Controversy over CRISPR challenger NgAgo irreproducibility reported

Does the new gene editing method NgAgo work or not? If not, what happened? The answers to both questions seem to depend on who you ask and what you read.

Fang Shimin (方是民) NgAgo

Wikipedia image

As much as CRISPR has been the revolutionary in the genetic modification technology arena over past methods, could CRISPR itself in the next few years become obsolete having been replaced by other new technologies such as the upstart NgAgo? I doubt it.

The odds for NgAgo making a run in this field may have gone down lately, at least based on a comment left by Sheng Qiang on my original post on NgAgo:

“A war of word broke out on the reproducibility of Han’s work these days, especially on the Mitbbs website. The doubters, represented by Zhouzi Fang, said that no labs have repeated Han’s work, especially the Figure 4 results. The supporters claimed that 20 labs in China already repeated Han’s work, yet no data have been shown to support the claim. The doubters suspect that this is another STAP cell incident for China. To be fair, we should probably give more time for labs around the world to repeat Han’s work, which was trumpeted in the Chinese media to be a Nobel prize worthy scientific breakthrough. Let’s just hope that this will not go down the same path as the STAP cells.”

The Zhouzi Fang mentioned seems to most likely be Fang Shimin (方是民), pictured above, who has a Wikipedia page here that mentions his role as a popular science writer who campaigns against pseudoscience and fraud. It also discusses a number of controversies in which he has been involved. I wonder if he might be like Japan’s juuichijigen who played a key role in uncovering STAP. I don’t know.

I’m hoping to learn more about this NgAgo situation so that we all can better judge what the status of NgAgo research might be. The notion that this could be another STAP-like situation would be very unfortunate, but it seems there’s not enough information now to judge and that’s a serious thing to assert. I agree with the commenter that more time is needed before we can be sure what’s what here.

So what is out there on discussions over NgAgo as to whether it works or not?

I did find this page on an “NgAgo” search onMitbbs (which when Google crudely translates it) seems to fit with what the commenter says about a war of words, but I have no idea if that page is reliable.

I also found this Chinese-language science news site reporting on the NgAgo controversy.

This Google group page on NgAgo also has some researchers reporting it doesn’t work for them, but others said it did work.

Overall, I’d say the jury is out, but it’s clear there are strong opinions both ways on NgAgo.

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.