REVIEW

Dynamic stem cell heterogeneityTeresa Krieger1,2 and Benjamin D. Simons1,2,3,*

ABSTRACTRecent lineage-tracing studies based on inducible genetic labellinghave emphasized a crucial role for stochasticity in the maintenanceand regeneration of cycling adult tissues. These studies haverevealed that stem cells are frequently lost through differentiationand that this is compensated for by the duplication of neighbours,leading to the consolidation of clonal diversity. Through thecombination of long-term lineage-tracing assays with short-termin vivo live imaging, the cellular basis of this stochastic stem cell lossand replacement has begun to be resolved. With a focus onmammalian spermatogenesis, intestinal maintenance and the haircycle, we review the role of dynamic heterogeneity in the regulation ofadult stem cell populations.

KEY WORDS: Adult stem cell, Gene heterogeneity, Germ line,Intestine, Lineage tracing, Tissue homeostasis

IntroductionIn multicellular organisms, groups of cells specialize within tissuesand organs to perform specific tasks and functions. In the courseof adult life, these functional cells can become exhausted andprogressively lost. To compensate for the ongoing loss ofdifferentiated cells, new functional cells must be generated so thattissues remain in homeostasis. Themaintenance and repair of cyclingadult tissues usually rely upon the turnover of a small population ofcells – termed adult stem cells – that possess the ability to self-renew,giving rise to differentiated cells while maintaining their number(Watt and Hogan, 2000; Fuchs and Chen, 2013).The capacity of self-renewal has long been considered the defining

feature of adult stem cells (Clermont and Leblond, 1952, 1953;Leblond and Stevens, 1948). To achieve homeostasis, stem cellproliferation and differentiationmust be perfectly balanced, such that,following division, on average one daughter cell stays in the stem cellcompartment, whereas the other differentiates either directly orthrough a limited series of divisions. Such fate asymmetry can beachieved as the invariant result of each and every stem cell division(termed ‘invariant asymmetry’). Alternatively, fate asymmetry maybe orchestrated at the level of the population (termed ‘populationasymmetry’), such that cell fate following each stem cell division isunpredictable or ‘stochastic’, and is specified only up to some definedprobability (Klein and Simons, 2011; Simons and Clevers, 2011).These alternative models (Fig. 1A), both of which may be instructedby intrinsic (cell-autonomous) or extrinsic (environmental) cues,suggest very different regulatory mechanisms.To address the factors that regulate stem cell self-renewal in adult

tissues, attention has focused on defining the molecular mechanisms

that control fate behaviour. By combining static marker-based assayswith the transcriptional profiling of fixed samples, significant progresshas been made in resolving key elements of the gene regulatorynetworks and signalling pathways that control stem cell activity andfate behaviour (Clements and Traver, 2013; Guruharsha et al., 2012;Holland et al., 2013; Laplante and Sabatini, 2012; Singh et al., 2012;Clevers and Nusse, 2012). However, it is becoming evident that stemcells function in dynamic and noisy environments in which levels ofgene expression may adjust or fluctuate in response to promoteractivity and extrinsic signals from the localmicroenvironment or niche(Morrison andSpradling, 2008). Therefore, to discriminate tissue stemcells from their more differentiated cell progeny and to define theirfunctional behaviour, it is essential to address dynamic aswell as staticmeasures. Historically, the importance of such an approach wasrecognized prior to the genomics revolution. Charles PhilippeLeblond, considered by many to be the father of modern stem cellbiology, emphasized the ‘time dimension in histology’, and did muchto advance early lineage-tracing methods using autoradiography andthe incorporation of thymidine analogues (Leblond, 1965). However,it was not until the advent of transgenic animal models that it becamepossible to reliably trace the lineage of individual cells and theirlabelled progeny over time (Kretzschmar and Watt, 2012).

In recent years, pioneering studies using in vivo live-imagingplatforms have begun to provide access to continuous-time lineagedata (Bertrand et al., 2010; Boisset et al., 2010; Lam et al., 2010;Yaniv et al., 2006; Ritsma et al., 2013; Rompolas et al., 2012),whereas methods based on single-cell deep sequencing now offerthe potential to resolve individual phylogenies, even in humantissues (Shapiro et al., 2013; Treutlein et al., 2014). By combiningthese lineage-tracing approaches with static marker-based assays,snapshots of clonal evolution over time can be integrated withpopulation-level measures to reveal how stem and progenitor cellscontribute to tissue maintenance. Efforts have also been made todevelop statistical and mathematical methods that can resolvestrategies of progenitor cell fate in development and tissuemaintenance (Klein and Simons, 2011). When applied to activelycycling adult tissues in both human and model organisms, includingthe epidermis (Clayton et al., 2007; Mascré et al., 2012),oesophagus (Doupé et al., 2012), intestine (Simons and Clevers,2011; Lopez-Garcia et al., 2010; Snippert et al., 2010; Hara et al.,2014; Amoyel et al., 2014) and germline (Klein et al., 2010; Shengand Matunis, 2011), these studies show a preference for populationasymmetric self-renewal, in which stem cells are continuously andstochastically lost and replaced by neighbours. This pattern of self-renewal results in ‘neutral drift’ dynamics, with the continual andstochastic loss of clones through differentiation compensated for bythe expansion of others so that the overall stem cell population sizeremains constant. In some cases, these studies have overturned long-held paradigms and refocused the search for the molecularregulatory mechanisms that underpin stochastic fate behaviour. Inparticular, they have prompted the question of how the balanceof stem cell proliferation and differentiation is regulated withindynamically changing environments (Simons and Clevers, 2011).

1The Wellcome Trust/Cancer Research UK Gurdon Institute, University ofCambridge, Tennis Court Road, Cambridge CB2 1QN, UK. 2Cavendish Laboratory,Department of Physics, J. J. Thomson Avenue, University of Cambridge,Cambridge CB3 0HE, UK. 3Wellcome Trust-Medical Research Council Stem CellInstitute, University of Cambridge, Cambridge CB2 1QR, UK.

*Author for correspondence (bds10@cam.ac.uk)

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These studies have also begun to question our understanding ofstem cell identity in adult tissues. When considering the identity ofstem cells, two key assumptions are usually implicit, but rarelychallenged. First, it is presumed that stem cells are defined by thesignature expression of molecular markers, distinct from their moredifferentiated cell progeny. Second, in the course of tissue turnover,stem cells and their progenitor cell progeny are thought to moveirreversibly through a differentiation hierarchy (Fig. 1B). However,

with the advent of more-refined lineage-tracing approaches, both ofthese assumptions have been called into question. Increasingevidence suggests that expression levels of key fate determinants arenot fixed, but drift over time or fluctuate in response totranscriptional activity and to extrinsic cues from the localmicroenvironment (Chambers et al., 2007; Chang et al., 2008;Pina et al., 2012; Levine et al., 2013; Imayoshi et al., 2013).Furthermore, progenitors expressing the same putative stem cellmarker can exhibit heterogeneous fate choices (Graf and Stadtfeld,2008), while stem cells with different expression profiles maybehave similarly in the long term. Finally, recent studies in disparatetissues have also shown that cells normally committed todifferentiation can, in the course of regeneration following thetargeted ablation of endogenous stem cell populations, re-acquirethe hallmark properties of tissue stem cells, including the potentialfor long-term self-renewal (Rompolas et al., 2012; van Es et al.,2012; Tata et al., 2013; Yanger et al., 2014; Tetteh et al., 2015).

Together, these findings question the traditional view of adultstem cell populations as discrete entities comprising functionallyequivalent cells. Instead, gathering evidence suggests that, in sometissues, stem cells may transit reversibly between discrete or acontinuum of states in which they become temporarily biasedtowards specific fates, but the final decision is made stochasticallyor governed locally by cell-extrinsic factors (Enver et al., 2009,1998; Chalancon et al., 2012). In this way, a transcriptionallyheterogeneous cell population may function, long-term, as a singleequipotent stem cell pool (Fig. 1C). Here, we review case studiesfrom three canonical cycling adult tissues types – the mammaliangermline, intestine and hair follicle – that exemplify the role ofheterogeneity and stochasticity in the regulation of adult stem cellbehaviour, as well as the conservation of self-renewal strategiesbetween seemingly disparate tissue types. These studies highlightthe value of a multifaceted approach to the study of tissuemaintenance that places emphasis on quantitative and dynamicmeasures of fate behaviour.

Examples of dynamic stem cell heterogeneityMammalian spermatogenesisInmammals, spermatogenesis takes place in the seminiferous tubulesof testes (Russell et al., 1993). In common with other cycling adulttissues, the testes contain adult stem cells – termed germline stemcells (GSCs) – that continually self-renew throughout adult life andare capable of rapid regeneration following injury. Throughout allstages of their development, germ cells are nourished by largesomatic Sertoli cells, which support a network of tight junctions thatseparate the basal and adluminal compartments of the seminiferoustubule (Fig. 2A). Spermatogonia (mitotic germ cells that includeGSCs) lie in close association with the basement membrane of theseminiferous tubule, and form the basal germ cell compartment.When meiosis begins, cells detach from the basement membrane,and translocate across the tight junctions. They then undergomeioticdivisions and spermiogenesis (Fig. 2B) before their release into thelumen as mature sperm. In mice, spermatogonia are subdivided into‘undifferentiated’ and ‘differentiating’ populations, with thedifferentiated cells expressing the receptor tyrosine kinase Kit.Furthermore, undifferentiated spermatogonia can exist as singlyisolated cells (termed Asingle or As) or as syncytial chains of cellsconnected by cellular bridges, consistingmainlyof two (Apair orApr),four (Aaligned-4 or Aal-4), eight (Aal-8) or 16 (Aal-16) cells (Fig. 2C) (deRooij and Russell, 2000).

In early studies, detailed analyses of fixed specimens led to theconjecture that stem cell activity is limited to the population of As

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Fig. 1. Proliferative hierarchies and patterns of stem cell self-renewal.(A) During tissue homeostasis, patterns of adult stem cell self-renewal can begrouped into four generic classes depending on: whether stem cell fate isregulated intrinsically (cell-autonomously) orwhether it relies onextrinsic signalsassociated with the niche/microenvironment; and whether fate asymmetry isenforced at each and every stemcell division or whether it is achieved only at thelevel of the population (Simons and Clevers, 2011). (B) Traditionally, adult stemcell populations are thought to reside at the apex of linear (i.e. ‘one-way’)proliferative hierarchies in which they give rise to one or more types of transit-amplifying cell progeny with strictly limited proliferative potential. (C) Recentstudies suggest a more flexible organization in which long-term self-renewalpotential, fate bias and proliferative activity may be moderated by niche locationand/or dynamical changes in transcriptional activity. In this scheme, stem cellsform a ‘dynamically heterogeneous’ pool in which cells may transfer reversiblybetween ‘states’ of variable survival and fate potential. In addition, progenitorsthat are normally committed to differentiation may re-acquire long-term self-renewal potential in crisis or injury, following exposure to niche factors.

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spermatogonia, whereas interconnected Apr and Aal syncytia wereirreversibly committed to differentiation, a hypothesis known as the‘As model’ (Huckins, 1971; Oakberg, 1971). Consistent with thismodel, post-transplantation colony formation and regenerationassays confirmed that the vast majority of stem cell activity isrestricted to the population of undifferentiated (Kit-negative)spermatogonia (Shinohara et al., 2000). More recently, theidentification of genetic markers that are enriched in or restrictedto As spermatogonia, including the transcriptional repressor ID4,the polycomb complex protein Bmi1 and the paired-box proteinPax7, allowed the potency of individual As cells to be assessed(Aloisio et al., 2014; Komai et al., 2014; Oatley et al., 2011). Thesestudies confirm that at least a fraction of As cells retains long-termself-renewal potential, lending further support to the As modelparadigm.However, recent lineage-tracing studies have questioned the

validity of the As model and offer a new perspective on the identityand function of adult stem cell populations in the germline and

indeed in other adult tissues. These studies focused on the fate andbehaviour of two separate compartments of undifferentiatedspermatogonia, characterized by the expression of the glial cell-derived neurotrophic factor family receptor α1 (GFRα1) and thetranscription factor neurogenin 3 (Ngn3). In undisturbed testes,these factors are expressed heterogeneously, with GFRα1expressed more widely in As cells and shorter syncytia (Apr anda few Aal), whereas Ngn3 is expressed in a largely complementarymanner (Fig. 2C). By developing an inducible genetic labellingtransgenic mouse model based on the Cre-loxP recombinationsystem with an Ngn3 promoter, the Yoshida lab showed that thevast majority of Ngn3-expressing cells proceed rapidly todifferentiation, maturation and loss, but that a small minority ofcells retain long-term self-renewal potential (Nakagawa et al.,2007). Furthermore, through the development of long-term‘scaling’ properties of the measured clone size distribution, afollow-up study showed that GSCs are not individually long-lived,but are stochastically lost through differentiation and replaced by

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Fig. 2. Stem cell dynamics during mammalian spermatogenesis. (A) Schematic showing the architecture and cellular organization of the mammaliantestis. Spermatogonia lie in close association with the basement membrane of the seminiferous tubule. When meiosis begins, they detach from thebasement membrane, translocate across the tight junctions between supporting Sertoli cells and undergo meiotic divisions and differentiation before theirrelease into the lumen as mature sperm. (B) Spermatogonia progress through a differentiation hierarchy while migrating from the basement membrane to thelumen. (C) In the undifferentiated compartment, spermatogonia can exist as singly isolated cells (termed Asingle or As) or as syncytial chains of two (Apair orApr), four (Aaligned-4 or Aal-4), eight (Aal-8) or 16 (Aal-16) cells. Undifferentiated spermatogonia are characterized by heterogeneous and complementaryexpression of GFRα1 and Ngn3, with As and smaller syncytial chains biased towards GFRα1 expression. Following upregulation of Ngn3, spermatogoniaare competent to transfer to the differentiated Kit+ compartment, in concert with the periodic seminiferous cycle. (D) Whole mount (top panels) of aseminiferous tubule showing GFRα1 expression (magenta) and GFP-labelled clones (green) at 14 days post-clonal induction. The fragmentation of an Aal-4

syncytial chain results in two Apr chains. Reproduced, with permission, from Hara et al. (2014). Schematic (bottom) showing the cellular basis for germ linestem cell maintenance: chance stem cell loss through differentiation, signalled by the downregulation of GFRα1, is perfectly compensated for by stem cellduplication achieved through the fragmentation of neighbouring GFRα1+ syncytia. Through this ongoing process of stem cell loss through differentiation andreplacement, stem cell-derived clones follow a ‘quasi’ one-dimensional pattern of ‘neutral drift’ where their chance extinction is compensated for by theexpansion of neighbours along the seminiferous tubule.

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neighbouring GSCs, leading to neutral drift dynamics of thesurviving clone size (Klein et al., 2010). Although these results areseemingly compatible with the As model, a subsequent in vivolive-imaging study by the same group revealed that the cellularbridges that connect cells within syncytia can break down, leadingto the infrequent ‘fragmentation’ of Ngn3-expressing syncytiainto single cells or shorter syncytia (Nakagawa et al., 2010). Suchflexible behaviour of Ngn3-expressing spermatogonia questionsthe premise of the As model that syncytia are irreversiblycommitted to differentiation. Instead, these results suggest thatthe entire pool of undifferentiated spermatogonia may contributeto stem cell activity.To address this issue, Yoshida and colleagues then combined

detailed in vivo live imaging with long-term genetic lineage tracingusing a pulse-labelling assay to follow the fate of individual GFRα1+spermatogonia and their differentiating progeny (Hara et al., 2014).Continuous live-imaging data totalling more than 1 year of filmingrevealed that just 5% of GFRα1-expressing As cell divisions arecomplete, with the vast majority leading to the generation of Apr

syncytia. Therefore, if the transition from As to Apr indeed signalledcommitment to differentiation, as conjectured by the As model, theGFRα1+ As population would become rapidly depleted over time.However, alongside the cell division rate of around once per 10 daysfor GFRα1-expressing cells (independent of syncytial length), thelive-imaging study also revealed fragmentation of GFRα1-expressing syncytia at a rate of around once per 20 days perinterconnecting bridge, providing a possible route to replenish theAs compartment.Together, these findings suggest a revised model of

GSC maintenance in which a morphologically heterogeneouscell population, comprising predominantly GFRα1-expressingspermatogonia (including As and syncytial chains), functions longterm as a single stem cell pool. In this paradigm, germ cellproduction involves a coordinated process in which the commitmentof cells to differentiation (signalled by the downregulation ofGFRα1 expression and upregulation of Ngn3) is perfectlycompensated for by the fragmentation of neighbouring GFRα1-expressing syncytia (Fig. 2D). To test this hypothesis, the measuredrates of cell division and syncytial fragmentation were used topredict the medium (weeks to months) and long-term (months toover 1 year) clonal evolution of labelled GFRα1-expressing cellsand their differentiating progeny. By collecting clone size andcompositional data at single cell resolution, compelling quantitativeevidence was obtained in support of the new model for germlinemaintenance. Through continual GSC loss and replacement, clonesundergo a neutral drift process in which their chance expansionthrough syncytial fragmentation is perfectly compensated for by thecontraction or loss of others through differentiation. At the sametime, this study established a cellular basis to understand the processof GSC loss and replacement that was revealed by the long-termscaling behaviour of the clone size distribution reported in theearlier Ngn3 lineage-tracing study.Although the cellular organization of themammalian germline is of

course unusual, these studies highlight several important features ofstem cell dynamics that may translate to other stem cell-supportedcycling adult tissues. First, maintenance of the stem cell compartmentinvolves the continual stochastic loss and replacement of stem cells,leading to a progressive consolidationof clonal diversity. Second, stemcell competence is not restricted to a homogeneous cell population,defined by a signature expression of molecular markers. Instead,through the reversible transfer of cells between morphologically andgenetically distinct states with differential survival probability, a

heterogeneous population is able to function long-term as a singleequipotent stem cell pool (see Fig. 1C). Third, it is only throughquantitative analysis that ‘neutral’ competition between equipotentstem cells can be discriminated from ‘non-neutral’ clonal dominanceassociated with engrained (i.e. long-term) heterogeneity of fatepotential. Fourth, the elucidation of short-term heterogeneity, andthe cellular basis for stem cell loss and replacement, is facilitated byaccess to continuous-time in vivo live imaging.

As well as presenting a new perspective on the identity of GSCsand the cellular basis for stem cell self-renewal, these studies alsoraise new mechanistic questions. What is the molecular regulatorybasis for stochastic GSC loss and replacement? What role is playedby the periodic seminiferous cycle that orchestrates the progressionof spermatogonia and spermatocytes through the differentiationpathway? How is the fragmentation of GFRα1+ syncytia soexquisitely correlated with the commitment to differentiation ofneighbours when GSCs are separated by legions of differentiatingprogeny? And, functionally, given the singular role of the germlinein the propagation of genetic information to the next generation,what are the implications of neutral drift clone dynamics for theinheritance of congenital disorders due to the acquisition and spreadof de novo mutations in GSCs (Goriely et al., 2003; Giannoulatouet al., 2013)? But before speculating on potential regulatorymechanisms and their implications, it is instructive to look forparallels of these dynamics. To this end, we consider a secondmammalian epithelial tissue that is characterized by a high degree ofturnover.

Intestinal maintenanceThe epithelium of the mammalian small intestine is organized intolarge numbers of self-renewing crypt-villus units (Fig. 3A). Villiform finger-like protrusions of the gut wall that project into thelumen to maximize available absorptive surface area. The villi arecovered by a simple post-mitotic epithelium, beneath whichcapillaries and lymph vessels mediate transport of absorbednutrients into the body. The base of each villus is surrounded bymultiple epithelial invaginations, termed crypts of Lieberkühn. Thecrypts play host to a population of rapidly proliferating intestinalstem cells (ISCs), which fuel the active self-renewal of theepithelium throughout adult life (Clevers, 2013). Thesemultipotent cells give rise to lineage-restricted transit-amplifyingcell progeny, which migrate along the walls of the intestinal cryptand generate the various differentiated absorptive and secretory celltypes.

The identity, multiplicity and behaviour of ISCs have remainedthe subject of continuing debate and controversy. Beginning withthe early pioneering studies of Leblond, which were based onincorporation of thymidine analogues, ISCs have been localized tothe base region of the crypt (Cheng and Leblond, 1974a,b).However, these early studies could not assess the long-term potencyor heterogeneity of individual crypt base progenitors. Subsequentlineage-tracing studies of individual clones marked by a chemicalmutagen (ethylnitrosourea) showed that, in the course of turnover,the entire intestinal epithelium could be derived from a singlemarked cell (Winton et al., 1988). This result provided strongevidence that the intestinal epithelium is maintained by multipotentprogenitor cells, which reside at the apex of a proliferative hierarchy.Later, with the advent of transgenics, long-term lineage-tracingstudies based on the clonal marking of targeted cell populationsidentified several putative ISC markers, including the leucine-richrepeat-containing G-protein-coupled receptor 5 (Lgr5), thepolycomb complex protein Bmi1 and the homeodomain protein

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Hopx (Barker et al., 2007; Sangiorgi and Capecchi, 2008; Takedaet al., 2011). These markers have been associated with differentsubpopulations of ISCs (Fig. 3B). Lgr5 expression is enriched in‘crypt base columnar cells’, which lie interspersed between large

mature secretary cells known as Paneth cells (Barker et al., 2007).By contrast, Bmi1 is morewidely expressed in the crypt base region,with a peak of expression around the boundary of the Paneth cellcompartment (row+4 from the base of the crypt), whereas Hopx ismore tightly expressed in the same region (Muñoz et al., 2012).

Although these studies confirm that the ISC compartmentcontains cells expressing Lgr5, Bmi1 and/or Hopx, suchqualitative studies cannot define the range, potential short-termheterogeneity and fate behaviour of ISCs. Once again, long-termlineage-tracing studies, allied with short-term in vivo live imaging,have provided the means to address the identity and functionalproperties of the ISC compartment. The first of these studies, a long-term lineage-tracing investigation based on the inducible geneticlabelling of intestinal cells using a Cre-loxP recombinase systemunder the control of a ubiquitous promoter, showed that, in commonwith GSCs, ISCs follow a pattern of population asymmetric self-renewal (as evidenced by scaling behaviour of the clone sizedistribution) in which ISC loss through differentiation is perfectlycompensated for by the duplication of a neighbouring ISC (Lopez-Garcia et al., 2010). Through this process of stochastic ISC loss andreplacement, stem cell-derived clones undergo neutral driftdynamics, expanding or contracting around the crypt base untilindividual clones become lost, or the crypt becomes monoclonal.

Although this study provided insight into the functional behaviourof the ISC compartment, by focusing on medium-term (weeks) andlong-term (months to 1 year) clonal dynamics, the size, molecularidentity and short-term potential of the ISC compartment could notbe resolved. However, subsequent pulse-chase lineage-tracingstudies based on Lgr5 expression (Snippert et al., 2010), combinedwith studies of the colony-forming efficiency of Lgr5-expressingcells co-cultured with Paneth cells (Sato et al., 2009), led to theconjecture that stem cell competence may be linked to Wnt factors,which signal through Lgr5 (de Lau et al., 2011). Thus, through‘neutral’ competition for Paneth cell contact following cell division,ISCs become displaced from the niche environment and enter into adifferentiation pathway (Fig. 3C).

Although, in principle, the short-term potency of crypt baseprogenitors can be assessed through the use of targeted promoters,difficulties associated with the toxicity and delayed action of the Crerecombinase, effects of the inducing agent and the slow acquisitionof fluorescent reporters make a definitive assessment problematic.Instead, to resolve potential heterogeneity of the stem cellcompartment, medium- and long-term lineage-tracing assays werecombined with short-term in vivo live imaging of clonally labelledtissue (Ritsma et al., 2014). By following the fate of marked Lgr5-expressing cells and their differentiating progeny over several daysof time-lapse imaging, van Rheenen and colleagues showed thatcells positioned at the base of the crypt (rows 0 to +2) experience asurvival advantage over cells positioned near the border of thePaneth cell niche (rows +3 to +4). Yet, through the reversibletransfer of cells between the border and base regions, theheterogeneous population of ISCs functions long-term as a singleequipotent stem cell pool (compare with Fig. 1C) (Lopez-Garciaet al., 2010; Kozar et al., 2013). Whether the short-term potency ofISCs correlates with the expression of the putative stem cell markersremains an intriguing unresolved issue.

Together, these findings highlight the fact that, despite obviousdifferences in anatomy and cellular organization, the dynamics andfate behaviour of GSCs and ISCs, as well as the means throughwhich they were elucidated, show striking and unexpected parallels.In both cases, the stem cell compartment is heterogeneous, withcells transferring reversibly between ‘states’ that are temporarily

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Fig. 3. Stem cell dynamics during intestinal maintenance. (A) Schematicshowing the cellular organization of the mammalian small intestine. In adults,stem cells at the intestinal crypt base exhibit multi-lineage potential, givingrise to transit-amplifying cell progeny, which migrate along the walls of thecrypt and differentiate into functional secretory and absorptive cell types. (B)On the basis of genetic lineage-tracing assays, the intestinal stem cellcompartment has been associated with several molecular markers, includingLgr5, Bmi1 and Hopx, that are expressed heterogeneously within the crypt.(C) Time-lapse in vivo clonal data depicting the process of dynamicheterogeneity. Upper panels: following genetic labelling, a clone marked byRFP containing three Lgr5-positive cells (GFP) at the base of the nicheexpands over the next 3 days to occupy both border and niche base regions.Lower panels: a clone containing two Lgr5-positive cells all at the nicheborder expands to occupy both border and base regions. Reproduced, withpermission, from Ritsma et al. (2014). Through this process of loss andreplacement, stem cell-derived clones follow a quasi one-dimensional patternof neutral drift in which their chance extinction is perfectly compensated forby the expansion of neighbours around the collar of the crypt, leading toscaling of the clone size distribution.

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biased or ‘primed’ for proliferation or differentiation. Yet, once aclone comprising an individually labelled stem cell and its progenyreflects the composition of the heterogeneous stem cell pool(a situation that will prevail on time scales comparable with the timeof transfer between different primed states), the subsequent clonalevolution will become statistically indistinguishable from that of aneffective single equipotent stem cell pool (Fig. 1C). In this scenario,quantitative clonal analyses of both the germline and intestine reveala process of stochastic stem cell loss and replacement that leads toneutral drift dynamics in which chance clonal contraction and loss iscompensated for by the expansion of neighbouring clones (Kleinand Simons, 2011).

Hair follicle cyclingWe have emphasized the functional similarities of stem cellmaintenance in the germline and intestine. But to what extentdoes their behaviour provide insight into other cycling tissue types?As mentioned above, quantitative clonal analyses based on geneticlineage-tracing approaches have provided evidence that populationasymmetric stem cell self-renewal may be an ubiquitous feature ofadult tissue maintenance, at least in actively cycling epithelialtissues (Klein and Simons, 2011; Simons andClevers, 2011). In eachdocumented case in which quantitative data on long-term clonalevolution have been available, their analysis is seen to be consistentwith the steady turnover of an equipotent stem cell population.However, as illustrated by the examples of the mammalian testis andintestinal crypt, long-term steady-state behaviour may mask thepresence of short-term dynamic heterogeneity and fate priming ofthe stem cell pool.In this context, the mammalian hair follicle provides an

interesting case study. The hair follicle is unusual as a cyclingtissue because it is not generated at a constant steady rate, butundergoes periodic bouts of regression and regeneration throughoutadult life. On the basis of label-retaining assays and lineage-tracingstudies using targeted promoters (Tumbar et al., 2004; Cotsareliset al., 1990), stem cell identity has been localized to a permanentand discrete region of the hair follicle known as the bulge (Fig. 4A).Otherwise dormant stem cells residing in the bulge regionsporadically enter into the cell cycle in response to signalsderived from the base of the niche, and give rise to progeny thatrepopulate the hair follicle. Alongside putative stem cell markerssuch as keratin 15 and the transcription factor Sox9, which areexpressed throughout the bulge region, other markers are expressedheterogeneously, such as the hematopoietic progenitor cell antigenCD34, which is expressed more strongly in the distal region whileLgr5 is enriched proximally (Rompolas and Greco, 2014) (Fig. 4B).To trace the dynamics of hair follicle stem cells during the phase of

regeneration, the Greco lab has recently employed a novel two-photon in vivo live-imaging approach, allowing deep penetrationinto the tissue (Rompolas et al., 2012).When combined with geneticlineage tracing, thismethod has enabled individual stem cell lineagesto be followed from their exact place of origin throughout the processof regeneration (Rompolas et al., 2013). This study found that stemcells located in the upper half of the hair follicle niche were morelikely to remain quiescent or to proliferate without committing to aspecific fate (Fig. 4A). By contrast, stem cells situated in the lowerbulge region were more likely to proliferate in response to activatingstimuli from the niche base, undergoing limited amplification beforedifferentiating. These observations suggest that, in commonwith theintestinal crypt, the location of a stem cell within the niche at theonset of a new regeneration cycle dictates its fate during the cycle(Rompolas et al., 2013).Whether stem cells in the lower bulge region

continue to harbour long-term self-renewal potential, as do stemcells at the niche border of crypt, or whether they have irreversiblyentered a differentiation pathway remains unclear. However, theflexibility and regenerative capacity of the progenitor populationshave been tested under injury conditions.

Using laser-induced cell ablation to specifically remove eitherthe bulge stem cells or the hair germ (stem cell progeny) at theonset of hair growth (Rompolas et al., 2013), further studies bythe Greco lab showed that, remarkably, in both cases the hairfollicle niche recovered the lost cell population, restored itsanatomical features and proceeded normally through the hair cycle(Fig. 4C). Differentiating hair follicle cells can thus regain stemcell competence in response to injury. Surprisingly, distantepithelial cells located above the bulge were also observed tobecome proliferative, and some descended rapidly into the niche(Fig. 4C). By limiting genetic labelling to epithelial cells outsideof the niche, it was confirmed that loss of the stem cell pool due toinjury can mobilize cells that do not normally participate in hairregeneration to repopulate the niche and sustain hair growth.Indeed, once these cells entered the niche, they displayedcharacteristics consistent with the fate of endogenous stem cellsin their new locations.

A

Epidermis

Stem cell niche

Hair shaft

B

QuiescenceProliferation without lineage commitment

Bulgeablated

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Hair germablated

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Hair germ

Sebaceousgland

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atin

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Fig. 4. Stem cell dynamics in the hair follicle. (A) Schematic of a mouse hairfollicle during the resting phase of the hair cycle. Stem cells reside in the bulgeand hair germ, whereas other epithelial cell populations occupy thecompartments located above the bulge. Clones derived from stem cellssituated in different parts of the niche have been observed to follow differentfates, with cells in the hair germ primed for differentiation, while cells in theupper part of the bulge aremostly quiescent. (B) Hair growth and stem cell self-renewal in the bulge is driven by a heterogenous progenitor population. Whilekeratin 15 and Sox9 are expressed throughout the compartment, CD34 isexpressed more strongly towards the upper end of the bulge, and Lgr5 isenriched proximally. (C) Following ablation of either the hair germ (left) or thebulge region (centre), the entire niche is repopulated through proliferation andmigration of the remaining stem cells. In response to ablation of the entire stemcell niche (right), non-hair epithelial cells migrate downwards to regenerate thestem cell compartment.

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The hair follicle study therefore provides evidence for both stemcell heterogeneity and flexibility under conditions of stress.Although, in this case, the recruitment of differentiating cells tothe stem cell niche has not yet been confirmed under normalphysiological conditions, the conversion of epithelial cells to bulgestem cells in response to crisis suggests that cells seeminglycommitted to a differentiation lineage are able to ‘reprogramme’ andassume long-term stem cell fate identity. Future studies will revealwhether stem cell heterogeneity and the flexibility of differentiatingprogeny represent a more ubiquitous feature of this and other adultstem cell populations.

Questioning stem cell identityThe emergence of stochastic stem cell fate behaviour, stem cellheterogeneity and priming in tissue maintenance questions ourunderstanding of adult stem cell identity and the definition ofcommitment. Even within an equipotent stem cell population,although all cells retain long-term self-renewal potential, chancestem cell loss and replacement mean that only a diminishingminority of clones actually persist long term. Yet it would make nosense to segregate cells prospectively according to their eventuallong-term fate. Similarly, in a dynamic heterogeneous stem cellpopulation, the long-term survival potential of individual cells mayitself vary over time. For example, in the intestinal crypt, an ISCpositioned at the border of the niche has a long-term survivalprobability that is several times smaller than an ISC positionedtowards the crypt base (Ritsma et al., 2014). However, if the borderISC or its ISC progeny transfers to the base region, the survivalprobability is proportionately readjusted. It would therefore seeminappropriate to designate only the base population as a stem celltype; instead, the entire compartment functions as just oneheterogeneous population.Furthermore, in defining stem cell behaviour, much of the

discussion in the literature has centred on the mode of division, andin particular on whether the fate outcome is symmetric orasymmetric (Watt and Hogan, 2000; Morrison and Kimble, 2006;Fuchs and Horsley, 2011; Watt and Huck, 2013). However, thisdesignation is useful only if fate behaviour is defined shortly priorto, or on, division. If fate outcome is linked to the proximity ofdaughter cells to the niche following division, as implicated in thegermline and intestine, the division mode may not be the primarydeterminant of daughter cell fate. In the search for a mechanism, itwould therefore be expedient to focus more on local environmentalcues that instruct fate behaviour.The potential for ambiguity in the definition of an adult stem cell

does not end there. Alongside the innate regenerative capacity of theendogenous stem cell population, evidence from regenerativestudies of hair follicles shows that cells normally committed todifferentiation in steady state are able to repopulate the stem cellcompartment and re-acquire long-term self-renewal potential inresponse to injury or stress. Indeed, such behaviour is far fromunique (Tetteh et al., 2015). Studies have shown that, following theablation of spermatogonia through busulphan administration, therecovery of the GSC compartment involves the large-scale transferof Ngn3-expressing cells to the GFRα1-expressing stem cellcompartment, as well as the expansion of the surviving GFRα1-expressing cell population (Hara et al., 2014; Nakagawa et al.,2010). Similarly, the targeted genetic ablation of Lgr5-expressingcells following exposure to diphtheria toxin leads to the transfer ofdifferentiating cells back into the stem cell compartment, and theregeneration of the stem cell pool (Tian et al., 2011). Furthermore,independent studies show that cells positive for the Notch ligand

Dll1, as well as quiescent Lgr5-expressing cells, which are bothlargely committed to differentiation into the secretory cell lineage inconditions of normal homeostasis, can re-establish multipotencyand contribute to long-term self-renewal following the ablation ofISCs through radiation damage (van Es et al., 2012; Buczacki et al.,2013). Finally, the regeneration of trachea following the geneticablation of basal cells (which include the resident stem cellpopulation) involves the de-differentiation of differentiated cellsknown as club cells (Tata et al., 2013). Together, these resultssuggest that the entry of cells into a differentiation pathway may notinvolve an abrupt ‘binary’ decision but may occur progressively,with cells retaining stem cell potential ready to be mobilized underappropriate cues. Such flexibility may strengthen the resilience oftissues to crisis or injury, enabling the ensemble of differentiatingprogeny to function as a ‘reserve’ stem cell population (comparewith Potten and Loeffler, 1990).

Taken together, these studies suggest that the fate potential of stemand progenitor cells may not be organized into a strict classical ‘one-way’ proliferative hierarchy involving functionally discrete cellpopulations. Rather, the arrangement of cell types may be moreaccurately represented as a continuum, in which both theproliferative and fate potential becomes gradually restricted. Insuch cases, transitions between different cell ‘states’ may occurreversibly even under physiological conditions, in response to niche-dependent factors, and can be promoted through injury or stress.

Stem cell-niche interactionsThe intestinal crypt and hair follicle bulge highlight the crucial roleplayed by interactions with the local microenvironment in definingthe proliferative capacity and fate behaviour of stem cells. In theintestinal crypt, the balance between stem cell loss and replacement,and size of the stem cell pool, are regulated by exposure to Panethcells as well as to factors from the adjacent stromal tissue. Throughcompetition for limited niche access, ISCs are able to self-renew,and they can recover their number during the regeneration of tissuefollowing the partial ablation of the stem cell compartment byradiation damage or other forms of injury (Tian et al., 2011;Metcalfe et al., 2014).

A similar strategy to regulate the size of the stem cell compartmentmayoperate in the germline. Although studies have not yet identifieda localized niche structure in themammalian testis, the association ofundifferentiated spermatogonia with the vasculature (Yoshida et al.,2007) suggests that intratubular domains may play host to a somaticcell type (or types) that creates a niche environment to support GSCs,much as Paneth cells do in crypts. Competition of spermatogonia foraccess to these limited niche domains may provide a simple androbust mechanism to regulate both the balance between syncytialfragmentation and differentiation, and the total size of the stem cellpool. Furthermore, if GFRα1 expression is linked to proximity to theniche, then the fragmentation of GFRα1+ syncytia may displacetheir neighbours from niche-maintaining sites, leading to loss ofGFRα1 expression and the upregulation of differentiation markerssuch as Ngn3.

In contrast to the mammalian testis, the effects of localized nichefactors on stem cell regulation have been defined in the Drosophilaovary and testis. In these cases, stem cell identity is traditionallythought to be restricted to the population of GSCs that directlycontact a central hub of stromal cells (Hardy et al., 1979; Kiger et al.,2000). These cells remain closely associated with their niche duringthe cell cycle through cadherin-mediated cell adhesion (Song andXie, 2002). Through a regulated process of spindle orientation, GSCdivision leads to a predominantly asymmetric fate outcome in which

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one daughter cell remains anchored to the hub and retains GSCidentity, while another is displaced from the niche and enters into adifferentiation pathway (Hardy et al., 1979). However, static lineage-tracing studies and ex vivo live imaging in the Drosophila testis andovary show that, even under normal physiological conditions,sporadic stem cell loss from the hub may be compensated for by thesymmetric duplication of neighbouring stem cells, and vice versa,leading to neutral drift dynamics of the clonal population (Sheng andMatunis, 2011; Kronen et al., 2014). Whether these rare events areassociated with chance loss or active displacement of ‘inferior’GSCs, or whether infrequent symmetric divisions are a routine partof the normal program of homeostatic turnover remains unclear. Inthis context, it is interesting to note that the second resident stem cellpopulation in Drosophila testis, the somatic cyst stem cells (CySC)that give rise to the cyst cells ensheathing developing germ, undergoloss and replacement at a much higher rate (Amoyel et al., 2014).Alongside the ability ofDrosophilaGSCs to undergo symmetric as

well as asymmetric cell divisions in normal homeostasis,differentiating germ cells also retain the ability to re-establishcontact with the hub and re-acquire stem cell function in the courseof regeneration following the depletion of the GSC pool by proteinstarvation or genetic ablation (Brawley and Matunis, 2004; Kai andSpradling, 2004). Although such behaviour has been traditionallyassociated with a process of de-differentiation, and is distinct fromGSC renewal through symmetric cell division, it is interesting to notethat theprocess of spindle orientation anddivision asymmetrymaynotbe essential for germline maintenance. In particular, studies based onthe targeted depletion of stat inGSCs, which leads to their detachmentfrom the hub, show that contact with CySCs alone is sufficient tomaintain GSC self-renewal and spermatogenesis (Leatherman andDinardo, 2010). Indeed, under these conditions, the maintenance ofDrosophila germ line may in fact parallel the process of dynamicheterogeneity that characterizes the mammalian system.Dynamic interactions with the niche may thus serve both to

constrain stem cell identity under physiological conditions andorchestrate the regeneration of the stem cell compartment followinginjury. Future studies might address the extent to which recruitmentto the stem cell pool upon injury is a reflection of underlying cellfate heterogeneity, or instead is the consequence of active cell fatereprogramming following catastrophic stem cell loss.

The role of stem cell quiescenceAlthough lineage-tracing assays provide a powerful read-out of thebehaviour and dynamics of cycling cells, they are notoriouslyinsensitive to the existence and potential function of long-termquiescent (slow-cycling) or dormant cell populations. Indeed, boththe germline and intestinal crypt have been associated with aquiescent progenitor cell population. In humans and other primates,detailed analyses of fixed specimens have identified asubpopulation of singly isolated spermatogonia, termed Adark onthe grounds of their histological appearance (de Rooij and Russell,2000). It has been speculated that this minority cell population mayplay a special role in the long-term maintenance of tissue,supporting the more rapidly cycling but transient spermatogonialcell population (Hermann et al., 2010). Similarly, studies of themouse intestinal crypt have identified a population of quiescent cellsmarked by the expression of telomerase reverse transcriptase (Tert)or Lgr5 (Muñoz et al., 2012; Montgomery et al., 2011).It is difficult to identify the potential function and significance of

theseminority quiescent cell populations, particularly in tissues such asthe germline and intestinal crypt, where active cycling cells are seen tomaintain life-long self-renewal (at least in mice). As shown by a recent

study of intestinal crypts, quiescence may not in itself be a signature ofstem cell function, at least under conditions of normal maintenance(Buczacki et al., 2013). However, in long-lived organisms, it may beadvantageous to hold a dedicated slow-cycling or dormant stem cellpopulation in reserve so that it may ‘drip-feed’ the cycling stem cellpool to compensate for progressive chance loss or ageing.

Alternatively, the reversible transfer of stem cells between anactive and quiescent state under physiological conditions (Fig. 1C),itself a manifestation of dynamic heterogeneity, may provide arobust mechanism to maintain a stem cell pool such that the overallturnover rate of the tissue is steady but slow. Equally, the sporadicentry of stem cells into a quiescent or dormant state in a cyclingtissue may provide an insurance mechanism to shield the widerpopulation from demands experienced by actively cycling cells, andthereby protect the long-term integrity of the tissue. Such behaviourwould mirror the strategy of phenotypic switching observed inbacterial populations (Balaban et al., 2004).

ConclusionsTaken together, these observations highlight the requirement todevelop an extended definition of stem cell identity, one thatadequately captures the heterogeneity of tissue stem cells and theflexibility of their progeny. In the studies discussed above, stem cellidentity consistently emerges not as the property of a discretepopulation, but as a functional state that a wider population of cellsmayenter, exit and re-enter according to the demands of the tissue.Allcells belonging to this wider population therefore have the capacityfor long-term self-renewal, but their proliferative potential at anygiven time may depend on their precise location, on signals from theniche environment, and on other extrinsic and intrinsic factors.

If stem cell identity is indeed a state that is accessible to a widepopulation of progenitors, it becomes crucial to determine howrecruitment to and exit from the stem cell pool is regulated at themolecular level. The dependence of self-renewal potential on theposition of a cell within the niche that was observed in intestine andhair follicle suggests that spatially localized signals may play animportant role in determining the state of progenitors; these couldresult from direct interactions with surrounding cells of the same ordifferent types, extracellular matrix components or solubleparacrine mediators (Jones and Wagers, 2008). Non-local signals,includingmetabolic and endocrine factors, may further contribute toaligning overall niche stem cell activity with the requirements ofdifferent tissues (Scadden, 2006).

In maintaining the stem cell pool at a constant size, it is not clearwhether the aberrant loss of a stem cell triggers recruitment of adifferentiating progenitor back to the niche, or whether stem cells exitthe compartment in response to the fate reversal ofdifferentiatingcells.Although the mechanisms that govern regeneration after injury maydiffer substantially from those operative in homeostasis, the examplesabove, however, suggest that stem cell recruitment may occur inresponse to stem cell loss from the niche. As repopulation of the nichehas been observed even following the ablation of the entire stem cellpool, it is conceivable that stem cell identity can be initiated by factorsderived from supporting cells or the extracellular matrix, rather thanfrom other stem cells. The physical structure of the niche, which isindependently maintained by the extracellular matrix, associatedvasculature and supporting cells, may therefore play amore active rolein regulating stem and progenitor cell fate than previously appreciated.

The observation that the recruitment of differentiating progenitorsback to the stem cell pool occurs under normal physiologicalconditions opens up an intriguing new possibility for the mechanismof ageing in tissues with high cellular turnover. Although the

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accumulation of mutations in nuclear and mitochondrial DNA isconsidered to be the most fundamental and irreversible cause ofageing, declining homeostasis in cycling tissues has also beenassociatedwith changes in the numbers or properties of stemcells andtheir niches (Liu and Rando, 2011; Jones and Rando, 2011). Inparticular, tissue ageing may be accompanied by a gradual loss offunctional stem cells, which has been thought to result from increasedrates of stem cell death, quiescence or differentiation (Nijnik et al.,2007; Inomata et al., 2009). However, paradigms that could explainhow the global process of ageing leads to the gradual loss of only asmall fraction of stem cells at any one time have so far been lacking.Following the discoveries of heterogeneity within stem cellcompartments and the ongoing interconversion between stem cellsand their differentiating progeny, it may be that ageing is a result not(only) of an increase in the active loss of stem cells, but (also) of adecrease in the recruitment of differentiating cells back to the niche.This would be consistent with a number of studies showing that stemcells from ageing animals can continue to function normally whenmaintained in a young niche or provided with young systemic factors(Katsimpardi et al., 2014; Ryu et al., 2006).Over the past few years, the co-existence of distinct progenitor cell

behaviours has been reported across a wide range of tissues, oftenthrough genetic lineage-tracing approaches (Challen et al., 2010; Luet al., 2012; Van Keymeulen et al., 2011). It remains an unresolvedissue whether these observations reflect an underlying heterogeneitywithin a single stem cell compartment, andwhether the continual inter-conversion of long-term stem cells and their differentiating progenyoccurs in these tissues under physiological conditions. Static lineagetracing alone can provide important clues to the degree of progenitorcell plasticity in some tissues, but owing to the lack of tools for thetargeted labelling of specific subpopulations of cells, it is frequentlyimpossible to identify unambiguously the source of labelled clones. Tounravel stem cell heterogeneity, lineage hierarchies and the de-differentiation capacity of differentiating cells, in vivo live-imagingapproaches will therefore be indispensable. In resolving the biologicalsignificance of heterogeneity within the stem cell compartment, weexpect that stem cell diversity and lineage plasticity will emerge asubiquitous features of adult tissue stem cell populations.

AcknowledgementsWe are grateful to Cedric Blanpain, Hans Clevers, Tariq Enver, Valentina Greco,Kenshiro Hara, Allon Klein, Anna Philpott, Jacco van Rheenen, ShahragimTajbakhsh, Doug Winton, Shosei Yoshida and all members of the Simons group forilluminating discussions.

Competing interestsThe authors declare no competing or financial interests.

FundingB.D.S. acknowledges the financial support of the Wellcome Trust [098357/Z/12/Z]as well as core grants from the Wellcome Trust [092096] and Cancer Research UK[C6946/A14492].

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