Review of Obokata STAP cells Nature papers

In two Nature papers (here and here) published today researchers report the astounding finding of reprogramming differentiated cells back to a pluripotent or even totipotent state simply by exposing the cells to extreme environmental stress, creating cells they called STAP cells. Update: see more thoughts on STAP stem cells here.

STAP cells: stressing the cell out

No genes. No proteins. No nuclear transfer. Just stressing the heck out of the cells, for example, by exposing them to acid. Nature writer David Cyranoski entitled his news piece on these papers “Acid bath offers easy path to stem cells”, which I thought was clever.

The authors report the creation of iPS like cells via sub-lethal stress and have named the cells stimulus-trigged acquisition of pluripotency (STAP) cells.

I agree with Cyranoski that these new papers on STAP cells are like to fuel a long-running debate. I also later in this post raise 6 key questions about this finding that should for now somewhat temper the over-exuberance that I’m hearing.

For now, the two papers are making a big splash. Stimulus-trigged fate conversion of somatic cells into pluripotency has first author Haruko Obokata and senior author Charles Vacanti. The second paper in the same issue of Nature and also with Obokata as first author has Teruhiko Wakayama as senior author: Bidirectional developmental potential in reprogrammed cells with acquired pluripotency.

What to make of these papers?

The second paper seems to be really just showing that the STAP cells are in fact not just pluripotent, but totipotent and can make extraembryonic tissues too. That seems surprising.

Obokata FIg. 1, STAP cells production process.
Obokata FIg. 1, STAP cells production process.

The first paper on STAP cells is really where we need to dig in deeper at this point I think.

The schematic above from the paper’s Fig. 1a shows the key protocol employed. By doing something akin to hitting the cells over the head with a sledgehammer of a pH 5.7 (physiological pH is more typically thought of as around 7.4), they report the blood cells of 1-week old mice turned on expression of an Oct-GFP reporter as they floated around in clusters in the media.

Obokata FIg. 2a, STAP cells
Obokata FIg. 2a, supposed colonies of STAP cells.

Puzzling data

One counterintuitive thing is that the team reported that they could make this happen with T cells and also a wider population of CD45+ cells of the spleen, but not from more primitive hematopoietic progenitor cells, which offhand I would have imagined should be more amenable to reprogramming. Importantly the team provided pretty good evidence that the STAP cells arose from the differentiated blood cells themselves rather than potentially from rare pre-existing primitive stem cells in the cell populations.

The Oct4-GFP+ cells glow green and they also reportedly functionally possess pluripotency. The cells not only express pluripotency factor proteins including Oct4 and have pluripotency-associated surface markers (see Fig. 2 including panel a shown above), but also could reportedly differentiate into ectoderm, endoderm, and mesoderm by in vitro differentiation assays and teratoma assays. The pluripotency factor transcriptional activation is mechanistically tied to promoter demethylation.

The STAP cells formed colonies over time after they were dissociated. The colonies definitely were not as pretty as regular mouse ES or iPS cells however (see Figure 2f-i).

Making STAP cells

In Fig. 3 they go on to show that not just blood cells, but a variety of other cells can be made into STAP cells including brain, skin, muscle, fat, bone marrow, lung, and liver of 1-week old Oct4-GFP mice.

In Fig. 4 they show that STAP cells (from cag-gfp reporter mice) can contribute to the germ-line via chimeric mouse generation. In Fig. 5 they report that they can make ES cell-like cells from STAP cells. My only hesitation with this particular data is that again the STAP colonies don’t look particularly ES cell-like, but I wonder how much does that matter given the rather solid functional and molecular assays? What do you think?

Unknown mechanisms

The authors themselves point out that the actual mechanism of reprogramming remains unclear at this time:

A remaining question is whether cellular reprogramming is initiated specifically by the low-pH treatment or also by some other types of sublethal stress such as physical damage, plasma membrane perforation, osmotic pressure shock, growth-factor deprivation, heat shock or high Ca2+ exposure.

After a relatively quick read, no particular red flags jump out at me from the STAP cell paper. It just seems too good and too simple of a method to be true, but the data would suggest so far at least that this team is onto something really important.

Open questions on STAP cells

But key open questions remain before anyone can really say just how important this is.

  • 1. Will it be reproducible by other labs?
  • 2. Will it work in human cells?
  • 3. Will it work in adult cells?
  • 4. What are the molecular mechanisms?
  • 5. Do these cells possess significant rates of mutations or epi-mutations, the latter being abnormalities in the epigenome?
  • 6. Are these cells tumorigenic (besides forming teratoma)?

In particular, if the answer to one or more of the first 3 questions is no, then the impact could be significantly muted.

Still the studies have both practical implications for potentially simple reprogramming of cells, and also suggests some fundamental concepts about cell and organismal biology that are intriguing including the idea that differentiated cells can be far more plastic than we all imagined. In addition it invokes the idea that when animals and hence their cells sustain injuries, a stem cell-like program may be induced.

Bottom line, I want to see what the future data addressing the 6 questions above tell us before getting too excited, but it’s definitely cool.

23 thoughts on “Review of Obokata STAP cells Nature papers”

  1. I’m a muscle researcher and I would like to say that it is almost impossible to reprogram muscle to ES cells. First, muscle development is very unique, a single fiber is composed by ~1000 progenitor cells that fuse during development. As a result, the muscle cell is the largest mammal cell, comprising many nuclei that positionate to membrane, but in a single syncytium. They stay in G0 all life long. All theses features make muscle very unlikely and hard to reprogram by stress (muscle indeed can support high level of stress).
    In order to dedifferentiate muscle cell, nuclei must undergo clivage to be again mononucleated, which is very complicated and can be achieved by overexpression of some transcription factors (like mdx1). Strong chromatin remodeling during muscle development is another concern. Although capable of redifferentiation, it remains unknown if they represent true iPS cells and I think they don’t.
    Interesting, intracelullar muscle pH can achieve 6.6 during exercise (measured by biopsy and probably even lower in vivo) as a result of H+ release by many metabolic processes – contraction to energy metabolism.
    As a muscle researcher, I do not believe that muscle specifically could be reprogramed by acid bath, it seems impossible.

  2. Can you find raw data of RNA-seq and ChIP-seq? BiosampleIDs are written in this paper, but there is no GEO or SRA accession ID.

  3. Can someone tell me if you could see any RNA-seq data in this paper? There is RNA-sequencing section in the Methods but I cannot find any RNA-seq data in the text or figures.

      1. Oh, OK, in the Letter. I was looking for the data in the Article, as the RNA-seq and ChIP-seq are listed also there. Thanks!

  4. Just want to say thank you for writing this. As soon as the snow if off the roads I’m going to show it to my HS Biology class to show them how real science journalism should look.

      1. They use HBSS, where the HCO3- is converted into soluble CO2 by the pH decrease (they set the pH using HCl), and might conceivably enter the cellto acidify it. However, I wonder if it doesn’t evaporate at 37oC, they don’t specify if they saturate the atmosphere with CO2 in the acid treatment.

  5. Despite the excitement from this story, there is a serious problem with the interchangeable use of the word/phrase “stress” and “external stimuli”.

    As someone who has spend a lot of time investigating the stress response, i must say if its really “No genes. No proteins. No nuclear transfer”, then please do not refer to this as any form of stress. There is a very prescribed adaptive response to stress that in addition to epigenetic regulation ultimately leads to the activation of specific genes aimed at the eradication of the stress or if the later can’t be achieved, killing the cell.

    The authors claimed they have tried heat stress, starvation, high-calcium, low pH and physical stress. all of these have very well studied stress adaptive responses. I will be very interested in some one highlighting genomic changes following the acid bath, as it appears other than for markers there are clearly no details.

    1. In vitro stress administered to cells is a much different type of stress that the whole organism may encounter.
      There is no adaption in the experiment by Vacanti, it is an in vitro stress, and acute.

      VSELs don´t proliferate in culture for a reason.

      VSELS are enriched in blood and at the locus of severe traumic stress: Myocardial Infarct and reaming hip bones for bone marrow,as two of several examples. The later being how VSELS were first identified.

      Whatever genomic changes there may be, in this paper, they play no role in vitality. Epigenetic changes are the operative here.

  6. Sensationalism is expected these days and given Haruko’s involvement the Japanese media are especially going nuts over this. To their credit though, they are going into far more technical detail on the process than the Western press and raising exactly the concerns you express above. Haruko herself is seriously modest in Japanese interviews and also emphasises the fact this is just an interesting starting point from which nothing practical may yet arise.

    The Western media on the other hand… “THIS WILL MAKE CLONING EASY!!”

    1. I’ve been told by the blood expert in the lab that the CD45neg cells include Tcells that carry recombinations at a lower rate. The STAP colonies are polyclonal, so they are derived from several different CD45neg cells, some of which may not be recombined.

      1. That was my thinking. Their results indicate that their “T cells” were not very pure. Plenty of Non-T cells were likely the source of the non-recombined band.

        1. OK had time to read it properly now. First thought they used the lymphocytes from Fig 1.i lane 3 as source for reprogramming, but they just refer to them as positive control….

  7. This is a very interesting discovery, even if it turns out to be mouse specific (doubt that). The most amazing of it is yet another lesson of that amazing cellular plasticity which seem to explode since 2006. I haven’t read the papers carefully yet, but it reminds me of MUSE cells (Multi-lineage differentiating Stress Enduring cell) of Mari Dezawa – adults stem cells, “produced” by stressing cells from different sources, or perhaps rather selected for by killing all other cells with stress. However Muse cells do not form teratomas. Also, perhaps the sorting methods used by Ratajczak for VSLEs, and/or MAPCs of Catherine Verfaillie induced stemness and those cells are not artifacts after all.

  8. I haven’t had a look at the paper in detail, but the first thing that struck me was “stress” induced pluripotency……..this reminded me straight away of the situation with VSELs, and the stress they are put under, which seem to be gradually being accepted, with studies published in numerous journals from several groups now. Is there a possible link?

  9. Nice review! Completely agree with your 6 points. As for question 3, the blurb from Nature News said the they originally tried with adult mouse cells and failed, which is when the senior author of the second paper suggested they try using neonatal cells.

    1. Thanks, Daniel. Nobody tries to temper the buzz at all. I’ll be curious to see more over-the-top headlines in the next 24 hours.

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