(2012) 31, 3009–3022 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc REVIEW Lgr5 and Lgr6 as markers to study adult roles in self-renewal and cancer

M Leushacke and N Barker

A*STAR Institute of Medical Biology, 8A Biomedical Grove, 06-06 Immunos, Singapore

The extended longevity of many imposes the imposes a strict requirement for daily self-renewal need for an effective tissue renewal capacity within the (Barker et al., 2008). Limited reservoirs of stem cells vital organs to maintain optimal function. Resident adult residing in anatomically-defined structures (the niche) stem cells are instrumental in delivering this renewal are considered instrumental in achieving adult tissue capacity by virtue of their characteristic ability to homeostasis (Barker et al., 2010a). Adult stem cells are maintain themselves long-term as a population (self- endowed with two essential characteristics that equip renewal), whilst also supplying all functional cell-lineages them with the ability to effect tissue homeostasis of the respective tissue (multipotency). The homeostatic throughout life. First, they ensure their continued activity of these adult stem cell reservoirs is tailored to existence within a tissue by generating more undiffer- meet the specific renewal requirements of individual entiated stem cells (‘self-renewal’) and second, they tissues through a combination of intrinsic genetic pro- produce all functionally differentiated cell lineages of gramming and local cues delivered from the surrounding the respective tissue (‘tissue-renewal’). Adult stem cells environment (the niche). Considerable phenotypic diver- are traditionally believed to divide slowly (referred to as sity therefore exists between adult stem cell populations in ‘quiescence’), a feature considered to protect the stem different organs, making it a considerable challenge to cell pool from premature exhaustion and DNA damage identify broadly applicable markers that facilitate their that may otherwise occur during frequent DNA identification and characterization. However, the 7- replication (Orford and Scadden, 2008). However, the transmembrane receptor, Lgr5 has recently gained notion that quiescence is a defining stem cell trait has prominence as a marker of Wnt-regulated adult stem cell recently been challenged by the identification of actively populations in the hair-follicle, intestine and . cycling adult stem cell populations in mammals. This A closely-related , Lgr6 marks adult stem cells raises the possibility that both proliferating stem cell responsible for fueling the renewal of the sebaceous gland reservoirs responsible for effecting daily tissue renewal and skin. The discovery of these markers has already and more quiescent ‘reserve’ populations may coexist in greatly improved our understanding of stem cell biology in some organs (Li and Clevers, 2010). Recent advances in these rapidly renewing tissues and has major implications IPS technologies highlight the influence of a few key for the identification and isolation of adult stem intrinsic genetic programs in defining stemness, but cell populations for exploitation of their regenerative extracellular cues derived from the local niche are medicine potential in the clinic. equally important in tailoring stem cell activities to Oncogene (2012) 31, 3009–3022; doi:10.1038/onc.2011.479; meet the requirements of their host tissue (Li and Xie, published online 17 October 2011 2005). The balance between self-renewal and differentia- tion is tightly controlled by a complex interplay of Keywords: Lgr5; Lgr6; adult stem cell; epithelial evolutionary conserved signaling pathways, including renewal; cancer Wnt, Notch, bone morphogenetic protein and Hedge- hog. The adult stem cells typically give rise to vigorously proliferating progeny with a limited lifespan, often referred to as transit-amplifying (TA) cells. Rapid cell Adult stem cells and tissue homeostasis division within this progenitor compartment serves to expand the cell pool available for supplying the various Cell turnover rates vary enormously between adult functional lineages of the host tissue. The repertoire of organs, with well-protected tissues such as the brain and terminally differentiated cells derived from the TA skeletal muscle exhibiting minimal renewal during our compartment exhibits a finite lifespan within their host lifetime (Fuchs, 2009). In contrast, the harsh operating tissue, to ensure optimal functionality. environment of the intestine, stomach and skin mucosa Identifying the adult stem cells, which may represent o1% of the total cell repertoire of a tissue, has proven Correspondence: Dr N Barker, A*STAR Institute of Medical Biology, challenging. In the absence of specific, well-validated 8A Biomedical Grove, 06-06 Immunos, Singapore 138648, Singapore. E-mail: [email protected] biomarkers, scientists have traditionally relied heavily Received 30 July 2011; revised 5 September 2011; accepted 13 September on indirect methodologies to reveal stem cell candidates. 2011; published online 17 October 2011 For example, the presumptive obligate quiescence of Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3010 adult stem cells is considered to endow this population project deep into the underlying submucosa (Figure 1a). with a characteristic ability to retain DNA labels long- This highly stereotypical organization effectively max- term in classic ‘pulse-chase’ experiments. However, imizes the absorptive potential of the small intestine. DNA label-retention by long-lived classes of terminally The contrasting epithelial architecture of the large differentiated cells in some tissues (Kiel et al., 2007) and intestine, visible as a flat surface interspersed with deep the rapid loss of DNA labels in any proliferating stem crypts, reflects its major role in compaction rather than cell populations highlights the danger of relying solely absorption (Barker et al., 2008). Constant mechanical on such indirect techniques. abrasion and environmental assault from ingested Transplantation has long been considered the ‘gold chemicals and pathogens takes a heavy toll on this standard’ for evaluating candidate stem cell populations epithelial lining, imposing a strict requirement for daily for which potential biomarkers are available (Snippert self-renewal. A massive cell production effort originat- and Clevers, 2011). However, phenotypic changes ing from small populations of adult stem cells sheltered bestowed on candidate cell populations as a conse- within the crypts ensures the daily replacement of some quence of their forced isolation from native, instructive 300 million cells dying on the surface epithelium (Barker niche signals may well artificially skew the cell behavior et al., 2008). Stem cell progeny born at the crypt base in this assay. Any observed stem cell capacity in this undergo rapid expansion by active proliferation to assay might therefore be more accurately referred to as generate the TA compartment, which populates the stemness ‘potential’ rather than true physiological majority of the crypt. These proliferating TA cells stemness when the cell is present in its undisturbed migrate upwards to the crypt-villus junction over a natural environment. An extreme example of how the period of 2 days, responding to various signaling cues microenvironment may influence the cellular behavior en-route by committing towards various functional cell was presented recently, when thymic epithelial cells were lineages. These cells subsequently exit the crypts onto shown to irreversibly adopt the fate of adult hair follicle the surface epithelium as mature enterocytes, goblet cells (HF) stem cells following transplantation into an and enteroendocrine cells, where they perform their inductive skin microenvironment (Bonfanti et al., 2010). specialized functions for a further 3–5 days before In vivo lineage tracing is a relatively recent technique undergoing programmed cell death and ultimately being designed to evaluate stem cell activity of candidate discarded into the intestinal lumen (Marshman et al., populations residing in their natural environment. This 2002; Barker et al., 2008; van der Flier and Clevers, involves the targeted introduction of a permanent 2009a). Paneth cells migrate in the opposite direction to genetic mark into the candidate population, allowing occupy the crypt base, a process driven by strong the visualization of both the modified stem cell and its repulsive forces created between high concentrations of descendents over time. Despite the fact that this EphB3 receptors on the Paneth cell surface and their technique is strictly dependent on the availability of corresponding ephrin-B1 ligands on the TA compart- single markers for the candidate population, it requires ment (Batlle et al., 2002). Up to 10 associated crypts are the generation of new genetically engineered mouse lines required to supply sufficient cells to counterbalance the and it cannot be applied for use in evaluating human cell death on a single villus in the small intestine. In the stem cell populations, it is undoubtedly the most small intestine, both the Paneth cells, which have a accurate in vivo demonstration of stem cell identity lifespan of around 3–7 weeks, and the immortal stem currently available for the mouse. cells that cohabit the crypt base, are excluded from this In vivo lineage tracing has been instrumental in weekly renewal cycle (Barker et al., 2010a). defining the 7-transmembrane receptors Lgr5 and Lgr6 Epithelial homeostasis in the adult intestine is as novel markers of multiple adult stem cell populations. orchestrated by several signaling pathways, including Here, we review the discovery and application of Lgr5 Wnt, Notch, bone morphogenetic protein and Hedge- and Lgr6 as markers to study adult stem cell roles in hog (for a review, see Sancho et al., 2003). The critical epithelial renewal and cancer in the hair-follicle, role of Wnt signaling was established when genetic gastrointestinal tract and skin. In addition, we discuss ablation of this pathway in mice was found to block recent developments on the functional importance of epithelial renewal in the small intestine (Korinek et al., Lgr5 homologs for intestinal stem cell maintenance 1998; Pinto et al., 2003; Ireland et al., 2004). Conversely, in vivo. stimulation of Wnt pathway activity by the administra- tion of R-spondin growth factors induces a massive increase in epithelial proliferation throughout the Trawling the Wnt target signature for intestinal stem intestinal tract (Kim et al., 2005). Deregulation of this cell markers signaling pathway in vivo has also been implicated in driving human colon cancer, highlighting its importance The mammalian intestinal tract can be sub-divided into in regulating epithelial homeostasis (Korinek et al., two major segments: the small intestine and the large 1997; Sancho et al., 2004). The canonical Wnt pathway intestine (colon), which are both lined by a sheet of exerts its effects through a complex intracellular cascade simple columnar epithelium. The epithelial layer of the originating at the cell surface and culminating in the small intestine is folded into millions of repeat units accumulation of b-catenin in the nucleus. The resulting comprising finger-like projections called villi and asso- assembly of potent Tcf/b-catenin transcription factor ciated pockets known as crypts of Lieberku¨hn that complexes drives a transcriptional program that is

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3011

Figure 1 Lgr5 þ adult stem cells contribute to epithelial tissue homeostasis in the small intestine. (a) Architecture of the small intestinal epithelium. Lgr5 marks proliferating Crypt Base Columnar (CBC) adult stem cells (indicated in red) that reside at the crypt base. (b) Flowchart of Lgr5 þ adult stem cell-driven tissue homeostasis in the intestine. Lgr5 þ adult stem cells generate all differentiated cell types of the intestinal epithelium, whilst self-renewing to maintain stem cell homeostasis. (c) Composition of the small intestinal stem cell niche. Paneth cells provide a cocktail of essential niche factors that act locally through surface receptors on neighboring Lgr5 þ stem cells. (d) Lgr5 homologs amplify canonical Wnt signals to maintain optimal stem cell function in vivo. Wnt ligands secreted by Paneth cells interact with Fzd/LRP receptor complexes on Lgr5 þ stem cells to maintain basal levels of Wnt signaling. Circulating R- spondins interact with Lgr5 homologs to amplify canonical Wnt signals on the stem cell compartment. instrumental in maintaining the intestinal stem cell pool, their specific expression pattern within the crypts: (i) TA as well as in promoting proliferation and migration compartment-enriched , typified by cell-cycle within the TA compartment and in defining cell fate regulators, such as CD44, c-Myb and c-Myc, (ii) genes choices within the crypts (Batlle et al., 2002; van de exclusively expressed on the Paneth cell compartment, Wetering et al., 2002; van Es et al., 2005). The identity of such as cryptdins (iii) genes demonstrating a more the transcriptional program was revealed by expression restricted expression pattern at the crypt base, repre- profiling of colon cancer cell lines exhibiting constitu- senting potential stem cell markers. tively active Wnt signaling (Korinek et al., 1997; van de There is a general consensus amongst the gut research Wetering et al., 2002). A core set of approximately 80 community that intestinal crypts do harbor limited target genes, referred to as the intestinal Wnt signature, reservoirs of adult stem cells, but the paucity of well- was found to be robustly expressed at various locations validated markers for this population has ensured within the murine small intestinal crypts (Van der Flier decades of intense debate over their precise identity. In et al., 2007). Target genes within this Wnt signature 2007, Hans Clevers and colleagues screened their panel could be broadly subdivided into 3 classes according to of intestinal Wnt target genes for candidate stem

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3012 cell markers, employing restricted expression at the robust expression of multiple Wnt target genes, high- crypt base as a selection criterium. This revealed the lighting the presence of active Wnt signaling within the 7-transmembrane gene Lgr5/GPR49, a presumed GPCR, intestinal stem cell compartment (van der Flier et al., as a promising candidate. Lgr5 expression was restricted 2009b). These expression analyses also revealed other to a population of slender cells interspersed between the stem-cell markers, including the secreted molecule differentiated Paneth cells at the crypt base in the small OLFM4 and the Wnt target Ascl2. Ascl2 is one of the intestine and on scattered cells at the base of the colonic mammalian homologs of the Drosophila achaete-scute crypts (Barker et al., 2007). These cells, known as crypt gene complex, which encodes related basic helix-loop- base columnar (CBC) cells, were discovered back in the helix transcription factors involved in determining the early 1970s using detailed electron microscopy studies cell fate. Conditional Ascl2 gene ablation in the mouse (Cheng and Leblond, 1974a, b). Largely on the basis of small intestinal epithelium resulted in rapid ablation of morphological considerations, Bjerknes and Cheng ori- the Lgr5 þ stem cells, whilst misexpression in non-stem ginally formulated the ‘stem cell zone’ model, proposing cells resulted in de novo crypt formation on the villi (van the CBC cells to represent the adult intestinal stem cell der Flier et al., 2009b). Collectively, this indicated a population (Bjerknes and Cheng, 1981). However formal fundamental role for this transcription factor in the proof of this model awaited the discovery of a specific control of intestinal stem cell biology. marker for this cell population.

Are Lgr5 þ CBC cells the only intestinal stem cell Validation of Lgr5 as a bona fide intestinal stem cell reservoir? marker An alternative school of thought, known as the þ 4 An in vivo lineage-tracing approach was adopted to model, describes intestinal crypt cells at the þ 4 position directly assess the stem cell potential of Lgr5 þ CBC (relative to the crypt base and directly above the cells. To facilitate this, an EGFP-ires-CreERT2 expres- terminally differentiated Paneth cells) as displaying stem sion cassette was placed under the control of the cell characteristics. Cells at this position were identified endogenous Lgr5 promoter by targeted knock-in into as cycling (every 24 h), yet retain nucleoside analoges the Lgr5 locus. The resulting mouse line, dubbed Lgr5- incorporated during crypt neogenesis (Potten et al., EGFP-ires-CreERT2 (hereafter referred to as Lgr5KI) 1974). Such apparently incompatible observations were was validated as a faithful EGFP reporter of endogen- proposed to result from asymmetric segregation of old ous Lgr5 expression in all tissues examined, including (labeled) and new (unlabeled) DNA strands during the intestine, where EGFP þ CBC cells were readily regular þ 4 cell division. The labeled template strand is observed at the crypt base (Barker et al., 2007). The retained in the cell destined to remain as a stem cell, same EGFP þ cells also selectively expressed a Tamox- whereas the newly synthesized strand is passed onto the ifen-inducible Cre enzyme, which could be used short-lived daughter cell. This ‘immortal strand hypoth- to introduce permanent, heritable genetic marks into esis’, originally postulated by John Cairns, is considered this population when the Lgr5KI mice were bred to protect the stem cell genome from potentially with conditional reporter lines such as Rosa26RlacZ dangerous mutations that may have occurred during (Soriano, 1999). This approach facilitated the tracing DNA synthesis (Cairns, 1975). The intestinal þ 4 cells of CBC-derived lacZ þ progeny within the intestine were also characterized as being extremely sensitive to over time (Barker and Clevers, 2007). One day post- irradiation-induced apoptosis, a feature considered to induction, lacZ reporter gene activity was restricted to protect the putative stem cell compartment from low numbers of Lgr5 þ CBC cells at the crypt base accumulating heritable genetic damage (Potten, 1977). throughout the intestine and the colon. At later time In 2008, Capecchi and colleagues employed the same points, ribbons of lacZ þ cells extended from the CBC strategy as Barker et al., to assess the compartment to the villus tip in the small intestine and ‘stemness’ of Bmi1 þ cells in the intestinal crypt. This comprised cells of all lineages (Barker et al., 2007) was prompted by the observation that Bmi-1 reporter (Figure 1b). Similar observations were made for the expression in vivo was predominantly restricted to the colon, although the rate of Lgr5-derived progeny crypt location housing the putative þ 4 stem cells migration towards the surface epithelium was lower (Sangiorgi and Capecchi, 2008). Lineage-tracing analy- than in the small intestine (likely reflecting a lower rate sis identified the Bmi1 þ cells in the proximal small of epithelial renewal in this region). These multi-lineage intestine as self-renewing, multipotent stem cells con- lacZ þ clones persisted throughout adulthood, allowing tributing to epithelial renewal during normal tissue the authors to conclude that the Lgr5-expressing cells homeostasis (Sangiorgi and Capecchi, 2008). Targeted are multipotent, self-propagating adult intestinal stem ablation of the Bmi1 þ population also impaired cells (Barker et al., 2007). In contrast to the widely- epithelial renewal, consistent with a loss of stem cell accepted notion that adult stem cells are typically function. The localization of these Bmi1 þ stem cells has quiescent, the Lgr5 þ stem cells were found to be actively drawn obvious comparisons with the þ 4 compartment proliferating, generating progeny every 24 h on average described by Potten and colleagues, although their (Barker et al., 2007; Schepers et al., 2011). Gene behavior in label retention and radiation sensitivity profiling of FACS-sorted Lgr5-EGFP þ cells revealed assays has yet to be assessed. Curiously, this transgenic

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3013 mouse model documented Bmi1 expression only in the provided from specified intestinal cell types within proximal small intestine, prompting the authors to the epithelium or from underlying sub-epithelial myofi- speculate that other, Bmi1-, stem cell populations must broblasts. exist in other regions. However, Lgr5 þ stem cells sorted from these purportedly Bmi1þ regions of the intestine were found to express elevated levels of Bmi1 in an Defining the intestinal stem cell niche independent study. The intestine is notorious for silencing transgenes that are expressed early during the Transverse slices through the crypt base in the small development. This has led to suggestions that the Bmi1- intestine reveal a striking symmetry between Lgr5 þ Cre line may not faithfully report endogenous Bmi1 CBC cells and the terminally differentiated Paneth cells expression in vivo (van der Flier et al., 2009b). High- (Snippert et al., 2010b). The pool of 12–14 Lgr5 þ stem resolution in situ analyses of endogenous Bmi1 and Lgr5 cells is alternately distributed between the Paneth cells, expression in the intestine are therefore needed to which effectively maximizes the heterotypic surface formally ascertain whether Bmi1 and Lgr5 mark contact between these two populations (Snippert et al., independent stem cell populations in the intestine. 2010b). These observations led Sato and colleagues to Many other markers of the þ 4 and CBC small speculate that Paneth cell interactions with Lgr5 þ stem intestinal stem cell compartments have been proposed cells may be important for maintaining stem cell over the last decade, including Prominin1, mTert, function in addition to their traditional roles in the Musashi1, DcamKL1 and Sox9 (for comprehensive innate immune response and host defense. This was reviews see (Barker et al., 2010a; Lin and Barker, confirmed when co-culturing of FACS-sorted intestinal 2011; Zeki et al., 2011). stem cells and Paneth cells was found to markedly enhance stem cell maintenance and outgrowth in vitro (Sato et al., 2010). Microarray analyses revealed Lgr5 þ stem cells as a source of renewable intestinal the Paneth cell compartment to be a major source of epithelia in vitro secreted growth factors including Wnt & EGF, and Notch ligands that are essential for organoid growth In 2009, Sato and colleagues published an in vitro in vitro (Sato et al., 2010). In agreement with these culture system capable of generating pieces of self- observations, a dramatic reduction in Lgr5 þ stem cell renewing intestinal epithelia from isolated Lgr5 þ stem numbers was found to accompany the ablation of the cell populations. The architecture of these three-dimen- Paneth cell compartment in vivo (Sato et al., 2010). sional organotypic structures was remarkably similar to Collectively, this established the Paneth cells as being a that of the normal intestinal epithelium, comprising crucial epithelial niche component for the Lgr5 þ stem multiple crypts containing basal Lgr5 þ cells and a cells in the small intestinal epithelium (Figure 1c). proliferative TA population associated with villus It is currently unknown whether niche signals originat- domains harboring differentiated cell types of all ing from the underlying submucosa contribute to intestinal lineages (Sato et al., 2009). Surprisingly, maintenance of intestinal stem cell function in vivo. Lgr5 þ stem cell outgrowth in this organotypic culture The Lgr5 þ stem cell niche in the colon is less well system proceeded in the absence of physical mesench- characterized. Classical lyzozyme-secreting Paneth cells ymal niche components, implying that Lgr5 þ intestinal are only rarely observed in the proximal colon, stem cells can act independently of positional cues indicating that other cell types must be the source of provided by the underlying submucosa. Nonetheless, niche factors in vivo. Cells expressing CD24, a surface maintenance of intestinal is absolutely de- antigen present on Paneth cells in the small intestinal pendent on the presence of a defined cocktail of secreted epithelium, have been reported in close proximity to the growth factors including R-spondin1, noggin and EGF Lgr5 þ stem cells at the base of the colonic crypts. (Sato et al., 2009). These factors are likely to be However, it remains to be established if this population provided by the niche in vivo. The Xenopus R-spondin is the source of Wnt and other essential growth factors, ortholog has been identified as secreted activator of the or whether the submucosa is providing the functional canonical Wnt/b-catenin signaling pathway (Kazans- niche environment in the colon (Sato et al., 2010). kaya et al., 2004) and transgenic expression of human R-spondin1 in mice induces a major increase in crypt cell proliferation in the small intestinal and colon Revealing the function of Lgr5 homologs on intestinal epithelium (Kim et al., 2005). EGF signaling has stem cells likewise been linked with intestinal crypt proliferation (Dignass and Sturm, 2001), whilst noggin is thought to Lgr5 and its close relatives Lgr4 and Lgr6 belong to the act primarily as a bone morphogenetic protein signal family of leucine-rich repeat, G-protein-coupled recep- inhibitor. Transgenic expression of noggin throughout tors that are evolutionarily related to the the intestinal epithelium induces the formation of hormone receptors FSH, LH and TSH (Hsu et al., numerous ectopic crypt units along the crypt-villus axis, 1998, 2000; Barker and Clevers, 2010). Lgr5 and Lgr6 implying that bone morphogenetic protein signaling have attracted much interest from the stem cell represses de novo crypt formation (Haramis et al., 2004). community following their discovery as specific markers The question arises whether these niche elements are of cycling adult stem cell populations in various tissues,

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3014 including the hair-follicle, gastrointestinal tract and but markedly abrogated R spondin-mediated enhance- skin. In contrast, Lgr4 expression is typically present ment of canonical Wnt signaling. Similarly, genetic on, but not restricted to, Lgr5/Lgr6 adult stem cell deletion of Lgr4/5 on intestinal crypt cultures phe- compartments in these tissues (for comprehensive nocopied withdrawal of R-spondin. Taken together, review, see Barker and Clevers, 2010). Lgr6 KO mice these studies identify Lgr4 and Lgr5 as optional Wnt co- are healthy and fertile, but both Lgr4 null and Lgr5 null receptors that facilitate enhancement of local canonical mutations are neonatal-lethal, defining an essential role Wnt signals by circulating R-spondins (Figure 1d). for Lgr4 and Lgr5 during embryogenesis (Mazerbourg Lgr6, which is not expressed in the gastro-intestinal et al., 2004; Van Schoore et al., 2005). However, their tract, was also found to be capable of amplifying function on the stem cell compartments of adult tissues canonical Wnt signals through functional interaction have only recently been investigated. Conditional with R-Spondin on Hek293T cells (Carmon et al., 2011; ablation of Lgr5 expression in the adult small intestine de Lau et al., 2011). However, conditional ablation of surprisingly yielded no obvious phenotype (de Lau Lgr6 in combination with Lgr5 and/or Lgr4 is required et al., 2011). In contrast, conditional ablation of Lgr4 to assess whether this homolog has a physiological caused a block in crypt proliferation and widespread function in potentiating canonical Wnt signals on other crypt loss within 5 days. An independent study reported adult stem cell compartments such as the skin. Surpris- a less-severe intestinal phenotype in young mice homo- ingly, G-protein signaling does not seem to be involved zygous for a hypomorphic Lgr4 allele, which die within in mediating Lgr5-homolog- derived Wnt signals, a month after birth (Mustata et al., 2011). This raising the question of whether their classification as phenotype was characterized by a modest reduction in GPCR’s is accurate. crypt proliferation and depletion of the Paneth cell Our current inability to isolate and culture pure compartment. However, there was no apparent effect on populations of human Lgr5 þ stem cells has hampered a either stem cell marker expression or on the Wnt target direct investigation of human Lgr5 homolog functions gene signature in vivo, implying that the stem cell in vivo. In cultured human colon cancer cell-lines, Lgr5 compartment remained viable. Isolated homozygous depletion was recently reported to enhance Wnt signal- Lgr4-hypomorphic crypts failed to initiate organoid ing, drive EMT and promote tumorigenesis (Walker outgrowth ex vivo, implying that epithelial renewal is et al., 2011). It remains to be established if such a impaired in the absence of robust Lgr4 expression function in antagonizing Wnt signaling is specific to (Mustata et al., 2011). The difference in severity of the human cancer cells, or whether it reflects Lgr5 func- Lgr4 phenotypes is at least in part a likely consequence tional differences between and mice. of reduced expression (in the hypomorph) versus complete ablation in the null allele. However, these studies collectively implicate Lgr4 as having an essential Homeostasis of Lgr5 þ intestinal stem cells function in maintaining optimal epithelial renewal in the small intestine. A defining characteristic of adult stem cells is their Simultaneous conditional deletion of Lgr4 and Lgr5 ability to self-renew almost indefinitely. This is in stark in adult mice markedly enhanced the small intestinal contrast to the majority of other somatic cells, which phenotype, characterized by almost complete crypt rapidly enter senescence when their telomere length is ablation within 3 days (de Lau et al., 2011). The severity reduced to a critical size during successive mitoses. and accelerated onset of this phenotype led to specula- Adult stem cells have been suggested to achieve near tion that Lgr4 and Lgr5 have essential, yet partially immortality by increasing telomerase expression to a redundant roles in maintaining small intestinal stem cell level that ensures a constant telomere length (Flores function in vivo. This was substantiated when expression et al., 2005). Such a mechanism would be particularly analyses of small intestinal crypts isolated 1 day post- beneficial for the Lgr5 þ intestinal stem cells, which are deletion of Lgr4 and Lgr5 revealed selective loss of expected to undergo many thousands of divisions during many stem cell-enriched genes, consistent with a rapid their lifetime. Consistent with this, a recent study demise of the stem cells. In addition, there was a robust documented elevated telomerase expression in FACS- reduction in over 50% of the intestinal Wnt target gene sorted intestinal Lgr5 þ cells as compared with their signature, consistent with a role for these Lgr receptors transit-amplifying daughter cells (Schepers et al., 2011). in mediating Wnt signaling at the crypt base (de Lau An independent study employed an mTERT-GFP et al., 2011). This preliminary link with Wnt signaling reporter mouse to identify intestinal stem cells on the was reinforced when mass spectrometry analyses re- basis of their presumed elevated telomerase activity vealed a direct physical interaction of Lgr4 and Lgr5 (Breault et al., 2008). In apparent contrast to Schepers with the /Lrp Wnt receptor complex (de Lau et al., mTert expression was not documented in the et al., 2011). Additional insight into the Wnt-signaling cycling Lgr5 þ stem cells, but was instead restricted to roles of Lgr4 and Lgr5 was provided when independent rare, quiescent cells at the crypt base that were reported studies revealed that members of the R-spondin family to function as stem cells following radiation-induced of secreted Wnt agonists are physiological ligands of damage (Montgomery et al., 2011). It is possible that Lgr4 and Lgr5 (Carmon et al., 2011; de Lau et al., 2011). distinct stem cell pools with varying dependencies on Depletion of Lgr4 in Hek293T cells had no effect on high telomerase expression do exist in the intestine, their ability to respond to exogenous Wnt3A ligands, although one would have predicted that an actively

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3015 dividing population such as the Lgr5 þ CBC’s would be spindle fiber orientation in cycling intestinal cells revealed more telomerase-addicted than a quiescent population. a typically perpendicular alignment of mitotic spindles Long-term maintenance of the adult stem cell pool is relative to the apical surface. This spindle fiber orientation essential for optimal tissue homeostasis throughout our was proposed to result in the asymmetric segregation of lifetime. In solid tissues this is typically considered to be DNA during cell division, with the template strand achieved by obligate asymmetric division, where each basally distributed to the daughter cell adopting a stem stem cell divides to generate two daughter cells that cell fate as predicted by the immortal strand hypothesis acquire divergent cellular fates—that is one stem cell (Quyn et al., 2010). However, these observations were and one TA cell. The stem cell daughter replenishes the challenged by a study analyzing the distribution of DNA stem cell pool, whilst the short-lived TA progeny labels within the Lgr5 þ stem cell compartment, which contribute to tissue renewal. Indeed, this strategy has concluded that newly synthesized DNA strands are been documented to occur in adult stem cell compart- randomly distributed between both daughter cells during ments from a range of organisms (Kemphues et al., stem cell division (Schepers et al., 2011). Hopefully, future 1988; Rhyu et al., 1994; Spana et al., 1995; Fuller and studies will shed more light onto the molecular mechan- Spradling, 2007). However, there is no a priori reason isms guiding intestinal stem cell division and determine why adult stem cells in different tissues are obliged to whether Lgr5 þ stem cell populations in different adult adopt this strategy to manage the delicate balance tissues employ common mechanisms to achieve tissue between self-renewal and tissue regeneration (Snippert homeostasis. and Clevers, 2011). Indeed, this balance can also be readily achieved by stochastic symmetric division within the stem cell pool to generate either two stem cells or two short-lived TA cells. Lgr5 as marker of stomach stem cells To investigate the specific dynamics of stem cell homeostasis in the intestine, Clevers and co-workers The epithelium of the stomach and intestine share a adopted an in vivo lineage-tracing approach using a new number of characteristics, including a highly stereo- multicolor Cre reporter mouse (Rosa-4color) to map the typical organization and regular self-renewal fueled by division fates of individual adult stem cells in the small limited reservoirs of adult stem cells located within intestine (Snippert et al., 2010b). The Rosa-4color line numerous epithelial pockets. In the stomach, the was created by targeting a construct comprising a epithelial pockets represent independent gastric units CAGG promoter, a LoxP-flanked NeoR-cassette (ser- comprising flask-shaped tubular glands, several of which ving as transcriptional roadblock), and the original lead into a single pit that opens out onto the gastric Brainbow-2.1 cassette (Livet et al., 2007) to the Rosa26 surface epithelium. Each gland can be further sub- locus. Lgr5KI/Rosa-4color mice were subsequently divided into isthmus, neck and base regions (Barker generated and induced with tamoxifen to activate et al., 2010a) (Figure 2a). The precise morphological stochastic expression of the different fluorescent repor- structure, cellular composition and tissue turnover of ter genes within the Lgr5 þ stem cell compartment individual gastric units varies considerably between (Snippert et al., 2010b). Short-term fate mapping of anatomically distinguishable gastric sub-domains individually marked stem cells at 24–48 h post-induction (Karam, 1999). In humans, the gastric lining can be demonstrated that symmetric division predominates subdivided into two major compartments: the pylorus within the intestinal stem cell compartment—that is, (composed of the pyloric antrum and canal) and the Lgr5 stem cells rarely generate two daughter cells that corpus domain. Elegant clonal marking experiments adopt divergent fates (stem cell vs TA cell). Longer-term tracing analysis of the Lgr5 stem cell population dynamics revealed a neutral drift towards clonality over time (visualized as a gradual conversion of multicolor stem cell populations to monochrome populations in single crypts). Collectively, these observations imply that Lgr5 stem cells double their numbers daily and stochastically acquire either stem cell or TA cell fates. An optimal balance between stem cell self-renewal and epithelial regeneration is therefore likely to be achieved by neutral competition between 10–14 symmetrically dividing stem cells within the limited niche at the crypt base (Snippert et al., 2010b). Additional support for this model was provided by an independent study employing a predominantly mathematical modeling strategy Figure 2 Lgr5 þ adult stem cells ensure epithelial tissue home- (Lopez-Garcia et al., 2010). However, contrasting ostasis of the pyloric stomach. (a) Architecture of the pyloric results were recently reported by Quyn and co-workers, epithelium. Proliferating Lgr5 þ adult stem cells reside at the gland base of pyloric gastric units. (b) Flowchart of Lgr5 þ adult stem who concluded that asymmetric division predominates cell-driven tissue homeostasis in the pylorus. Pyloric Lgr5 þ adult within the Lgr5 þ intestinal stem cell compartment stem cells generate all differentiated cell types of the pyloric (Quyn et al., 2010). Their quantitative analysis of mitotic epithelium, whilst self-renewing to maintain stem cell homeostasis.

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3016 documented the existence of multipotent adult stem cells Lgr5 þ adult stem cells in gastrointestinal cancer within the glandular stomach, but were unable to reveal their identity (Bjerknes and Cheng, 2002). Early studies The gradual acquisition of genetic/epigenetic mutations identified a region just below the pit, termed the isthmus, in key tumor-suppressor genes and is widely as the major site of gastric proliferation (Leblond and considered to be the primary route for metastatic cancer Stevens, 1948). The subsequent discovery of immature, in humans. Tumors arising in the same tissue often granule-free cells resembling embryonic stem cells within display marked phenotypic and functional heterogene- the isthmus prompted speculation that this region ity, which may reflect differences in the cell type housed the adult stomach stem cells (Karam and sustaining the original mutation (that is, different cells Leblond, 1993). However, a lack of specific markers of of origin), or may alternatively be dictated by the type of these candidate isthmus populations has precluded mutations occurring within a common target cell formal demonstration of their stem cell identity. (Visvader, 2011). The last decade has seen a major The widely accepted model championing the isthmus investment in developing mouse models and in vitro/ as the exclusive stem cell reservoir of the stomach was ex vivo assays to address the potential cell-of-origins of recently challenged, when a population of proliferating cancers as a prerequisite to the development of novel Lgr5 þ cells restricted to the base of the pyloric glands prognostic markers and preventative therapies. The was shown to function as self-renewing multipotent stem inherent longevity of adult stem cells has long been cells contributing to daily epithelial renewal (Barker considered to render them susceptible to accumulating et al., 2010b). A functional demonstration of the Lgr5 þ the necessary mutation spectrum to initiate tumor stem cell credentials was achieved by an in vivo lineage formation. However, formal demonstration of this for tracing, employing the same Lgr5KI/Rosa26RLacZ many solid tumors is still lacking. Indeed, IPS technol- mouse model used to establish the stem cell identity of ogy has elegantly proven the ease with which terminally intestinal Lgr5 þ cells. Stochastic activation of lacZ differentiated cells can re-acquire stem/progenitor-like reporter in isolated Lgr5 þ cells at the properties following the introduction of only a few gland base was evident 1–2 days post-tamoxifen genetic changes (Takahashi et al., 2007; Yu et al., 2007). induction. Over the course of the next 3–4 days, LacZ þ One should therefore assume that any somatic cell has progeny became visible throughout the gland, consistent the potential to trigger cancer formation in the presence with active cell division occurring within the basal Lgr5 of an appropriate mutation spectrum, until proven compartment. These LacZ þ clones continued to expand otherwise using relevant experimental models. until they extended to the surface epithelium after 8–10 days, providing an estimate of the self-renewal rate of the pyloric epithelium (Barker et al., 2010b). At later Lgr5 þ adult stem cells as the intestinal cancer origin time points, multiple gastric units populated exclusively by LacZ þ progeny were observed throughout the Colorectal cancer (CRC) is one of the most common pyloric region. Importantly, this lineage tracing was types of human cancer with around one million new maintained throughout the entire lifetime of the mouse, cases diagnosed per year worldwide (Cunningham et al., allowing the authors to conclude that basal Lgr5 þ cells 2010). CRC development follows a well-defined se- are functioning as multipotent, self-renewing stem cells quence of histological stages—from aberrant crypt contributing to daily epithelial homeostasis in this proliferation or hyperplasia to benign adenomas, to region of the stomach (Barker et al., 2010b) locally circumscribed carcinoma and finally to meta- (Figure 2b). In vitro, isolated pyloric Lgr5 þ cells were static carcinoma—corresponding to a gradual accumu- capable of generating pieces of self-renewing gastric lation of genetic mutations (Fearon and Vogelstein, epithelia with an architecture and cellular make-up 1990) (Figure 3). Activating Wnt pathway mutations similar to that of normal pyloric gastric units. Like their are responsible for initiating adenoma formation in intestinal stem cell counterparts, the pyloric stem cells the intestinal tract, predominantly by loss-of functional demonstrated a strict in vitro requirement for active Wnt mutations in the adenomatous polyposis coli (APC) signaling (Barker et al., 2010b). Expression profiling of tumor-suppressor gene (80%) or oncogenic mutations endogenous pyloric Lgr5 þ stem cells also revealed in the Wnt effector b-catenin (Powell et al., 1992; robust activation of the intestinal Wnt target gene Korinek et al., 1997; Rubinfeld et al., 1997; van de program, consistent with exposure to local canonical Wetering et al., 2002). However, the identity of the cell Wnt signals at the gland base (Van der Flier et al., 2007). sustaining the original Wnt pathway mutation has However, the origin of endogenous Wnt ligands and proven difficult to ascertain (Shih et al., 2001; Preston other niche components for Lgr5 þ pyloric stem cells et al., 2003). remains to be established. Future studies should also The contribution of adult intestinal stem cells to CRC address how Lgr5 þ stem cell homeostasis is achieved in initiation in mice was recently addressed using the Lgr5- the pylorus and aim to establish the relationship EGFP-IRES-CreERT2 mouse line to drive the condi- between the basal stem cell pool and the disparate tional deletion of APC exclusively in the intestinal stem proliferation zone at the isthmus. Hopefully, real-time cell compartment (Barker et al., 2009) (Figure 3a). This imaging of Lgr5-driven in vivo lineage tracing using the resulted in the rapid transformation of the Lgr5 þ stem Rosa-4color mice will provide sufficient clonal resolu- cells, which persisted and fueled the growth of large tion to achieve this in the near future. adenomas spanning the lumen in both the small intestine

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3017

Figure 3 A model for cancer initiation and progression in the intestine (a) Tumor-initiating mutations in the Wnt pathway (yellow lightening arrow) occur in an Lgr5 þ stem cell (red) of the healthy intestinal epithelium. (b) The transformed Lgr5 þ stem cell survives and supplies transformed (b-cateninhigh) progeny to the TA compartment. (c) Mutant TA cells are unable to exit onto the villus and instead expand sideways to establish an early benign adenoma harboring a limited reservoir of Lgr5 þ cells (candidate CSCs). These Lgr5 þ tumor cells reside in tumor ‘niches’ containing Paneth-like cells. (d, e) The gradual accumulation of additional mutations (yellow lightening arrows) in KRAS, TGF-b RII/Smad4 and P53 within long-lived Lgr5 þ tumor stem cells drives cancer progression from benign adenoma to metastatic carcinoma over time. and colon within a few weeks (Barker et al., 2009) Lgr5 as a potential colon CSC marker (Figure 3b). In contrast, APC deletion in non-stem cell populations using a different Cre driver failed to sustain The existence of discrete populations of tumor-propa- adenoma growth. Conditional expression of oncogenic gating cells (often referred to as CSCs) within human b-catenin in Bmi1 þ /Prominin1 þ intestinal stem cell colon tumors is still the subject of much speculation pools was also found to promote intestinal neoplasia (Clevers, 2011). These CSC pools, which are not in mice (Sangiorgi and Capecchi, 2008; Zhu et al., 2009). necessarily related to the tumor-initiating cells, are More recently, Merlos-Suarez and colleagues revealed thought to be instrumental in fueling cancer growth by that tumor-initiating cells in human colorectal cancers supplying the various differentiated cells that constitute have similar expression profiles to healthy human the tumor mass, and by gradually accumulating the intestinal stem cells. Notably, Lgr5 expression was mutation spectrum responsible for driving tumor highly enriched in both populations, further validating evolution towards metastasis (Visvader, 2011). Reliable this as a bona-fide stem/ (CSC) marker CSC biomarkers are essential for validating and (Merlos-Sua´rez et al., 2011). Collectively, these observa- characterizing candidate populations as a prerequisite tions indicate that stem cells are likely to be the for the development of more effective cancer therapies predominant cell-of-origin of colorectal cancer. targeting their eradication. CD133 gained prominence as one such candidate biomarker when serial transplan- tation studies using immunodeficient mice demonstrated Lgr5 þ adult stem cells as the stomach cancer origin a marked enrichment of tumor-propagating potential within the minor CD133 þ tumor population in compar- Gastric cancer is another leading cause of worldwide ison with unfractionated tumor (O’Brien et al., 2007; mortality, particularly within Asia. The underlying Ricci-Vitiani et al., 2007). These studies estimated genetics of gastric cancer initiation and progression are o2.5% of the colon cancer population to display CSC less well-defined than for CRC, but it is well documen- potential in vivo. However, it should be noted that the ted that inappropriate activation of the Wnt signaling native tumor microenvironment is likely to be instru- pathway occurs in a subset of gastric cancers arising mental in conferring CSC properties to resident cell predominantly in the pylorus (Park et al., 1999; populations (Visvader and Lindeman, 2008). If true, Clements et al., 2002). Indeed, both mice and humans then the behavior of isolated tumor cells in cross- harboring germline APC mutations develop gastric species transplantation assays may not always be an cancer in addition to CRC (Offerhaus et al., 1999; accurate readout of endogenous CSC identity (Kelly Tomita et al., 2007). The recent discovery of an active et al., 2007). stem cell population in the pyloric region has facilitated The restricted expression pattern of the intestinal stem a direct evaluation of their contribution to Wnt-driven cell marker Lgr5 in intestinal adenomas has prompted gastric cancer in vivo. Using a similar conditional speculation that it may also be selectively expressed on deletion approach to that described above, targeted the intestinal CSC populations. Adenomas generated by ablation of APC tumor suppressor activity in the Lgr5 þ targeted transformation of intestinal stem cells in stem cell compartment was found to initiate adenoma Lgr5KI mice harbored a limited reservoir (o10%) of growth in the pylorus (Barker et al., 2010b). This study Lgr5-EGFP þ cells, which appeared to reside within established the Lgr5 þ pyloric stem cell as a potential cell crypt-like domains in close proximity to cells displaying of origin of Wnt-driven stomach cancers. However, their Paneth cell features (Barker et al., 2009) (Figure 3c). contribution to Wnt-independent tumor initiation in the Takahashi et al., subsequently reported restricted Lgr5 stomach remains to be addressed. expression within the most basal layer of human

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3018 adenoma cells localized at the tumor-host interface 2004). In telogen HF, the Lgr5 þ cells were shown to (Takahashi et al., 2011), and elevated Lgr5 expression reside exclusively in the lower bulge and hair germ (Jaks has been documented on more metastatic colon cancer et al., 2008) (Figure 4a). These cells were shown to cell-lines (Uchida et al., 2010) More robust evidence for actively proliferate (at least during the anagen phase of the CSC identity of human Lgr5 þ populations was the HF), establishing the Lgr5 þ pool as a separate provided by Batlle and colleagues, who reported that population to the label-retaining stem cells present in the Lgr5 and other normal intestinal stem cell genes are upper bulge regions. Following transplantation, Lgr5 þ selectively expressed on human colon CSC populations. cells were capable of generating all skin components, Strikingly, the presence of these Lgr5 þ tumor cell including functional HFs, sebaceous glands and the populations significantly correlated with a poor prog- interfollicular epidermis (Jaks et al., 2008). However, nosis for CRC cancer patients (Merlos-Sua´rez et al., in vivo lineage tracing demonstrated that telogen Lgr5 þ 2011). stem cell activity was strictly confined to HF regenera- The Lgr5KI/ROSA4-color-tracing tools now avail- tion under physiological conditions (Figure 4b). These able in the research community should facilitate a direct Lgr5 þ HF stem cells were later identified as a potential evaluation of Lgr5 þ CSC potential in intestinal cancers cell-of-origin of basal cell carcinoma following muta- by intra-tumor lineage tracing. In simplistic terms, this tional activation of Hedgehog signaling in a wounding would be accomplished by selectively activating a new environment (Kasper et al., 2011). Collectively, these fluorescent reporter gene within a pre-existing Lgr5 þ observations establish that proliferating stem cells intestinal tumor population to facilitate genetic tracing marked by Lgr5 coexist with a more quiescent pool in of their activity and fate during subsequent tumor the adult HF. Like the intestine, these stem cell growth. Although not without its own technical populations are likely targets for oncogenic mutation challenges, this approach would circumvent many of as a route to human skin cancers. the technical limitations associated with cross-species transplantation as a means of accurately assessing Lgr5 CSC identity in situ. Lgr6 as marker of skin stem cells

Like the intestinal lining, our skin must undergo regular renewal fueled by resident adult stem cells in order to Lgr5 as marker of hair-follicle stem cells maintain optimal barrier functions in the face of a constant battery of environmental assault. The existence The hair-follicles present on most areas of our skin of such epidermal adult stem cell populations has long undergo many hundreds of regeneration cycles during been inferred by fate-mapping (Ghazizadeh and Taich- our lifetime. Each regeneration cycle comprises an active man, 2001) and ex vivo culture studies (reviewed in growth phase (anagen), driven by dedicated adult stem (Green, 2008), but a lack of markers has precluded their cell populations residing in the permanent portion of the formal identification. Expression mapping of Lgr6, a HF, followed by involution (catagen), and finally a close homolog of Lgr5, revealed expression in the resting phase (telogen). Accordingly, each HF comprises developing hair-pegs of embryonic skin and in a a permanent domain (including the sebaceous gland and restricted domain of the adult hair-follicle, just below the underlying bulge domain) and a cycling proportion the sebaceous gland (Snippert et al., 2010a) (Figure 4a). that transiently forms during early anagen (Fuchs, In vivo lineage tracing using a Lgr6-GFP-ires-CreERT2/ 2009). Various studies employing independent trans- Rosa26R-lacZ model established the embryonic Lgr6 þ plantation and in vivo transplantation approaches have cells as being the origin of the HF, sebaceous gland and established the bulge as a major stem cell reservoir for the epidermis (Snippert et al., 2010a). In contrast, the HF (Rochat et al., 1994; Taylor et al., 2000; Oshima induction of lineage tracing in adult mice documented et al., 2001; Trempus et al., 2003; Blanpain et al., 2004). a more restricted contribution to the sebaceous gland One bulge population is characterized by the ability to and the interfollicular epidermis (Snippert et al., 2010a) retain DNA labels, indicative of relative quiescence. (Figure 4b). Unlike their Lgr5 þ bulge cousins, the Lineage-tracing studies demonstrated that this label- Lgr6 þ stem cells appear to reside in a Wnt-independent retaining population is transiently activated to undergo niche in adult skin (Fuchs and Horsley, 2008; Snippert symmetrical division during anagen to expand the bulge et al., 2010a). Lgr6 lineage-tracing analysis during stem cell reservoir (Blanpain et al., 2004; Zhang et al., wound repair demonstrated that adult Lgr6 þ skin 2009). A proportion of these newly-generated stem cell stem cells permanently contribute to newly generated progeny subsequently exit the bulge during late telogen epidermis. Importantly, Lgr6 þ cells were also found to populate the hair-germ. In the following anagen to contribute to HF neogenesis within the wound phase, these hair germ cells actively proliferate, leading epithelium (Snippert et al., 2010a), implying plasticity to the formation of transit-amplifying cells, which of the Lgr6 þ stem cells under damage conditions further differentiate to fuel HF regeneration (Greco (Figure 4b). The development of epithelial culture et al., 2009; Zhang et al., 2009). Expression profiling of systems facilitating the growth of human skin from these CD34 þ bulge stem cells subsequently revealed isolated Lgr6 þ adult stem cells will hopefully represent a enrichment of the intestinal stem cell marker Lgr5 major breakthrough in treating human skin disorders in (Blanpain et al., 2004; Morris et al., 2004; Tumbar et al., the clinic.

Oncogene Lgr5 and Lgr6 markers to study adult stem cell roles M Leushacke and N Barker 3019

Figure 4 Dedicated adult stem cell compartments ensure epithelial homeostasis in adult hair-follicles and skin and contribute to tissue regeneration following injury. (a) Cartoon of a resting HF plus associated epidermis, depicting the location of label-retaining, Lgr5 þ and Lgr6 þ adult stem cell populations. (b) The relative contribution of Lgr5 þ and Lgr6 þ adult stem cells to skin homeostasis under physiological conditions and in response to injury.

Perspectives proliferation and symmetrical division are unique to Lgr5/Lgr6 adult stem cell populations remains to be The discovery of Lgr5 and Lgr6 as markers of established. The same Lgr5/Lgr6 reporter mice various adult stem cell populations in the mouse has have facilitated the isolation and in-depth characteriza- contributed greatly to our understanding of adult tion of adult stem cell populations in the gut and stem cell biology in rapidly self-renewing tissues and skin, revealing additional markers that will likely prove the contribution of adult stem cells to epithelial cancer. invaluable in helping to identify and purify their Our ability to visualize and track stem cell activity in situ human counterparts for clinical applications. It is using dedicated reporter mice and next generation hoped that the new epithelial culture methods will be in vivo lineage-tracing tools has revealed how an optimal instrumental in exploiting the regenerative medicine balance between adult stem cell homeostasis and tissue potential of these human stem cell populations for renewal is achieved in vivo and helped define the role of treating human disorders in the gastrointestinal tract local niche interactions in regulating this process. and skin. Many of the findings appear to contradict long- established adult stem cell dogmas such as obligate asymmetric stem cell division and characteristic cellular Conflict of interest quiescence governed by predominantly stromal niches. Whether such unexpected characteristics as active The authors declare no conflict of interest.

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