A senior co-author on controversial, and now retracted, stem-cell papers has quietly posted new tips on how the research can be replicated.

Two papers claiming that stressing the body’s cell could produce embryonic-like stem cells, a process called stimulus-triggered acquisition of pluripotency (STAP), were heralded when published in Nature in January but thrashed soon after when problematic images and figures were soon found.

That might not have been so worrisome if the experiments, which the authors called easy to do, were replicated, but various external groups tried and failed to do so. Co-authors in Japan responded with a tip sheet. Soon after that, the lead author on the paper laying out the fundamental STAP technology, Charles Vacanti of the Brigham and Women’s Hospital in Boston, Massachusetts, released his own, quite different, list of tips for reproducing STAP. Still no one succeeded in replicating the findings.

Since April, Hitoshi Niwa, a well-respected mouse-stem-cell specialist at the RIKEN Center for Developmental Biology (CDB) in Kobe and a co-author on the papers, has been giving a focused, last-ditch effort to replicate the experiment; on 27 August, he reported no luck so far and suggested that light emission from dying cells, known as autofluorescence, might have been confused with fluorescent tags meant to signal conversion to the embryonic-like state.

During that period, the lead author on both papers, the CDB’s Haruko Obokata, was found guilty of misconduct and both papers were retracted. Obokata’s supervisor at the CDB, Yoshiki Sasai, committed suicide, and Vacanti stepped down as chairman of Brigham and Women’s department of anaesthesiology, perioperative and pain medicine. The CDB itself has halved in size.

One might have thought that STAP was finished. But Vacanti is not one to give up so easily.

Even when he finally agreed to retract the papers, he maintained, in a post on his department’s website, that “there has been no information that cast doubt on the existence of the stimulus-triggered acquisition of pluripotency (STAP) cell phenomenon itself.” Vacanti said that he was confident that Niwa would “replicate the core STAP cell concept that my brother Martin and I originally hypothesized, and trust that it will be verified by the RIKEN as well as independently by others.”

Now, in a note posted without fanfare on Vacanti’s department’s website and dated 3 September — one week after Niwa announced failure to replicate the findings — Vacanti has offered his second revision to the STAP protocol.

In comparison with his first revised protocol in March (‘Refined protocol for generating STAP cells from mature somatic cells’), the new one (‘REVISED STAP CELL PROTOCOL. 09.03.14’) highlights the use of ATP in the solution, in combination with two stresses — exposure to acid and physical pressure on the cell membranes — that he used in the previous recipe. “In recent months, our lab decided to re-explore the utility of a low pH solution containing ATP in generating STAP cells,” Vacanti writes in the revised protocol. “We found that while pH alone resulted in the generation of STAP cells, the use of a low pH solution containing ATP, dramatically increased the efficacy of this conversion. When this acidic ATP solution was used in combination with mechanical trituration of mature cells, the results were even more profound” (emphasis original).

“We made a significant mistake in our original declaration that the protocol was ‘easy’ to repeat,” the protocol continues. “This was our belief at the time, but it turned out to be incorrect. Many of the steps described appear to be a function of the technique of the individual investigator. Consequently, the revised protocol below should increase the likelihood of success.”