Teruhiko Wakayama alleged yesterday that Haruko Obokata, his co-author of two controversial Nature papers, switched mice in experiments to create stem cells. Credit: Asahi Shimbun via Getty Images

A co-author of two controversial papers claiming a new method of generating embryonic-like stem cells has presented genetic data showing that the cells used to make the claim were not what they were said to be. The finding was supported by a second source, which suggested that cells made by ‘stimulus-triggered acquisition of pluripotency’ (STAP) were probably nothing more than conventional embryonic stem cells and were possibly the product of switched samples.

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Both announcements, made on 16 June, increase pressure on the study leader, biologist Haruko Obokata of the RIKEN Center for Developmental Biology (CDB) in Kobe, Japan, to prove that the STAP phenomenon exists.

After numerous problems were identified, Obokata and others agreed to retract two papers describing the STAP techniques they had published in Nature1,2 in January. (Nature’s news and comment team is editorially independent of its research editorial team.) But the question remained of whether STAP — in which stresses such as acid exposure or physical pressure are enough to turn bodily cells into embryonic-like cells — is a real phenomenon, as Obokata has steadfastly maintained.

Teruhiko Wakayama, a pioneer in mouse cloning who is currently at the University of Yamanashi, headed the CDB laboratory where Obokata created the STAP cells. During the experiments, Wakayama gave Obokata newborn mice from his laboratory. She claims that she took spleen cells from these mice and exposed them to acid to create STAP cells, which she then gave to Wakayama. Wakayama took the purported STAP cells and made self-renewing stem-cell lines. He also injected them into mouse embryos to make ‘chimeric’ mice, thus purportedly demonstrating the cells’ pluripotency, or ability to turn into any cell type.

After various problems in the papers emerged, Wakayama started to wonder whether the cells he received had truly been made by the STAP method (see 'Mismatch alleged in acid-bath stem-cell experiment'). He sent the eight stem-cell lines that had been presented in the paper to be analysed at the National Institute of Radiological Sciences (NIRS) in Chiba, just east of Tokyo. Geneticists at the NIRS targeted the sites where a gene encoding green fluorescent protein (GFP), used by researchers to mark the expression of certain genes, had been inserted into the mouse genomes.

In the mice that Wakayama gave to Obokata, the gene was on chromosome 18. But in the purported STAP cells, it was on chromosome 15. This strongly suggests that the cells came from different mice. “In my laboratory, there are neither mice nor embryonic stem cells with GFP on the fifteenth chromosome,” Wakayama told Nature.

Wakayama is cautious about the interpretation of these results. “We cannot say with certainty that STAP cells never existed. While the management of experimental mice is extremely strict at CDB, it is always possible that Obokata brought in baby mice from somewhere,” he says.

But similar tests carried out by RIKEN CDB and published at the same time further call into question the origin of the STAP cells. The CDB looked at GFP insertion sites and genetic background in six other STAP cell lines that Obokata had kept in her laboratory. “The results are in agreement with the results of the analyses of samples held by Prof. Wakayama,” CDB director Masatoshi Takeichi wrote in an announcement posted on the centre’s website on 16 June. Takeichi notes that the cells with GFP in chromosome 18 are “of unknown provenance”. The CDB is now investigating the source of these cells.

In response, Obokata’s lawyer, Hideo Miki, has been quoted as saying that such a mix-up was impossible. “She doesn’t think it could have happened, not intentionally or mistakenly,” says Miki.