Title: Tissue-Resident Adult Stem Cell Populations of Rapidly Self-Renewing Organs
Abstract: The epithelial lining of the intestine, stomach, and skin is continuously exposed to environmental assault, imposing a requirement for regular self-renewal. Resident adult stem cell populations drive this renewal, and much effort has been invested in revealing their identity. Reliable adult stem cell biomarkers would accelerate our understanding of stem cell roles in tissue homeostasis and cancer. Membrane-expressed markers would also facilitate isolation of these adult stem cell populations for exploitation of their regenerative potential. Here, we review recent advances in adult stem cell biology, highlighting the promise and pitfalls of the candidate biomarkers of the various stem cell populations. The epithelial lining of the intestine, stomach, and skin is continuously exposed to environmental assault, imposing a requirement for regular self-renewal. Resident adult stem cell populations drive this renewal, and much effort has been invested in revealing their identity. Reliable adult stem cell biomarkers would accelerate our understanding of stem cell roles in tissue homeostasis and cancer. Membrane-expressed markers would also facilitate isolation of these adult stem cell populations for exploitation of their regenerative potential. Here, we review recent advances in adult stem cell biology, highlighting the promise and pitfalls of the candidate biomarkers of the various stem cell populations. The maintenance and repair of adult tissues relies on small populations of resident stem cells. These specialized cells are defined by their ability to maintain themselves over very long periods of time (“self-renewal”) and to generate all the differentiated cell types of the tissue (“multipotency”). In addition to these defining characteristics, adult stem cells are often assumed to be quiescent within the niche, dividing infrequently to generate one stem cell copy and a rapidly cycling cell. The rapidly cycling cells (transit-amplifying cells) then undergo a limited number of cell divisions before terminally differentiating into the functional cells of that tissue. This stem cell-driven tissue renewal is particularly evident in the epithelial lining of the intestine, stomach, and skin, which is constantly exposed to a barrage of environmental assault. The ability to identify and isolate these adult stem cell populations would undoubtedly accelerate our understanding of stem cell roles in tissue homeostasis and cancer and would also facilitate exploitation of their massive regenerative potential in the clinic. Much effort has, therefore, been invested around the world in identifying reliable adult stem cell biomarkers, fueling impressive advances in adult stem cell research over the last few years. The mammalian intestinal tract can be divided into two anatomically and functionally distinct segments: the small intestine and the colon (Gregorieff and Clevers, 2005Gregorieff A. Clevers H. Wnt signaling in the intestinal epithelium: from endoderm to cancer.Genes Dev. 2005; 19: 877-890Crossref PubMed Scopus (308) Google Scholar). These segments share the same basic structure, comprising an outer layer of smooth muscle interfaced with the enteric nervous system responsible for mediating the rhythmic peristaltic movements that force digested food along the tube, a middle layer of connective tissue (the stroma) harboring nerves and lymphatic vessels, and an inner absorptive epithelial lining called the mucosa. The architecture of this epithelial lining differs markedly between the small intestine and colon, reflecting their distinct functions in vivo. The inner surface of the small intestine is designed to maximize the available surface area for absorption, which it achieves by having numerous finger-like protrusions called villi that project into the lumen, in close association with invaginations called crypts of Lieberkühn. In contrast, the surface of the colonic mucosa is essentially flat, with multiple crypts that penetrate deep into the underlying submucosa. The absence of villi in this region reflects the major role of the colon in stool compaction rather than absorption. The precise cellular composition of the intestinal epithelium varies between the different anatomical regions of the small intestine (duodenum, jejunum, and ileum) and the colon. Absorptive enterocytes (which also secrete hydrolytic enzymes) are abundant throughout the entire small intestine, although their absolute numbers are greatest in the duodenum, where the villi are longest. Mucus-secreting Goblet cells are most predominantly found in the ileum and the colon, reflecting a need to lubricate the passage of stool as it becomes more compact during its progression toward the anus. Paneth cells, which play a major role in regulating the local microbial environment by secreting various antimicrobial peptides and lysozyme, are largely restricted to the crypts of the small intestine. Other, more rare cell types include the hormone-secreting enteroendocrine cells, together with the less well-defined brush/tuft/caveolated cells, cup cells, and the M cells residing on lymphoid Peyer's patches (Barker et al., 2008Barker N. van de Wetering M. Clevers H. The intestinal stem cell.Genes Dev. 2008; 22: 1856-1864Crossref PubMed Scopus (207) Google Scholar). The crypts and villi are lined with a layer of a simple, columnar epithelium that is completely renewed every 3–5 days throughout our entire lifetime (Leblond and Stevens, 1948Leblond C.P. Stevens C.E. The constant renewal of the intestinal epithelium in the albino rat.Anat. Rec. 1948; 100: 357-377Crossref PubMed Google Scholar). This remarkable self-renewal rate is necessitated by the extremely harsh conditions that exist within the intestinal lumen, with the epithelial cells being subject to a constant barrage of mechanical, chemical, and pathogen-driven attacks. Small populations of crypt-resident adult stem cells are instrumental in driving this self-renewal process. The crypts are the real “engine” of the self-renewal process of the intestinal epithelium, with each one responsible for generating in excess of 250 new epithelial cells per day. This engine is fueled by the resident stem cells, thought to be located close to the crypt base (Figure 1A ). These stem cells produce a population of vigorously proliferating progenitors called transit-amplifying (TA) cells, which rapidly expand through multiple rounds of cell division as they move upwards as a coherent column toward the crypt/villus border (Heath, 1996Heath J.P. Epithelial cell migration in the intestine.Cell Biol. Int. 1996; 20: 139-146Crossref PubMed Scopus (90) Google Scholar). During this upward migration, these TA cells begin to differentiate and subsequently exit the crypt onto the villus after 2 days as mature, functional epithelial cells. They continue migrating along this epithelial conveyer belt until they reach the villus tip, where they die and are shed into the lumen for excretion. Up to ten crypts are needed to supply new cells to a single villus in order to balance the extreme turnover rate present at the villus tip. The much longer-lived crypt-resident Paneth cells are excluded from this upwardly mobile epithelial conveyer belt. Instead, they migrate in the opposite direction to occupy the crypt base, where they live for 6–8 weeks. Early genetic marking studies of chimeric mice and rare mosaic human patients first demonstrated that individual adult crypts are monoclonal, having their origin in a single multipotent stem cell that is active during early intestinal development (Bjerknes and Cheng, 1999Bjerknes M. Cheng H. Clonal analysis of mouse intestinal epithelial progenitors.Gastroenterology. 1999; 116: 7-14Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, Hermiston et al., 1993Hermiston M.L. Green R.P. Gordon J.I. Chimeric-transgenic mice represent a powerful tool for studying how the proliferation and differentiation programs of intestinal epithelial cell lineages are regulated.Proc. Natl. Acad. Sci. USA. 1993; 90: 8866-8870Crossref PubMed Scopus (39) Google Scholar). The existence of a self-renewing, multipotent stem cell population in adult crypts was formally proven by tracking inheritance patterns of genetic marks introduced at random into single crypt cells via somatic mutation (Bjerknes and Cheng, 2002Bjerknes M. Cheng H. Multipotential stem cells in adult mouse gastric epithelium.Am. J. Physiol. Gastrointest. Liver Physiol. 2002; 283: G767-G777PubMed Google Scholar, Winton et al., 1990Winton D.J. Gooderham N.J. Boobis A.R. Davies D.S. Ponder B.A. Mutagenesis of mouse intestine in vivo using the Dlb-1 specific locus test: studies with 1,2-dimethylhydrazine, dimethylnitrosamine, and the dietary mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline.Cancer Res. 1990; 50: 7992-7996PubMed Google Scholar). Clones of marked cells comprising all epithelial lineages were observed in individual crypts, consistent with the original mutation having been introduced into a multipotent stem cell. Similar results were obtained by studying inheritance patterns of spontaneous mitochondrial mutations in adult human crypts (Taylor et al., 2003Taylor R.W. Barron M.J. Borthwick G.M. Gospel A. Chinnery P.F. Samuels D.C. Taylor G.A. Plusa S.M. Needham S.J. Greaves L.C. et al.Mitochondrial DNA mutations in human colonic crypt stem cells.J. Clin. Invest. 2003; 112: 1351-1360Crossref PubMed Google Scholar). This study revealed the coexistence of both mutant and wild-type clones in single crypts, thus establishing the presence of multiple adult stem cells. However, given that these mutations were introduced at random, the identity of the mutant stem cell was not revealed. Even today, the identity of the adult intestinal stem cells is intensely debated. Two opposing models of intestinal stem cell identity dominate the literature: the +4 model and the stem cell zone model (Figure 1A). The +4 model was originally proposed when early cell tracking experiments predicted a common cell origin at position 4–5, just above the differentiated Paneth cell compartment (Cairnie et al., 1965Cairnie A.B. Lamerton L.F. Steel G.G. Cell proliferation studies in the intestinal epithelium of the rat. I. Determination of the kinetic parameters.Exp. Cell Res. 1965; 39: 528-538Crossref PubMed Google Scholar). Additional experimental support for this model was provided by Potten and colleagues, who reported that radiation-sensitive, label-retaining cells reside immediately above the uppermost Paneth cell, at positions ranging from +2 to +7, but on average at +4 (Potten, 1977Potten C.S. Extreme sensitivity of some intestinal crypt cells to X and gamma irradiation.Nature. 1977; 269: 518-521Crossref PubMed Google Scholar). Their sensitivity to radiation was considered to be a desirable stem cell characteristic, preventing potentially carcinogenic genetic abnormalities from becoming established within the long-lived stem cell population. Retention of nucleotide labels such as BrdU or H3-Thymidine is also considered to be a reliable surrogate stem cell trait, usually indicative of quiescence under physiological conditions. However, label-retaining cells in the intestine were invariably shown to be actively proliferating (every 24 hr). Long-term retention of labels incorporated during crypt cell neogenesis (early in development or following radiation-induced damage) was instead proposed to result from asymmetric segregation of old (labeled) and new (unlabeled) DNA strands during subsequent cell divisions (Potten et al., 2009Potten C.S. Gandara R. Mahida Y.R. Loeffler M. Wright N.A. The stem cells of small intestinal crypts: where are they?.Cell Prolif. 2009; 42: 731-750Crossref PubMed Scopus (68) Google Scholar). This finding predicted that the labeled template strand would be retained in the cell destined to remain a stem cell, while the newly synthesized strand, complete with any replication errors, is passed onto the short-lived daughter cell. This immortal strand theory (Cairns, 1975Cairns J. Mutation selection and the natural history of cancer.Nature. 1975; 255: 197-200Crossref PubMed Google Scholar) is considered to protect the stem cell genome from accumulating potentially dangerous mutations. However, experimental support for this model is still limited (Shinin et al., 2006Shinin V. Gayraud-Morel B. Gomes D. Tajbakhsh S. Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells.Nat. Cell Biol. 2006; 8: 677-687Crossref PubMed Scopus (249) Google Scholar), and it only remains valid if there is no symmetric cell division occurring within the stem cell niche. An alternative school of thought, referred to as the stem cell zone model, evolved following the discovery that the crypt base is not exclusively populated by differentiated Paneth cells. Electron microscopy studies in the early 1970s revealed the presence of slender, immature, cycling cells, referred to as crypt base columnar cells (CBC), wedged between the Paneth cells (Cheng and Leblond, 1974Cheng H. Leblond C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types.Am. J. Anat. 1974; 141: 537-561Crossref PubMed Google Scholar). Following 3H-Thymidine exposure, surviving CBC cells were reported to actively phagocytose other dying, radiolabeled CBC cells at the crypt base. The resulting radioactive phagosomes, initially restricted to occasional CBC cells, were subsequently observed within more differentiated cells belonging to the four major lineages higher up the crypts. This rudimentary lineage tracing result was interpreted as evidence for the CBC cells being the common origin of all four major epithelial cell lineages. However, the phagosome-labeled examples of the four cell lineages were only observed in separate crypts, precluding formal demonstration of CBC cell multipotency. Bjerknes and Cheng continued to champion the CBC cells as true adult stem cells, providing additional, yet still indirect, evidence of CBC cell multipotency in more refined genetic marking studies (Bjerknes and Cheng, 1999Bjerknes M. Cheng H. Clonal analysis of mouse intestinal epithelial progenitors.Gastroenterology. 1999; 116: 7-14Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, Bjerknes and Cheng, 2002Bjerknes M. Cheng H. Multipotential stem cells in adult mouse gastric epithelium.Am. J. Physiol. Gastrointest. Liver Physiol. 2002; 283: G767-G777PubMed Google Scholar). They reported the existence of both long-lived and short-lived clones of marked cells within the crypts of these mice. Only the long-lived clones comprising all four major cell lineages consistently included a marked CBC cell. This was interpreted as further evidence for marked CBC cells being the self-renewing, multipotent origin of these clones. Based on these observations, Bjerknes and Cheng subsequently formulated their current stem cell zone model (Bjerknes and Cheng, 1981aBjerknes M. Cheng H. The stem-cell zone of the small intestinal epithelium. I. Evidence from Paneth cells in the adult mouse.Am. J. Anat. 1981; 160: 51-63Crossref PubMed Google Scholar, Bjerknes and Cheng, 1981bBjerknes M. Cheng H. The stem-cell zone of the small intestinal epithelium. III. Evidence from columnar, enteroendocrine, and mucous cells in the adult mouse.Am. J. Anat. 1981; 160: 77-91Crossref PubMed Google Scholar, Bjerknes and Cheng, 1999Bjerknes M. Cheng H. Clonal analysis of mouse intestinal epithelial progenitors.Gastroenterology. 1999; 116: 7-14Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In this model, the CBC stem cells reside in a stem cell-permissive environment (the stem cell zone) at the crypt base. These proliferating stem cells regularly generate progeny, which subsequently exit the niche at the “common origin of differentiation” around position +5, where they commit (presumably via committed progenitors) toward the various functional lineages (Figure 1B). Maturing Paneth cell progenitors migrate downwards, with the oldest Paneth cells residing at the very base of the crypt. Progenitors of all other lineages mature as they migrate upwards onto the villus epithelium. An array of markers has been proposed for the putative adult stem cell populations (Figure 1C), but the validity of the vast majority of these candidates is not supported by direct evidence for stemness as assessed by transplantation or lineage tracing (Table 1). Instead, many studies have relied on IHC/in situ approaches to define candidate stem cell markers according to positional information alone, leading to a great deal of confusion and controversy in the ISC field.Table 1A Summary of the Promise and Pitfalls of Current Adult Intestinal Stem Cell MarkersMarker+4/CBCEvidenceWeaknessReferencesLabel retention+4 (on average), but also reported within CBC compartmentMarked cells exhibit desirable stem cell traits such as radiosensitivityNo functional demonstration of stemness. Often incorrectly assumed to reflect quiescencePotten, 1977Potten C.S. Extreme sensitivity of some intestinal crypt cells to X and gamma irradiation.Nature. 1977; 269: 518-521Crossref PubMed Google ScholarMusashi-1+4 and CBCHighly expressed throughout the stem cell compartmentNo functional demonstration of stemness. Also likely to be expressed in TA compartmentPotten et al., 2003Potten C.S. Booth C. Tudor G.L. Booth D. Brady G. Hurley P. Ashton G. Clarke R. Sakakibara S. Okano H. Identification of a putative intestinal stem cell and early lineage marker; musashi-1.Differentiation. 2003; 71: 28-41Crossref PubMed Scopus (283) Google Scholar, He et al., 2007He X.C. Yin T. Grindley J.C. Tian Q. Sato T. Tao W.A. Dirisina R. Porter-Westpfahl K.S. Hembree M. Johnson T. et al.PTEN-deficient intestinal stem cells initiate intestinal polyposis.Nat. Genet. 2007; 39: 189-198Crossref PubMed Scopus (203) Google ScholarProminin-1CBCSupported by in vivo lineage tracingLikely expressed throughout the stem cell compartment and the TA compartmentZhu et al., 2009Zhu L. Gibson P. Currle D.S. Tong Y. Richardson R.J. Bayazitov I.T. Poppleton H. Zakharenko S. Ellison D.W. Gilbertson R.J. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation.Nature. 2009; 457: 603-607Crossref PubMed Scopus (274) Google Scholar, Snippert et al., 2009Snippert H.J. van Es J.H. van den Born M. Begthel H. Stange D.E. Barker N. Clevers H. Prominin-1/CD133 marks stem cells and early progenitors in mouse small intestine.Gastroenterology. 2009; 136 (e.1): 2187-2194Abstract Full Text Full Text PDF PubMed Scopus (99) Google ScholarSOX9CBCLow-level expression in the CBC compartment. Sox9lo cells also demonstrate stem cell traits in vitroSox9 expression also seen in non-stem cell populations, including Paneth cellsFormeister et al., 2009Formeister E.J. Sionas A.L. Lorance D.K. Barkley C.L. Lee G.H. Magness S.T. Distinct SOX9 levels differentially mark stem/progenitor populations and enteroendocrine cells of the small intestine epithelium.Am. J. Physiol. Gastrointest. Liver Physiol. 2009; 296: G1108-G1118Crossref PubMed Scopus (42) Google Scholar, Mori-Akiyama et al., 2007Mori-Akiyama Y. van den Born M. van Es J.H. Hamilton S.R. Adams H.P. Zhang J. Clevers H. de Crombrugghe B. SOX9 is required for the differentiation of paneth cells in the intestinal epithelium.Gastroenterology. 2007; 133: 539-546Abstract Full Text Full Text PDF PubMed Scopus (124) Google ScholarLgr5/Gpr49CBCSupported by in vivo lineage tracing and organoid cultureNo reliable antibodies available for detecting endogenous membrane expressionBarker et al., 2007Barker N. van Es J.H. Kuipers J. Kujala P. van den Born M. Cozijnsen M. Haegebarth A. Korving J. Begthel H. Peters P.J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (1174) Google Scholar, Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. et al.Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (519) Google ScholarOLFM4CBCCBC stem cell-specific expression as determined by microarray/in situCannot be used to isolate stem cell populationsVan der Flier et al., 2009Van der Flier L.G. van Gijn M.E. Hatzis P. Kujala P. Haegebarth A. Stange D.E. Begthel H. van den Born M. Guryev V. Oving I. et al.Transcription factor Achaete scute-like 2 (Ascl2) controls intestinal stem cell fate.Cell. 2009; (in press)PubMed Google ScholarASCL2CBCCBC stem cell-specific expression as determined by microarray/in situ. Essential for stem cell survival in-vivoCannot be used to isolate stem cell populationsVan der Flier et al., 2009Van der Flier L.G. van Gijn M.E. Hatzis P. Kujala P. Haegebarth A. Stange D.E. Begthel H. van den Born M. Guryev V. Oving I. et al.Transcription factor Achaete scute-like 2 (Ascl2) controls intestinal stem cell fate.Cell. 2009; (in press)PubMed Google ScholarBMPR1α/P-PTEN+4Overlapping expression with LRC populationNo functional demonstration of stemness. Also reported to mark enteroendocrine cellsHe et al., 2007He X.C. Yin T. Grindley J.C. Tian Q. Sato T. Tao W.A. Dirisina R. Porter-Westpfahl K.S. Hembree M. Johnson T. et al.PTEN-deficient intestinal stem cells initiate intestinal polyposis.Nat. Genet. 2007; 39: 189-198Crossref PubMed Scopus (203) Google Scholar, He et al., 2004He X.C. Zhang J. Tong W.G. Tawfik O. Ross J. Scoville D.H. Tian Q. Zeng X. He X. Wiedemann L.M. et al.BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling.Nat. Genet. 2004; 36: 1117-1121Crossref PubMed Scopus (451) Google Scholar, Bjerknes and Cheng, 2005Bjerknes M. Cheng H. Re-examination of P-PTEN staining patterns in the intestinal crypt.Nat. Genet. 2005; 37 (author reply 1017–1018): 1016-1017Crossref PubMed Scopus (14) Google ScholarWIP1+4 (& less frequently in the CBC zone)Overlapping expression with LRC populationNo functional demonstration of stemnessDemidov et al., 2007Demidov O.N. Timofeev O. Lwin H.N. Kek C. Appella E. Bulavin D.V. Wip1 phosphatase regulates p53-dependent apoptosis of stem cells and tumorigenesis in the mouse intestine.Cell Stem Cell. 2007; 1: 180-190Abstract Full Text Full Text PDF PubMed Scopus (47) Google ScholarmTERT+4 (very infrequently)Overlapping expression with LRC populationNo functional demonstration of stemness. Only marks minor subset of LRC populationBreault et al., 2008Breault D.T. Min I.M. Carlone D.L. Farilla L.G. Ambruzs D.M. Henderson D.E. Algra S. Montgomery R.K. Wagers A.J. Hole N. Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells.Proc. Natl. Acad. Sci. USA. 2008; 105: 10420-10425Crossref PubMed Scopus (44) Google ScholarSOX4+4Expression restricted to +4 cell positionNo functional demonstration of stemnessVan der Flier et al., 2007Van der Flier L.G. Sabates-Bellver J. Oving I. Haegebarth A. De Palo M. Anti M. Van Gijn M.E. Suijkerbuijk S. Van de Wetering M. Marra G. et al.The Intestinal Wnt/TCF Signature.Gastroenterology. 2007; 132: 628-632Abstract Full Text Full Text PDF PubMed Scopus (190) Google ScholarsFRP5+4Expression restricted to +4 cell positionNo functional demonstration of stemnessGregorieff et al., 2005Gregorieff A. Pinto D. Begthel H. Destree O. Kielman M. Clevers H. Expression pattern of Wnt signaling components in the adult intestine.Gastroenterology. 2005; 129: 626-638PubMed Google ScholarDCAMKL-1+4Overlapping expression with LRC populationNo functional demonstration of stemness. Also reported to mark differentiated Tuft cellsGiannakis et al., 2006Giannakis M. Stappenbeck T.S. Mills J.C. Leip D.G. Lovett M. Clifton S.W. Ippolito J.E. Glasscock J.I. Arumugam M. Brent M.R. et al.Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches.J. Biol. Chem. 2006; 281: 11292-11300Crossref PubMed Scopus (92) Google Scholar, May et al., 2008May R. Riehl T.E. Hunt C. Sureban S.M. Anant S. Houchen C.W. Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice.Stem Cells. 2008; 26: 630-637Crossref PubMed Scopus (112) Google Scholar, Gerbe et al., 2009Gerbe F. Brulin B. Makrini L. Legraverend C. Jay P. DCAMKL-1 expression identifies Tuft cells rather than stem cells in the adult mouse intestinal epithelium.Gastroenterology. 2009; 137 (author reply 2180–2181): 2179-2180Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, Bezencon et al., 2008Bezencon C. Furholz A. Raymond F. Mansourian R. Metairon S. Le Coutre J. Damak S. Murine intestinal cells expressing Trpm5 are mostly brush cells and express markers of neuronal and inflammatory cells.J. Comp. Neurol. 2008; 509: 514-525Crossref PubMed Scopus (35) Google ScholarBmi1+4Supported by in-vivo lineage tracingOnly present in 10% of proximal SI. Likely overlap with Lgr5+ CBC stem cellsSangiorgi and Capecchi, 2008Sangiorgi E. Capecchi M.R. Bmi1 is expressed in vivo in intestinal stem cells.Nat. Genet. 2008; 40: 915-920Crossref PubMed Scopus (408) Google Scholar Open table in a new tab The majority of the published intestinal stem cell markers are reported to be specific for the +4 cell population, reflecting the prevailing view that these are the true adult stem cells. However, the stem cell zone model has seen a renaissance recently, following the identification of leucine-rich G protein-coupled receptor 5 (LGR5) (GPR49) as a specific CBC marker. Lgr5 encodes an orphan GPCR of unknown function related to the sex hormone receptors (Barker and Clevers, 2010Barker N. Clevers H. Leucine-rich repeat-containing G-protein-coupled receptors as markers of adult stem cells.Gastroenterology. 2010; 138: 1681-1696Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Lgr5 was identified during an in situ screen of a Wnt target gene panel for restricted expression at the crypt base (Barker et al., 2007Barker N. van Es J.H. Kuipers J. Kujala P. van den Born M. Cozijnsen M. Haegebarth A. Korving J. Begthel H. Peters P.J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (1174) Google Scholar). The majority of the Wnt target genes were either broadly expressed throughout the crypt or were restricted to the differentiated Paneth cells at the crypt base. This observation reflects the crucial roles of Wnt signaling in maintaining both the proliferative TA compartment and in driving maturation of the Paneth cells in vivo. In contrast, Lgr5 expression was restricted to the proliferative CBC cells, establishing LGR5 as the first specific marker for this candidate stem cell population. The stem cell potential of these LGR5+ CBC cells was subsequently assessed by in vivo lineage tracing using an Lgr5-EGFP-ires-CreERT2/Rosa26RlacZ mouse model. Lgr5-EGFP reporter gene activity in these mice faithfully recapitulated the endogenous Lgr5 expression pattern. Following stochastic induction of LGR5-CRE activity using tamoxifen, lacZ reporter gene activity was initially observed in isolated CBC cells. At later time points, this lacZ genetic mark was found in cells of all lineages throughout the crypt-villus epithelium, demonstrating the multipotency of the LGR5+ CBC cells. Importantly, this tracing was maintained throughout the lifetime of the mouse, identifying the LGR5+ CBC cells as self-renewing, multipotent adult stem cells responsible for epithelial homeostasis under physiological conditions (Barker and Clevers, 2007Barker N. Clevers H. Tracking down the stem cells of the intestine: strategies to identify adult stem cells.Gastroenterology. 2007; 133: 1755-1760Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). This approach also identified LGR5+ cells at the base of the colonic crypts as adult stem cells. Gene expression profiling of FACS-sorted LGR5-EGFP+ cells revealed the adult stem cell “transcriptome.” This list included multiple Wnt target genes, reflecting Wnt signaling activity within the stem cell compartment, as well as other stem cell markers such as OLFM4 and ASCL2 (van der Flier et al., 2009avan der Flier L.G. Haegebarth A. Stange D.E. van de Wetering M. Clevers H. OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells.Gastroenterology. 2009; 137: 15-17Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Genetic ablation of ASCL2 expression in vivo resulted in silencing of the stem cell signature and rapid stem cell death, revealing a crucial role for this transcription factor as a master regulator of stemness (van der Flier et al., 2009bvan der Flier L.G. van Gijn M.E. Hatzis P. Kujala P. Haegebarth A. Stange D.E. Begthel H. van den Born M. Guryev V. Oving I. et al.Transcription factor achaete scute-like 2 controls intestinal stem cell fate.Cell. 2009; 136: 903-912Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Using a novel intestinal epithelial culture method, single LGR5-EGFP+ cells isolated from the small intestine were shown to be capable of generating self-renewing intestinal organoids in vitro (Sato et al., 2009Sato T. Vries R.G. Snippert H.J. van de Wetering M. Barker N. Stange D.E. van Es J.H. Abo A. Kujala P. Peters P.J. et al.Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (519) Google Scholar). These epithelial organoids had a remarkably similar architecture and comp