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The decade of stem cell plasticity – transdifferentiation buzz


I clearly remember the time when at the beginning of the new millennium the era of stem cell plasticity or transdifferentiation was declared. Three “transdifferentiation papers” made a big buzz as “Breakthrough of the Year [1]” according to Science journal: bone marrow turned in muscle [2] and liver cells [3], neural stem cells turned into hematopoietic tissue [4]. It was a very exiting time for stem cell research. Very inspiring. That’s basically was the main reason why I left clinical medicine and turned into stem cell research. People were in a hurry to set up preclinical studies and launch clinical trials based on transdifferentiation as the mechanism of action. People relied on experimental data claiming that bone marrow stem/ progenitor cells can repair the heart by differentiating into the functional cardiomyocytes. It was almost a decade ago.

The second wave of new exiting findings came a few years later and was like a “cold shower” for “transdifferentiation fans”. The discovery of a fusion phenomenon explained, at least in part, the “transdifferentiation magic”. It seems like bone marrow cells can not differentiate directly into other tissues, such as liver or brain, but simply fused [5] with mature cells in a particular organ, displaying acquired tissue-specific qualities and phenotype. Another challenge came up from cell culture artifacts [6] and detection techniques flaws in ex-vivo induced transdifferentiation.

I’m going to cite some of the recent reviews [7] below, because they are reflecting exactly the same things what I was thinking about lately. I’ll start from the great review [8] by Neil Theise [9] – one of the pioneers and activists of the “stem cell plasticity movement”.

It would seem that finally, after a decade, sufficient data have accumulated to indicate that adult cells have a range of differentiative capacity far beyond that previously considered possible. Abundant single-cell transplant experiments overwhelmingly support this view.

He indicated four pathways or forms for cell plasticity – (1) as a step of cell maturation within canonical lineages, (2) de- (re-) differentiation (in nonmammalian vertebrates) in embryogenesis and carcinogenesis, (3) direct differentiation across lineage or germ layer (most commonly used as “transdifferentiation” term), (4) cell fusion. Since first two pathways are very well known in biology for a long time, I’m going to focus only on last two, because a lot of new things were discovered only in the past decade and have/had “cell therapeutic promise”.

The earliest critique of plasticity data focused on the important issue of the lack of single-cell experiments. The first reported experiments all transplanted large populations of donor cells. Was it truly a multipotent adult stem cell that was responsible for marrow reconstitution and nonhematopoietic tissue engraftment? Or was it simply a less fundamental dogma being challenged: organ-specific stem cells might be located not only in the organ, but also in the marrow.

Theise analyzed and summarized 24 reports, published since the seminal 2001 Krause paper [10] and assessing the plasticity on a single cell level. Experiments involved hematopoietic, mesenchymal or multipotent adult stem/ progenitor cells.

Plasticity on a clonal basis was demonstrated in all but one of these articles. Not all experiments investigated whether observed plasticity arose from fusion or direct differentiation, although of those that did, direct differentiation was more common.
With this number of positive results articles and only one report of negative results, one may question why there is such controversy in the field. Although one may reasonably question the physiologic importance of adult stem cell plasticity for routine but robust repair of organs, it would seem difficult to question that the potential for multiorgan plasticity is real. And if it is, should it not warrant full attention and funding in order to exploit and expand it for therapeutic or industrial purposes?

Wow, one versus 24 and still there’s a debate? Who do you think is so brave to oppose all of this “transdifferentiation beauty’?

Close examination of the single negative result article of a single-cell plasticity experiment may shed light on these questions. This is the oft quoted “Little evidence for developmental plasticity of adult hematopoietic stem cells [11]” article by Wagers et al., from the Weismann laboratory at Stanford. To my knowledge, this is the only attempt to actually replicate our group’s single-cell bone marrow transplantation experiment that investigated and demonstrated trilineage engraftment from a clonally expanded, single hematopoietic stem cell. In their report in Science, stated to be an attempt to “rigorously test” for such clonal and trilineage engraftment, they identified a single hepatocyte and seven Purkinje neurons derived from the single transplanted c-kit+thy1.1lolin−Sca1+ cell in several recipient mice so examined.
The rigor of this report was questioned, however, in two follow-up letters published several months later in the same journal, one from us [12] and one from the Blau research group [13].

I love this piece, so I can’t resist:

I explore this article in detail, not to cavil, but because one would be hard-pressed to find a discussion of plasticity in the extensive critical literature, even now, that does not prominently make reference to the negative finding of this “Little evidence . . .” report. Besides our own single-cell transplant article, the other substantial reports of clonally demonstrated plasticity go largely unmentioned, as though only these two articles exist for comparison. And yet, in their own reply to our “Comment” [14] (published in Science together 5 months after their article came out), the Weissman group stated “our data are not directly comparable to those of Krause et al. and do not implicitly refute their observations”.

However, the prestige of the journal in which it appeared, let alone the prestige of the laboratory from which it emerged, guaranteed “Little evidence . . .” a central and enduring role in all discussions of the entire field. A few months after its publication, this exchange took place between two well-known stem cell investigators regarding the Weissman group’s negative results article and was recorded by journalist Cynthia Fox [15]:
Investigator 1: “Very poor editorial judgment.”
Investigator 2: “That paper has done damage, but I think it will all wash out.”
I hope that it has finally begun to “wash out,” but I fear that significant damage has indeed been done.

I won’t go for more details, I’d just encourage you to read this review.
Another interesting excerpt from Peter’s J. Quesenberry review [16]:

A large number of subsequent studies have shown that host tissue, usually under injury circumstances, can be partially replaced by cells derived from transplanted marrow. This led to the stem cell plasticity controversy, a rather meaningless exercise that we have addressed previously in a perspective titled “Ignoratio Elenchi” (irrelevant conclusions or red herrings). Proposals were made that for results in this area of investigation to be taken seriously, they had to be “robust,” they had to be on a clonal basis (which only shows heterogeneity), they had to show function (which was never adequately defined), and, the biggest red herring of all, they could not involve cell fusion. Why this latter point became an issue is unclear, but it unfortunately became a major negative feature of grant and manuscript reviews. There were a few “negative” studies that appeared to be designed to obtain negative results and that represented marginal science.

In continuing studies, virtually every tissue in the body has been found to be subject to marrow conversions or stem cell plasticity. There have been >30 articles on marrow-to-lung conversions and, although the percent conversions varied widely, all studies have demonstrated this. Many studies have also addressed whether cell fusion was the mechanism underlying the observed plasticity.

Excerpt from Diane’s Krause review [17]:

…there is definitive proof that marrow-derived epithelial cells exist in vivo. Because they are very rare, sensitive and stringent detection techniques are needed for their analysis. Future research needs to elucidate the functionality of these cells as well as the BM cell source and the mechanisms underlying their appearance.

So, what conclusions can we make, recapping the decade of stem cell plasticity? What did we learn and what is the current status? What are the unanswered questions? I’ll try to wrap it up as the following:

1. There is no agreement in cell plasticity terminology and definitions. Researchers can not define what is transdifferentiation. Some of them consider it as a change in cell differentiation status from one lineage to another within one germ layer, some of them consider it only across germ layers (from mesoderm to ectoderm, for example), some of them simply avoid to use this term. Theise uses “direct differentiation” rather than “transdifferentiation”. Krause wrote about this:

Transdifferentiation or plasticity refers to one committed cell becoming another type of committed cell, and this has not been definitively proven to date.

By the way, do you make a distinction between plasticity, transdifferentiation, direct differentiation, direct transdifferentiation and changing of cell fate? Or use them as synonyms?
2. There is no doubt that the phenomenon of cell plasticity or transdifferentiation exists in mammals.
3. We still don’t know the physiological role of this phenomenon. Does it occurs spontaneously at some extent in steady-state organism or only as a response to tissue injury? Please keep in mind that besides regeneration, transdetermination and transdifferentiation can play a significant role in mataplasia and cancer.
4. We still don’t know the mechanisms of how plasticity or transdifferentiation actually occurs. The possible mechanisms could be the following:

    A. cell fusion;
    B. genetic material transfer by microvesicles [16];
    C. phagocytosis of apoptotic bodies [18];
    D. cell cycle alteration-induced extracellular signaling molecules [19].

5. We still don’t know what population of bone marrow cells that have plasticity qualities and are able to transdifferentiate. Stem cells and what kind (hematopoietic or mesenchymal), progenitors and what kind, mature hematopoietic cells (monocytes/macrophages [20]). Or maybe all of them are able to do so and it’s context-dependent?

It has not been demonstrated so far which type of BM cell is able to “adopt the morphology and protein expression of an epithelial cell.” There are no data that definitively prove whether hematopoietic cells are able to adopt the phenotype of epithelial cells in the lung. Wagers et al. [21] demonstrated that this is not the case, but their data are not conclusive, as stated here. Theoretically, other, nonhematopoietic BM cells, like MSC, might also be capable of differentiating into epithelial cells, but this has not been definitively proven, either.

6. The frequency of transdifferentiation event in adult organism is very rare. So, it’s very hard to detect and therefore there are a lot of artifacts due to technical limitations.
7. We still don’t know what is the role of transdifferentiation/ fusion in disease healing and tissue repair.
8. Artificially induced transdifferentiation in cell culture, could be an artifact [6] and the function of such cells could be altered [22].
9. Because of the rarity and unknown physiological meaning, the possibility of transdifferentiation can not justify therapeutic potential of infused bone marrow cells. In other words, we can not currently use plasticity/ transdifferentiation magic as a mechanism of action of therapeutic cell products.

I’m planning to research more on the issue of transdifferentitation in human, detected after bone marrow transplantation procedures. So, stay tuned!

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Reference: Special issue of Experimental Hematology – Hematopoietic Stem Cell Plasticity [7] (all reviews for free!)