See below for update – this research was the subject of an investigation into fraud conducted by the Riken institute. Primary papers in Nature have now been retracted.
In 2012 Prof Sir John Gurdon and Shinya Yamanaka shared the Nobel Prize in Physiology or Medicine for the discovery that mature, specialised cells could be reprogrammed to become immature cells capable of developing into all tissues of the body. Their findings have revolutionised our understanding of how cells and organisms develop and given scientists the powerful research tool that is the induced pluripotent stem cell (iPS cell).
In 2006 Shinya Yamanaka and colleagues reported an elegant but complicated method for inducing pluripotency in adult cells and creating these iPS cells. The technique involves re-introducing four specific transcription factor genes (originally Oct4, Sox2, cMyc and Klf4), that are important in early stem cells, into adult cells. Although the success rate of creating iPS cells was only 0.1%, this technique was widely hailed as a success story in the journey to making stem cell therapy viable in the clinic and moving away from controversial uses of embryonic stem cells.
Earlier this year, however, a group of different researchers from Japan reported a surprisingly simply alternative method for iPS production. In a two papers published in the Journal Nature (Obakata, H. et al, 2014) this team showed that exposure to stress, including low pH, can make cells even more malleable than traditional iPS cells, and do it faster and more efficiently.
“It’s amazing. I would have never thought external stress could have this effect,” says Yoshiki Sasai, a stem-cell researcher at the RIKEN Center for Developmental Biology in Kobe, Japan, and a co-author of the latest studies. It took Haruko Obokata, a young stem-cell biologist at the same centre, five years to develop the method and persuade Sasai and others that it works. “Everyone said it was an artefact — there were some really hard days,” says Obokata.
Obokata was regularly carrying out routine culture of adult cells as part of her PhD work when she noticed that some would start to look like stem cells when squeezed through a capillary tube. It may seem a little backward, but shape is often a useful indicator of reduced or increased pluripotency in stem cells – she had the idea that stressing cells might make them pluripotent. Like any good scientist, she decided to follow her curiosity and investigate this further. Eventually she found that bacterial toxin, low pH and physical squeezing were all able to coax the cells into showing signs of pluripotency – but she faced an uphill battle to convince her fellow researchers.
To truly be considered pluripotent, Obokata had to show that these cells could turn into all cell types. The easiest way to do this is to fluorescently label iPS cells in the mouse embryo. If the introduced cells are pluripotent, they should differentiate along with the rest of the mouse embryo and the resultant mouse should have glowing cells in each of its tissues. Eventually Obokata managed to produce a fully fluorescent mouse, but this wasn’t enough for some of her critics.
To convince sceptics, Obokata had to prove that the pluripotent cells were converted mature cells and not pre-existing pluripotent cells. So she made pluripotent cells by stressing T cells, a type of white blood cell whose maturity is clear from a rearrangement that its genes undergo during development. She also caught the conversion of T cells to pluripotent cells on video. Obokata called the phenomenon stimulus-triggered acquisition of pluripotency (STAP).
The results could fuel a long-running debate. For years, various groups of scientists have reported finding pluripotent cells in the mammalian body, such as the multipotent adult progenitor cells described by Catherine Verfaillie, a molecular biologist then at the University of Minnesota in Minneapolis. But others have had difficulty reproducing such findings. Obokata started the current project in the laboratory of tissue engineer Charles Vacanti at Harvard University in Cambridge, Massachusetts, by looking at cells that Vacanti’s group thought to be pluripotent cells isolated from the body. But her results suggested a different explanation: that pluripotent cells are created when the body’s cells endure physical stress. “The generation of these cells is essentially Mother Nature’s way of responding to injury,” says Vacanti, a co-author of the latest papers.
Obokata has already reprogrammed a dozen cell types, including those from the brain, skin, lung and liver, hinting that the method will work with most, if not all, cell types. On average, she says, 25% of the cells survive the stress and 30% of those convert to pluripotent cells — already a higher proportion than the roughly 1% conversion rate of iPS cells, which take several weeks to become pluripotent. She now wants to use these results to examine how reprogramming in the body is related to the activity of stem cells. Obokata is also trying to make the method work with cells from adult mice and humans.
“The findings are important to understand nuclear reprogramming,” says Shinya Yamanaka, who pioneered iPS cell research. “From a practical point of view toward clinical applications, I see this as a new approach to generate iPS-like cells.”
It’s incredibly important to question new research like this. In fact, the RIKEN research institute in Japan (where the authors work) has just opened an investigation into this work after some labs raised concerns over reproducibility.
None of ten prominent stem-cell scientists who responded to a questionnaire from Nature has had success. A blog soliciting reports from scientists in the field reports eight failures. But most of those attempts did not use the same types of cells that Obokata used.
Some researchers do not see a problem yet. 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.
The jury is definitely still out in terms of reproducibility, and we must be careful not to immediately discount novel discoveries. Perhaps more worrying are concerns raised by several blogs into potential irregularities in some of Obokata’s previous work.
In an earlier paper published by Obokata in 2011 concerning the potential of stem cells in adult tissues, a figure showing bars meant to prove the presence of a certain stem-cell marker appears to have been inverted and then used to show the presence of a different stem-cell marker. A part of that same image appears in a different figure indicating yet another stem-cell marker. The paper contains another apparent unrelated duplication.
Charles Vacanti, an anaesthesiologist at Harvard Medical School in Boston and a co-author on the 2011 paper, told an investigation started by Nature that he learned only last week of a “mix up of some panels”. He has apparently already contacted the journal to request a correction and states that it seems to have been an honest mistake and “did not affect any of the data, the conclusions or any other component of the paper.”
I guess we should watch this space…
Obokata, H., Sasai, Y., Niwa, H., Kadota, M., Andrabi, M., Takata, N., Tokoro, M., Terashita, Y., Yonemura, S., Vacanti, C.A., et al. (2014a). Bidirectional developmental potential in reprogrammed cells with acquired pluripotency. Nature 505, 676-680.
Obokata, H., Wakayama, T., Sasai, Y., Kojima, K., Vacanti, M.P., Niwa, H., Yamato, M., and Vacanti, C.A. (2014b). Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature 505, 641-647.
Gurdon, J.B. (1962). The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles. Journal of Embryology and Experimental Morphology 10:622-640.
Takahashi, K., Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663-676.