理化所发育生物学中心小保方晴子被质疑的论文提出了一种颠覆性新技术，被该课题组命名为STAP细胞，文章全面介绍了，她们如何将小鼠血液中的白细胞，通过简单地酸和挤压处理，就可诱导成为能分化为任何组织的培养样干细胞。论文发表后一周，就有人发现了论文中存在的多个问题。4月1日，理化研究所调查委员会确定小保方晴子在《自然》论文中存在明确的学术不端问题。4月8日，小保方晴子对调查结论提出上诉请求。5月6日，理化研究所发布了一份21页的报告，来自同一调查委员会逐项驳斥了小保方晴子的上诉要求，并最后得出结论，“没有任何必要对调查委员会3月31日的结论进行重新调查”。理化研究所将准备对小保方晴子及共同作者的做出最终处罚。
理化研究所所长诺贝尔奖获得者野依良治昨天的声明中说，他们已经决定不重新调查关于学术不端的问题，并已经建议小保方晴子撤回涉及学术不端的论文。小保方晴子的律师HideoMiki告诉日本时报说自己对这一决定极端不满。
野依良治昨天的声明中也提到一些委员会成员（4／6）自己存在学术不端的问题，说他们相信委员会已经出色地完成了调查，并得出适当的调查结论。至于他们自己的学术不端问题，将分别进行独立调查。
昨天，理化研究所已经成立了一个独立的委员会，将决定对小保方晴子进行那种纪律处罚。理化研究所执行主任MinoruYonekura昨天下午在新闻发布会上说，委员会将大概一个月后提出最后决定。中国香港大学胚胎学和干细胞学家KennethKa-HoLee也恰逢今天在F1000Research在线发表了他们在徒劳验证小保方晴子论文方面的细节。但是文章的结论是他们无法重复出结果，可能是由于研究方法的问题。（In conclusion, we have not been able to replicate Obokataet al.’s findings to produce STAP stem cells from somatic cells. It appears that the method for producing STAP stem cells is not as simple and straight forward as has been reported.）
http://news.sciencemag.org/asiapacific/2014/05/riken-makes-verdict-two-problematic-stem-cell-papers-final
Nevertheless, we have tried to replicate the first stages of Obokata’s findings using CD45+ splenocytes isolated from Oct4-GFP neonates, but could not activate the expression of the Oct4-GFP transgene. This is despite using their most updated protocol for producing STAP cells, which was reported inProtocol Exchange(Obokataet al., 2014c). We also tried using Oct4-GFP lung fibroblasts instead of splenocytes, but again we failed to detect Oct4-GFP expression after acid-treatment. Occasionally, there were cells that appeared GFP positive, but we later determined them to be autofluorescence from apoptotic cells. We made sure that the pH was exactly maintained at pH 5.7 during the experiments by measuring the pH before and after cell treatment. This is because theProtocol Exchangeprotocol placed a lot of emphasis on maintaining an optimal pH during the acid treatment of the cells. We found that there was a pH 0.1 increase after the acid buffer was added to treat the cells – so our starting pH was actually 5.6 to compensate. At the end of acid bath stimulation, we also measured the pH of the buffer to confirm that it was still pH 5.7. Therefore, our inability to produce STAP cells could not be attributed to changes in the pH during the cell stimulus procedures.
Another possibility why we could not replicate Obokata’s results might be the difference in the strains of Oct4-GFP transgenic mice used. We acquired our transgenic mice from The Jackson Laboratory (CBA-Tg (Pou5f1-EGFP) 2Mnn/j) while Obokata used transgenic mice generated byOhboet al., 2003. Their transgenic mice were developed from a C57BL.6J background, and carry the EGFP cDNA under the control of an Oct4 18-kb genomic fragment (consisting of a minimal promoter and proximal and distal enhancer). Perhaps the transgene in these mice is more easily activated than in our Jackson Laboratory mice. This could potentially explain why Obokata’s transgenic splenocytes, but not our transgenic splenocytes, expressed the EGFP reporter following acid bath treatment. Nevertheless, in the context of generating STAP stem cells, it is not the expression of the transgene that is important but rather the expression of the endogenous Oct4 gene - and related endogenous stemness genes, Sox2 and Nanog. Expression of these genes could not be demonstrated using qPCR analysis following splenocyte acid treatment and culture.
In conclusion, we have not been able to replicate Obokataet al.’s findings to produce STAP stem cells from somatic cells. It appears that the method for producing STAP stem cells is not as simple and straight forward as has been reported.
