The STAP paper sage continues – the Knoepfler post

Stem cell scientist and blogger Paul Knoepfler (from my alma mater, UC Davis), has written a nice summary of the STAP situation as it sits.  See his very useful post here.  He points out the ironic truth that Obokata and her co-authors agreed to retract the Nature STAP cell letter, but not the Nature article even though the Nature letter is not the one found by the RIKEN Center to contain figures that were manipulated.   Dr. Knoepfler wrote an editorial to the journal Nature in which he called for the journal to retract BOTH papers.  This is pretty much the view of the scientific community in general, at least from where I sit.  

Once the STAP papers came out, a host of labs tried to recapitulate the experiments described in the papers.  These are some very successful stem cell labs with very talented people.  They pretty universally had trouble recapitulating the results of Obokata and others.  Now that’s not definitive proof that something’s wrong.  Some experiments are really hard to do and it takes time to learn how to do them even if you are really good.  However, even after the detailed STAP protocol was made available, people still had trouble getting it to work.  Now things started to look hinkey.  Further mining of the papers began to show some really deep problems – things that did not make sense.  When clarification was asked for, the problems began to look even bigger.  This is the point at which the RIKEN Center became involved.

I think we should give the RIKEN Center some credit.  After all, looking into a signature publication from your own institute and the workings of one of your own is not easy.  But investigate they did, and the results were not pretty.  They did not sugar-coat their findings, but reported them forthrightly.  According to Dr. Knoepfler, RIKEN is currently determining a punishment for what it called “Dr. Obokata’s misconduct.”  If misconduct produced the Nature article then it should be retracted.  If there is some good science in that paper, then let the authors re-do it and resubmit it.  But as it stands, I think Dr. Knoepfler is completely correct when he writes, “the whole STAP story is fundamentally flawed.”

Nature should request and then demand a retraction from the authors.  If they do not get this approval, it seems to me that they are well within their rights to either retract the papers on their own pending further review or take legal action to get the papers retracted.  Most of the stem cell community, bloggers included, just want to put this whole affair behind us.

STAP Cells: The Plot Thickens Even More

You might remember that Charles Vacanti and researchers at the RIKEN Institute in Japan reported a protocol for reprogramming mature mouse cells into pluripotent stem cells that could not only integrate into mouse embryos, but could also contribute to the formation of the placenta. To convert mature cells into pluripotent cells, Vacanti and others exposed the cells to slightly acidic conditions or other types of stressful conditions and the cells reverted to a pluripotent state.

Even though Vacanti and others published these results in the prestigious journal Nature, as other scientists tried to replicate the results in these papers, they found themselves growing more and more frustrated. Also, some gaffes with a few of the figures contributed to a kind of pall that has hung over this research in general.

The original makers of these cells, stress-acquired acquisition of pluripotency or STAP cells, have now made a detailed protocol of how they made their STAP cells publicly available at the Nature Protocol Exchange. Already. it is clear that a few things about the original paper are generating many questions.

First of all, Charles Vacanti’s name does not appear on the protocol. He was the corresponding author of the original paper. Therefore the absence of his name raises some eyebrows. Secondly, the authors seem to have backed off a few of their original claims.

For example one of the statements toward the beginning of the protocol says, “Despite its seeming simplicity, this procedure requires special care in cell handling and culture conditions, as well as in the choice of the starting cell population.” Whereas the original paper, on the first reading at least, seemed to convey that making STAP cells was fairly straightforward, this seems to no longer be the case, if the words of this protocol are taken at face value.

Also, the protocol notes that cultured cells do not work with their protocol. The authors write, “Primary cells should be used. We have found that it is difficult to reprogram mouse embryonic fibroblasts (MEF) that have been expanded in vitro, while fresh MEF are competent.”  This would probably explain inability of several well-regarded stem cell laboratories to recapitulate this work, since the majority of them probably used cultured cells. This, however, seems to contradict claims made in the original paper that multiple, distinct cell types could be converted into STAP cells.

Another clarification that the protocol provides that was not made clear in the original paper is that STAP cells and STAP stem cells are not the same thing. According to the authors, the protocol provided at Nature Protocol Exchange produces STAP cells, which have the capacity to contribute to the embryo and the placenta. On the other hand, STAP stem cells, are made from STAP cells by growing them in ACTH-containing medium on feeder cells, after which the cells are switched to ESC media with 20% Fetal Bovine Serum. STAP stem cells have lost the ability to contribute to extra-embryonic tissues.

Of even greater concern is a point raised by Paul Knoepfler at UC Davis. Knoepfler noticed that the original paper argued that some of their STAP cells were made from mature T cells. T cells rearrange the genes that encode the T cell receptor. If these mature T cells were used to make STAP cells, then they should have rearranged T cell receptor genes. The paper by Vacanti and others shows precisely that in a figure labeled 1i. However, in the protocol, the authors state that their STAP cells were NOT made from T-cells. In Knoepfler’s words: “On a simple level to me this new statement seems like a red flag.”

Other comments from Knoepfler’s blog noted that the protocol does not work on mice older than one week old. Indeed, the protocol itself clearly states that “Cells from mice older than one week showed very poor reprogramming efficiency under the current protocol. Cells from male animals showed higher efficiency than those from female.”  Thus the universe of cells that can be converted into STAP cells seems to have contracted by quite a bit.

From all this it seems very likely that the STAP paper will need to go through several corrections. Some think that the paper should be retracted altogether. I think I agree with Knoepfler and we should take a “wait and see” approach. If some scientists can get this protocol to work, then great. But even then, multiple corrections to the original paper will need to be submitted. Also, the usefulness of these procedure for regenerative medicine seems suspect, at least at the moment. The cells types that can be reprogrammed with this protocol are simply too few for practical use. Also, to date, we only have Vacanti’s word that this protocol works on human cells. Forgive me, but given the gaffes associated with this present paper, that’s not terribly reassuring.

Results of STAP Cell Paper Questioned

Reports of Stimulus-Triggered Acquisition of Pluripotency or STAP cells has rocked the stem cell world. If adult cells can be converted into pluripotent stem cells so easily, then perhaps personalized, custom stem cells for each patient are just around the corner.

However, the RIKEN institute, which was heavily involved in the research that brought STAP cells to the world has now opened an investigation into this research, since leading scientists have voiced discrepancies about some of the figures in the paper and others have failed to reproduce the results in the paper.

Last week, Friday (February 14, 2014, spokespersons for the RIKEN centre, which is in Kobe, Japan, announced that the institute is looking into alleged irregularities in the work of biologist Haruko Obokata, who works at the institution. Obokata was the lead author listed on two papers that were published in the international journal Nature. These papers (Obokata, H. et al. Nature 505, 641–647 (2014), and Obokata, H. et al. Nature 505, 676–680 (2014) described a rather simple protocol for deriving pluripotent stem cells from adult mouse cells by exposing them to acidic conditions, other types of stresses such as physical pressure on cell membranes. The cells, according to these two publications, had virtually all the characteristics of mouse embryonic stem cells, but had the added ability to form placental structures, which is an ability that embryonic stem cells do not have. The investigation initiated by the RIKEN centre comes at the behest of scientists who have noticed that some of the images used in these papers might have been duplicated from other papers. Also, several scientists have notes that they have been unable, to date, to replicate her results.

These concerns came to a head last week when the science blog PubPeer, and others, noted some problems in these two Nature papers and in an earlier paper from 2011. Obokata is also the first author of this 2011 paper (Obokata, H. et al. Tissue Eng. Part A 17, 607–15 (2011), and this paper contains a figure that seems to have been used for one of the figures in the 2014 paper. Also, there is another figure duplication.

Harvard Medical School anesthesiologist Charles Vacanti who was the corresponding author of one of the Nature papers has said that has learned last week about a data mix up in the paper and has contacted the journal to request a correction. “It certainly appears to have been an honest mistake [that] did not affect any of the data, the conclusions or any other component of the paper,” says Vacanti. Note that Vacanti is a co-author on both papers and a corresponding author on one of them.

In the other paper, Obokata serves as the corresponding author and this paper contains an image of two placentas that appear to be very similar. Teruhiko Wakayama works at Yamanashi University in Yamanashi prefecture, and he is a co-author on both of these papers. According to Wakayama, he sent more than a hundred images to Obokata and suggests that there was confusion over which to use. He says he is now looking into the problem.

Additionally, ten prominent stem-cell scientists have been unable to repeat Obokata’s results. One particular blog listed eight failures from scientists in the field. However, most of those attempts did not use the same types of cells that Obokata used.

Some scientists think that this could simply be a case of experienced scientists working with a system that they know very well and can manipulate easily, unlike outsiders to this same laboratory. For example, Qi Zhou, a cloning expert at the Institute of Zoology in Beijing, who says most of his mouse cells died after treatment with acid, says that “setting up the system is tricky; as an easy experiment in an experienced lab can be extremely difficult to others, I won’t comment on the authenticity of the work only based on the reproducibility of the technique in my lab,” says Zhou.

However, others are more deeply concerned. For example, Jacob Hanna, a stem-cell biologist at the Weizmann Institute of Science in Rehovot, Israel, however, says “we should all be cautious not to persecute novel findings” but that he is “extremely concerned and sceptical”. He plans to try for about two months before giving up.

It could be that the protocol is far more complicated that thought. For example, even Wakayama has been having trouble reproducing the results. To be sure, Wakayama and a student of his were able to replicate the experiment independently before publication, but only after being coached by Obokata. But since he moved to Yamanashi, he has had no luck. “It looks like an easy technique — just add acid — but it’s not that easy,” he says.

Wakayama says that his own success in replicating Obokata’s results has convinced him that her technique works. “I did it and found it myself,” he says. “I know the results are absolutely true.”

Clearly one way to clear this up is for the authors of this groundbreaking paper to publish a detailed protocol on how to make STAP cells. This should clear up any problems with the papers. Vacanti says he has had no problem repeating the experiment and says he will let Obokata supply the protocol “to avoid any potential for variation that could lead to confusion”.

The journal Nature has said that there are aware of the problems with the papers and looking into the matter.

For now, that’s where the issue sits. Frustrating I know, but until we know more we will have to just “wait and see.”

Human STAP cells – Troubling Possibilities

Soon after the publication of this paper that adult mouse cells could be reprogrammed into embryonic-like stem cells simply by exposing them to acidic environments or other stresses , Charles Vacanti at Harvard Medical School has reported that he and his colleagues have demonstrated that this procedure works with human cells.

STAP cells or stimulus-triggered acquisition of pluripotency cells were derived by Vacanti and his Japanese collaborators last year. These new findings show that adult cells can be reprogrammed into embryonic-like stem cells without genetic engineering. However, this technique worked well in mouse cells, but it was not clear that it would work with human adult cells.

Vacanti and others shocked the world when they published their paper in the journal Nature earlier this year when they announced that adult cells in mice could be reprogrammed through exposure to stresses and proper culture conditions.

Now Vacanti has made good on his promise to test his protocol on human adult cells. In the photo below, provided by Vacanti, human adult cells were reprogrammed to a pluripotent state by exposing them to stresses, followed by growth in culture under specific conditions.

Human STAP cells
Human STAP cells

“If they can do this in human cells, it changes everything, said Robert Lanza of Advanced Cell Technologies in Marlborough, Massachusetts. Such a procedure promises cheaper, faster, and potentially more flexible cells for regenerative medicine, cancer therapy and cell and tissue cloning.

Vacanti and his colleagues say they have taken human fibroblast cells and tested several environmental stressors on them to recreate human STAP cells. He will not presently disclose which particular stressors were applied, he says the resulting cells appear similar in form to the mouse STAP cells. His team is in the process of testing to see just how stem-cell-like these cells are.

According to Vacanti, the human cells took about a week to resemble STAP cells, and formed spherical clusters just like their mouse counterparts. Vacanti and his Harvard colleague Koji Kojima emphasized that these results are only preliminary and further analysis and validation is required.

Bioethical problems potentially emerge with STAP cells despite their obvious potential. The mouse cells that were derived and characterized by Vacanti’s group and his collaborators were capable of making placenta as well as adult cell types. This is different from embryonic stem cells, which can potentially form all adult cell types, but typically do not form placenta. Embryonic stem cells, therefore, are pluripotent, which means that they can form all adult cell types. However, the mouse STAP cells can form all embryonic and adult cell types and are, therefore, totipotent. Mouse STAP cells could form an entirely new mouse. While it is now clear if human STAP cells, if they in fact exist, have this capability, but if they do, they could potentially lead to human cloning.

Sally Cowley, who heads the James Martin Stem Cell Facility at the University of Oxford, said of Vacanti’s present experiments: “Even if these are STAP cells they may not necessarily have the same potential as mouse ones – they may not have the totipotency – which is one of the most interesting features of the mouse cells.”

However the only cells known to be naturally totipotent are in embryos that have only undergone the first couple of cell divisions immediately after fertilization. According to Cowley, any research that utilizes totipotent cells would have to be under very strict regulatory surveillance. “It would actually be ideal if the human cells could be pluripotent and not totipotent – it would make everyone’s life a lot easier,” she opined.

Cowley continued: “However, the whole idea that adult cells are so plastic is incredibly fascinating,” she says. “Using stem cells has been technically incredibly challenging up to now and if this is feasible in human cells it would make working with them cheaper, faster and technically a lot more feasible.”

This is all true, but Robert Lanza from Advanced Cell Technology in Marlborough, Massachusetts, a scientist with whom I have often deeply disagreed, noted: “The word totipotent brings up all kinds of issues,” says Robert Lanza of Advanced Cell Technology in Marlborough, Massachusetts. “If these cells are truly totipotent, and they are reproducible in humans then they can implant in a uterus and have the potential to be turned into a human being. At that point you’re entering into a right-to-life quagmire”

A quagmire indeed, for Vacanti has already talked about using these STAP cells to clone human embryos. Think of it: the creation of very young human beings just for the purpose of ripping them apart and using their cells for research or medicine. Would we allow this if the embryo were older; say the age of a toddler? No we would rightly condemn it as murder, but because the embryo is very young, that somehow counts against it. This is little more than morally grading the embryo according to astrology.

Therefore, whole Vacanti’s experiments are exciting and novel, they hold chilling possibilities. Lanza is right, and it is doubtful that scientists would show the same deference or sensitivities to the moral exigencies he has shown.