Attempts to Recapitulate STAP Cells Fail


Japanese stem cell scientist Haruko Obokata was the first author of two papers that appeared in the journal Nature earlier this year that described the derivation of pluripotent stem cells from mature cells without the use of genetic manipulation. Instead, these cells were subjected to environmental stresses such as physical pressure or exposure to acid that, according to these papers, caused the cells to express genes associated with pluripotency. Culturing of these cells led to the derivation of pluripotent stem cells lines. Thus were born STAP or stimulus-triggered acquisition of pluripotency cells. Needless to say, these results were hailed as a remarkable advance in stem cell biology.

Unfortunately, as soon as the papers were published, several high-level laboratories tried to recapitulate these results and universally failed. Even more troubling were some of the inconsistencies that came to the forefront in the published papers that the reviews had apparently missed or were ignored by the journal. The RIKEN center where this work was done even launched an internal investigation that concluded that Dr. Obokata was guilty of scientific misconduct.  Obokata gave approval to formally retract her Nature papers.  However, the RIKEN Center gave Obokata and her colleagues until the end of November to prove that she could reproduce STAP cell derivation.

Now the jury is in – Obokata has been unable to replicate her results. In the original experiments, Obokata used a gene fusion that caused the cells to glow green if they expressed genes related to pluripotency. In her replication of her original experiments, Obokata produced such green glowing cells when she subjected to environmental stresses. However, this is only a preliminary test that only involved a few such cells. More rigorous tests that were conducted, however, failed. In this case, Obokata’s stressed adult cells were introduced into a mouse embryo to see whether they could contribute to the development of various tissues during animal development. Obokata’s stressed cells, however, were unable to integrate into the developing embryos. Since this is the ultimate test for pluripotency, and since these cells were not able to pass this test, it seems virtually certain that Obokata’s original results were completely bogus.

With her signature conclusions in tatters, Obokata has resigned from the RIKEN center. In a very emotional resignation letter, Obokata wrote she could not “find words enough to apologize… for troubling so many people at RIKEN and other places.”  The RIKEN president, Ryoji Noyori, wrote in an accompanying statement that Dr. Obokata had been subjected to horrible psychological stress as a result of this affair.  Noyori added that he accepted her resignation to hopefully save her from suffering further from a severe “mental burden.”  One the co-authors of the STAP papers, Japanese stem cell scientist Yoshiki Sasai, committed suicide a few weeks after the retraction of the paper.

Hopefully, RIKEN and the other scientists who were involved in this venture move on and continue with the business of pushing back the frontiers of science.  It is entirely possible that intentional fraud was involved, but ultimately, we will never know.  For now, it is clear that sloppiness and a lack of skepticism about one’s own results contributed to this fiasco.  I think most people simply want to put this whole sordid event behind them.  However, there are pointed lessons to be learned and we will be better investigators if we learn them.

For one, peer review is not omnipotent.  Post-publication review is important and will continue to be important.  Secondly, journals need to be willing to solicit outside opinions to ensure the quality of high-level publications.  Third, the majority of scientists publish in journals that most people will never read.  Their work is not glamorous, but instead document tedious, high-quality, detailed, scientific research.  The majority of such work will never appear in Nature or Science or Cell, but that’s alright because good solid research is still good solid research regardless of where it appears.  It is really too bad that the push for high-visibility publications can cause people to publish too quickly before results have been properly vetted.  The STAP episode might be a reminder for journals to take greater care with the review of original research.

The STAP paper sage continues – the Knoepfler post


Stem cell scientist and blogger Paul Knoepfler (from my alma mater, UC Davis), has written a nice summary of the STAP situation as it sits.  See his very useful post here.  He points out the ironic truth that Obokata and her co-authors agreed to retract the Nature STAP cell letter, but not the Nature article even though the Nature letter is not the one found by the RIKEN Center to contain figures that were manipulated.   Dr. Knoepfler wrote an editorial to the journal Nature in which he called for the journal to retract BOTH papers.  This is pretty much the view of the scientific community in general, at least from where I sit.  

Once the STAP papers came out, a host of labs tried to recapitulate the experiments described in the papers.  These are some very successful stem cell labs with very talented people.  They pretty universally had trouble recapitulating the results of Obokata and others.  Now that’s not definitive proof that something’s wrong.  Some experiments are really hard to do and it takes time to learn how to do them even if you are really good.  However, even after the detailed STAP protocol was made available, people still had trouble getting it to work.  Now things started to look hinkey.  Further mining of the papers began to show some really deep problems – things that did not make sense.  When clarification was asked for, the problems began to look even bigger.  This is the point at which the RIKEN Center became involved.

I think we should give the RIKEN Center some credit.  After all, looking into a signature publication from your own institute and the workings of one of your own is not easy.  But investigate they did, and the results were not pretty.  They did not sugar-coat their findings, but reported them forthrightly.  According to Dr. Knoepfler, RIKEN is currently determining a punishment for what it called “Dr. Obokata’s misconduct.”  If misconduct produced the Nature article then it should be retracted.  If there is some good science in that paper, then let the authors re-do it and resubmit it.  But as it stands, I think Dr. Knoepfler is completely correct when he writes, “the whole STAP story is fundamentally flawed.”

Nature should request and then demand a retraction from the authors.  If they do not get this approval, it seems to me that they are well within their rights to either retract the papers on their own pending further review or take legal action to get the papers retracted.  Most of the stem cell community, bloggers included, just want to put this whole affair behind us.

RIKEN Institute Investigation into STAP Paper Concludes Misconduct was Committed


The STAP paper that generated so much excitement in Nature has been subjected to some pretty substantial knocks. Several labs have tried to replicate the experiments from this paper, and no one has consistently succeeded. Also, a detailed protocol was released, but the claims of this protocol contradict those in the published paper. Also, one of the authors on the original STAP paper, has even said that he no longer believes the results of his own paper.

The RIKEN institute, where this research was conducted, convened an internal investigation to determine what went wrong. Even though they do not call for the paper to be retracted, they do conclude that deliberate falsification did occur in the paper. There report can be read, in English, here.

The report examines six problems with the original paper:
1. Unnatural appearance of colored cell parts shown by arrows in d2 and d3 images of Figure 1f.
2. In Figure 1i, lane 3 appears to have been inserted later.
3. A part of the Methods section on karyotyping appears to have been copied from another paper.
4. A part of the procedures described in the Methods section on karyotyping appears to be different from the actual procedures used in the experiment.
5. The images for Figures 2d and 2e appear to be incorrect, and closely resemble images in Dr. Obokata’s PhD dissertation.

The first problem is chalked up to what happens to microscope pictures when they are compressed into JPEG files and sent with an electronic copy of a manuscript. Having had figures sliced, diced, shrunk and compressed, blurred, and converted to black and white after submitting them to journals, I can vouch for Dr. Obokata on this one. Therefore, they do think that this one is a problem.

Problem 2 they think is due to true tampering. Lanes in gels, western, southern and northern blots are sometimes cut and pasted in papers, but Nature, apparently has a policy about this and their policy is that this is a no-no. Also, they conclude that the gel lane pasting “created the illusion that the data of two different gels belonged to only one gel, but may also lead to the danger of misinterpretation of the data.” I think they are completely correct on this one.

Problem 3 was probably a dunderheaded mistake. They think that Dr. Obokata plagiarized the protocol, but in all honesty, it could have simply been the result of being in a hurry and having a deadline that you have to meet to finish your Ph.D. and get your papers submitted by a certain date. To my reading, this one sounds like a lack of sleep and being in a hurry. But honestly lady, couldn’t you have at least cited the other paper from which you took the protocol in the first place?

Problem 4 they think is a simple case of someone else did the work and you didn’t check with them first before including it in the paper.  Thus it is an oversight and not a case of falsification. On this one, I think the senior author has to bear a lot of the blame. It’s his butt on the line if the paper has anything wrong in it, and he simply did not read the submitted paper carefully enough before submitting it.

Problem 5 is a pretty flagrant case of bait-and-switch. The original figure in the paper was supposed to be STAP cells made from spleen. However, Dr. Obokata said that these were pictures of bone marrow blood cell-making stem cells instead of spleen stem cells. Also the pictures she substituted came from her Ph.D. dissertation, and were of cells that had not been treated with acid, but had been subjected to shear forces by forcing them through a narrow pipette. This is a different experiment than the one she reported. Also, her statements that she had forgotten that these figures of cells treated completely differently are hard to believe. I think we are justified in calling this one a whopper.

Problem 6 is a mislabeling of two figures of cells that came from the same experiment. It is a classic case of the paper being rewritten before publication, the figures being completely reworked, and the labeling getting all messed up. This one is not falsification but it is negligence.

All in all, the paper is a mess. Whatever might have been observed has been fogged over by fraud, negligence, and too many cooks in the paper-writing kitchen. This sounds like too many people were involved in the preparation of the paper and they did not properly talk to each other. This is a black eye for the Riken Institute, which has done so much very fine work. They are to be commended for speedily convening the investigation and for expeditiously examining the evidence. However, large efforts need to have one clearing house for data and all that data needs to be checked, checked and rechecked after every rewrite and before submission.

I think the papers clearly need to be retracted. The investigation does not make that recommendation, but it is the honorable thing to do under the circumstances.

STAP Paper Author Urges that the STAP Paper Be Withdrawn


Japanese scientist, Teruhiko Wakayama, a professor at Japan’s University of Yamanashi, who was part of the research team that described the production and characterization of STAP cells, has called for his own headline-grabbing study on stem cells to be withdrawn from publication. Wakayama says that the main findings of this paper have been thrown into doubt.

When the STAP cells came out in January it was hailed as a game-changer that could herald a new era of medical biology. The paper was published in the prestigious journal Nature and was also widely covered in Japan and across the world.

Since that time, however, there have been reports that several other scientists have been unable to replicate the Japanese team’s results. Also there seem to be some disparities with some of the paper’s data and images.

“It is no longer clear what is right,” Wakayama told public broadcaster NHK.

STAP or stress-triggered acquisition of pluripotency cells seemed to represent a simple way to reprogram mature animal cells back into an embryonic-like state that would allow them to generate many types of tissue.

From these STAP cell papers, various editorials dreamed big and suggested that just about any cell in your body could be simply and cheaply reprogrammed back into embryonic cell-like cells, and be used to replace damaged cells or grow new organs for sick and injured people.

Wakayama even said, “When conducting the experiment, I believed it was absolutely right.” However, now he is not so sure. He continued: “But now that many mistakes have emerged, I think it is best to withdraw the research paper once and, using correct data and correct pictures, to prove once again the paper is right. If it turns out to be wrong, we would need to make it clear why a thing like this happened.”

A spokesperson from the journal Nature has said that they were aware of, “issues relating to this paper,” and that an investigation was underway. However, at this point, the journal had no further comment to make.

Robin Lovell-Badge, a stem cell expert at Britain’s National Institute for Medical Research, cautioned against premature assumptions on whether the research was flawed. “I have an open mind on this,” he told Reuters. “I’m waiting to hear from several serious stem cell labs around the world on whether they have been able to reproduce the methods.”

Wakayama’s co-researcher Haruko Obokata, the first author on the STAP paper, became an instant celebrity in Japan after she spoke during a Nature media briefing to science reporters all over the world about her findings.

The Japanese team was joined by other researchers from Brigham and Women’s Hospital and Harvard Medical School in the United States in this research. They took skin and blood cells from mice, grew them, and then subjected them to stresses that brought the cells “almost to the point of death.” They exposed the cells to a variety of stresses, including trauma, low oxygen levels, and acidic environments.

One of these “stressful” situations used by these researchers was to bathe their cells in a weak acid solution for around 30 minutes. Within days, the scientists said they had found that the cells had not only survived but had also recovered by naturally reverting into a state similar to that of an embryonic stem cell.

Unfortunately, other research teams have yet been able to replicate the findings, and the RIKEN Center for Developmental Biology in Japan, where Obokata works, said last week it had “launched an independent inquiry into the content of the paper.

That inquiry will be conducted by a panel of experts from within and outside RIKEN, it said, and would be published as soon as it was concluded.

A spokesperson from the RIKEN Institute declined to comment on Wakayama’s call for the paper to be withdrawn.

STAP Cells: The Plot Thickens Even More


You might remember that Charles Vacanti and researchers at the RIKEN Institute in Japan reported a protocol for reprogramming mature mouse cells into pluripotent stem cells that could not only integrate into mouse embryos, but could also contribute to the formation of the placenta. To convert mature cells into pluripotent cells, Vacanti and others exposed the cells to slightly acidic conditions or other types of stressful conditions and the cells reverted to a pluripotent state.

Even though Vacanti and others published these results in the prestigious journal Nature, as other scientists tried to replicate the results in these papers, they found themselves growing more and more frustrated. Also, some gaffes with a few of the figures contributed to a kind of pall that has hung over this research in general.

The original makers of these cells, stress-acquired acquisition of pluripotency or STAP cells, have now made a detailed protocol of how they made their STAP cells publicly available at the Nature Protocol Exchange. Already. it is clear that a few things about the original paper are generating many questions.

First of all, Charles Vacanti’s name does not appear on the protocol. He was the corresponding author of the original paper. Therefore the absence of his name raises some eyebrows. Secondly, the authors seem to have backed off a few of their original claims.

For example one of the statements toward the beginning of the protocol says, “Despite its seeming simplicity, this procedure requires special care in cell handling and culture conditions, as well as in the choice of the starting cell population.” Whereas the original paper, on the first reading at least, seemed to convey that making STAP cells was fairly straightforward, this seems to no longer be the case, if the words of this protocol are taken at face value.

Also, the protocol notes that cultured cells do not work with their protocol. The authors write, “Primary cells should be used. We have found that it is difficult to reprogram mouse embryonic fibroblasts (MEF) that have been expanded in vitro, while fresh MEF are competent.”  This would probably explain inability of several well-regarded stem cell laboratories to recapitulate this work, since the majority of them probably used cultured cells. This, however, seems to contradict claims made in the original paper that multiple, distinct cell types could be converted into STAP cells.

Another clarification that the protocol provides that was not made clear in the original paper is that STAP cells and STAP stem cells are not the same thing. According to the authors, the protocol provided at Nature Protocol Exchange produces STAP cells, which have the capacity to contribute to the embryo and the placenta. On the other hand, STAP stem cells, are made from STAP cells by growing them in ACTH-containing medium on feeder cells, after which the cells are switched to ESC media with 20% Fetal Bovine Serum. STAP stem cells have lost the ability to contribute to extra-embryonic tissues.

Of even greater concern is a point raised by Paul Knoepfler at UC Davis. Knoepfler noticed that the original paper argued that some of their STAP cells were made from mature T cells. T cells rearrange the genes that encode the T cell receptor. If these mature T cells were used to make STAP cells, then they should have rearranged T cell receptor genes. The paper by Vacanti and others shows precisely that in a figure labeled 1i. However, in the protocol, the authors state that their STAP cells were NOT made from T-cells. In Knoepfler’s words: “On a simple level to me this new statement seems like a red flag.”

Other comments from Knoepfler’s blog noted that the protocol does not work on mice older than one week old. Indeed, the protocol itself clearly states that “Cells from mice older than one week showed very poor reprogramming efficiency under the current protocol. Cells from male animals showed higher efficiency than those from female.”  Thus the universe of cells that can be converted into STAP cells seems to have contracted by quite a bit.

From all this it seems very likely that the STAP paper will need to go through several corrections. Some think that the paper should be retracted altogether. I think I agree with Knoepfler and we should take a “wait and see” approach. If some scientists can get this protocol to work, then great. But even then, multiple corrections to the original paper will need to be submitted. Also, the usefulness of these procedure for regenerative medicine seems suspect, at least at the moment. The cells types that can be reprogrammed with this protocol are simply too few for practical use. Also, to date, we only have Vacanti’s word that this protocol works on human cells. Forgive me, but given the gaffes associated with this present paper, that’s not terribly reassuring.

Results of STAP Cell Paper Questioned


Reports of Stimulus-Triggered Acquisition of Pluripotency or STAP cells has rocked the stem cell world. If adult cells can be converted into pluripotent stem cells so easily, then perhaps personalized, custom stem cells for each patient are just around the corner.

However, the RIKEN institute, which was heavily involved in the research that brought STAP cells to the world has now opened an investigation into this research, since leading scientists have voiced discrepancies about some of the figures in the paper and others have failed to reproduce the results in the paper.

Last week, Friday (February 14, 2014, spokespersons for the RIKEN centre, which is in Kobe, Japan, announced that the institute is looking into alleged irregularities in the work of biologist Haruko Obokata, who works at the institution. Obokata was the lead author listed on two papers that were published in the international journal Nature. These papers (Obokata, H. et al. Nature 505, 641–647 (2014), and Obokata, H. et al. Nature 505, 676–680 (2014) described a rather simple protocol for deriving pluripotent stem cells from adult mouse cells by exposing them to acidic conditions, other types of stresses such as physical pressure on cell membranes. The cells, according to these two publications, had virtually all the characteristics of mouse embryonic stem cells, but had the added ability to form placental structures, which is an ability that embryonic stem cells do not have. The investigation initiated by the RIKEN centre comes at the behest of scientists who have noticed that some of the images used in these papers might have been duplicated from other papers. Also, several scientists have notes that they have been unable, to date, to replicate her results.

These concerns came to a head last week when the science blog PubPeer, and others, noted some problems in these two Nature papers and in an earlier paper from 2011. Obokata is also the first author of this 2011 paper (Obokata, H. et al. Tissue Eng. Part A 17, 607–15 (2011), and this paper contains a figure that seems to have been used for one of the figures in the 2014 paper. Also, there is another figure duplication.

Harvard Medical School anesthesiologist Charles Vacanti who was the corresponding author of one of the Nature papers has said that has learned last week about a data mix up in the paper and has contacted the journal to request a correction. “It certainly appears to have been an honest mistake [that] did not affect any of the data, the conclusions or any other component of the paper,” says Vacanti. Note that Vacanti is a co-author on both papers and a corresponding author on one of them.

In the other paper, Obokata serves as the corresponding author and this paper contains an image of two placentas that appear to be very similar. Teruhiko Wakayama works at Yamanashi University in Yamanashi prefecture, and he is a co-author on both of these papers. According to Wakayama, he sent more than a hundred images to Obokata and suggests that there was confusion over which to use. He says he is now looking into the problem.

Additionally, ten prominent stem-cell scientists have been unable to repeat Obokata’s results. One particular blog listed eight failures from scientists in the field. However, most of those attempts did not use the same types of cells that Obokata used.

Some scientists think that this could simply be a case of experienced scientists working with a system that they know very well and can manipulate easily, unlike outsiders to this same laboratory. For example, Qi Zhou, a cloning expert at the Institute of Zoology in Beijing, who says most of his mouse cells died after treatment with acid, says that “setting up the system is tricky; as an easy experiment in an experienced lab can be extremely difficult to others, I won’t comment on the authenticity of the work only based on the reproducibility of the technique in my lab,” says Zhou.

However, others are more deeply concerned. For example, Jacob Hanna, a stem-cell biologist at the Weizmann Institute of Science in Rehovot, Israel, however, says “we should all be cautious not to persecute novel findings” but that he is “extremely concerned and sceptical”. He plans to try for about two months before giving up.

It could be that the protocol is far more complicated that thought. For example, even Wakayama has been having trouble reproducing the results. To be sure, Wakayama and a student of his were able to replicate the experiment independently before publication, but only after being coached by Obokata. But since he moved to Yamanashi, he has had no luck. “It looks like an easy technique — just add acid — but it’s not that easy,” he says.

Wakayama says that his own success in replicating Obokata’s results has convinced him that her technique works. “I did it and found it myself,” he says. “I know the results are absolutely true.”

Clearly one way to clear this up is for the authors of this groundbreaking paper to publish a detailed protocol on how to make STAP cells. This should clear up any problems with the papers. Vacanti says he has had no problem repeating the experiment and says he will let Obokata supply the protocol “to avoid any potential for variation that could lead to confusion”.

The journal Nature has said that there are aware of the problems with the papers and looking into the matter.

For now, that’s where the issue sits. Frustrating I know, but until we know more we will have to just “wait and see.”

Histones Might Hold the Key to the Generation of Totipotent Stem Cells


Reprogramming adult cells into pluripotent stem cells remains a major challenge to stem cell research. The process remains relatively inefficient and slow and a great deal of effort has been expended to improve the speed, efficiency and safety of the reprogramming procedure.

Researchers from RIKEN in Japan have reported one piece of the reprogramming puzzle that can increase the efficiency of reprogramming. Shunsuke Ishii and his colleagues from RIKEN Tsukuba Institute in Ibaraki, Japan have identified two variant histone proteins that dramatically enhance the efficiency of induced pluripotent stem cell (iPS cell) derivation. These proteins might be the key to generating iPS cells.

Terminally-differentiated adult cells can be reprogrammed into a stem-like pluripotent state either by artificially inducing the expression of four factors called the Yamanaka factors, or as recently shown by shocking them with sublethal stress, such as low pH or pressure. However, attempts to create totipotent stem cells capable of giving rise to a fully formed organism, from differentiated cells, have failed.  However, a paper recently published in the journal Nature has shown that STAP or stimulus-triggered acquisition of pluripotency cells from mouse cells have the capacity to form placenta in culture and therefore, are totipotent.

The study by Shunsuke Ishii and his RIKEN colleagues, which was published in the journal Cell Stem Cell, attempted to identify molecules in mammalian oocytes (eggs) that induce the complete reprograming of the genome and lead to the generation of totipotent embryonic stem cells. This is exactly what happens during normal fertilization, and during cloning by means of the technique known as Somatic-Cell Nuclear Transfer (SCNT). SCNT has been used successfully to clone various species of mammals, but the technique has serious limitations and its use on human cells has been controversial for ethical reasons.

Ishii’s research group focused on two histone variants named TH2A and TH2B, which are known to be specific to the testes where they bind tightly to DNA and influence gene expression.

Histones are proteins that bind to DNA non-specifically and act as little spool around which the DNA winds.  These little wound spools of DNA then assemble into spirals that form thread-like structures.  These threads are then looped around a protein scaffold to form the basic structure of a chromosome.  This compacted form of DNA is called “chromatin,” and the DNA is compacted some 10,000 to 100,000 times.  Histones are the main arbiters of chromatin formation.  In the figure below, you can see that the “beads on a string” consist of histones with DNA wrapped around them.

DNA_to_Chromatin_Formation

There are five “standard” histone proteins: H1, H2A, H2B, H3, and H4.  H2A, H2B, H3 and H4 form the beads and the H1 histone brings the beads together to for the 30nm solenoid.  Variant histones are different histones that assemble into beads that do not wrap the DNA quite as tightly or wrap it differently than the standard histones.  Two variant histones in particular, TH2A and TH2B, tend to allow DNA wrapped into chromatin to form and more loosely packed structure that allows the expression of particular genes.

When members of Ishii’s laboratory added these two variant histone proteins, TH2A/TH2B, to the Yamanaka cocktail (Oct4, c-Myc, Sox2, and Klf4) to reprogram mouse fibroblasts, they increased the efficiency of iPSC cell generation about twenty-fold and the speed of the process two- to threefold. In fact, TH2A and TH2B function as substitutes for two of the Yamanaka factors (Sox2 and c-Myc).

Ishii and other made knockout mice that lacked the genes that encoded TH2A and TH2B. This work demonstrated that TH2A and TH2B function as a pair, and are highly expressed in oocytes and fertilized eggs. Furthermore, these two proteins are needed for the development of the embryo after fertilization, although their levels decrease as the embryo grows.

Graphical Abstract1 [更新済み]

In early embryos, TH2A and TH2B bind to DNA and induce an open chromatin structure in the paternal genome (the genome of sperm cells), which contributes to its activation after fertilization.

These results indicate that TH2A/TH2B might induce reprogramming by regulating a different set of genes than the Yamanaka factors, and that these genes are involved in the generation of totipotent cells in oocyte-based reprogramming as seen in SCNT.

“We believe that TH2A and TH2B in combination enhance reprogramming because they introduce a process that normally operates in the zygote during fertilization and SCNT, and lead to a form of reprogramming that bears more similarity to oocyte-based reprogramming and SCNT” explains Dr. Ishii.