From stem cell break through to major concerns: the japanese story

[icon name=”user” class=”” unprefixed_class=””]  By Joanne P. Shelby-Klein BSN RN

When reports of a possible successful Stimulus Triggered Acquisition of Pluripotent cells (STAP) were published in the Nature Journal in January 2014, the scientific community became very excited at the possibility of a simple way to produce embryonic stem cells to be used in future medical treatments. Within weeks the excitement turned to concern as questions were raised about the accuracy of the research methods and results as reported by Dr. Haruko Obokata and her research team. Japan’s RIKEN Research Institute launched an immediate investigation into the concerns with a five person expert panel. This panel found six major problems that needed further examination. Two of the problems found were unintentional mistakes. The remaining four included a charge of plagiarism and including an image of a special electrophoresis gel that appeared to be added in at a later date.

RIKEN investigated every aspect of the project including DNA samples of all the cell types used. Lab notebooks, emails, documents and remaining data left in the equipment used were all carefully examined. They also conducted many lengthy face to face interviews with all people involved in the research. It was found that three different types of embryonic stem cells were mixed in with the STAP cells. Whether it was accidental or deliberate contamination was hard to figure out because they were unable to determine who handled the experiments and added the cells. The committee then offered an opinion that the experiment was not properly performed and some of the reported DNA figures did not match with original data. They concluded that the two figures were fabricated and pointed to scientific misconduct. They also concluded that both the questionable figures came from Dr. Obokata , that she was responsible for them and their fabrication. They also concluded that the co-authors and associates involved with the project overlooked the fabrication as well as other breaches of conduct and procedures. It was concluded that two key researchers, Dr. Wakayama and Dr. Sasai, played a major role in the research misconduct as they had major responsibilities for the final data presented and written.

As the RIKEN investigation moved forward in March of 2014, the quality and accuracy of Dr. Obokata’s Doctoral Thesis, completed in 2011, was being questioned and investigated by the University that granted it, Wasaba University in Tokyo. It was reported that the first twenty pages of her thesis were copied from the US National Institute of Health primer on stem cells with no credit given to the source as would be normal procedure in scientific writing. They also found one photograph was taken from a commercial website and no credit give to the source. Dr. Obokata wanted to retract the thesis but her request was not granted by the University.

In May of 2014 the RIKEN Institute instructed Dr. Obokata to retract the article in Nature 505: 641-647. In July the two STAP articles published in Nature were retracted at the request of Dr. Obokata and the co-authors. In June of 2014, a second preliminary committee of investigation was launched to look further into the complaints and allegations because of the diversity and number of serious questions still unanswered.

By September of 2014 the full investigation was under way by a committee of seven experts from outside of the RIKEN Institute. The Committee met fifteen times between September 22 and December 23, 2014. The committee carefully reviewed the findings in the preliminary report and discussed the best way to perform the investigation. They looked at the original data, from summer to autumn 2012, that lead to the concerns and charges of research misconduct. Once again they examined the original data and compared it to the data and files used in the article manuscript preparation. Once again, lab notebooks, progress reports and emails of the researchers were thoroughly reviewed and compared to the research regulations and the original procedure approved. All people involved in the research were interviewed and questioned multiple times for their roles and findings in the project.

Investigation finding number one The three lines of STAP cells listed as such in the papers actually came from embryonic stem cells with two possible reasons for this occurring. Either the STAP cells culture dishes were contaminated with embryonic stem cells during the growing process or the embryonic stem cell cultures were contaminated with STAP cells before putting them on the culture dish.

Finding two: Can the person who contaminated the cell cultures be identified? The committee reviewed the procedures for performing the cultures and identified that Dr. Wakayama and Obokata both handled the specimens. However, the procedure required the cultures to grow for seven days in an incubator separate from the lab, with a minimal number of people entering the room during the day. Of concern is the fact that at night, any number of people could have entered the lab, recognized the stem cell culture dish and contaminated them. Every possibility was examined and no specific evidence for direct contamination was found. This means that the committee could not identify who might have contaminated the samples.

Finding three: Concerns about figures, data and text of the papers. The committee found a discrepancy in dates of the STAP cell and Embryonic Stem cell data. Dr. Obokata offered an explanation by saying the cell growth experiments were performed separately. The embryonic cells were done in spring/summer 2011 and the STAP cells started in January and February 2012. The lab notebooks showed no notes that support this and Dr. Obokatas work schedule and attendance do not show that she could have done the experiment every three days. Dr. Obokata admitted that she followed Dr. Wayakama’s advice to use a figure from another paper to measure cell growth. Cell growth would normally have been measured every three days. Dr. Obokata admitted she measured only at the beginning, as well as sporadically, and not when the cultures were re-plated as the procedure demanded.

Evaluation of the data storage showed that Obokata handled the data inappropriately, allowing opportunity for mistakes to happen and making the traceability of the research more difficult if not impossible. In addition, only part of the data had been deliberately selected and assembled into panels that showed support for the authors model so that readers were misled into accepting the authors conclusions. In other words data was fabricated. According to the investigators, Dr. Wayakam should have been monitoring and guiding Dr. Obokata in the collecting of this data and this caused him to have some responsibility in this failure even though he did not actively participate in the data collection failure.

Also it was found that two different histological images (microscopic tissue samples) were found to be labeled inconsistently when compared to the DNA samples. The investigators could not prove beyond a shadow of a doubt that the samples and images were not simply mislabeled by mistake and not pure misconduct.

The committee requested, on numerous occasions, that Dr. Obokata present original data. Every attempt was made to locate the data on every hard disk attached to every microscope in the lab. No original data was found. They came to the conclusion that the images on the microscope show different samples, not the same one. The samples may have been obtained with a different camera, different exposure time or processed in a different way. Keep in mind that sample collection and data collection must be done following the same process for the sample and research to be valid. The committee could not prove that the lack of original data and labeling errors was simply made from common labeling or imaging mistakes. Some of these issues could have been avoided by making the best effort possible to handle the data and accurately use appropriate software. In addition, it is possible that Dr. Obokata may not have had appropriate training in using the equipment, but the committee could not reach a definite conclusion because of the lack of original data.

Finding Number Four The investigators reviewed the possibility of concealment of data. Dr. Obokata reported specific data on recombination of STAP cells or STAP stem cells. When this was attempted to be replicated by the co-authors, the same results did not occur. She then asked a member of the Wayakama lab team to repeat the same experiment and again the results could not be replicated. This appeared to indicate inconsistencies in the data from the very first experiment. The investigators reported that Dr. Niwa felt that the recombination data included in the manuscript to be sent to Nature be carefully considered before including. Dr.Niwa was concerned after the manuscript was published that other researchers would not be able to reproduce the experiment. He also felt the original experiment steps were insufficient and needed to be explained further before it could be repeated. Again, based on available materials, logs and data from the original experiment the committee could not rule misconduct.

The second Investigating committee released its final report ,as summarized above, in December of 2014. The findings and conclusions were established by using the RIKEN Regulations on the Prevention of Research Misconduct from September 2012, regulation 61. The committee also used the MEXT(Japanese Ministry of Education, Culture, Sports, Science and Technology) guidelines for research misconduct issued on August 26, 2014 to help arrive at their conclusions. It’s important to know that RIKEN had revised its regulations on the effective date of November 25,2014, while the investigation was continuing. However the committee used the definitions of the original 2012 regulations to define research misconduct as “fabrication, falsification, or plagiarism.” Based on these definitions the committee concluded that Dr. Obokata fabricated data concerning the stem cell growth curve and represents research misconduct. Dr. Niwa and Dr. Wakayama were not found to be guilty of research misconduct. The research misconduct, such as that done by Dr. Obokata, serves as a set back and blockage to the pursuit of strong and necessary research science.

The committee also concluded that the research misconduct found to date in the published and retracted pages is only a smart part of the problems in the papers. Evidence pointed to STAP cells being contaminated with embryonic stem cells or contaminated in such a way that the scientific method could not explain it. This evidence does not back up the main premise of the papers on STAP cells. This may have happened because of the limited ability of the investigators as well as their scope of authority. Limited ability and authority is almost always a frustration to investigators who must look into what happens during a research study. The investigating committee recognized this limitation and based their findings and conclusions on what they and other researchers would have done in similar circumstances and the information available.

More importantly, the investigators asked what could be done to minimize and hopefully prevent this type of research misconduct from happening again. The MEXT guidelines and regulations support the belief that research misconduct is a matter of researcher ethics and responsibility to both scientists and patients. Preventing research misconduct depends both on the self-discipline of the researchers and also on the ability of the research facility and scientific community to govern itself within the guidelines and regulations established by the scientific community.

The thoroughness of the investigation and the time frame in which it was conducted is impressive. The RIKEN researchers showed dedication and determination to complete an appropriate self- regulating course of action when a problem occurred, action that can and should happen if any research lab identifies a problem. The problems identified in the STAP story could happen in any lab and research facility. Preventing these problems from occurring is the responsibility of the lab itself by making sure that staff is properly educated and effective management is in place. Plagarism or copying someone elses work and calling it your own is a very serious situation. Fabricating data to support a hypothesis is an equally serious charge. All of this points to a need for ongoing education in research ethics and possible consequences of not following the accepted practices. Failing to follow regulations and evidence/best practices in research can cause serious problems including a lack of trust in future research. The healing process, to the research community, from the STAP papers has started, thanks to the prompt action and thorough investigation by the RIKEN Institute. Research work will continue on. It’s important to all of us.

Resources:

  1. http://www.riken.jp/en/pr/topics/2015/20150126_2/
  2. Nature Volume:505Pages: 676–680Date published:(30 January 2014)DOI: doi:10.1038/nature12969 Received 10 March 2013 Accepted20 December 2013 Published online 29 January 2014 Retraction (July, 2014)
  3. http://www.fiercebiotechresearch.com/story/scientist-lets-not-discredit-acid-bath-stem-cell-studies-yet/2014-02-24
  4. http://phys.org/news/2014-12-japan-lab-dismisses-ground-breaking-stem.html
  5. Report on STAP Cell Research Paper Investigation, December 25, 2014
  6. Research Publication Investigative Committee Isao Katsura, Chair
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