New Nature papers debunk STAP cells

Today marks nearing the completion of a full circle for one of science’s biggest controversies: the STAP cell fiasco. Today STAP cells are completely refuted with the publication of two new papers in Nature and we know much more–with some notable gaps still–about what went wrong.

In January of last year, an international team of collaborators from RIKEN in Japan and Harvard/Brigham & Women’s Hospital (including the lab of Charles Vacanti where the STAP idea reportedly originated) here in the US published two Nature papers making the extraordinary claim that ordinary cells could be reprogrammed into embryonic stem cell (ESC)-like cells.

And it could be done simply, cheaply, and quickly using various forms of cellular stress including low pH. I was highly skeptical when I read the papers, but tried to keep an open mind. This sounded cool, even if also too good to be true.

I published a review of the papers here on this blog on the day they were published and I included six key open questions that would be required to assess the real impact of these papers. Over the next few weeks I posted an increasingly skeptical series of posts questioning STAP.

Others in the larger community including anonymous scientific sleuth JuuichiJigen and some on PubPeer were skeptical as well. In fact, they started noticing issues with the data and text of the papers.

RIKEN and Nature began investigations. Ultimately the papers were retracted in relatively quick fashion. While a lot of harm was done even so and tragedy would strike later, the rapid refutation of STAP attenuated the overall damage.

For more background on the key STAP events check out this comprehensive STAP history timeline. Ken Lee’s lab took the lead in scientific refutation of STAP and published their work in F1000 here after Nature rejected it under unclear circumstances.

I also started a novel, but admittedly somewhat basic attempt at crowdsourcing global efforts at STAP replication. Very quickly we came to a consensus that autofluorescence was likely a key stumbling point for the STAP papers as the authors probably misinterpreted it as real signal from a GFP pluripotency reporter.

Suspicions grew elsewhere that STAP cells might really be ESCs or some other pluripotent stem cells, possibly mixed with trophoblastic stem cells (TSC). Ultimately, STAP first author Haruko Obokata was found by RIKEN to have committed misconduct and she is no longer working at the institution. RIKEN underwent a big shakeup as a result of STAP as well. STAP co-author and highly respected biologist Yoshiki Sasai committed suicide, which was one of the most tragic and sobering events I’ve seen in science during my career. In Japan there had been a media frenzy on the STAP problems. In the US things on the STAP front were and continue to be quieter. As recently as about a year ago, Vacanti and co-author Koji Kojima publicly expressed complete confidence in STAP and put up a refined protocol on the web.

So what was the real deal with STAP?

Today Nature published two articles thoroughly refuting STAP cells and providing some further insights.

In one of the papers, STAP cells are derived from ES cells, the authors used whole genome sequencing (WGS) to examine archived STAP cell-related samples and other cells present in the laboratories where the STAP work was conducted. Using essentially a form of genomic fingerprinting, the team reports conclusive evidence that STAP cells were in actuality ESCs:

In summary, our investigations based on WGS of STAP-cell related materials reveal that all of these materials are derived from previously established ES cell lines and refute the evidence shown in the two Nature papers that cellular stress can reprogram differentiated cells into pluripotent cells.

You can see Figure 1b from this study showing the WGS comparison that the genomic characteristics of various cell lines.

STAP refutationThe matching patterns between two STAP-derived lines FLS3 and CTS1 and the supposedly unrelated FES1 ESC line are particularly striking. It now seems almost certain that a number of STAP cells are in reality FES1-related ESC lines and that the STAP cells were not created by cellular stress.

The other new paper from another team, Failure to replicate the STAP cell phenomenon, comes to similar conclusions and further clarity arises:

“In summary, our replication attempts and genetic analysis indicate that existing STAP protocols are neither robust nor reproducible. To substantiate future claims of reprogramming and alternative states of potency, we urge a rigorous application of several independent means for validating functional pluripotency and genomic profiling to confirm cell line provenance. Ultimately, the essential standard of robustness and reproducibility must be met for new claims to exert a positive and lasting influence on the research community.”

This second team led by George Daley at Brigham and Women’s spans the globe, but importantly they did some of the work actually in Vacanti’s lab, still finding no evidence that STAP is real. They wrote, “Working within the Vacanti laboratory where the concept of STAP cells originated, and assisted by a co-author of the STAP papers…”

Seven laboratories were involved in this second STAP replication effort: Daley, Deng, Hanna, Hochedlinger, Jaenisch, Pei and Wernig. This is an all-star team of stem cell research labs.

One bottom line from the paper is that this team collectively worked very hard to try to get STAP to work, but it didn’t:

“In summary, 133 replicate attempts failed to document generation of ES-cell-like cells, corroborating and extending a recent report.”

Like the other team, these scientists analyzed the STAP cells including their genomes. They found inconsistencies between their new findings and the claims in the original STAP papers:

“In the original STAP reports, the authors stated that they mixed CD451 cells from male and female mice owing to the small number of CD451 cells retrieved from individual neonatal spleens. However, our analysis indicates that CD451 cells were female, whereas the derived cells (STAP cells, STAP stem cells and FI-SCs) were all male, a clear inconsistency.”

These authors also found indications of trophoblastic stem cells (TSC) being mixed into the STAP samples. TSC may explain the reported totipotency of some derivations of supposed STAP cells.

Nature itself explained why it published these new papers (in the Brief Communications Arising or BCA format):

“Why is Nature publishing these pieces? The main reason is to update the scientific record. The wording of the STAP retraction notices left open the possibility that the phenomenon was genuine. It said: “Multiple errors impair the credibility of the study as a whole and we are unable to say without doubt whether the STAP-SC phenomenon is real.” The two BCAs clearly establish that it is not.”

We are just about, but not quite at the end of the STAP story it seems. In my opinion there is still more to be learned about what went so wrong. How did the ESCs and in some cases TSCs end up in the cell culture mix? Accidental contamination? Intentional attempt to bolster the seductive hypothesis?

We may never know, but today there is a great deal more clarity overall at least.

The publication of these two new papers is a very positive step, but it is important to stress that absent post-publication review, rapid and open team science, and social media efforts, the STAP cell myth may have continued to have been believed by many in the research world until this day when these debunking papers were published. That delay would likely have caused immeasurable damage. Thus, there were important roles both for traditional scientific correction via journals and new, transformative types of rapid post-publication review.

Obokata Can’t Reproduce STAP Cells

Obokata press conference

Signaling a nightmare ending to what was originally a fairy tale scientific story, the Japanese press (e.g. here) are reporting that Haruko Obokata has herself been unable to make STAP cells again as part of a RIKEN team testing STAP under watchful supervision.

In advance of a scheduled RIKEN press conference tomorrow, newspapers including Asahi Shimbun are reporting now that RIKEN will announce STAP was not replicated even with the involvement of Obokata.

Earlier this year RIKEN had said their internal investigation had found Obokata committed misconduct in her STAP research published in January as two papers in Nature that were later retracted.

Only three months ago, senior STAP paper author Charles Vacanti and collaborating scientist Koji Kojima of Brigham and Women’s Hospital reaffirmed their belief in STAP and posted a new STAP protocol on-line. They said STAP was a phenomenon in which “we have absolute confidence” and stressed the importance of adding ATP into the mix to make STAP, which had not been previously emphasized. It’s unknown if Obokata’s STAP replication efforts included trying the newer Vacanti protocol with ATP.

It’s unclear at this time what this somewhat final failed replication effort will mean for the STAP authors.

Harvard STAP cell authors release new protocol, affirm belief in phenomenon

Even as everyone was going through the Science and Nature reviews of the rejected STAP papers this week, something else on the STAP front that happened last week.

What’s up?

Well, the STAP Nature papers are retracted, RIKEN CDB is going to be reorganized, RIKEN CDB has tried at least 22 times to make STAP cells and every time it didn’t work, and senior STAP author Charles Vacanti in theory is now on sabbatical as of September 1st. Even so Vacanti and fellow STAP researcher Koji Kojima, released yet another new STAP protocol on September 3.

The new protocol is intended to have a better chance to work for others.

Their first bullet point is admission of an earlier misstep in saying it was “easy” to make STAP cells. Apparently they concede it’s actually kind of hard to make STAP cells.

One could ask where is the line between hard and impossible?

Their second point is to suggest that people try making STAP cells not just with low pH, but also adding in ATP.

Then they dive into detailing an actual protocol in a step-by-step fashion. They call it the most effective STAP protocol “du jour”.

They close with a paragraph that I think contains a key typo (emphasis mine):

We have developed this most recent protocol to address concerns that to date, to our knowledge, other groups have been able to generate STAP cells using our previously published or posted protocols. While we are confident that the original protocols published, will work if performed with meticulous attention to detail, we have tried to develop a protocol based on new information, that should be much more effective in demonstrating a phenomenon in which we have absolute confidence.

I think they meant in the first sentence to say “other groups have been unable to generate STAP cells…”

In fact as far as I know, nobody has been able to make STAP cells in replication efforts including Obokata herself.

Vacanti and Kojima conclude by saying that they still firmly believe in STAP.