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STAP細胞の再現に香港のグループが成功か?
投稿日 2014年4月2日 投稿者: marugametorao
http://marugametorao.wordpress.com/2014/04/02/stap%e7%b4%b0%e8%83%9e%e3%81%ae%e5%86%8d%e7%8f%be%e3%81%ab%e9%a6%99%e6%b8%af%e3%81%ae%e3%82%b0%e3%83%ab%e3%83%bc%e3%83%97%e3%81%8c%e6%88%90%e5%8a%9f%e3%81%8b/
香港のグループがSTAP細胞の再現に成功か?
私がKnoepfler Lab Stem Cell Blogに投稿した疑問点や小保方さん、丹羽さんにメールした内容に対する回答が、Vacantiが公開したSTAP細胞の作製法の中に記載されていた。やはり、Key issueの一つはtrituration procedureであった。
（ I appreciate very much if you answer my question below. I sent the following e-mail to Dr Obokata. In your published today’s protocol, you did not describe the trituration procedures in detail. Is it not important?）
Protocol for generating STAP cells from mature somatic cells
https://research.bwhanesthesia.org/site_assets/51520d191eea6679ce000001/cterm/Refined_STAP_protocol-9a685fc86fec5ca857ad58ae75462d07.pdf
https://research.bwhanesthesia.org/research-groups/cterm/stap-cell-protocol
A5. As a final extremely important step in the trituration process, make two fire polished pipettes with very small orifices as follows: Heat the standard 9” glass pipette over a Bunsen burner and then pull and stretch the distal (melting) end of the pipette, until the lumen collapses and the tip breaks off, leaving a closed, pointed glass tip. Wait until the pipette cools, and then break off the closed distal tip until a very small lumen is now identifiable. Repeat this process with the second pipette, but break the tip off a little more proximally, creating a slightly larger distal lumen. The larger lumen should be about 100-150 microns in diameter, while the other pipette should have a smaller lumen of about 50-70 microns. Now triturate the cell suspension through the pipette with the larger lumen for 10 minutes. Follow this with trituration through the pipette having the smaller lumen (50-70 microns) for an additional 15 minutes. Continue to triturate the suspension until it passes easily up and down the fire polished pipette of the smaller bore. This is a very important step. Do not skip this step, or take a shortcut. Again, remember to precoat each pipette with media. Also, during trituration, try to avoid aspirating air and creating bubbles or foam in the cell suspension.
その方法に基づいて、香港の研究グループがSTAP細胞の再現に一部成功した模様だ。しかし、数回以上の再確認が必要である。
Blogger Reports STAP Success
A stem-cell researcher claims to have reproduced stimulus-triggered acquisition of pluripotency by following a revised protocol posted online last week.
http://www.the-scientist.com/?articles.view/articleNo/39601/title/Blogger-Reports-STAP-Success/
Last week, Chinese University of Hong Kong’s Kenneth Lee said he would blog his group’s attempts to replicate the controversial stimulus-triggered acquisition of pluripotency (STAP) technique described in two January Nature papers using a refined protocol that Charles Vacanti of Harvard Medical School and Brigham & Women’s Hospital had posted online. Vacanti, one of the authors on the original STAP publications, has vigorously defended the technique even though other labs have failed to replicate the results. “The data and conclusions are honest and valid,” he told reporters in early March.
Lee now claims he has succeeded at reproducing STAP using Vacanti’s protocol—well, sort of. In posts at ResearchGate, Lee presented confocal and qPCR analyses from the transgenic fibroblasts his team mechanically triturated and bathed in acid over a period of three days, per the latest instructions. Only one day into this procedure, Lee’s team noticed that lots of stressed-out cells were dying. “We expect more and more necrotic cells will form during culture,” Lee wrote on March 28, adding: “The third day of culture is the critical period as reported by Vacanti.”
Today (April 1), Lee posted his team’s day three qPCR analysis, which showed that the trituration-only treated cells expressed higher levels of the pluripotency regulator Oct4 than did the trituration plus acid-bathed cells or the controls. “I am shocked and amazed by the qPCR results for the 3 day-old control and STAP cultures,” he wrote.
But other commenters at ResearchGate have questioned whether autofluorescence may be skewing Lee’s STAP results. And the irony of Lee posting such positive news today was not lost on Sergey Kiselev from the Russian Academy of Sciences’ Vavilov Institute of General Genetics, who—citing an earlier misconduct investigation surrounding STAP—wrote: “Great joke for April Fools! . . . Go on, Ken, please.”
Correction (April 1): An earlier version of this post stated the cells were titrated when in fact they were triturated. The Scientist regrets the error. (引用終了)
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