June 3, 2020

The Niche

Knoepfler lab stem cell blog

Reactions to RIKEN’s STAP cell protocol

A small group of the STAP authors via RIKEN have now released a detailed protocol also available at Nature Protocol Exchange for making STAP stem cells entitled “Essential technical tips for STAP cell conversion culture from somatic cells”. This comes about 5 weeks after publication of their Nature STAP stem cell papers.

2020 update: With the 4th and perhaps final Obokata paper retraction this year, is the STAP saga finally over?

Drs. Obokata, Sasai, and Niwa have added more detail to this protocol compared to the methods in the Nature article. They say further that a protocol paper will be published in a journal with even more detail later. A key question that popped to mind is why Dr. Charles Vacanti was not an author on this methods piece? Will he be an author on the actual methods paper? If not, why not?

The authors start off the protocol with some background summary about STAP and then a cautionary note:

“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.”

To paraphrase, I’d say the gist is, “Caution: STAP cells are extremely difficult to make.”

It would seem from this protocol (see quote below) that STAP is unlikely to work in a broad number of cell types even though the original paper sure seemed to highlight that it did work in many different cell types.

“The types of cells used for STAP conversion are also critical, and the use of cells from other sources (e.g., the use of cultured fibroblasts after passaging) may also result a failure to achieve STAP conversion. We have reproducibly observed STAP cell conversion when proper procedures are followed in  the correct sequence”

However, they do say that primary MEFs can be made into STAP cells.

The authors also reinforce their argument of a key distinction between STAP cells and STAP stem cells, which I think many did not fully appreciate from the original papers. So everyone has to be more careful with nomenclature here. STAP cells are not the same as STAP stem cells.

The first 8 steps in the new protocol create the “STAP cells”.

Then the authors list a separate procedure for making “STAP stem cells” or FGF4-induced stem cells “FI stem cells” after that from the same starting “STAP cells”:

“STAP stem cells, which are converted from STAP cells in ACTH-containing medium (see Procedure), lose the ability to contribute to extra-embryonic tissues. FI stem cells, which are generated from STAP cells in FGF4-containing medium, in contrast retain the capacity to contribute to both embryonic and extra-embryonic lineages in blastocyst injection assay, although their embryonic contribution is relatively low.”

So just to be clear they report that the first 8 protocol steps alone are reported to create totipotent cells.

Then to make STAP stem cells they list just 2 additional needed steps of growing cells in ACTH-containing media on feeders and then switch to ESC media with 20% FBS. It seems quite simple.

STAP cell generation from T cells?
STAP cell generation from T cells?

The oddest thing about the new detailed STAP protocol seems to relate to the T cell receptor.

Strangely, the authors seem to indicate in an “IMPORTANT” note after these 2 STAP stem cell inducing steps that in fact it seems that their STAP stem cells (and presumably STAP cells?) are not actually arising from T lymphocytes:

“We have established multiple STAP stem cell lines from STAP cells derived from CD45+ haematopoietic cells. Of eight clones examined, none contained the rearranged TCR allele, suggesting the possibility of negative cell-type-dependent bias (including maturation of the cell of origin) for STAP cells to give rise to STAP stem cells in the conversion process. This may be relevant to the fact that STAP cell conversion was less efficient when non-neonatal cells were used as somatic cells of origin in the current protocol.”

Doesn’t this contradict Fig. 1i (see above) of the original Nature article that showed T cell receptor rearrangement? Also the authors even in this new protocol paper refer to the cells of origin as “lymphocytes”.

I’m not an immunologist and perhaps I’m missing something so I’d be curious if others with a deeper knowledge of T cells could weigh in here on the meaning of the author’s statement vs. the original paper.

On a simple level to me this new statement seems like a red flag, but perhaps others can clarify the meaning and whether this suggests the authors are making STAP cells actually not from lymphocytes but rather from some kind of hematopoietic progenitor/stem cell.

OK, so what about the first 8 steps to make the STAP cells themselves?

Nothing particularly unusual struck me about it other than perhaps overall the numerous apparently crucial subtleties to the protocol.

I need to go through the new protocol in more depth and compare it to the original STAP paper, but to me this methods piece–even assuming it helps people independently replicate STAP and indeed it could well do that–would seem to narrow the likely impact of STAP cells/ STAP stem cells.

Jeanne Loring nicely summed things up in a comment on this blog a few minutes ago:

Now that RIKEN has released a detailed protocol, I hope someone can follow it and reproduce the results. I’m disappointed that it doesn’t work in mice older than one week and that the isolation and treatment of the cells has to be so specific. Since I’m not interested in reprogramming baby mouse cells, this is of no use to me. Does that mean that I believe in STAP cells? I may after someone else is successful after following the exact protocol. But do I care? No.

Did you all find anything else notable about this new detailed protocol? Other broader reactions?

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