Last month, we posted an article that asked how one could declare human cells pluripotent, when the most robust tests are neither ethical nor feasible. Here are some of our favorite responses. If you’ve got more to say, please add your own comments.
Peter Andrews, Sheffield University
I rather think the discussion is becoming like the Middle Ages’ discussions about how many angels can stand on the head of a pin. Does it matter? Sometimes in science it helps to have terms that are not so precisely defined – indeed the term ‘gene’ is an example. In fact it can mean a variety of subtly different things – which in fact makes it generally useful. When people wanted more precise terms, new ones were invented, like the ‘cistron’ based upon a very specific assay.
The same may be true of pluripotency. To me it means what it says – the ability of a cell to be capable of generating many cell types by differentiation. When we come to ES (and related EC) cells, we can actually find a very broad range of capacities – ranging from cells that have completely lost their ability to differentiatiate (nullipotent) to those with a very broad range – ultimately all somatic cell types. But we know very little about the molecular basis of pluripotency and what controls the range of cells into which a stem cell can differentiate. On the face of it at the moment I think we have little or no way of identifying which ES cells can generate a whole mouse in the tetraploid assay and which cannot even form the germ line in chimeras. In the face of this type of uncertainty, I would advocate retaining ‘pluripotency’ as a somewhat vague, term meaning ability to differentiate into a lot of cell types, and then as the need arises invent new terms with precise definitions based on specific assays – very much as the concept of the gene and its associated terminologies evolved.
Shinya Yamanaka, Kyoto University
This is an important, but difficult question. First of all, we don’t know whether human ES cells are really ES cells or not. Because the lack of chimera experiments, we will not be able to answer this question. This means we lack a positive control. I have been telling my students that one of the worst experiments you can do is one without positive and negative controls.
In human ES cell field, all the scientists are forced to perform bad experiments without positive control. The best we can do is to describe how the cells are similar to human ES cells. This includes not only teratoma formation, but also surface marker, gene expression, DNA methylation, telomerase activity. You are right that some iPS cells can make teratomas, but do not give rise to germline transmission. However, these cells have different gene expression and DNA methylation.
I don’t think it is governments to make definition of pluripotency. It should be scientific community.
Paul Tesar, Laboratory of Molecular Biology, National Institutes of Health, NINDS
Since I’m associated with NIH, I won’t comment on the recent nomenclature alteration.
I do, however, think that the definition of pluripotency sits at the heart of modern biology. Currently it is more of a semantic argument but I think further study will clarify the issue. Existing methodologies such as blastocyst injection and teratoma formation are inclusive but not exclusive when defining pluripotency. Additionally, they require secondary characteristics that are not necessarily involved in pluripotency. For example, cells that do not incorporate into the ICM, maybe because of cell adhesion or cell cycle differences, can not be examined by blastocyst injection. This does not mean that they are not pluripotent. Likewise, cells that do not rapidly proliferate when transplanted to an ectopic site will not form a teratoma. Can quiescent cells be pluripotent? Does growth or cell adhesion have to be linked to pluripotency? I think, thus far, pluripotent cells have satisfied one or the other of these basic assays but it is becoming harder to pinpoint the defining characteristic of pluripotency.
It sounds a bit outlandish but one could imagine something like a ‘pluripotency score’ which could be computed from a variety of cellular characteristics. It is difficult to define what exactly would need to be input, but in a current sense one could imagine looking across the genome at a large number of histone and DNA modifications. The ‘pluripotency score’ would basically be the probability that the chromatin is immediately capable of changing to form a panoply of differentiated tissues. SCNT and iPS cells have shown us that most, if not all, cells are capable of being pluripotent, but only after reprogramming. A much deeper understanding of multiple aspects of cell biology are necessary for something like a ‘pluripotency score’ to be a reliable and predictive measure, but at least it provides a framework to move forward instead of walking away or simply arguing semantics.
William Gunn, Tulane University
I would like to share what the consensus view is trending towards in my field, multipotent stromal cells(aka mesenchymal stem cells, MSCs).
I think the subtlety that is most often missed when talking about differentiation capacity is that differentiation is a cell-intrinsic process, but it’s only assayed at the level of a whole culture. In other words, you’re assaying a heterogeneous population of cells for phenotypes that different subpopulations possess to various degrees.
Further, these populations interact through cell-cell contacts and paracrine signaling, forming microenvironments which change constituency over time. Outside of ESCs and HSCs, it’s an open question whether there’s really one cell in a stem cell culture that could make all the various tissues, or if the pluripotency we see is a result of a mixture of progenitors of the various types that we just haven’t learned how to distinguish yet. When exactly these progenitors may have become committed to a lineage isn’t known.
The heterogeneity and dynamic nature of pluripotent cells is what has been confounding the studies which try to pin down markers of pluripotency or “stemness”, and I’m not sure we’ll get a satisfying answer until we develop the tools to study these cells on the single-cell level.
Evidence supporting this can be found in the work of Kuznetsov et al,
back in 1997: https://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=9286749&ordinalpos=41&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum