就被接受了。可见Nature的审稿比另外两家都更不严谨，感觉
: 是只要看到题材火热就立刻给发了。
: 当然，Science 自己也发过一些被领域广泛质疑的报道（大家还记得砷细菌么?)
:
| o********r
发帖数: 775 | 8 Science隔了一天又爆了Nature的最初审稿意见。基本上三个reviewes也是一片质疑，
奇怪的是AE给了major revision而不是rejection。Nature这一次问题的确很大。
【在 s******y 的大作中提到】
: 哈哈哈。挺搞笑的。
: 不过，Science 爆的是他们自己这个杂志对这个文章的审稿意见吧，应该也征求了那些
: 审稿人的同意，所以不好说是违背professional guideline。而且好几个杂志其实是公
: 开把审稿意见和文章一起发表的。
: 从Science 的说法看来，这个文章先投到Cell, 被据了，然后投到Science, 也被据了
: ，而且Science 的reviewer 还详细的指出某个图片貌似有作假的嫌疑。然后这个文章
: 投到了Nature, 居然很快就被接受了。可见Nature的审稿比另外两家都更不严谨，感觉
: 是只要看到题材火热就立刻给发了。
: 当然，Science 自己也发过一些被领域广泛质疑的报道（大家还记得砷细菌么?)
:
| s******9
发帖数: 283 | 9 最后CNS三大期刊一怒之下互爆，观众们看到高大上杂志节操扫地一定非常解恨。
【在 o********r 的大作中提到】
: Science隔了一天又爆了Nature的最初审稿意见。基本上三个reviewes也是一片质疑，
: 奇怪的是AE给了major revision而不是rejection。Nature这一次问题的确很大。
| s******y
发帖数: 28562 | 10 太奇怪了，Nature怎么会同意把最初审稿意见交给Science 看呢？
【在 o********r 的大作中提到】
: Science隔了一天又爆了Nature的最初审稿意见。基本上三个reviewes也是一片质疑，
: 奇怪的是AE给了major revision而不是rejection。Nature这一次问题的确很大。
| | | K**4
发帖数: 1015 | 11 应该是那个harvard老板动用了关系搞定了
【在 o********r 的大作中提到】
: Science隔了一天又爆了Nature的最初审稿意见。基本上三个reviewes也是一片质疑，
: 奇怪的是AE给了major revision而不是rejection。Nature这一次问题的确很大。
| o********r
发帖数: 775 | 12 看那个PDF貌似某个co-author泄露出来的decision letter
【在 s******y 的大作中提到】
: 太奇怪了，Nature怎么会同意把最初审稿意见交给Science 看呢？
| s******y
发帖数: 28562 | 13 这样也行？Science 不怕Nature 打官司？
【在 o********r 的大作中提到】
: 看那个PDF貌似某个co-author泄露出来的decision letter
| h********n
发帖数: 4079 | | s******y
发帖数: 28562 | 15 象这样的？
【在 h********n 的大作中提到】
: 支持互爆打脸
| D*a
发帖数: 6830 | 16 貌似是先泄露到retraction watch还是什么的吧？他们这网站要能活下去，肯定要保护
线人吧
【在 s******y 的大作中提到】
: 这样也行？Science 不怕Nature 打官司？
| v***r
发帖数: 1046 | 17 AE估计滚蛋了，这些编辑屁都不懂，听说当年的业绩和经手的文章的影响挂钩,看大牌
发文章.
【在 o********r 的大作中提到】
: Science隔了一天又爆了Nature的最初审稿意见。基本上三个reviewes也是一片质疑，
: 奇怪的是AE给了major revision而不是rejection。Nature这一次问题的确很大。
| P*A
发帖数: 7996 | 18 不是哪家比别家审稿更严谨，只是这家正好有这个作者的铁哥们，而别家有别的作者的
铁哥们而已
【在 s******y 的大作中提到】
: 哈哈哈。挺搞笑的。
: 不过，Science 爆的是他们自己这个杂志对这个文章的审稿意见吧，应该也征求了那些
: 审稿人的同意，所以不好说是违背professional guideline。而且好几个杂志其实是公
: 开把审稿意见和文章一起发表的。
: 从Science 的说法看来，这个文章先投到Cell, 被据了，然后投到Science, 也被据了
: ，而且Science 的reviewer 还详细的指出某个图片貌似有作假的嫌疑。然后这个文章
: 投到了Nature, 居然很快就被接受了。可见Nature的审稿比另外两家都更不严谨，感觉
: 是只要看到题材火热就立刻给发了。
: 当然，Science 自己也发过一些被领域广泛质疑的报道（大家还记得砷细菌么?)
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