STAP Cell Update: New STAP-like paper, Obokata, Vacanti, Real Origin of STAP cells, & More

The STAP cell mess that began in January of this year has in some ways quieted down.

In a broader sense, I believe that STAP is now and will be in the future viewed as a scandal that revealed some less than ideal aspects to the world of biomedical science and publishing.

Where does STAP stand today?

A New STAP-Like Paper?electric iPSC

The most recent development is the publication of a new paper pointed out by a number of people to me as perhaps STAP-like. It is entitled “Electromagnetic Fields Mediate Efficient Cell Reprogramming into a Pluripotent State”. It was published in the journal ACS Nano.

This Baek, et al. paper suggests that you can dramatically more efficiently create induced pluripotent stem cells (iPSC) by exposing somatic cells to an electromagnetic field (see graphical abstract above). My reaction? Let’s see if another lab can reproduce this, but I’m not terribly optimistic. Derek Lowe weighed in on this paper here. The Pubpeer folks have some concerns too and the authors have responded (which is a good thing) there as well.

STAP stem cellsObokata Thesis in Jeopardy

At this time, first author Haruko Obokata is faced with more immediate issues such as her future at RIKEN and her thesis. She must correct her Waseda University thesis or it may be revoked. The University did an abrupt U-turn on this as earlier they had said that while the Obokata thesis had problems it was not that big a deal. Now they are requiring a correction. Given the apparent massive plagiarism in it and re-used figures, I don’t see how a correction is possible frankly.

Vacanti still believes in STAP, issues new protocol

Obokata’s former mentor at Harvard/Brigham Women’s, Charles Vacanti, recently reaffirmed his belief in STAP and along with his lab member Koji Kojima, published yet another STAP protocol this time detailing that the addition of ATP might help other labs make it work. I’m skeptical. I do find it fascinating that Vacanti still believes in STAP despite all the evidence to the contrary. Otherwise in the STAP news, it’s interesting to speculate that during his sabbatical that he may continue working on STAP.

Nature‘s role in STAP

I still think that Nature has not come to terms with its role in STAP. As has been said many times, no journal, editors, or reviewers can catch all problems in a paper, but given the released STAP reviews of previous versions of the STAP papers including one at Nature that wasn’t initially accepted and received pretty harsh reviews, it sure seems the overall review process at Nature should have done better. All things considered, I kinda doubt we’ll hear anything else from the journal on STAP. If the trend of a surging number of overall retractions at Nature continues, however, there may be more of an impetus for change.

Remaining STAP mystery: where did STAP cells really come from?

If acid and other stressors (now perhaps including electricity) do not really make pluripotent or totipotent stem cells, then where did the alleged STAP cells/STAP stem cells come from that seemed in the mouse assays to have pluripotency or totipotency? There have been some indications that STAP cells have a different genetic make up or transcriptomic profile than they were “supposed to” as the authors reported these features in the retracted STAP papers. Were STAP cells actually a mixture of ES cells and trophoblastic stem cells? Some kind of iPS cells? We still do not know.

“Magical” STAP papers were blistered by Nature’s own reviewers, but then accepted just months later

The reviews of a STAP paper submitted to and rejected by the journal Science in 2012 were posted at Retraction Watch yesterday. They filled in some gaps in the puzzle of the series of events that led to such flawed science being published in Nature in January 2014, but the reviews also raised more questions.

Today, more STAP paper reviews have surfaced.

ScienceInsider posted a piece with additional STAP paper reviews with these coming from Nature reviewers commenting on what would later become accepted and published by Nature only months later in seemingly only moderately revised form.

The Nature reviews (you can read them here on the Science website) are very critical of the STAP papers and raise a host of important, largely still unanswered questions about STAP.

STAP magic

My overall sense is that the three reviewers did a thorough and fair job of reviewing these STAP papers. It sure seems that none of the three reviewers were even remotely close to being comfortable with these papers being published in Nature. In each case it would seem that a major revision would have been necessary prior to even having a remote chance at publication. One of the reviewers summed up a STAP cell article as essentially reporting an unproven, “magical” approach (see screenshot above).

The ball is now firmly in Nature‘s court to facilitate a thorough understanding of the STAP situation. It seems reasonable to expect more from Nature than its one editorial that shrugged off any significant responsibility including this key portion:

“We have concluded that we and the referees could not have detected the problems that fatally undermined the papers. The referees’ rigorous reports quite rightly took on trust what was presented in the papers.”

Nature‘s own reviewers’ comments would seem to directly challenge this statement.

I’m not going to go through all of these criticisms and questions raised in these reviews of the originally submitted Nature STAP papers point-by-point, but the overall consensus was that these papers were seriously flawed. This fits well with the gestalt of the reviewer comments on the rejected STAP/SAC paper at Science.

If you look at the published STAP cell Nature papers and think about the details mentioned in these acidic reviews of the original forms of the same papers, there is a sense that not much fundamentally was improved in the papers during that intervening period of months.

The big question remains then: how did these STAP papers go from being rebuffed based on scathing reviews at Nature on April 4, 2013 to acceptance by the same journal on December 20, 2013 and publication about a month later?

Full Reviews of Rejected STAP Paper Point to Early Signs of Big Trouble

Before the two STAP cell papers were published in Nature in January of 2014, much of the same data was reportedly submitted as single papers to other high-profile journals including Science.

In these cases, the proto-STAP papers as we might call them were rejected.

But why?

Until now we largely could only speculate.

However, the reviews of the 2012 proto-STAP manuscript at Science can now be read at Retraction Watch.

Retraction Watch

As a result of reading the Science reviews, today we know what the reviewers at Science thought in 2012 of this proto-STAP paper and this sheds much light on what went so terribly wrong with STAP overall. There were many big red flags. Keep in mind that the Nature reviewers would not know about the Science reviews unless by chance one or more of the reviewers for Nature had also participated in the review process at Science.

This early generation STAP paper was entitled “Stress altered somatic cells capable of forming an embryo”.

There was no “STAP” acronym at that point. Instead, the stress-produced stem cells were called “SACs”, an acronym presumably standing for “stress-activated somatic cells” or “stress-altered stem cells”. Therefore, let’s call this proto-STAP paper, the SAC paper.

All three Science reviewers had serious doubts about the SAC paper and pointed out numerous specific concerns.

For example, Reviewer 1 right away early in their review pointed out that the SAC phenomenon was probably not real and was instead explainable by two simple experimental problems: stress-associated GFP reporter activation and cell culture cross contamination.

Crucially, this same reviewer noticed the gel splice, later present in the accepted Nature STAP article Figure 1. However, apparently the STAP/SAC team did not take that concern or most of the other reviewer issues to heart.

Reviewer 2 was extremely skeptical of SAC as well, listing twenty-one specific problems/issues to be addressed. Unfortunately, it seems that most of these concerns also remained unaddressed in the later accepted Nature STAP papers. It is fair to say that although 21 issues seems like a lot that these concerns seem reasonable and not overly harsh.

What else did the reviewers say?

Both Reviewers 1 and 2 had the shared concern that pluripotency-related gene expression seemed abnormally high in the SAC cells. Way way too high.

Reviewer 2 wanted much more data before being convinced. For example, they wrote:

Given the novelty of the claims, a thorough characterization of the SACs is warranted, as is some probing of the mechanisms. This would necessitate a more sophisticated genomic analysis of SACs, through microarray or RNA-seq, and genome-wide DNA methylation analysis — analyses that other pluripotent stem cell lines have been routinely subjected to and for which methods for smaller cell numbers have been developed.

Reviewer 3 was not as detailed with their concerns, but more generally identified some areas of concern such as those articulated in this paragraph:

the methods and cell protocols used must be described in far more detail. For example, the section on Oct4 should state how many cells were sorted and describe the appearance of the cells. Is it possible that rare populations of cells pre-exist or are already apparent on day 1 (thus, what are the “dots” of Fig. 1?). The authors will argue that, indeed, under certain circumstances, they were able to reprogram terminally differentiated cells, and that this was attributable to TCR recombination. I think, ideally, that the cells should be experimentally tagged and traced. This would unequivocally clarify the source of the cells and, further, would exclude the possibility that some cells pre-existed in a pluripotent state.

Critically, it is necessary to determine whether SAC cells can propagate stably in culture and whether such cells can be passaged.

 

Experimental tagging and tracing of the cells would have been a major step forward for clarifying whether the SAC/STAP phenomenon was real. STAP/SAC cells should have been made in parallel to iPS cells as well for direct comparison.

One has to wonder how the Nature reviewers and editors could not have picked up on so many problems that were apparent to the Science reviewers. Every review at a different journal of the identical paper will be distinct of course, but this data seemed inherently flawed in a systematic way. This was no ordinary paper either. It was a no-brainer that this kind of paper with revolutionary claims required extraordinary, very meticulous editorial oversight. It is therefore not an unreasonable expectation that the Nature review process of the STAP papers should have picked up on some of these serious problems.

Nobody likes to get a harsh review of a submitted manuscript, but it’s crucial after you calm down in that situation to consider that some of the comments by the reviewers likely raised legitimate, important issues to address before resubmission. This way you can avoid problems and improve your paper. Apparently to a large extent that didn’t happen between the SAC paper and STAP paper stages.

In the end these Science reviews of the rejected SAC paper indicate that the STAP manuscript and data were problematic in fundamental ways back in 2012.

Holding Institutions Responsible for Research Misconduct: the recent case of a death of stem cell scientist

Zubin MasterBy Zubin Master

Scientist Yoshiki Sasai, age 52, committed suicide and was found dead on August 5, 2014. Sasai was deputy director of the Center for Developmental Biology (CDB) at RIKEN in Kobe, Japan, and coauthor on two recently retracted Nature papers about a reportedly easier way to make induced pluripotent stem cells. The papers were retracted due to duplication and manipulation of images done by the main researcher and lead author on the two papers – Haruko Obokata. Although cleared of any direct involvement, Sasai was under immense pressure and heavily scrutinized by the media, public and peers. This involved speculation about Sasai’s alleged intentions to orchestrate a media frenzy, and for being overly ambitious and motivated to win future grants overlooking the integrity of the science.

According to colleagues at RIKEN, Sasai was receiving counseling since the scandal broke headlines and he was also hospitalized for about a month in March (1). He was ultimately found hanging in a stairwell of a neighboring building close to the CDB and beside him were three letters addressed to CDB management, his laboratory, and Obokata. On August 12, Kazuhiro Nakamura, the family lawyer explained the contents of Sasai’s suicide note left for the family. Sasai was “worn out by the unjust bashing in the mass media and the responsibility he felt towards RIKEN and his laboratory” (2). But unsubstantiated claims in the media were not the only source of stress for Sasai. The speculation in tabloids might have also influenced how RIKEN and other colleagues behaved towards Sasai. In June, a report released by an independent RIKEN reform committee criticized CDB leaders for hyping the science and did not interview Sasai about such accusations. Their final recommendation was to dismantle CDB. According to the family lawyer, this was a tremendous shock for Sasai (2).

In this blog, I want to discuss the responsibilities research institutions have over research integrity and misconduct. Zimbardo’s Stanford prison experiments (3) and other social psychology research has taught us that ethical behavior is not only shaped by dispositional attribution (an internal moral character), but it is also influenced by many situational (environmental) features. Similarly, our understanding about the causes of research misconduct is shifting from the idea of a few “bad apples” to the realization that the immense pressure to publish and translate research findings, and poor institutional support are factors that influence research misconduct. This is not to excuse misbehavior by researchers; rather, it is about moral responsibility, and research institutions are also accountable in cases of research misconduct. While scholars on research integrity are aware of the responsibility of research institutions, the institutions themselves have taken few measures to promote research integrity and prevent misconduct; they remain virtually blameless in high profile cases of research misconduct (4). The tragic death of Dr. Yoshiki Sasai should cause us to consider the role of institutions. How well do research institutions handle investigations? Do they take measures to protect researchers and others involved in the case? How do institutions promote research integrity and prevent misconduct? I think the short answer to these questions is that institutions still do the minimum to promote the responsible conduct of research and likely react punitively to individual researchers by removing the supposed bad apples and then taking minimal corrective measures. This institutional myopic view of research misconduct needs to change, and institutions need to be held morally accountable along with scientists who commit misconduct.

The Case

There has been no shortage of news surrounding misconduct in stem cell research even beyond the STAP situation. I have given lectures to science trainees in stem cell research (5) and blogged about several relatively recent scandals (6; 7). The pattern of misconduct seen in stem cell research might be due to the heightened attention it receives in the media and interest by the public. Moreover, ways to detect misconduct are becoming relatively more widely used by journals and other scientists, and publishing in top-tier journals like Science and Nature certainly draws attention.

In January of this year, Haruko Obokata in the CDB, RIKEN reported that she was able to convert mouse cells to a pluripotent state simply by exposing cells to stress, the procedure called stimulus triggered acquisition of pluripotency (STAP). Soon after its publication, allegations of plagiarism, and figure manipulation and duplication were reported. Additionally, other researchers were unable to reproduce the STAP experiments. In April, an investigative committee at RIKEN found Obokata had committed research misconduct. While she admitted to being sloppy, Obokata continued to defend the results. The investigative report also concluded that while Sasai was not directly involved in misconduct, he bore “heavy responsibility” (8). A reform committee, chaired by a University of Tokyo emeritus professor Teruo Kishi, faults Sasai and a former CDB researcher, now at another university, for accepting Obokata’s data without question or further examination (9). The reform committee found that inadequate oversight extended to the highest leadership of CDB due to the desire for a major breakthrough and the committee recommended that CDB be completely reformed.

It is evident that Yoshiki Sasai, a leader in the scientific community and at CDB, was under massive pressure. I will neither speculate on the toll this incident had on Sasai or whether the investigation was handled well and the recommendations sound. Instead, I want to shift focus to look more at the research environment scientists work in more generally and consider what research institutions do (or can do) to promote research integrity.

Shifting the Culture of Science.

The espoused norms of science we learn in the past seem incongruent with current practices today. The ideals of science – openly sharing materials/methods, being motivated by discovery and not personal gain, judging one’s own work and others rigorously through strict standards – are being replaced with secrecy, self-promotion, and fierce competition (10, 11, 12). Competition in science creates a pressure to publish, and perhaps more recently to translate and commercialize research (13, 14). A recent survey by New Scientist reported that of 111 stem cell scientists who responded, 56% felt stem cell research was put under more intense scrutiny than other areas of biomedical science, and of those that replied positively, 56% said that this affects their work (15). Moreover, almost 17% of the stem cell researchers reported that they felt pressure to submit a paper for publication they believed was incomplete or needed verification. Combining the pressure to produce results in a hyper competitive and a bleak job market creates a stressful environment for anyone. It remains empirically unclear however, whether such a competitive environment is a recipe for research misconduct. Given the culture of science today, what are institutions doing to create a healthier environment?

Globally, several research institutions promote research integrity but differ in their approach. Some have robust policies, training, and provide resources while I suspect many only have a suite of policies and a non-transparent mechanism to address allegations of misconduct. I believe research institutions can do several things to promote research integrity and help prevent misconduct.

For starters, institutions can raise awareness and help promote a culture of research integrity by educating trainees, faculty, and research administrators and staff. Education can provide scientists with the tools they need to deal with ethical issues in a constructive manner when they arise. Michael Kalichman explains that the primary goal of education should be to “foster a research culture in which conversations about the responsible conduct of research are expected and acceptable” (16). Efforts to restore and rehabilitate researchers found to engage in misbehavior is likely going to incorporate education as part of the program. Education about the responsible conduct of research needs to be more than a minimum requirement scientists have to undertake and institutions are obligated to offer.

In addition to fostering a culture of integrity, some scholars advocate that research institutions can perform random or for-cause (when misconduct is suspected) data audits (17). However, it seems scientists have little appetite for such audits because they fear it would inhibit scientific freedom, and be burdensome and costly. While there is virtually no evidence to demonstrate whether such a policy decreases research misbehavior within an institution, it does not have to be a taxing effort on scientists or research administration (17).

Institutions should further make transparent a mechanism of how allegations should be handled, but also to provide resources like an ombudsperson as trusted broker for researchers to confidentially discuss potential problems amicably prior to raising a formal allegation requiring investigation. And if a formal investigation is needed, institutions must make serious efforts to ensure there are no reprisals against the complainant, witnesses, investigators, or the accused. In cases where scientists are under tremendous duress during an investigation, appropriate accommodations for researchers should be made.

UC San Diego is one example of an institution that has made significant efforts to promote the responsible conduct of research by having several research ethics policies, a hotline to report research fraud, a transparent mechanism to address potential allegations, and providing courses, seminars and resources to faculty and students (18). The tools are available – university and college administrators need to seek them out and implement them at their respective institutions.

Empirical research on institutional integrity climates is beginning to be performed (19). While journals, funding agencies, integrity scholars, scientific societies and other players can all do their part to promote a culture of research integrity, research institutes are well poised to promote integrity not only within their organization, but also to the larger institution of science. Research institutions should do more than simply remove the “bad apples” as they too bear some moral responsibility over research misconduct.

Acknowledgements: I would like to thank Dr. Paul Knoepfler, Ms. Lisa Willemse, and Ms. Tina Muratovic for helpful feedback.

This piece was also co-posted at the AMC blog and on the Signals blog.

Guest Post was written by Dr. Zubin Master, Assistant Professor at the Alden March Bioethics Institute, Albany Medical College and Research Associate at the University of Alberta’s Health Law Institute.

Web Links

1. http://www.japantimes.co.jp/news/2014/08/05/national/embattled-stap-study-co-author-dies-after-apparent-suicide-bid/#.VADIBE10ymQ

2. http://www.nature.com/news/stem-cell-pioneer-blamed-media-bashing-in-suicide-note-1.15715?WT.ec_id=NEWS-20140819

3. http://www.prisonexp.org/

4. http://mitpress.mit.edu/books/research-misconduct-policy-biomedicine

5. http://ori.hhs.gov/images/ddblock/march_vol22_no2.pdf

6. http://www.signalsblog.ca/rush-to-publish-and-the-repercussions-of-carelessness-in-science/

7. http://www.amc.edu/BioethicsBlog/post.cfm/not-another-stem-cell-scam-alleged-fabrication-false-affiliation-plagiarism-and-poor-publication-ethics-all-in-a-single-shot

8. http://www.nature.com/news/research-integrity-cell-induced-stress-1.15507

9. http://news.sciencemag.org/asiapacific/2014/06/japanese-stem-cell-debacle-could-bring-down-center

10. http://jama.jamanetwork.com/article.aspx?articleid=194592

11. http://www.ncbi.nlm.nih.gov/pubmed/12712166

12. http://www.jstor.org/discover/10.1525/jer.2007.2.4.3?uid=2129&uid=2&uid=70&uid=4&sid=21104081253391

13. http://genomemedicine.com/content/1/2/22

14. http://genomemedicine.com/content/4/2/17

15. http://www.newscientist.com/data/doc/article/dn25281/stemcellsurveypdf1.pdf

16. http://www.ncbi.nlm.nih.gov/pubmed/24073608

17. http://www.tandfonline.com/doi/abs/10.1080/08989621.2013.822259?journalCode=gacr20#preview

18. http://blink.ucsd.edu/research/policies-compliance-ethics/responsible-conduct.html

19. http://link.springer.com/article/10.1007%2Fs11948-012-9409-0

Perspectives: RIKEN itself fails to reproduce STAP & big CDB shake up expected soon

Nikkei is reporting that the RIKEN internal attempt to replicate so-called STAP (acid bath) cells has failed. Update: apparently, although RIKEN calls the efforts preliminary, the team tried to make STAP an amazing 22 times and 22 times it failed.

The rumors for weeks in the stem cell gapevine that RIKEN itself could not get STAP to work, even with the help of Dr. Haruko Obokata, have been confirmed by Nikkei. Obokata was first author on both Nature STAP papers, which were retracted. One of Obokata’s mentors and a STAP paper co-author, Dr. Yoshiki Sasai, recently committed suicide at least in part likely due to issue related to STAP. See a tribute to Dr. Sasai here.

STAP cell embryo

The best that the RIKEN team could get in the replication attempt, according to Nikkei sources, was a “faint” hint of pluripotent markers “nowhere near” to that observed in iPS cells or embryonic stem cells. STAP cells were supposed to be not just pluripotent, but also totipotent, allowing for contribution to an entire embryo (see image at left from the STAP work). Now that claim seems light years from reality. For more background on STAP and a timeline of the events see here.

This may be the final straw that pushes RIKEN to formally declare that STAP cells do not exist, a conclusion that I think the world of science has mostly already come to some time ago.

This latest twist is no surprise and just adds to the continuing STAP sadness, but hopefully is another step toward an end for the STAP disaster.

Unfortunately, however, this could get even worse.

Nikkei also reports that tomorrow RIKEN will announce a major shakeup at the RIKEN Center for Developmental Biology (CDB), where much of the original STAP work took place, that could mean as much as half of the scientists at the CDB moving to other RIKEN units and in a few cases losing their jobs (update: new reports and sources indicate that most RIKEN employees leaving the CDB will be transferred to other RIKEN units and not lose their jobs). That seems grossly unfair as most of the affected people would probably have had nothing to do with STAP. This anticipated personnel cut would be just another level of tragedy to the STAP story.