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Stem cell plasticity: Recapping the decade, mapping the future

  • Neil D. Theise

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    • Corresponding Author InformationOffprint requests to: Neil D. Theise, M.D., Division of Digestive Diseases, Beth Israel Medical Center, Baird Hall, 17-61, First Avenue at 16th Street, New York, NY 10003

Departments of Pathology and Medicine, Beth Israel Medical Center, Albert Einstein College of Medicine, New York, NY., USA

Received 8 April 2010; received in revised form 8 April 2010; accepted 13 April 2010. published online 03 May 2010.
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Article Outline

In slightly more than a decade of stem cell plasticity research, 24 peer-reviewed articles have demonstrated plasticity across organ and/or embryonic lineage boundaries at the single-cell level, with only 1 article showing negative results. These data, taken together with data about reversibility of gene restrictions that have also accumulated during the same period, indicate that postnatal cells, even “terminally differentiated” ones, have a degree of plasticity not appreciated previously. This review looks back at the four known pathways of cell plasticity and at previously described “plasticity principles” of Genomic Completeness, Cellular Uncertainty, Stochasticity of Cell Origin and Fate, relating these to issues of experimental design and discourse that are key to understanding and evaluating plasticity data. Although the physiologic roles played by such plasticity may still be debated, the manipulations of these phenomena for therapeutic or industrial purposes should finally be considered ripe for exploration. For the future, plasticity, indeed all stem cell biology, must be considered as part of a larger web of cell-to-cell and cell-to-matrix interactions that function fully only at the tissue level; thus, the success of stem cell biology necessarily must involve assembling data from cell and molecular biology research into systems of interactions that might be reasonably called “tissue biology.” Interdisciplinary collaborations with complexity and chaos theorists, using mathematical/computer modeling of cell behaviors, will be vital to fully exploring stem cell behaviors in the coming decades.

 

As the last millennium was coming to a close, the journal Science declared three articles on adult stem cell plasticity, together, the “Breakthrough of the Year” [1]. This trio, from the research groups of Malvilio, Vescovi, and Petersen, showed marrow becoming muscle (breaking organ restriction of mesoderm-derived stem cells) [2], neural stem cells giving rise to hematopoiesis (ectoderm to mesoderm) [3], and hematopoietic stem cells becoming liver cells (mesoderm to endoderm) [4].

The turn of the millennium was an exciting time for stem cell research, but the excitement was rapidly tempered by the nascent plasticity field's entanglement with extrascientific concerns of the day, concerns that ranged from the personal to the political [5].The personal opposition was to be expected: this was a classical paradigm shift and resistance of individuals would inevitably arise in proportion to how scientists' own lives and world views were dependent on the unraveling status quo.

The political concerns, however, were more surprising, exploding in response to protests from some (not all) fundamentalist religious communities opposed to research on human embryos and empowered by the rise to power in the United States of political elements beholden to them. The American anti-abortion lobby, newly empowered, seized on the adult stem cell plasticity findings to shift their argument from a moral one (“human embryonic stem cell research is murder”) to a practical one (“if adult stem cells can do everything, then embryonic stem cell research is unnecessary”).

Sadly, the scientific establishment responded in kind: if the argument was now framed as “adult, therefore not embryonic,” the establishment answer became “not adult, therefore embryonic.” To many, including the National Academy of Science, editors of leading science journals, and scientists on grant review panels, it seemed necessary to downplay the successes of adult stem cell research, because any success would be used to stymie embryonic stem cell research. Thus, rather than a robust and thriving synergy between adult and embryonic stem cell fields taking plasticity research and regenerative medicine forward into the new millennium, there was widespread confusion for scientists, politicians, and lay people alike.

Yet, despite these difficulties, the field has somehow, however fitfully, moved forward. This review will recap the decade of discovery in adult stem cell plasticity and elaborate on some of the biological implications of the findings, including the ways in which experimental design may color, both positively and negatively, experimental results and their interpretations. Hopefully, with this introduction and summary, the reader will understand some of the limitations of past experiments, develop criteria with which to evaluate past and future reports, and join me in a degree of expectant optimism while mapping the possibilities for future adult stem cell research.

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Cell plasticity 

The phrase stem cell plasticity (with or without qualifiers such as “adult” or “postnatal”) is overly specific. The theme is truly the plasticity of all cells, not just stem cells, because plasticity pathways do not always begin with or pass through a stem cell phenotype. Four cell plasticity pathways are now recognized (Fig. 1) 6, 7.

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  • Figure 1 

    Pathways of cell plasticity. 1: Canonical cell lineage differentiation pathways of both development and adult tissue maintenance and repair. Shifts between one pathway or another respond to the tissue or systemic needs of the organism, either as preprogrammed processes or as a repair/regeneration reaction to injury. 2: Dedifferentiation and redifferentiation may be seen when adult cells return to a blastema-like state in response to injury, then differentiating into differentiated cell populations as part of repair or regeneration. This is most commonly recognized in amphibians, but is also well-documented in fetal mammals (including humans) and in some adult mammals, as well. Such a process may be demonstrable in human neoplasias. 3: The change from one lineage to another in response to cues from the microenvironment is the pathway most often referred to as “plasticity” in the last decade and engendered the most controversy. The degree to which this takes place in living organisms depends on whether there is injury and, if it is present, its nature and severity. Although the physiologic role of this process may still be debated, evidence in adult mammals that it does take place to some degree is well-documented. How to manipulate it for therapeutic and/or industrial purposes is the pressing question. 4: Nuclear reprogramming by cell-to-cell fusion (a), sometimes followed by nuclear fusion (b) is also well-documented. Moreover, postfusion reduction division (c) has also been confirmed. Like the pathway of direct differentiation, the degree to which cell-to-cell fusion, with or without subsequent reduction division, plays significant physiologic roles is not yet clear.

The first pathway is often not considered plasticity at all because it comprises all the well-documented lineages whereby the fertilized egg becomes all of the major cells of the developing embryo, fetus, and postnatal organism and whereby tissues are generally maintained (Fig. 1(a)). “Plasticity” has so often been used to describe unexpected cell flexibility, but an objective stance would have to acknowledge that all the canonical lineages are true plasticity events in that at every step of lineage development, cells of one type give rise to cells of another type, however closely related.

The second pathway is seen predominantly in nonmammalian vertebrates and in mammalian malignancies. In this pathway, a differentiated cell type dedifferentiates into a stem cell-like phenotype from which it can then regenerate cells of varying lineages (Fig. 1(b)). This is most well-recognized in amphibians in which the surviving cells at an amputation site become a blastema, which then gives rise to a new limb [8]. This pathway is not restricted to amphibians, however. Amputation of digits and limbs in developing mammalian (including human) fetuses can lead to similar limb regeneration, mediated in part by homeobox protein MSX-1, and may even be induced in postnatal animals through bone morphogenic protein−dependent processes 9, 10. Also, Heber-Katz and Gourevitch have verified that the postnatal MRL mouse has amphibian-like capacities for tissue regeneration, the mechanism of which involves repression of extracellular matrix formation through metalloproteinase overexpression by mononuclear cells homing to the wound [11]. Extracellular matrix breakdown fragments have also been shown to recruit multipotent stem/progenitors to sites of such injury [12]. Such de- to redifferentiation is also routinely seen clinically in human malignancies [13].

The third and fourth pathways are the ones most commonly meant by the term plasticity. It still remains unclear what role they routinely play physiologically: are these events only displayed in response to particular injuries? Or do they contribute in any way to normal tissue maintenance, at any stage of life?

It is pathway three, the pathway of direct differentiation across assumed lineage “boundaries,” that triggered all the controversy: cells of one lineage becoming cells of another lineage, across organ or embryonic tissue barriers, by changing gene expression in response to microenvironmental cues (Fig. 1(c)) 14, 15. We know this process depends on complex tissue signaling: mobilization of stem/progenitor cells from the marrow or from other organs; homing of the cells, usually to sites of tissue injury, in response to cytokine/chemokine signaling (e.g., stromal cell−derived factor-1 [SDF-1] and stem cell factor [SCF]); and response to extracellular signaling, presumably by cell-to-cell and cell-to-matrix interactions at the engraftment site, leading to a new differentiation state 16, 17.

The fourth pathway, like the third, was largely unexpected. This pathway is mediated by cell-to-cell fusion events 18, 19, 20, wherein a circulating cell arrives at the site of engraftment, perhaps by specific recruitment via cytokine/chemokine signaling, but also, perhaps through mechanical aspects, such as trapping of large monocytes in small capillary or sinusoid vascular spaces in damaged tissue (Fig. 1(d)) [21]. The engrafting cell merges with a pre-existent, differentiated target cell to become a tetraploid “heterokaryon.” The resulting tetraploid cell may be binucleated or have one nucleus if nuclear fusion follows cell fusion. Either way, the contributed genome changes its gene expression patterns in response to cues from the cytoplasm and/or nucleoplasm. Reduction division to diploid progeny may then follow [22].

The first demonstration of physiologic fusion was in the FAH-null mouse model of hereditary tyrosinemia type I in the laboratory of Markus Grompe [18]. In this model, the fatal genetic defect was cured by transplantation with wild-type bone marrow. The first such report of cure was hailed as a proof of principle for cell plasticity by direct differentiation, but a closer look showed that rescue was actually from fusion between donor-derived monocytes streaming into the damaged liver and pre-existing, diseased hepatocytes [23]. The donated monocyte nuclei then shifted their gene expression to produce hepatocyte-specific proteins, thereby also correcting the genetic defect.

These experiments were a disease-curing, physiologic version of the groundbreaking heterokaryon experiments of Helen Blau two decades before. Blau and colleagues fused differentiated human and mouse cells, documenting how gene expression in the donated nucleus changed toward that of the recipient cell type [24]. This line of investigation led Blau et al., anticipating the more recent “new plasticity” paradigms, to suggest that “differentiation is an actively maintained state,” rather than a irreversibly, rigidly programmed one [25].

Beyond the extraordinary nature of the fusion finding was the extraordinarily polemical way in which the research was presented, not as a new, important scientific discovery in its own right, but as a way to “disprove” plasticity by direct differentiation 5, 26. Even the studies' own investigators chose, at first, to emphasize the research as a negative result for plasticity rather than an independently exciting pathway [18].

Nonetheless, political posturing aside, both pathways have now been confirmed. A key article to highlight in this regard comes from the laboratory of Darwin Prockop in which green fluorescent protein (GFP) expressing mesenchymal stem cells (MSCs) were cocultured with heat-shocked, small airway epithelial cells [27]. Observing changes in real-time showed MSCs becoming epithelia by direct differentiation, but fusion was also witnessed. Most intriguingly, the fusion took place through extension of a pseudopod from MSC to injured epithelial cell, GFP blowing through this link followed by coalescence, indicating an active, presumably receptor/ligand mediated process rather than just fusion through mechanical pressure.

The key concept to keep in mind, deserving of italics for emphasis, is that whether the direct differentiation or fusion pathways of plasticity occur physiologically and to what extent depends on whether there is tissue injury, the nature and severity of such injury [21]. We will return to convincing demonstrations of both of these plasticity mechanisms later in this review.

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Single-cell test 

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.

The first reported single hematopoietic stem cell experiment was reported in by my research group in Cell, in May 2001, just in time for the plasticity debates to kick into high gear [28]. I have been able to identify 23 subsequently published experiments involving hematopoietic, mesenchymal and “multipotent adult progenitor cells”; these are summarized in Table 1 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

Table 1. Single-cell plasticity experiments with marrow-derived stem cells
YearFirst authorCell typeExperimentDifferentiationFusion vs direct differentiation
2001Krause [28]2-day homing, lin− HSCBM TxTrilineageNot investigated
2001Halleux [29]hMSCIn vitroOsteocytes, adipocytes, chrondrocytesNot investigated
2002Jiang [30]MAPCBlastocyst Tx and BM TxTrilineageNot investigated
2002Grant [31]Sca-1+ ckit+ lin− HSCBM TxRetinal endotheliumNot investigated
2002Wagers [32]ckit+ thy1.1lo lin− Sca1+ HSCBM TxVery rare hepatocytes, Purkinje cellsNot investigated
2003Masuya [33]Sca-1+ ckit+ lin− CD34− BMBM TxRenal mesangial cellsDirect differentiation
2003Keene [34]Murine MAPCBlastocyst TxNeurons, glial cellsNot investigated
2003Spees [27]hMSCIn vitroBronchial epitheliumBoth
2004Jang [35]2-day homing, lin− HSCIn vitroHepatocytesDirect differentiation
2004Hess [36]Sca-1+ ckit+ lin− CD34− BMBM TxMicroglia, Neural Perivascular cellsNot investigated
2004Kawada [37]mMSC (negative results for HSC)In tissue TxCardiac myocytesNot investigated
2005Yoon [38]hBMSCIn vivo/In vitroCardiac myocytes, smooth muscle, endotheliumBoth
2005Lange [39]rMSCIn vitroHepatocytesDirect differentiation
2006Visconti [40]Sca-1+ ckit+ lin− CD34− BMBM TxCardiac valve fibroblastsDirect differentiation
2006Lang [41]Sca-1+ ckit+ lin− CD34− BMBM TxInner ear fibrocytesDirect differentiation
2006LaRue [42]Sca-1+ ckit+ lin− CD34− BMBM TxTumor fibroblastsDirect differentiation
2006Ebihara [43]Sca-1+ ckit+ lin− CD34− BMBM Tx, In vitroFibroblastsDirect differentiation
2006De Bari [44]hMSCBM Tx and in tissue Txchondrocytes, osteoblasts, adipocytes, skeletal myocytesNot investigated
2006Tropel [45]mMSCIn vitroNeuronsDirect differentiation
2007Fujita [46]Sca-1+ ckit+ lin− CD34− BMBM TxMyocardial infarct myofibroblastsNot investigated
2007Cogle [47]ckit+ thy1+ lin− Sca1+ HSCBMTxColonic adenomas, Lung squamous cell cancerDirect differentiation
2007Chamberlain [48]hMSCIn utero (sheep)
BM Tx		HepatocytesDirect differentiation
2008Miyata [49]Sca-1+ ckit+ lin− CD34- BMBM TxHepatic stellate cellsDirect differentiation
2009Sera [50]Sca-1+ ckit+ lin− CD34− BMBM TxAdipocytesDirect differentiation

BM = bone marrow; hMSC = human mesenchymal stem cells; HSC = hematopoietic stem cell; MAPC = multipotent adult progenitor cells; mMSC, mouse mesenchymal stem cells; rMSC = mesenchymal stem cells; trilineage = differentiation along several endodermal, mesodermal, and ectodermal lineages; Tx = transplant.

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Case for plasticity 

Plasticity on a clonal basis was demonstrated in all but one of these articles. In some, clonality was entirely within the mesodermal lineage (e.g., cardiac, smooth, and skeletal muscle, adipocytes, fibroblasts of various organs, with and without injury, and tumors, chondrocytes, glial cells, endothelial cells, osteoblasts, hepatic stellate cells, oligodendrocytes), although some showed endodermal (e.g., pneumocytes, hepatocytes, cholangiocytes, enterocytes) and/or ectodermal (e.g., neuroectoderm, neurons, epithelial of the skin and adnexa) differentiation. Plasticity events were induced by various means: engraftment after whole-body irradiation to accomplish the initial marrow transplantation, direct transplantation into sites of injury, injection of marked cells into blastocysts, and coculture with cells of injured organs. 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?

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Case against… 

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” article by Wagers et al., from the Weismann laboratory at Stanford [32]. 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 [51] and one from the Blau research group [52].

In our single-cell transplant experiment, donor and recipient mice were age-matched at 4 to 6 weeks old. Wagers et al. [32] used 6- to 12-week-old donor and 10- to 14-week-old recipient mice. Age impacts significantly on stem cell functioning; younger donors and younger recipients outperform older ones.

In our experiment, levels of engraftment ranged from 30% to 91% in the five mice that we examined for engraftment. Wagers et al. [32] reported that their mice had 0.03% to 71.6% peripheral blood engraftment, but engraftment levels in the four mice analyzed for plasticity were not specified, nor did they indicated the level of blood engraftment at the time that the mice were sacrificed for visceral and brain examination.

Donor cells were not the same. Wagers et al. [32] used purely phenotypic sorting to select for c-kit+thy1.1lolin−Sca1+ cells. We obtained our donor cells through a functional assay for marrow homing and slow cycling after negative selection for mature hematopoietic lineage markers (lin-negative selection). Of possible import is that our lin-negative selection included AA4.1 antibody that removed hematopoietic progenitors, an exclusion not performed by Wagers et al. This difference alone might account for the different results, with our selection possibly isolating an “earlier,” more readily plastic stem cell.

A response to this last question might be that Wagers et al. [32] also performed parabiosis experiments that would also have included the bone marrow subfraction we tested, but this method itself, although widely used in studies of marrow-derived stem cells has its own technical issues that have, to my knowledge, remained unexplored: the extensive acute and ongoing chronic wound healing at the site of skin, subcutis, and vascular grafting, with concomitant extensive production of granulation tissue (which we now know to be extensively marrow-derived), may have acted as a significant sink for any circulating progenitors that might have otherwise engrafted elsewhere.

Wagers et al. [32] used a GFP-positive donor developed by that laboratory, a complete characterization of which has not, to my knowledge, been published. Moreover, the detection of GFP relied on direct fluorescence alone, which is perhaps not the most sensitive and specific method of detection [53]. (For more discussion of this issue, see Methods of Detection.)

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 29, 30, 31, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 go largely unmentioned, as though only these two articles exist for comparison. And yet, in their own reply to our “Comment” (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” [54].

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 [55]:

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.

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Principles of plasticity 

Even before the stem cell debates began to rage in earnest, my collaborator, Diane Krause of Yale University and I began to think about the implications of the new plasticity data. We formulated these into three “principles of cell plasticity” that sketched the outlines of this new field, offering guidelines for experimental design, evaluation of data, and thereby, we hoped, clarifying the nature of the new paradigm that was taking shape (Table 2) 56, 57, 58.

Table 2. Principles of cell plasticity
Genomic CompletenessAny cell with the entire genome intact (i.e., without deletions, translocations, duplications, mutations) can potentially become any other cell type.
Cellular UncertaintyAny attempt to analyze a cell necessarily alters the nature of the cell at the time of isolation, thereby altering outcomes of subsequent differentiation events.
Stochasticity of Cell LineagesDescriptions of progenitors and progeny of any given cell must be expressed stochastically.

Plasticity principle number 1: Genomic completeness 

This first principle states that any cell containing the entire genome intact—without deletions, mutations, translocations, multiplications—can potentially become any other cell type of the organism from which it has been isolated. This perhaps extreme statement of plasticity potential of all cells acknowledges two bodies of knowledge, one old, one new.

The first is that the original experiments upon which the dogma of restricted differentiation was founded were not designed to demonstrate the full range of differentiative potential. These historic experiments involved transplanting cells from one part of a developing embryo into another region of the embryo. Up to a certain point in development, transplanted cells would take on the differentiation behaviors of the new environment, after that time, the cells no longer reprogrammed but continued to become cell types as determined by their site of origin. Thus was enshrined the concept that during development cells become increasingly restricted in terms of differentiation potential, most cells becoming “terminally differentiated” in postnatal tissues. The underlying epigenetic mechanisms of this progressive and seemingly irreversible restriction were found in the latter half of the 20th century and the dogma was significantly reinforced. However, the dogma was based on experiments that were not well-suited to uncover the less frequent, more subtle possibilities still inherent within adult cells. Likewise, because the question being asked of molecular biologists was “how does gene restriction take place,” the reversibility of gene restrictions took longer to uncover. But during the same era in which the new cell plasticity events were being discovered, the parallel, molecular field of gene restrictions was revealing that epigenetic restrictions were reversible both experimentally and physiologically. Indeed, new such mechanisms are now being identified routinely, many of which are tissue- and cell-type specific, meaning that these are not simply artifacts of the laboratory, but can be coordinated, physiologically important processes. Review of this literature is beyond the scope of this article, but has been accomplished in somewhat greater detail elsewhere 59, 60.

When we first published this Genomic Completeness principle, the proof of principle experiment had already been performed with the cloning of Dolly [61]. Most surprising, perhaps, was not just the general epigenetic reprogramming in the course of nuclear transfer in response to cytoplasmic signaling in the recipient ovum, but that even X-inactivation was reversed, cells of adult cloned animals having random X-inactivation [62].

To come back to the political debates, these issues have now come around to demonstrate how inseparable advances in adult stem cell research may be from embryonic stem cell and cloning research. Only through studying embryonic stem cells could it have taken only a handful of years to recognize the importance of Nanog, OCT-4, and SOX-2 for the maintenance or reconstitution of developmental plasticity [63]. This embryonic stem cell research was indispensable for the development of induced pleuripotent stem cell technology, the current headline-making corner of stem cell research, through insertion of extra copies of pleuripotency genes 64, 65, 66. Given that cytoplasmic signaling in the ovum accomplished this trick in nuclear transfer, I look forward to future discoveries of microenvironmental cues that might trigger the same induction of pleuripotentiality without a need for gene insertion.

Plasticity principle number 2: Cellular uncertainty 

Intentionally echoing Heisenberg's Uncertainty Principle, we suggested that any attempt to observe a cell alters the state of that cell at the time of characterization and potentially alters the likelihood of subsequent differentiation events 55, 56, 57, 58. The implications of this are easy to grasp. As Richard Lewontin so elegantly stated: “the internal and the external co-determine the cell” [67]. It is not only genomic programming that determines the form and behavior of a cell, but microenvironmental influences are equally important. The differentiation state of a cell arises where the internal molecular dynamics meet the extrinsic molecular and mechanical influences. Given that any experimental technique we now have available to us is at least minimally invasive (venopuncture), if not quite violent (tissue disaggregation), it is disingenuous to consider cell biology experiments to have “isolation steps” that are separate from “conditioning steps.” Every isolation step is inherently a conditioning step.

This is not trivial and colors all our attempts to understand today's stem cell biology. For example, in its most dramatic form, the most common stem cell isolation for human stem cell therapies is sorting by CD34 expression. However, this necessarily involves binding of anti-CD34 antibodies to the epitope on the cell surface. We still have little understanding of what CD34 does as a signaling molecule [68]. We therefore have no information about what the act of antibody binding entails for the cell. (We know, for example, that different anti-CD45 monoclonal antibodies can variously up- or downregulate CD45 functioning [69].) If we do not know what CD34 does physiologically, we cannot know the effects of binding of anti-CD34 antibodies in the course of stem cell isolation. Are we, in fact, isolating stem cells? Or are we creating stem cells through isolating CD34-positive cells? Or something in between?

When we first proposed this principle, we included the word potentially because, like Heisenberg in his first discussions of quantum uncertainty, we thought cellular uncertainty might be a result of current technological limitations. Heisenberg soon reasoned, correctly, that it was not a technological limitation, but an inherent property of the universe. Through applications of complexity theory to cellular uncertainty, it now seems unlikely that this principle is contingent on technological limitations; rather, as disturbing as this is to traditionalists, it is inherent in the nature of the cell. This principle, by clarifying the contingent nature of the cell erodes the exclusive centrality of cell theory, the foundational doctrine of what we call Western biology and medicine; this theme, however intriguing, is also beyond the scope of this essay, but is reviewed elsewhere [70].

Plasticity principle number 3: Stochasticity of cell origin and fate 

The logical extension of the first two principles is that once a cell is in hand to be studied, one can never be absolutely certain about the cell lineage that produced that cell or about where it might go, in terms of differentiation, once it is manipulated. Instead, it appears that cells are inherently stochastic and the origin and fate of cells must be expressed stochastically 56, 57, 58. For many years, the determinism vs stochasticity question was passionately debated within cell biology in general, but in stem cell biology in particular.

From our own perspective, the presence of even contingent cellular uncertainty coupled with the reversibility of gene restrictions made some degree of stochasticity mandatory. But in recent years, stochasticity of gene expression could be directly identified and quantified. It is clear, for example, that quantum effects can erupt from the subatomic into the molecular realm 71, 72. But also, through active observation of elements of the nucleosome, such as in the work of the Cremer laboratory of the Ludwig-Maximilians-University, the play of randomness in the system can be demonstrated 73, 74. These investigators fluorescently labeled euchromatic and heterochromatic chromosomal domains in living cells and then observed their movements in real time. As expected, the heterochromatic regions were essentially unmoving, particularly those localized to the nuclear membrane. However, euchromatic regions moved freely, sometimes outside of the chromatic domain, sometimes within. This movement was best described mathematically as a “random walk.”

Given that access to transcription factors depends on exposure in the nucleoplasm, the randomly changing exposure as euchromatic genes move between the interior and the exterior of the chromosomal domain means that there is inherent stochasticity to gene expression. It is not to say that it is completely random, but there is an observable constrained randomness (constraint arising from structural/architectural aspects of adjacent chromatin regions).

Heterogeneity in any isolated cell population, even beginning with single cells that are then clonally expanded, would also be unavoidable. Colvin, Quesenberry, and collaborators have produced a detailed body of work that calls into question common and basic experimental methods in this regard by demonstrating that a cell's differentiation capacity varies depending, for example, on its point in the cell cycle [75]. Among other things, this implies that if cells are not synchronized before an experiment, or if repetitions of single-cell experiments do not take cell cycle into consideration, stochasticity will be experimentally unavoidable, even heightened.

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Complex and chaotic systems theories: 21st century tissue biology 

But perfected experimental designs cannot eliminate stochasticity completely. Indeed, the bulk of evidence, made possible by recent refinements of our ability to measure molecular events in single cells, now strongly favors the less conservative, stochasticity side of the debate [76]. The key to reconciling experimentally apparent deterministic behaviors of cell lineages with inherent stochasticity is the application of systems biology approaches, characterizing cell and molecular behaviors as complex or chaotic dynamical systems 77, 78, 79, 80, 81, 82, 83.

Misapprehensions about cell behavior are, in part, dependent on the reductionist methodologies of the past centuries. Although their success is inarguable, thinking of biological systems as machines with parts has its limitations. Understanding the pieces does not automatically lead to understanding the whole. It is time that, just as we have developed fields of cell and molecular biology, we have a field characterized as tissue biology 60, 84, 85. One sign of this is how the plasticity data depend on photomicroscopic images of tissue sections. Indeed, a telling rejection of our first plasticity submission included critiques from several reviewers that we had little true data: because our data were histologic images, not gels and culture results. But soon, such microscopy proliferated—there is scant plasticity data that is independent of tissue-level behaviors.

Likewise, the importance of the tissue level of scale is that in vivo stem cell behaviors is entirely dependent on the web of signaling between all adjacent cells and the matrix, a web of behaviors that can only fully be described by careful modeling in accord with standards in the systems biology field. In such a light, it becomes clear that we must think in a new way. It is not that if determinism fails, then everything is random: this was the either/or fallacy that tripped up Einstein in his later years. Rather, there is an inherent, but limited degree of randomness, sometimes referred to as “quenched disorder,” in these processes, a degree of randomness that allows biological systems to be responsive, adaptive, and alive 70, 77, 80.

With the arrival of sophisticated complexity and chaos theorists and mathematical modelers in the stem cell world, apparent differentiative stability is now well-understood, in fact, to be dependent on stochastic behaviors. The experimental data consistently support these understandings.

A few sampled titles of recent articles should vividly convince nonmathematically inclined stem cell biologists that something is afoot and that cross-disciplinary communications should be encouraged, however unnerving: “transcriptome-wide noise controls lineage choice in mammalian progenitor cells” [86], “chaotic expression dynamics implies pluripotency: when theory and experiment meet” [87], “stochasticity and the molecular mechanisms of induced pluripotency” [88], “stochasticity in gene expression: from theories to phenotypes” [89], and “reprogramming cell fates: reconciling rarity with robustness” [90]. I list these here, rather than just leaving them as reference citations, in the hope that stem cell researchers unfamiliar with the burgeoning interface with systems biology will be intrigued enough to explore. This is what 21st-century medicine and biology will be about after four centuries of primarily reductionist successes. It is clearly time to start reassembling the pieces into a “tissue biology” whole.

One example of this progress is worth highlighting. In 1994, Morrison and Weissman stated unequivocally regarding Sca-1−positive stem cells: “The ability to predict the longevity of reconstitution based on lineage marker expression indicates that reconstitution potential is deterministic, not stochastic” [91].These experiments and interpretations were appropriate in the context of experimental techniques and concepts of the time. But only 10 years later, we have a new, more subtle view, made possible by vastly improved single-cell molecular analysis techniques and application of contemporary mathematical methods: Chang and colleagues, specifically looking at Sca-1−positive cells, reveal that the appearance of determinism in the earlier experiments represents metastable states arising from inherently stochastic gene expression processes [92].

Our imaginings of the limits on cell behaviors reveal more about the limitations of our imaginations rather than those of the cells, which are among the most exquisitely sensitive, subtle, and dynamic arrangements of matter in the universe.

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Issues of experimental design and discourse 

To state the obvious: experimental design has been and will remain crucial for plasticity research [93]. That this requires restatement is a measure of just how distorted the discourse around adult cell plasticity became through the political confusions shadowing it from the start. However, review of some examples is useful, not only in distinguishing high-profile polemics from actual science, but to suggest ways in which future research can benefit by improving experimental design and interpretation of data. Many of these examples will involve marrow to liver plasticity as these are closest to my own area of expertise.

Nature and severity of injury 

Early reports of hepatobiliary derivation from marrow-derived cells rapidly encountered resistance in a series of negative results articles that employed very different models of injury. Reproducibility is of course a key criterion for verification of data. But reproducibility should include replication of the original experimental methods, not simply challenges from unrelated models that may not, in fact, reflect on the original experiments.

For example, Petersen et al. identified marrow-derived hepatic progenitors (“oval cell”) in their initial article [4], which we soon confirmed in humans [94]. However; some follow-up reports did not find the same result. Menthena et al. employed three well-recognized approaches to generating oval cells, none of which, however, were precise matches to Petersen et al.’s first experiments [95]. Petersen et al. responded by focusing on one of these methods, i.e., poisoning the hepatocytes' ability to replicate by administration of pyrrolizidine alkaloids (e.g., retrorsine, monocrotaline) and then performing partial hepatectomy. With hepatocytes no longer able to regenerate in such models, the stem cell compartment activates and oval cells proliferate.

Given that these toxins affect all rapidly proliferating cells, not just hepatobiliary cells, Petersen et al. questioned whether the toxin might also inhibit the newly transplanted marrow, thus suppressing the marrow to liver pathway [96]. Indeed, their intuition was correct: replicating the experiment of the Menthena et al. article yielded no marrow-derived stem cells, but by reversing the order of injuries, hepatotoxin first followed by transplantation and partial hepatectomy, 20% of oval cells were marrow-derived.

The degree of injury is also important and few commentaries comparing different reports will look critically at this feature of experimental design. Turning to the marrow−lung literature is illustrative. Herzog et al., in the laboratory of Diane Krause, closely examined the relationship of radiation dose to this question [97]. The results were clear: at low levels of radiation (400 and 600 cGy) no marrow-derived pneumocytes were identified, but at higher levels (900 cGy, cumulatively in two split 450-cGy doses or as a single dose), pneumocyte engraftment was found. Severity of injury in this model is crucial to the presence or absence of plasticity events.

As for hepatocytes from marrow, the rescue of the FAH-null model by monocyte fusion alone and not by direct differentiation of marrow-derived progenitors was also definitively authoritative for many as a negative finding for plasticity. However, it has been established that secreted factors are important for the homing of marrow cells to sites of injury, SDF-1 and SCF 16, 17. We have recently explored expression of SDF-1 and SCF in the FAH-null mouse model as well as other models of liver injury commonly used in plasticity research (radiation and the biliary toxin DDC) and found all three models provoked very different SDF-1 and SCF responses; in particular, not only does serum SCF not rise significantly in the FAH-null model, but it actually declines significantly [21]. Of course, hepatocytes derived from direct differentiation of marrow progenitors were not identified in that approach; a major signal for their recruitment was absent. Moreover, the histologic displays of each injury reveal such profoundly striking differences that one would be hard-pressed to imagine how a reasonably objective comparison could see them as equivalent ways to test any single phenomena.

Thus, confirming that both the nature and severity of an injury (including its timing and its systemic effects beyond the organ of interest) determines the degree to which plasticity is seen. To assume that plasticity data from one model of injury can ever provide clear commentary on the findings of a different model (differing either in kind or in severity) is inappropriate if one is truly interested in scientific rigor. Absolutist negative statements, such as those repeated during the decade by Willenbring and Grompe [26], which privilege their own FAH-null model as the single and ultimate arbiter of stem cell behaviors in any setting, must make the reader cautious, if not, in fact, suspicious of an author's polemical intent.

Methods of detection 

How one identifies plasticity events is crucial. Herzog et al., in this same article, demonstrated how important a panel of positive and negative markers is for accurate identification even in thin tissue sections [97]. In our original lung from marrow articles, we used a single marker for distinguishing pneumocytes (either immunostaining for keratin or fluorescence in situ hybridization for surfactant B messenger RNA) from background leukocytes, both of which would have Y chromosomes as a marker of marrow origin in gender-mismatched transplant experiments [28]. For greater rigor, Herzog et al. costained for CD45, a leukocyte marker, and found that some, though not all, apparent Y-positive pneumocytes were actually recipient pneumocytes overlapping with donor-derived leukocytes even in tissue sections only 3-μm thick. Thus, our initial investigations demonstrating marrow-derived pneumocytes, while confirmed by Herzog's results in principle, probably overestimated the degree of engraftment. These issues are more fully discussed in this issue of the Journal in the article by Kassmer and Krause [98].

Transgenic markers are also commonly used in plasticity research, such as GFP-positive donor cells transplanted into marker-negative, wild-type recipients. Several transgenic mouse strains with “ubiquitous GFP expression” are in use; however, the “ubiquitous” does not, in fact, mean “in every cell,” but rather “in every cell type,” with the number of cells in any organ being highly variable. Swenson et al. compared three such strains and found marked differences, organ by organ, in expression [53]. In the strain with the highest circulating blood cell GFP expression, liver, kidney, and small intestine had very limited expression. Another strain had strong expression in all four organs, but still, a minority of cells in each organ was actually positive. Another had weak staining in the viscera, but even in the blood, expression was varied from animal to animal of the same strain. Methods of detection (direct fluorescence, immunofluorescence, immunohistochemistry, varied monoclonal antibodies) also resulted in variable detection rates.

Before one even begins a transplantation experiment, full evaluation of the expression in an uninjured donor transgenic animal, along with evaluation of different detection methods are the first controls that must be performed. If one cannot consistently detect labeled hepatocytes in the donor, how can one expect to sensitively detect hepatocytes derived from that animal's marrow in a transplanted recipient?

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Summary 

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. Parallel developments in fields of epigenetics, revealing extensive mutability of gene restrictions, undermine the ability to dismiss the findings out of hand. Understanding how subtle aspects of experimental design determine positive and negative outcomes—and a careful evaluation of all data in the light of these implications—should mitigate the political and polemical pressures which have so far inhibited the field.

The one argument that remains somewhat potent is the question of the physiologic role played by plasticity. This remains an open question. However, even if the physiologic role is minute, even that hint of behavior implies that laboratory analysis may reveal ways in which it can be usefully exploited, for the benefits of regenerative medicine and of industry. It would be premature, on that basis alone, to reject the field as unworthy of significant investment and pursuit.

Certainly, hundreds of clinical trials around the world (although still infrequently in the United States), often with autologous stem cells, have been inspired by the cell plasticity data. Results are not conclusive, but they remain intriguing. What becomes ever more clear is that such therapeutic experiments, while having begun in response to plasticity reports, are now providing even more data that it is not plasticity alone that is a pathway to regeneration, but the extensive signaling between the transplanted cells and the complex array of elements—intact and disordered—in the tissues into which they are placed that yields interesting outcomes. Again, we are face-to-face with a web of possibilities, pointing to one sure aspect of future work: stem cell biology functions at the level of “tissue biology.” The field must turn to systems approaches to fully explore how our bodies develop, maintain, and heal themselves.

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Conflict of Interest Disclosure 

No financial interest/relationships with financial interest relating to the topic of this article have been declared.

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PII: S0301-472X(10)00156-6

doi:10.1016/j.exphem.2010.04.013

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