Symptoms: Notes from the Biological Literature (3)

Stephen L. Talbott

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Posted: January 8, 2015   (Article 26)

In 2006 two Japanese researchers, S. Yamanaka and K. Takahashi, shook up the medical research world by announcing that they had “reprogrammed” adult mouse skin cells (in particular, fibroblasts) to become induced, pluripotent stem cells. Their method: add to the cultured adult cells just four well-chosen genes that result in the over-expression of four gene-regulatory proteins.

This was a time when the debate over the use of stem cells obtained from human embryos was fierce, so the idea that a stem cell might be created (“induced”) from a normal adult skin cell produced quite a sensation.

Laboratories the world over have been running with this discovery ever since, and there were already significant developments by the time I found myself giving a talk at a conference involving a number of molecular biologists. Not having familiarized myself with the work on induced pluripotency, I did not discuss it in my talk, but I did emphasize what seemed to me a decisively important point: there are no “central controllers” or “master regulators” in the cell.

During the discussion period afterward, a researcher at one of the nation’s major stem cell research laboratories aggressively challenged me by stating flatly: “You’re wrong. There are master regulators in the cell. We prove it every day in our laboratory, where we insert just three or four transcription factors in a differentiated cell and cause it to revert to a pluripotent stem cell”. (For some definitions, see the box at right.)

At the time I could only vaguely allude to research results that, as I was peripherally aware, were already coming in — and then add, “Wait and watch, and you will see. I have no doubt whatever that your master regulators are an illusion”.

Well, since that time I have been amused, though hardly surprised, to note the rising tide of complication and unpredictability besetting pluripotency research. It’s the usual story: the original results soon have to be qualified, and the number of qualifications grows rapidly. Stepping back in order to understand perplexing experimental results, researchers must take an ever wider view, whereupon they discover one contextual factor after another they have previously overlooked. The upshot of the brief history since 2006 looks something like this:

• We’ve been greeted by a steadily growing list of distinct ways to initiate the process by which differentiated cells become more like stem cells. There are many possible “master regulators”, it seems, few if any of which seem to be absolutely necessary as initial triggers, to the exclusion of others. This raises a question without any reasonable answer: “Which are the real master regulators?”
• Another steadily growing list enumerates different features of the cell that need to be recognized and attended to, because the original recipe for inducing pluripotency left them out of sync with the intended program. The regulatory function, in other words, was only partly carried out. The question here is, “How masterful are the master regulators?”
• The same question arises again due to the fact that the master regulators tend to have whatever effects they do have only in a tiny percentage of the cells to which they are applied. The great majority of cells pursue diverse pathways of their own choosing in response to the artificial assault on their integrity. Are master regulators that fail to regulate still master regulators?
• There is now a growing realization that the very concept of pluripotency as a well-defined state is problematic. The concept needs to be expanded, diversified, and rendered more flexible, because manifestations of pluripotency in the organism are fluid and context-dependent.

The details supporting this list have been reported so frequently in the literature that I needn’t review them here. Instead, just a few select observations:

Pointing to the “striking differences” between true embryonic stem cells and induced pluripotent stem cells, Sancho-Martinez et al.* wrote in a 2011 paper entitled “The Labyrinth of Nuclear Reprogramming”:

In summary, the stem cell and regenerative medicine fields seem to dance an unpredictably syncopated groove in which every new discovery leading to the general enthusiasm of the community is followed by another one alerting us to the potential pitfalls that early clinical translation might face.

One of the reasons for wanting to create stem cells from differentiated cells is that the stem cells can then be transformed back into one or more differentiated cell types. The hope is that, by placing appropriate stem cells in bodily locations where tissue has been damaged — say, in the heart after a heart attack — those cells would be transformed so as to regenerate the damaged tissue.

But this was never going to be as easy as was first imagined. As two molecular biologists from Cambridge, Massachusetts, have written in Science, individual induced pluripotent stem cell lines “display highly variable biological properties. This makes their propensity to differentiate into specific functional cell types unpredictable” (Soldner and Jaenisch 2012*).

The most recent and most striking summary of the current state of affairs comes from a December 11, 2014 article in Nature, “The Black Box of Reprogramming”, by David Cyranoski*. Noting, no doubt correctly, that the use of induced pluripotent stem cells in therapy has come closer to reality, he quickly adds that “There is just one hitch”:

No one, not even the dozen or so groups of scientists who intensively study reprogramming, knows how it happens. They understand that differentiated cells go in, and pluripotent cells come out the other end, but what happens in between is one of biology’s impenetrable black boxes.

The problems, as Cyranoski reports them, all have to do with context. For example, the researchers must work with collections of cells, but even in the most carefully controlled tissue cultures, no two cells are exactly the same. The differences may seem small, but the results testify to their significance: in experiments only about one in a thousand cells becomes a “true” pluripotent cell — “and even these may differ from one another in subtle but important ways. What is more, the path to reprogramming may vary depending on the conditions under which cells are being grown, and from one lab to the next. This makes it difficult to compare experimental results, and it raises safety concerns should a mix of poorly characterized cells be used in the clinic”.

Apparently the cleanly efficient master regulators my conference challenger claimed already to have in hand subsequently abandoned their posts.

The importance of context here, as in all aspects of molecular biology, has been widely acknowledged. In a 2010 paper*, Martin Pera and Patrick Tam, from medical schools at the University of California and the University of Sydney, respectively, wrote that the heterogeneity and plasticity of stem-cell populations “are modulated by extrinsic signaling”. According to them, “An emerging view of the stem-cell state holds that it is not an invariant and cell-autonomous state but, instead, should be considered as the dynamic response of the cell lineage as a whole to the external environment”.

Consistent with this, molecular biologists have now produced what seems to approximate an entirely new, “F-class” of stem cells with its own special properties. They achieved this simply by prolonging the cells’ exposure to the selected pluripotency factors. And there are already suggestions of a third kind of pluripotent state. Two of the researchers doing this work (Wu and Belmonte 2014*) refer to “a spectrum of distinct [pluripotent] cell types”.

All this notwithstanding, the usual desire for “mechanisms” and “control” figures centrally in the biologists’ quest. After all, says Alexander Meissner at Harvard University (quoted by Cyranoski), “The one thing we know is that it’s not magic, there is a mechanism. That’s good news — we should be able to find it”, although he adds that it is “almost disappointing” to see such little progress from year to year.

No one’s looking for magic. But one might hope that years of disappointment would lead to some fundamental re-thinking about the kinds of “mechanism” being looked for. Nevertheless, efforts to conceive the cell in machine-like terms continue unabated. Jacob Hanna, a stem-cell biologist at the Weizmann Institute of Science in Israel, captures the idea well:

Yes, we can make induced pluripotent stem cells and yes we can [then] differentiate them, but I think we feel that we do not control them enough ... Controlling cell behaviour at will is very cool. And the way to do it is to understand their molecular biology with great detail.

Surely understanding is the right thing to aim at. But will it prove to be “cool”, as in “controlling”? That is not what the research trajectory described above suggests. But this does not mean we must throw up our hands and resign ourselves to impenetrable mystery.

To speak of the importance of context, or of the whole organism, is to deny neither the importance of detailed analysis nor the possibility of a certain predictability. If we have gotten to know a person very well, observing his behavior in a great many circumstances, both routine and extraordinary, we begin to gain a picture of his character. This allows us to say of many of his actions, “Yes, that is the sort of thing we would expect of him” — or, “That was done in the way only he would have done it”.

Something similar is true of any given species, as every competent naturalist sooner or later discovers. But recognizing character — a crucial holistic task that biologists continually engage in, even if they are not paying attention to it and even if they care nothing about consciously disciplining it as a scientific activity — does not give billiard-ball predictability or control.

Yes, there are things we can do that produce guaranteed results. Decapitate someone or feed him a large dose of strychnine, and you can count on a morbid outcome. But this tells us little about the coherence of organic processes. And while it is true that, even within the normal contexts of life, some signals are “louder” than others, producing a more or less predictable result in a wider range of circumstances, neither does this present us with a picture of mechanistic control.

Consider the effort to obtain pluripotent stem cells from specialized cells of the adult. Molecular biologists went to great lengths to isolate a minimal set of factors that might signal to the cell as “loudly” as possible, “Begin reverting to a more pluripotent state”. This actually does happen naturally within certain contexts of the human body. (See, for example, the section Which comes first: the cell or its niche? in “Who Are You and Who Am I and Who Are We?”)

However, in the case of induced pluripotency, the signals arrive out-of-context. They are not in harmony with everything else going on. The cell nevertheless, being something of a whole in its own right, tries to make what sense it can of the signals. These signals do, after all, possess a strong suggestive power; they normally occur in a context requiring the maintenance of pluripotency. But the contextual contradictions make for inevitable problems and an unpredictability far exceeding that of most routine developmental processes.

This is not to say anything other than the obvious. If biologists had been thinking in a proper holistic manner — looking from the context toward the local effect instead of in the reverse direction — they would have greeted the initial Yamanaka paper by immediately expecting the kind of research narrative that in fact ensued. I would have had no occasion to say at that conference, “Wait and see”, because the point at issue would have been a matter of trivial understanding. Or, to put it in slightly different terms: the proper understanding would have been there if biologists had decided to take their own mantra — “context matters” — as a serious revelation about the nature of the organisms they study, rather than as an excuse for their failure to achieve precise and “cool” local control.

Of course, the reality of the organism forces the researcher more and more toward the holistic truth, even if clumsily and at unnecessary expense. Contextual details get filled in as the desire for control is pursued, simply because that’s the only way to get things to work better. But it would be good if biologists realized the full fact of the matter: to reckon with more and more of the context means to bring more and more of the organism’s life into the experimental situation. It is progressively to lose the idea of isolated, perfectly controllable mechanisms and gain the reality of an actual organism insistently narrating its own life story amid all the circumstances of its life.

Even in the most ideal case, the end result will not be anything like absolute control. Rather, it will be a gratifying and, one hopes, morally serious familiarity with the character of a cell type or tissue or organism. It will be the ability to say, “This is the sort of thing such a cell might do”, and, if our concern is truly therapeutic: “Here is how we might cooperate with and heal the available context so as to help the organism realize more fully its own potentials for a healthy existence”.

In my previous posting, Of Humans and Our Microbial Guests: A Dynamic and Living Balance, I emphasized that the dramatic new discoveries about the human microbiome can be understood only within the full human context. If we want whatever predictive and therapeutic powers we may be able to gain by manipulating the microbiome, we will have to work from the broadest and most living understanding of that context. It will not do to content ourselves with freezing the context for experimental purposes, observing the result produced by varying one element of the context, and then proclaiming that element the “cause” of the result.

Such an approach may be fine in the laboratory or engineering workshop, where the controlled context can be maintained. But it doesn’t work as soon as you let the organism begin living its own life. It is, after all, always improvising, telling a story, coordinating its activity in pursuit of the unique needs of its own kind of life. This is not the same as merely being moved by physical causes. Rather, it is a way of turning the lawful basis of its physical life toward its own ends.

As it happens, an article entitled “Ditch the Term Pathogen” appeared in Nature the day after I posted “Of Humans and Our Microbial Guests”. The coincidence couldn’t have been a happier one. The authors suggest, in perfect concordance with my own argument, that a focus on particular microbes as “pathogens” tends to cut short the process of understanding. This is because part of what it means for a microbe to be pathogenic has to do with the host organism’s way of engaging with it. Pathology is always the quality of a relationship, and this relationship often has a tremendous range of possibilities.

The focus on pathogens as inherent “culprits” tends to shift research away from discovering how to help the host organism deal more healthily with the potentially unwelcome guest. Instead, heavy artillery is brought to bear against the “invader” — an invader that may have been peacefully present all along, until its host presented it with conditions inviting pathogenicity.

The authors mention, for example, that much of the world’s attention in the Ebola crisis has been focused on the sick and dying, “even though crucial clues to curbing the outbreak may be found in those who remain healthy despite being exposed to the virus”. How and under what circumstances the interactions between a microbe and a host become damaging is the thing to investigate, and that, of course is a matter of context. One of the article subheadings reads, “Context is Everything”.

There is no shortage of examples. Some generally harmless microbes began to show up as “disease organisms” only in the second half of the twentieth century, when immune-suppressing chemotherapy came into vogue and intravenous catheters provided a pathway from the skin to the blood. In one out of three people, Staphylococcus aureus causes no harm, despite being associated with skin infections and respiratory disease in many others. And so on. But let’s allow the authors to speak for themselves:

Researchers probing the human microbiome (the community of microorganisms that live in and on our bodies) using genomics are being forced to recognize that myriad factors and interactions shape its composition. It varies in different people, at different times in development and in association with disease.

Yet much of the research on infectious diseases continues to be dominated by reductionist approaches; one variable is altered while all others are assumed to hold constant. Microbiologists tend to view the microbe as the key variable in disease and treat the host as a constant. Immunologists generally see the microbe as a constant and the host response as the variable (for instance, immunologists frequently inject microbes into normal and genetically manipulated laboratory animals, to assess the factors that shape the host response). These two groups go to different conferences, read and publish in different journals, and receive funds from different granting panels.

What is needed is the simultaneous analysis of microbial and host variables using new analytical tools.

In other words, relationship and context are indeed everything. But how radical will the demand for “new analytical tools” turn out to be? And will all the necessary tools really be analytical in nature? Is this even conceivable in a world where living context — the dynamism and coherence of indivisible relationships — is what matters? Surely much more remains to be said.

This document: https://bwo.life/org/comm/ar/2015/lit-notes3_26.htm

Steve Talbott :: Symptoms: Notes from the Biological Literature (3)