PS: Knoepfler Lab Stem Cell Blogから引用；
（最初の段階で細胞蛍光が見られるのは死滅細胞によるautofluorescenceではないかという疑問が挙げられていたが、当然のことながら、PI ( propidium iodide)を利用し検査が行われていたことを笹井氏が説明していた。下記のKnoepfler教授の「今までのSTAP細胞大失態からのトップ10の教訓」はためになることが書かれている。
1．Top 10 lessons from STAP cell fiasco so far
Posted on April 16, 2014
STAP stem cells The ultimate fate of the two Nature STAP cell papers remains in troubled limbo.
One of the senior authors, Dr. Sasai, held a news conference yesterday in Japan that included a call for the papers to be retracted.
He was variously quoted as believing in STAP or alternatively as just thinking it was an unproven hypothesis.
Whether STAP cells or STAP stem cells are real or not, overall STAP has been a disastrous situation. Can biomedical science learn anything from this STAP fiasco? Maybe some thoughts below.
■ Organize, keep records, and annotate your images. It seems that the STAP papers were plagued by confusion over images and the way image data was handled or changed. More broadly in science, sometimes research projects generate tons of data even if they are not “big data” genomics projects. In fact, it is not unusual for just one line of cell or developmental biology research to generate hundreds of image files. Each one might have a different exposure time or other varying attributes and researchers might legitimately adjust some images that are too faint, etc. It is wise to use a system where lab members catalog images and a naming paradigm that includes the date. Any changes to images must also be documented in writing and original unmodified forms kept as trackable backup files.
■ Don't always believe your eyes. It seems that some of the STAP authors believe in STAP, but I wonder if “STAP” to them means simply cells glowing green? The reality is that for cells to be STAP they must have functions and pass a whole host of tests, not simply glow green (even if that green is real and not autofluorescence–see next point below) from a Oct4-GFP reporter. Just because you “see the green light” doesn’t mean it is STAP. Human beings including scientists are very visual creatures. Who doesn’t find certain microscopy images captivating? Seriously, a microscopy image can be like a piece of fine art. But sometimes data in the form of a visual image can be deceiving. The more general expression “I see the light!” is about discovery, but usually more about a discovery of beliefs rather than facts.
■ Danger, autofluorescence ahead. And speaking of “seeing the green light”, it was only days into the STAP craziness when a stem cell biologist told me in confidence that s/he believed STAP could be largely a mistake due to misinterpretation of autofluorescence as real signal. I still haven’t seen compelling evidence against the notion that the greenness of STAP is just autofluorescence in certain images and FACS data. Perhaps this STAP mess will make the entire biomedical research community more cognizant of the dangers of misreading cellular autofluorescence and the need to check for it.
■ Cells are not always what you think they are. It seems quite possible that some of the cells involved in the STAP cell research were not what some of the researchers thought they were. Cell line contamination is a common problem at least in part because different kinds of cells are stored and sometimes simultaneously grown in labs. Cells also grow at different rates so contamination of one cell type with even a few different cells can burgeon into a big problem over a surprisingly short period of time.
■ To be a good reviewer, data should always trump big names in importance. One of the problems exemplified by the STAP papers is that big name authors can sometimes sway reviewers inappropriately to be lenient on papers. In the end, as a good reviewer, you have to keep focused on the data, not the reputation of the authors.
■ Weigh the risks, benefits and responsibilities of being an author yourself. If you are possibly going to be an author on any given collaborative paper, use caution. Read the paper carefully, ask to see data if you have concerns, think about what it means for you to be an author of this paper, and if in doubt, at least consider saying ‘no’. In this time when most everyone wants more publications, sometimes paradoxically it is best not to be an author. Of course, sometimes potential co-authors or even corresponding authors don’t know about problems in papers and such problems can be hard to find so the decision as to whether to be an author can be tricky. Finally, take the specifics of those “author contributions” sections seriously as to what you did or did not do for any given paper. I wonder at this time how many of the STAP authors, if they could go back in time, would choose not to be author on those papers?
■ To editors, be extra-cautious about those “sexy” papers. A paper like either of the STAP ones is certainly exciting on first read and could have big impact. You might call them “editor-bait”. Heck, despite the controversy the STAP papers have already been cited many times in just a couple months by other papers. However, these kinds of high-profile papers are high risk for journals and editors too. As with the reviewer caution above, editors should not be swayed by big name authors if the story seems too good to be true and if anything, the more excited an editor is about a paper the more cautious they should be in how they handle it. Paradoxical? Perhaps, but I think it’s true.
■ To journals, give all manuscripts a thorough automated checkup. EMBO now reportedly has an automated screening process for manuscripts for image issues (manipulation, duplication, etc) and EMBO editors have indicated that the STAP papers would not have passed. Did Nature not have such screening in play when the STAP papers were reviewed? Does it now hopefully have such a system? Which journals automatically test for plagiarism of text or images? Clearly this kind of automated manuscript checkup should be standard procedure for all journals.
■ To scientists, don’t fall in love with your hypothesis. STAP almost feels like a fairy tale love story gone bad. I’m not talking about the love of two folks for each other like Cinderella and the Prince in a Disney movie, but rather the way scientists can sometimes fall in love with an idea. Avoiding this trap is naturally easier said than done because ideas can be super exciting.
■ Check the hype. There is nothing wrong with being excited about a paper or its potential impact, but be cautious about crossing the line to outright hype. Not everything is a “breakthrough” and that’s OK. Good, strong science doesn’t have to be a stunning breakthrough to have a positive impact. Scientists, journals, and institutions need to walk a fine line between advocating for our work publicly (which is needed) and overstating its importance, especially to the public or reporters. Many media folks are prone to hyping science as well. I believe that STAP was hugely hyped by many of the parties involved.
Any other things possibly to be learned in a positive way from all the STAP calamity?
Kenneth Ka-Ho Lee The Chinese University of Hong Kong
Thank you all for your kind comments. I said I was not going to live blog anymore but I also hate to leave the STAP story incomplete.
Previously (1st of April), we have demonstrated that pipetting Oct4-GFP lung fibroblasts through different size glass pipettes for 3 days can elicit Oct-4 and Nanog expression by approximately 10 folds and Sox2 by 2 folds. The expression level was not high enough to label them as pluripotent stem cells.
We have now completed a second set of experiments. We did not do any acid-bathing this time - only mechanical pipetting. We pipetted the cells for 30 mins and then grew them on non-adhesive culture plates. On successive days, the cells were pipetted twice (5 mins) a day. The same culture condition as in the first set of experiment. We established by qPCR that this hash treatment induced Oct-4 expression by 2.5 folds. It did not induce Sox2 and Nanog expression (Table 1).
Since there were still cells alive at the end of 3 days, we decided not to do only more pipetting and just let these cells grow on the non-adhesive culture plates for a further 3 days. At the end of 6 days of culture, the cells were harvested for qPCR. We found that Oct-4 expression was maintained by approximately 2.5 folds. Like in day 3, Sox2 and Nanog expressions were not induced (Table 2).
Hypothesis: Perhaps treating these cells with Valproic acid to open up the histones might facilitate a better outcome in terms of expression of the stemness markers?
There is a Chinese saying that: when we are about to die, we can release a final burst of energy that allow us to finish off some of the things that we want to do before we leave this world “迴光返照”. Could it be that the fibroblasts that we mechanical pipetted to the point of death “reawakens” and start randomly express some of the stem cell markers? These cells are definitely not STAP cells. Perhaps I should call them “April fool” cells or even “reawakening” cells (Joke!).
Even better, I could call them “Easter” cells since Jesus was reawakened from death and the Easter holidays are fast approaching us (Joke!)?
I am going to take a page from Paul Knoepfler's Blog and ask you to vote.
What should I call the cells that I created here?
1. STAP cells
2. April Fool Cells
3. Reawakening cells
4. Easter cells