Embryonic Stem Cell Contamination Responsible for STAP Research Snafu

STAP or stimulus-triggered acquisition of pluripotency cells were allegedly derived from mature, adult cells by simply subjecting those cells to environmental stresses. These environmental stresses, such as low pH treatments and so on, were thought to cause cells to express genes that pushed them into an embryonic stem cell-like state. Researchers from the RIKEN institute reported these reports in the prestigious international journal Nature, and these advances were hailed as a stupendous advancement in stem cell biology.

However, as soon as stem cell scientists tried to repeat the results from these papers and failed, trouble started. Major laboratories had no success in recapitulating the results in the RIKEN institute papers, and, on-line post-publication reviews noticed some nagging problems in the published papers. RIKEN institute launched an investigation into the matter, and concluded that the lead researcher in these papers was guilty of scientific misconduct.

Now, new work as suggested that the whole thing was the result of contamination of the RIKEN group cells with embryonic stem cells. How that contamination occurred, however, remains unknown.

The RIKEN institute investigation was instigated by the institute and was carried out by a committee composed of seven outsiders. The committee analyzed DNA samples and laboratory records from two research teams who had participated in the STAP cell research. Those Nature papers have been retracted, but were once thought to provide a shortcut to producing pluripotent stem cells. The latest investigation suggests that the STAP findings resulted from contamination by embryonic stem cells. The investigation found signs of three separate embryonic stem cell lines, and they noted that it is difficult to imagine how contamination by three distinct lines could be accidental, but that they could also not be certain that it was intentional.

“We cannot, therefore, conclude that there was research misconduct in this instance,” the committee wrote. It did, however, find evidence that lead investigator Haruko Obokata, the lead author of the STAP papers, who formerly worked at the RIKEN Center for Developmental Biology in Kobe, Japan, had fabricated data for two figures in the original STAP publications.

The First Patient Treated with iPSC-Derived Cells

Nature News has reported that a Japanese patient was received the first treatment derived from induced pluripotent stem cells.

Ophthalmologist Masayo Takahashi from the Riken Center for Developmental Biology and her team used genetic engineering techniques to reprogram skin fibroblasts from this patient into induced pluripotent stem cells. These cultured iPSCs were then differentiated into retinal pigment epithelium cells. Takahashi’s colleagues, led by Yasuo Kurimoto at Kobe City Medical Center General Hospital, then implanted those retinal pigment epithelium cells into the retina of this female patient, who suffers from age-related macular degeneration.

It is unlikely that this procedure will restore the woman’s vision. However, because age-related macular degeneration is a progressive process, Takahashi and her research team will be examining if this procedure prevents further deterioration of her sight. Takahashi’s Riken team has extensively tested this procedure in laboratory animals and recently received human trial clearance. Takahashi’s team will also be looking particularly hard at the side effects of this procedure; such as immune reaction or cancerous growth.

“We’ve taken a momentous first step toward regenerative medicine using iPS cells,” Takahashi says in a statement, according to Nature News. “With this as a starting point, I definitely want to bring [iPS cell-based regenerative medicine] to as many people as possible.”

STAP Author Agrees to Retract Both Nature Papers

STAP cells or Stimulus-Triggered Acquisition of Pluripotency cells were allegedly derived from adult mouse cells by subjecting those adult cells to a variety of environmental stresses. Even though the derivation of STAP cells was not terribly efficient, the ability to make pluripotent stem cells without viruses or the introduction of new genes seemed to be a godsend for stem cell scientists. Unfortunately, further testing and inquiries into STAP cells has revealed multiple problems and several labs have been completely unable to recapitulate the results of the researchers who reported the derivation of STAP cells. These problems have led many scientists to question the factuality of STAP cell derivation.

STAP cells took another hit this week when genetic tests of STAP cells indicated that those cells do not match the mice from which they were allegedly derived, according to a report from Nature News Blog.

The derivation of STAP cells were initially reported by Haruko Obokata from the RIKEN center and her colleagues. Given the remarkable nature of the claims in those papers, many scientists were skeptical and moved to test the protocols utilized by Obokata and others in those paper to make STAP cells from adult mouse cells. Unfortunately, these independent tests universally flopped, and an internal investigation by the Riken Center came to the conclusion that Dr. Obokata was guilty of research misconduct, which she has denied.

Teruhiko Wakayama, a scientist from Yamanashi University and one of the co-authors on the STAP papers, subjected some of the cell lines that he derived using the STAP approach back in March to a battery of genetic tests. He was dismayed to discover that some of these cell lines did not match the adult mice from which they were supposed to have been generated. This raises the possibility that the STAP cells are the result of contamination, which is a perennial problem in cell culture laboratories. Wakayama did not observe any anomalies with the lines reported in the Nature papers, but, just to be safe, he sent those and other lines to an independent, and unnamed, lab for further examination and corroboration.

These independent tests, according to reports from Japanese media sources, have found that none of the STAP cell lines match the mouse strains they were supposed to be from. This calls “into question whether the STAP phenomenon has ever been demonstrated.”

Last week, the Nature News Blog reported that Dr. Obokata had agreed to retract one of the two STAP papers, even though the retraction has yet to appear in print. Now, according to the ScienceInsider, Obokata has consented to retracting both Nature papers. The ScienceInsider added this will not end the STAP story, since Riken is doggedly trying to determine whether the STAP phenomenon exists and as some critics are asking how these flawed papers were published in the first place.

“The science of the two papers was rigorously, robustly peer-reviewed as part of our usual editorial procedures. Any inaccuracies in the presentation of data that may have come to light since the peer review are being investigated,” a spokesperson from Nature told ScienceInsider.

Lead Author On STAP Papers Publicly Apologizes in Press Conference

On April 9th, the Japanese scientist at the center of a controversy over studies purporting to turn mature cells to stem cells simply by bathing them in acid or subjecting them to mechanical stress, Haruko Obokata, publicly apologized for her errors associated with the published work.

In a press conference in Osaka, Japan, with a crowd of voracious reporters flashing their cameras, Obokata blamed her scientific immaturity and lack of awareness of research protocols for the errors that were found in her two high-profile papers on the studies that were published in the journal Nature in January, which included the use of a duplicated image. With Obokata were two lawyers who are representing her.

To her credit, Obokata took full responsibility for the errors in the papers and apologized to her co-authors for the messy situation in which they presently find themselves. She also apologized to the RIKEN Center for Developmental Biology, where she did her work, for the embarrassing press this ordeal had brought upon it. Additionally, she sought forgiveness from the RIKEN committee whose report earlier this month found her guilty of scientific misconduct. At the time, Obokata had attacked the report.

This is Obokata’s first public statement in more than two months, and she held the press conference to apologize for the errors and to make the case that her research, despite the caveats and mistakes, was still valid. Also, Obokata wanted to establish that the inaccuracies in the papers were not deliberate. The day before the press conference, Obokata submitted a formal appeal to RIKEN for their committee to retract its misconduct findings. She insisted that the “stimulus-triggered activation pluripotency” or STAP phenomenon, as it has been dubbed, exists. RIKEN has 50 days to respond to her appeal.

In the STAP work, lead author Obokata, along with Japanese and US colleagues, described remarkable experiments in which she reprogrammed mature mouse cells to an embryonic state merely by stressing them. Unfortunately, she her two papers soon fell under suspicion and last month a RIKEN-appointed investigative committee found in a preliminary report that they contained numerous errors. A further report on 1 April by the RIKEN committee concluded that two of the errors in this paper constituted a case of scientific misconduct. Obokata aggressively responded on the same day in a written statement in which she expressed “shock and anger” at these conclusions. She also thought that the committee had unfairly come to their conclusions without giving her a chance to explain herself. On this day, however, Obokata’s seemed to sing a very different tune in which she pleaded for forgiveness and presented several apologies. However, she steadfastly maintains that her primary findings are true.

Obokata continues to insist that the two problems that the committee declared cases of scientific misconduct (the duplicated image and the swapping of a diagram of an electrophoresis gel) were honest mistakes, and that she had not been given enough time to explain her side to the committee.

After her brief introductory remarks, Obokata’s lawyer gave a 20-minute presentation to make the case that neither problem constituted misconduct. Defining fraud as fabrication, he countered that in both cases Obokata had the original data that should have been used but merely added the wrong data by mistake. For the more damning finding — an image of teratomas that had appeared in her doctoral dissertation and then again in the recent papers — the committee had found that she had changed a caption, which made it look intentional. The lawyer however traced the image back to a slide, part of a presentation that Obokata had continually updated and reused, until its origin became obscured. In one of her many apologies, Obokata said, “If I had gone back to carefully check the original data, there wouldn’t have been this problem.”

After the lawyer’s presentation, Obokata responded to journalists’ questions for more than 2 hours. Why had she only handed two laboratory notebooks over to the committee looking into her research? She said that she said she had four or five more that the committee hadn’t requested. Obokata denied that she ever agreed to retract the papers. Had she asked to retract her PhD dissertation? No, she merely sought advice on how to proceed Obokata’s dissertation is under investigation at Waseda University, where she studied for her doctorate).

Obokata also denied the possibility that the STAP cells had resulted from contamination from embryonic stem cells, saying that she had not allowed embryonic cells in the same laboratory and that she had carried out tests which precluded that possibility.

She said that she had created STAP cells more than 200 times, adding that she knows someone who has independently achieved it but refused to give the name (citing privacy). She believes that a RIKEN group trying to demonstrate STAP cells will help her. She has not, she said, been asked to participate in those efforts. She added that she would consider doing a public replication experiment but that it was not up to her whether she could.

Two hours into the questioning, her lawyer cut off journalists, citing concern for Obokata’s frail emotional state, and said she had to return to the hospital where she has been staying. She bowed, apologized, then bowed again and left with the reporter’s cameras flashing away as she retreated.

I feel genuinely sorry for this young lady.  Her career in science is essentially over.  It is within the realm of possibility that her mistakes were unintentional and were the result of a hurry to publish.  In this case, her adviser does bear some of the blame for her mistakes.  However, at this point it seems more likely that her mistakes were probably intentional.  If that is the case she should have known that such a high-prolife paper describing such a novel finding would be subjected to intense scrutiny and repeated attempts to verify it.  I am reminder of Moses’ admonition to the tribes that had settled on the East side of the Jordan River if they do not help the other tribes fight for their lands.  In Numbers 32:22-24, Moses said, “then when the land is subdued before the Lord, you may return and be free from your obligation to the Lord and to Israel. And this land will be your possession before the Lord.  But if you fail to do this, you will be sinning against the Lord; and you may be sure that your sin will find you out. 24 Build cities for your women and children, and pens for your flocks, but do what you have promised.”

Indeed your sin will find you out, and if Ms. Obakata intentionally attempted to deceive her colleagues, then it would appear that her sin has found her out.  At the moment I am willing to give her the benefit of the doubt, but if further evidence emerges that the whole thing is bogus, then I will retract my half-hearted support.  It is entirely possible that she found something novel and interesting that happens to cells when they are stresses.  However, it seems equally clear that a conversion into an embryonic stem cell-like state is probably not one of these things.  I reiterate my original belief – the original STAP paper should be retracted.

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.”

Stimulus-Triggered Acquisition of Pluripotency Cells: Embryonic-Like Stem Cells Without Killing Embryos or Genetic Engineering

Embryonic stem cells have been the gold standard for pluripotent stem cells. Pluripotent means capable of differentiating into one of many cell types in the adult body. Ever since James Thomson isolated the first human embryonic stem cell lines in 1998, scientists have dreamed of using embryonic stem cells to treat diseases in human patients.

However, deriving human embryonic stem cell lines requires the destruction or molestation of a human embryo, the smallest, youngest, and most vulnerable member of our community. In 2006, Shinya Yamanaka and his colleges used genetic engineering techniques to make induced pluripotent stem (iPS) cells, which are very similar to embryonic stem cells in many ways. Unfortunately, the derivation of iPSCs introduces mutations into the cells.

Now, researchers from Brigham and Women’s Hospital (BWH), in Boston, in collaboration with the RIKEN Center for Developmental Biology in Japan, have demonstrated that any mature adult cell has the potential to be converted into the equivalent of an embryonic stem cell. Published in the January 30, 2014 issue of the journal Nature, this research team demonstrated in a preclinical model, a novel and unique way to reprogram cells. They called this phenomenon stimulus-triggered acquisition of pluripotency (STAP). Importantly, this process does not require the introduction of new outside DNA, which is required for the reprogramming process that produces iPSCs.

“It may not be necessary to create an embryo to acquire embryonic stem cells. Our research findings demonstrate that creation of an autologous pluripotent stem cell – a stem cell from an individual that has the potential to be used for a therapeutic purpose – without an embryo, is possible. The fate of adult cells can be drastically converted by exposing mature cells to an external stress or injury. This finding has the potential to reduce the need to utilize both embryonic stem cells and DNA-manipulated iPS cells,” said senior author Charles Vacanti, MD, chairman of the Department of Anesthesiology, Perioperative and Pain Medicine and Director of the Laboratory for Tissue Engineering and Regenerative Medicine at BWH and senior author of the study. “This study would not have been possible without the significant international collaboration between BWH and the RIKEN Center,” he added.

The inspiration for this research was an observation in plant cells – the ability of a plant callus, which is made by an injured plant, to grow into a new plant. These relatively dated observations led Vacanti and his collaborators to suggest that any mature adult cell, once differentiated into a specific cell type, could be reprogrammed and de-differentiated through a natural process that does not require inserting genetic material into the cells.

“Could simple injury cause mature, adult cells to turn into stem cells that could in turn develop into any cell type?” hypothesized the Vacanti brothers.

Vacanti and others used cultured, mature adult cells. After stressing the cells almost to the point of death by exposing them to various stressful environments including trauma, a low oxygen and acidic environments, researchers discovered that within a period of only a few days, the cells survived and recovered from the stressful stimulus by naturally reverting into a state that is equivalent to an embryonic stem cell. With the proper culture conditions, those embryonic-like stem cells were propagated and when exposed to external stimuli, they were then able to redifferentiate and mature into any type of cell and grow into any type of tissue.

To examine the growth potential of these STAP cells, Vacanti and his team used mature blood cells from mice that had been genetically engineered to glow green under a specific wavelength of light. They stressed these cells from the blood by exposing them to acid, and found that in the days following the stress, these cells reverted back to an embryonic stem cell-like state. These stem cells then began growing in spherical clusters (like plant callus tissue). The cell clusters were introduced into developing mouse embryos that came from mice that did not glow green. These embryos now contained a mixture of cells (a “chimera”). The implanted clusters were able to differentiate into green-glowing tissues that were distributed in all organs tested, confirming that the implanted cells are pluripotent.

Thus, external stress might activate unknown cellular functions that set mature adult cells free from their current commitment to a particular cell fate and permit them to revert to their naïve cell state.

“Our findings suggest that somehow, through part of a natural repair process, mature cells turn off some of the epigenetic controls that inhibit expression of certain nuclear genes that result in differentiation,” said Vacanti.

Of course, the next step is to explore this process in more sophisticated mammals, and, ultimately in humans.

“If we can work out the mechanisms by which differentiation states are maintained and lost, it could open up a wide range of possibilities for new research and applications using living cells. But for me the most interesting questions will be the ones that let us gain a deeper understanding of the basic principles at work in these phenomena,” said first author Haruko Obokata, PhD.

If human cells can be made into embryonic stem cells by a similar process, then someday, a simple skin biopsy or blood sample might provide the material to generate embryonic stem cells that are specific to each individual, without the need for genetic engineering or killing the smallest among us. This truly creates endless possibilities for therapeutic options.