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.

Testing Cord Blood Stem Cells as a Treatment for Cerebral Palsy


The Cord Blood Registry (CBR) has announced partnerships with the University of Texas Health Science Center at Houston and Georgia Regents University to establish FDA-regulated clinical trials to test the efficacy of intravenous infusions of umbilical cord blood in children with cerebral palsy.

According to statistics from the Center for Disease Control (CDC), one in every 323 children in the United States has been diagnosed with cerebral palsy or related disorders that affect movements, balance, and posture.

In these proposed clinical trials, a child who has been diagnosed with cerebral palsy-type disorders will receive intravenous infusions of their own umbilical cord blood that was banked at the time of their birth.

Because cerebral palsy results from abnormal brain development or brain damage to the motor centers of the developing brain, umbilical cord blood treatments might provide the means to help the brain heal itself. These umbilical cord blood treatments will take place along side more traditional treatments such as surgery, medications, orthopedic braces, and physical, occupational, and speech therapies.

Encapsulated Stem Cells to Treat Diabetes


A research group from the Sanford-Burnham Medical Research Institute in La Jolla, San Diego, California has used pluripotent stem cells to make insulin-secreting pancreatic beta cells that are encapsulated in a porous capsule from which they secrete insulin in response to rising blood glucose levels.

“Our study critically evaluates some of the potential pitfalls of using stem cells to treat insulin-dependent diabetes,” said Pamela Itkin-Ansari, an adjunct assistant professor with a joint appointment at UC San Diego. “We have shown that encapsulated hESC-derived pancreatic cells are able to produce insulin in response to elevated glucose without an increase in the mass or their escape from the capsule. This means that the encapsulated cells are both fully functional and retrievable.”

For this particular study, Itkin-Ansari and her colleagues used glowing cells to ensure that their encapsulated cells stayed in the capsule. To encapsulate the cells, this group utilized a pouch-like encapsulation device made by TheraCyte, Inc. that features a bilaminar polytetrafluoroethylene (PTFE) membrane system. This pouch surrounds the cells and protects from the immune system of the host while giving cells access to nutrients and oxygen.

With respect to the cells, making insulin-secreting beta cells from embryonic stem cell lines have met with formidable challenges. Not only are beta cells differentiated from embryonic stem cells poorly functional, but upon transplantation, they tend to be fragile and poorly viable.

To circumvent this problem, encapsulation technology was tapped to protect donor cells from the ravages of the host immune system. However, an additional advance made by Itkin-Ansari and her colleagues is that when they encapsulated islet-precursor cells, derived from embryonic stem cells, these cells survived and differentiated into pancreatic beta cells. In fact, islet progenitor cells turn out to be the ideal cell type for encapsulation, since they are heartier, and differentiate into beta cells quite efficiently when encapsulated.

In their animal model tests, these cells remained encapsulated for up to 150 days. Also, as an added bonus, because the progenitor cells develop glucose responsiveness without significant changes in mass, they do not outgrow their capsules.

In order to properly get this protocol to work in humans, Itkin-Ansari and her group has to scale up the size of their capsules and the number of cells packaged into them. Another nagging question is, “How long will an implanted capsule last in a human patient?

“Given the goals and continued successful results, I expect to see the technology become a treatment option for patients with insulin-dependent diabetes,” said Itkin-Ansari.

To date, Itkin-Ansari and others have been able to successfully treat diabetic mice. The problem with these experiments is that they mice were made diabetic by treatment with a drug called beta-alloxan, which destroys the pancreatic beta cells. Human type 1 diabetic patients have an immune system that is sensitized to beta cells. Even though the encapsulation shields the beta cells from contact with the immune system, will this last in human patients with an aggressive immune response against their own beta cells? It seems to me that induced pluripotent cells made from the patient’s own cells would be a better choice in this case than an embryonic stem cell line.

Nevertheless, this is a fine piece of research for diabetic patients.

Scar-less Healing in the Fetus


In early fetal development, skin wounds undergo regeneration and healing without scar formation. Unfortunately, this wound healing mechanism later disappears, but by studying the fetal stem cells capable of this scarless wound healing, researchers may be able to apply these mechanisms to develop cell-based approaches able to minimize scarring in adult wounds.

Michael Longaker, Peter Lorenz, and co-authors from Stanford University School of Medicine and John A. Burns School of Medicine, University of Hawaii, Honolulu, describe a new stem cell that has been identified in fetal skin and blood that may have a role in scarless wound healing. In the article “The Role of Stem Cells During Scarless Skin Wound Healing,” the authors propose future directions for research to characterize the differences in wound healing mechanisms between fetal and adult skin-specific stem cells.

“This work comes from the pioneers in the field and delineates the opportunities towards scarless healing in adults,” says Editor-in-Chief Chandan K. Sen, PhD, Professor of Surgery and Director of the Comprehensive Wound Center and the Center for Regenerative Medicine and Cell-Based Therapies at The Ohio State University Wexner Medical Center, Columbus, OH.

New Method Derived Skeletal Muscle Cells from Pluripotent Stem Cells


A University of Wisconsin research team led by Masatoshi Suzuki has devised a new protocol for the production of large quantities of skeletal muscle cells from pluripotent stem cells.

Suzuki and his team used embryonic stem cells lines and induced pluripotent stem cells to generate large quantities of muscles and muscle progenitor.

Suzuki adapted a technique used to make brain cells to derive his muscle cells in culture. He grew the stem cells as floating spheres in high concentrations of two growth factors: fibroblast growth factor-2 (FGF2) and epidermal growth factor (EGF). This combination of growth factors directed the stem cells to differentiate into skeletal muscle cells and muscle progenitors.

To replace damaged or diseased muscles in the clinic, physicians will require large quantities of muscle cells. Therefore, there was an ardent search to design a technique that was efficient, but also fast and relatively simple. Even though several protocols have been devised to differentiate pluripotent stem cells into muscle cells, not all of these protocols are practical for clinical use. For example, some protocols are simply too cumbersome for clinical use. Still others make use of genetically engineered cells that have not been approved for clinical use.

Earlier, Suzuki transplanted lab-engineered skeletal muscle into mice that had a form of amyotrophic lateral sclerosis. These animals had better muscle function and survived better than the control animals.

The muscle progenitors generated in Suzuki’s laboratory could potentially play a similar role in human patients with Lou Gehring’s disease. Suzuki’s method can grow muscle progenitor cells, which can grow in culture, from induced pluripotent stem cells, which are derived from the patient’s own cells. Such cells could be used as a model system to study the efficacy of particular treatments on the patient’s muscles, or they could be used to treat patients who have muscle defects.

“Our protocol can work in multiple ways and so we hope to provide a resource for people who are exploring specific neuromuscular diseases in the laboratory,” said Suzuki.

The advantages of Suzuki’s protocol are manifold. First, the cells are grown in a defined medium devoid of animal products. Secondly, the stem cells are grown as spheres, and these grow faster when grown as spheres than they do with other techniques. Third, 40-60 percent of the cells grown in this culture system differentiate into skeletal muscle cells or muscle progenitor cells. This is a very high proportion of muscle cells when compared to other protocols.

Suzuki hopes that by toying with the culture system, he and his colleagues can increase this proportion of muscle cells that form from the initial stem cell culture. This would enhance the potential of using these cells for clinical purposes.

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.