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Scientist Said He May Have Made STAP Cells—Just As Riken Called Fraud

Wed, 04/02/2014 - 1:23pm
Cynthia Fox

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In light of a press conference yesterday announcing two counts of deliberate misconduct, Riken Institute brass want co-authors of the Nature “acid bath” stem cell papers to retract one—after appeal. But two developments may complicate this.

First, lead author Haruko Obokata refuses to accept it.

Second, Hong Kong stem cell researcher Kenneth Lee just became the first scientist outside some co-authors to publicly claim he, too, may have made “acid bath” stem cells.

The catch: he made them without acid. And it was cells he was using as controls that reacted properly.

“Yes, it is very real,” he said via email. “The `negative’ control came up positive! If we did not set up a negative control we would have missed it.”

Cloning pioneer Teru Wakayama is also excited, even though he is a co-author who wants the papers retracted regardless. “This is important information about Dr. Lee's interesting results,” he emailed, adding that he understands they are preliminary.

Lee’s Trick: Trituration?

Kenneth Lee's preliminary data showing he may have repeated controversial STAP experiments. (Source: Lee lab)In a graph (see right) he posted on ResearchGate yesterday, Lee showed that on Day Three of his attempt to stress neonatal mouse fibroblasts into a stem-cell state—the point of the Nature work he was trying to reproduce—the stress of acid plus trituration didn’t bring out demonstrable pluripotency (embryonic-stem-cell) gene expression.

But robust trituration alone did, Lee said. Trituration is physical pipette manipulation. It can injure some cells, and stress others by isolating them. Harvard University's Charles Vacanti strongly emphasized the trituration part of the Nature papers (which he co-authored) in an additional protocol he posted days ago. By contrast, the original papers had strongly emphasized the acid, and simply required some trituration as a matter of course (e.g., mincing the spleen to isolate CD45+ blood cells).

But instead of Vacanti’s acid-plus-trituration recipe, it was trituration alone that appeared to switch on the pluripotency genes Oct4 and Nanog in Lee’s once-mature cells.

“Dear all,” Lee said on ResearchGate. “I am shocked and amazed by the qPCR results for the 3 day-old control and STAP cultures. Totally speechless! ENJOY!”

Less amazed was Vacanti, who has repeatedly noted in interviews that trituration, via progressively smaller-bore pipettes, worked first for his crew through the years—and still best for some cells. Via email he explained why he thought trituration alone may have worked for Lee’s fibroblasts. The Nature papers mainly described different cells: CD45+ blood cells.

“The protocol I posted on our laboratory website was intended to be an efficient guide to assist in the creation of STAP cells from a wide variety of cell sources,” Vacanti emailed yesterday. “I am certain that for each particular cell or tissue type, age or species, different stresses will be more effective than others. We believe that trituration alone, or in combination with other stresses such as low pH (acid), is extremely effective. Dr. Obokata's data suggested that (low) pH (acid) was effective for the particular cell type on which she focused.”

Lee, who is with the Chinese University of Hong Kong, earlier tried the acid approach on neonatal CD45+ blood cells, as the papers dictate, and repeatedly failed.

Shortly after Lee’s recent trituration post, respected scientists on Twitter and elsewhere—including Lee himself—warned his data are far too preliminary to draw any conclusions. They are not peer-reviewed yet. The pool of cells is tiny. Oct4 and Nanog expression is much higher in real embryonic stem cells. (Updated note: Lee's expression, however, was similar to that claimed in the first STAP Nature paper.) Pluripotency-gene expression may just be some kind of weird last gasp cells make when, stressed a certain way, they die.

But it may help ensure that, for a while longer, “the STAP Saga continues,” as Lee noted.

Wakayama agrees. “I still very much want to see the same results from an outside lab,” he emailed. “I want to believe in STAP cells.”

Press Conference of 70,000 Comments

On one Japanese video-sharing website alone late Monday night (US EST), more than 105,000 people watched—and made a whopping 70,000 comments on—the press conference releasing the Riken Institute's final report investigating acid bath stem cells, or STAP cells. STAP (“Stimulation Triggered Acquisition of Pluripotency”) cells are embryonic-like-stem cells made largely by stressing ordinary cells with coffee-mild acid. That claim was made by a Harvard/Riken team in two January 29 Nature papers. But after being called “Copernican,” and captivating world press, the papers aroused numerous scientist-blogger concerns of irreproducibility and fraud. (Leading the charge: University of California Davis biologist Paul Knoepfler, and anonymous biologist Juuichi Jigen.)

At the press conference (and in an English draft of the final report acquired by Bioscience) two instances of serious “research misconduct” were described by a Riken-selected committee. First, it found “deliberately” manipulated images damaged proof that Obokata's starting cells were differentiated. (Fig. 1i in the first paper, supposed to show the Tcrb rearrangement—marking more mature lymphoctytes—persisting back to the stem cell state.) This was “falsification.”

Second, the committee found four images helping prove the mature cells reverted to stem cells—in vitro assays proving “pluripotency”—were taken from Obokata’s unrelated thesis (Fig. 2d and 2e in the first paper). This crippled pluripotency claims, and was called “fabrication.”

The committee said both counts of misconduct were committed by Obokata. But two co-authors were criticized for missing it: Riken Center for Developmental Biology (CDB) deputy director Yoshiki Sasai, and cloner Wakayama of Yamanashi University.

Emailed Wakayama: “Although Dr. Obokata was a postdoc of Dr. Vacanti, and I thought that Dr. Vacanti would take responsibility for Dr. Obokata’s results, I totally understand that any data, generated from my lab, was my responsibility. I must take care, even when it is a visitor’s data. I was a too (naïve) scientist. If I can (offer an) excuse, Dr. Obokata brought the teratoma data when she came to my lab. It already existed before she joined my lab.” (The “teratoma” data were among those taken from Obokata’s unrelated thesis).

At the press conference, a statement from Sasai reportedly said he was “deeply regretful,” but that STAP work had seemed consistent with other studies.

Hours Later: Lee

Then, mere hours after that press conference was over, Lee posted his ResearchGate data finding he may have made STAP cells via trituration.

Is the pluripotency gene expression he saw just a side effect of some dying cells? Will it be repeatable? Lee will try. Meanwhile, Obokata in a statement insisted her errors were accidental, and that STAP cells exist. She will appeal. The English version of her response is not out, but part of it appears in The Yomiuri Shimbun.

Wakayama says the “Japanese government wanted a quick answer from Riken”—hence the relatively sudden final report. But many questions remain unanswered. One is a find Wakayama made last week that the wrong mice were used in some STAP back-up work. This may invalidate a claim in the first paper’s methods section that some STAP cells—and subsequent chimeric mice—were made from mouse 129. It also calls into question other STAP line origins. Wakayama says he gave 20 STAP lines for evaluation to Riken and an outside team. Results may take weeks.

Furthermore, Riken will keep trying to make STAP cells for a year, says a statement by Riken CDB’s director. And late last night, Lee, noting “these pluripotent markers could be the bi-product of un-regulated gene expression by dying or stressed cells,” urged other scientists to start triturating their cells, too.

So will the Nature paper(s) be retracted? Many say it is likely. Key proof has been discredited by the image tampering and switching. Will people keep trying to make STAP cells anyway? Recent events indicate this is also likely. Those addicted to the STAP Stem Cell Sagas may well rest easy. As one exhausted Japanese commenter noted, it is beginning to seem possible that “the torture never stop.” But many are encouraged by the persistent need of some scientists to gain clarity in the matter.

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