October 20, 2020

The Niche

Knoepfler lab stem cell blog

Are STAP Stem Cell Nature Papers Compromised?

Are the Nature STAP stem cell papers compromised? Could Nature‘s investigation conclude with something as serious as retraction or rather a mild slap on the wrist? Somewhere in between? (note you may find this update post from 2020 to be of interest: Whatever happened to the STAP cell scientists including Haruko Obokata?)

Why is this question even being raised?

A large number of potentially serious issues with these papers have been identified and discussed on PubPeer here and here, as well as elsewhere. These have included confirmed data duplication in the Nature letter.

A potential resolution to these purported problems could be achieved if the authors and Nature publicly release unmodified, original versions of the images and data in these two papers.

I’m not holding my breath on that one, but it seems like a path to clarity.

Nature‘s own policies on images are relevant here and it seems that legitimate questions can be at the very least raised as to whether these papers violated aspects of these policies in quite a few ways.

Post-publication reviewers have found numerous problems even just with Figure 1 of the Obokata, et al. Nature article. Let’s use this as an example. We’ll go through just that one figure step-by-step. I’ve posted the figure legend at the bottom of this post for reference.

  • Figure 1a is their experimental model.
  • Figure 1b reports that an Oct4-GFP reporter comes on specifically after low-pH treatment in spheres of cells in suspension. Concerns have been raised that the green signal may have been enhanced and/or could be autofluorescence, and the 3 panels may not have been captured and/or manipulated in an equivalent manner to each other.
  • Figure 1c is FACS analysis of the Oct4-GFP reporter turning on at day 7. The upper 2 panels are low-pH treated cells. By day 7 this data would suggest that basically 100% of cells became GFP+, a stunning reported finding.
  • Figure 1d is a quantification of viable cells by relative GFP status reporting a conversion to a GFP+ state in about 50% of cells by d7. This would seem to conflict with Figure 1c.
  • Figure 1e shows cells growing on a plate and some are GFP+. The figure panels are of such low magnification that there’s not much to be sure about here. Like the rest of the data in this figure overall, there are no positive controls.
  • Fig. 1g rectangles, supposed stap stem cell culture
    Fig. 1g rectangles, supposed STAP stem cell culture, but there are manipulations.
  • Figure 1f, after simply increasing the brightness, looks very unusual (see above). First, it is of very poor quality and has extreme pixelation–is that simply due to live cell imaging capture? Panels d2 and d3 look entirely different in nature from the d0 and d1 panels. There are strange flat gray rectangles, especially in d2 and the rest of the d2 and d3 panels are divided up in relatively large squares and rectangles. Are some of these simply combinations of low-signal pixels due to jpegs/compression artifacts or what? That is possible but if so, why are these artifacts not evident in panels d0 and d1, which only have an even sea of small, normal appearing color variegated (rather than flat gray) pixels? The green signal in d2 and d3 panels seems washed out. What is the pink signal showing up? Update: To be clear, I do not personally believe that Figure 1f has “cutting and pasting” as some have suggested, but the low quality and strange, relatively inconsistent appearance of the panels makes one wonder if the 4 panels were not processed in an equivalent manner.
  • Figure 1g shows EM of cells of 3 different types. Since only 1 cell of each type is shown, not much can be concluded here.
  • Figure 1h shows that the GFP+ cells are small. Why does the left edge of the green histogram peak abruptly end at 4 microns? Was a gate applied to remove anything smaller? One kind of gets the feeling that there could have been many GFP+ cells/cellular fragments/dead cells <4 microns. Why are there no X axis values smaller than 4 or larger than 10?
  • Figure 1i has some odd things such as straight lines surrounding lane 3 that are visible when the brightness is boosted. This has been a big issue on PubPeer. In addition, many people have been asking why the STAP cells made from T cells in lanes 4 and 5 would contain a prominent unrecombined (upper) band? Were the “T cells” used not very pure?

Again this is just one figure in one of the two papers. Numerous other concerns have been raised about other figures in both papers. 

What do I think at this point?

On one level I really dislike all this microscopic analysis of data problems in the STAP papers, but on the other hand this is such a huge finding, the reputation of the stem cell field is on the line at some level, and trust has been shaken in these papers that it seems that this kind of post-publication analysis here is on the whole needed and appropriate even if very uncomfortable.

The more I look at these two STAP papers, the more concerned I get. Also, many of the figures would have benefited from having murine iPS cell induction images and data shown in parallel as controls. That would have provided crucial context.

The bottom line for me now is that at some level a part of me still clings to a tiny and receding hope this has all been overblown due to simple misunderstandings, but that seems increasingly unlikely. The  definite numerous problems with the 2011 Obokata/Vacanti paper also have reduced confidence.

What will Nature‘s investigation conclude? No one can be sure, but I am predicting that Nature is taking this extremely seriously and if appropriate, will not minimize important issues when it makes its findings public. Nature‘s own integrity as a journal is at play here too and they know that.

For reference, Figure 1 legend verbatim:

a, Schematic of low-pH treatment. b, Oct4-GFP+ cell clusters appeared in culture of low-pH-treated CD45+cells (middle; high magnification, right) on day 7 (d7) but not in culture of control CD45+ cells (left). Top: bright-field view; bottom, GFP signals. Scale bar, 100 μm. c, FACS analysis. The x axis shows CD45 epifluorescence level; y axis shows Oct4-GFP level. Non-treated, cultured in the same medium but not treated with low pH. d, GFP+ (green) and GFP (yellow) cell populations (average cell numbers per visual field; ×10 objective lens). n = 25; error bars show average ± s.d. e, Snapshots of live imaging of culture of low-pH-treated CD45+ cells (Oct4-gfp). Arrows indicate cells that started expressing Oct4-GFP. Scale bar, 50 μm. f, Cell size reduction in low-pH-treated CD45+ cells on day 1 before turning on Oct4-GFP without cell division on day 2. In this live imaging, cells were plated at a half density for easier viewing of individual cells. Scale bar, 10 μm. g, Electron microscope analysis. Scale bar, 1 μm. h, Forward scattering analysis of Oct4-GFPCD45+ cells (red) and Oct4-GFP+CD45 cells (green) on day 7. Blue line, ES cells.i, Genomic PCR analysis of (D)J recombination at the Tcrb gene. GL is the size of the non-rearranged germline type, whereas the smaller ladders correspond to the alternative rearrangements of J exons. Negative controls, lanes 1, 2; positive controls, lane 3; FACS-sorted Oct4-GFP+ cells (two independent preparations on day 7), lanes 4, 5.”

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