Aminimal definition of stemness states that a stem

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1 Perspective Tracking Down the Stem Cells of the Intestine: Strategies to Identify Adult Stem Cells NICK BARKER and HANS CLEVERS Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, The Netherlands Aminimal definition of stemness states that a stem cell should be able to maintain itself over long periods of time, while producing all differentiated cell types of the pertinent tissue. Two strategies allow for a direct demonstration of these characteristics. The first and most widely pursued strategy involves the isolation of candidate stem cell populations, followed by in vitro culture and/or transplantation into recipient animals. This approach has been highly successful in the description of the hematopoietic ( bone marrow ) stem cell, and more recently of cancer stem cells in leukemia 1 and in solid tumors. 2 5 In a dramatic example of this approach, Visvader et al 6 showed that a single isolated mammary gland stem cell can regrow an entire mammary gland. In the second strategy, candidate stem cells are genetically marked in situ, facilitating visualization of the modified stem cell and its offspring over time. As an example, Cotsarelis et al 7 marked hair follicle stem cells in mice using a progesterone-activatable version of the Cre recombinase enzyme, expressed specifically in these stem cells using a transgenic keratin-15 promoter. Activation of the Cre enzyme by progesterone irreversibly activated the genetic marker R26R-LacZ by excision of a road-block DNA sequence specifically in the stem cells. These cells and their offspring can then be visualized by a simple blue stain for LacZ activity. In many tissues, the technology is not available to isolate or genetically mark adult stem cells to definitively demonstrate their stemness. In such cases, long-term retention of DNA labels is often used as a surrogate marker of stemness. The propensity of stem cells to retain DNA labels is commonly believed to reflect the fact that stem cells tend to be quiescent, whereas their direct descendants, the vigorously proliferating transit-amplifying cells, rapidly dilute out any incorporated DNA label. As an alternative mechanism behind label retention, Cairns 8 formulated the immortal strand hypothesis 35 years ago. He proposed that stem cells may selectively retain their old DNA strands, while donating the newly synthesized DNA strands to their transit amplifying daughters. To this date, the immortal strand hypothesis has been the subject of controversy. 9,10 Few convincing examples have been reported, and its molecular machinery remains unidentified. Stem Cells of the Small Intestine The crypts of the small intestine have long been known to harbor a functional stem cell compartment, yet a paucity of unique molecular markers has hampered the definitive identification of the stem cells proper. Although there is general agreement that every crypt contains 4 6 independent stem cells, 2 schools of thought exist as to their exact identity. For more than a century, the small intestinal crypt has been viewed as a tube of proliferating cells bounded from below by Paneth cells. 11 Since the late 1950s, the prevailing school of thought has therefore placed the stem cells at position 4 relative to the crypt bottom, with the first 3 positions being occupied by the terminally differentiated Paneth cells. Potten et al 12,13 have provided experimental support for the 4 stem cell model, by reporting that radiation-sensitive, labelretaining cells reside specifically at this position. The second school of thought has been based on the identification 30 years ago of crypt base columnar (CBC) cells, small cycling cells hidden between the Paneth cells 14 (Figure 1A). Originally based on morphologic considerations, 15 but more recently also on clonal marking techniques, 16 Leblond, Cheng, and Bjerknes 17,18 have proposed that the CBC cells, residing within a stem cell zone at the crypt base, may represent the true stem cells (Figure 1B). Neither of these 2 hypotheses on stem cell identity is supported by direct definitive evidence for stemness along the lines discussed in the first paragraph. For such evidence, the identification of a unique stem cell marker would be of paramount importance. Wnt Target Genes as Candidate Stem Cell Markers The Wnt signaling pathway has a unique and central role in the physiology and pathology of the intestine. Not only is it the dominant force behind the proliferative activity in intestinal crypts, its mutational activation is the principal cause of colon cancer. 22,23 We have determined the genetic program that 2007 by the AGA Institute /07/$32.00 doi: /j.gastro GASTROENTEROLOGY 2007;133:

2 Figure 1. CBC cells located at the crypt base in the small intestine. (A) Electron microscopy identifies the CBC cells as small, slender cells interspersed between the Paneth cells. (Reprinted with permission from Cheng H, Leblond CP. Origin, differentiation and renewal of the 4 main epithelial cell types in the mouse small intestine. I. Columnar cell. Am J Anat 1974;141: ) (B) Leblond, Cheng, and Bjerknes proposed that CBC cells are likely to be the origin of the various cell types on the small intestinal epithelium. (Adapted from Cheng H, Leblond CP. Origin, differentiation and renewal of the 4 main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the 4 epithelial cell types. Am J Anat 1974;141: ) is inappropriately activated in human colon cancer cells upon mutation of the Wnt regulatory APC gene, and found that the same genetic program is physiologically expressed in proliferative cells in healthy crypts. 24 The program consists of a core of some 80 Wnt target genes. 25 We reasoned that the possibility existed that a subset of these genes could be uniquely expressed in crypt stem cells. To identify such markers, we performed histologic expression studies for each of the 80 Wnt target genes. Although the great majority of the genes were expressed throughout the proliferative crypt compartment, we detected several genes with a much more restricted expression within crypts. One of these, the Lgr5/Gpr49 gene, was expressed in a particularly unique fashion. The Lgr5 gene encodes an orphan G proteincoupled receptor (GPCR), characterized by a large leucine-rich extracellular domain (Figure 2A). It is closely related to GPCRs with glycoprotein ligands, such as the TSH-, FSH- and LH-receptors (Figure 2B). Lgr5 was on our original list of Tcf4 targets in colorectal cancer, but has since been observed to be overexpressed in cancers of the ovary and liver. 26,27 Moreover, it is highly expressed in stem cells of another Wnt-driven self-renewing structure, the hair follicle. 7 In situ hybridization on small intestine of an APC min mouse revealed expression in a limited number of crypt bottom cells, as well as in adenomas. 28 This expression pattern clearly differed from that obtained with any other of the 80-gene Wnt signature. The Lgr5 gene marked small cells interspersed between Paneth cells, reminiscent of the cycling CBC cells, described by Leblond et al

3 Perspective continued tion of the tongue and lower jaw causes newborns to swallow large amounts of air, leading to early neonatal death, a phenotype confirmed in our mice. In the heterozygous Lgr5-LacZ mice we observed a dynamic and complex expression pattern before birth (Barker et al, A dynamic expression pattern for LGR5 during embryogenesis [2007] in preparation). In contrast, expression in adult mice was restricted to rare, scattered cells in the eye, brain, hair follicle, mammary gland, reproductive organs, stomach, and intestinal tract. In the small intestine, we confirmed the Lgr5 expression in the CBC cells between the Paneth cells (Figure 3B). By morphology, the slender Lgr5 ve CBC cells with their scant cytoplasm and flat, pizza slice shaped nuclei were readily distinguishable from the adjacent Paneth cells. The CBC cells invariably expressed the Ki67 cell-cycle marker. By BrdU labeling, we determined that the average cycling time of CBC cells is in the order of 1 day, somewhat counterintuitive to the notion that these could be stem cells. In colon, Lgr5 expression was observed in cells of similar number and shape at the bottom Figure 2. Lgr5/Gpr49 is an orphan G-protein coupled receptor related to the glycoprotein hormone receptors LHR, FSHR, and TSHR. (A) Predicted structure of Lgr5, comprising a large amino-terminal extracellular domain containing leucine-rich repeats for ligand interaction, a 7-transmembrane domain, and a short c-terminal domain for signal transduction. (B) The phylogenetic relationship between Lgr5 and family members (reproduced with permission from Luo CW, Hsueh AJ. Genomic analyses of the evolution of LGR genes. Chang Gung Med J 2006;29:2 8).31 Although Bursicon has been identified as the ligand for Drosophila LGR2, there are currently no known ligands for the remaining group B receptors Lgr4, Lgr5, and Lgr6. To describe its expression in greater detail, we obtained a knock-in allele, in which LacZ was integrated just N-terminal to the first transmembrane domain of Lgr5, essentially creating a null allele. While our study was in progress, Morita et al29 published the Lgr5-/- phenotype. A malforma- Figure 3. Lgr5 expression is restricted to the CBC cells of the small intestine. (A) GFP expression in CBC cells of Lgr5-EGFP knock-in mice. (B) LacZ expression in CBC cells of Lgr5-LacZ knock-in mice. (C) Cartoon depicting the presence of 4-6 Lgr5-positive CBC cells intermingled with the Paneth cells at the base of the small intestinal crypts. 1757

4 of the crypts. CBC cells, however, had never been observed in the colon. Encouraged by these observations, we generated another knock-in allele, in which we integrated a cassette into exon I which encoded green fluorescent protein (GFP) and a tamoxifen-inducible version of the Cre recombinase enzyme. The GFP pattern observed in adult tissues faithfully recapitulated the Lgr5-LacZ expression pattern. By confocal imaging, we could visualize live GFP cells in small intestine (Figure 3A) and colon. Immuno-electron microscopy illustrated the unique ultrastructural anatomy of the GFP ve CBC cells. Typically, the CBC cells were relatively broad at their base and contained a flat, wedge-shaped nucleus and very little cytoplasm or organelles. A slender extension of apical cytoplasm extended between the neighbouring Paneth cells to the crypt lumen and carried some microvilli. Following the genetic marking strategy that had been successfully used by Cotsarelis et al 7 in the hair follicle, we then crossed the knock-in allele with the Cre-activatable R26R-LacZ reporter. 30 Injection of tamoxifen should activate the CreERT2 fusion enzyme uniquely in the CBC cells. Cre-mediated excision of the roadblock sequence in the R26R-LacZ reporter should then irreversibly mark these cells. Moreover, although potential progeny of the CBC cells should no longer express GFP, the activated LacZ reporter should function as a genetic mark, facilitating lineage tracing. The strategy worked surprisingly well. Adult mice were injected with a low-dose tamoxifen pulse to activate the R26R-LacZ reporter stochastically and at low frequency in CBC cells. The mice were humanely killed at 1, 5, 12, or 60 days postinduction. On day 1, occasional CBC cells in the crypts of small intestine and colon were observed to express the LacZ marker. 28 At later time points, parallel ribbons of blue cells emanated from these marked CBC cells and ran up the side of adjacent villi. As predicted, the ribbons reached the villus tips 5 days postinduction (Figure 4). The CBC cells were capable of long-term maintenance of the self-renewing epithelium; in day 60 intestines, the frequency of blue crypts and ribbons was essentially identical to that seen in the first week postinduction. The blue ribbons of 60 day-induced intestine contained enterocytes, goblet cells, Paneth cells, and enteroendocrine cells at normal ratios. Using mutational marking, Cheng and Bjerknes 16 previously reported the existence of different types of long-lived epithelial clones, namely, columnar (enterocyte) clones, mucous (goblet) clones, and mixed clones. The clones observed in our study were exclusively of the mixed variety. We made essentially identical observations in the colon. Blue clones emanated from the crypt bottom and contained colonocytes as well as goblet cells, and essentially remained unchanged during the 60 days of chase. Thus, the Lgr5 ve cells in small intestine and colon fulfilled the stem cell requirements in being long lived, as well as being capable of generating all cell types of the epithelium. Figure 4. Whole-mount photograph of small intestine from Lgr5- EGFP-Ires-CreERT2/Rosa26-LacZ mice induced for 5 days with lowdose tamoxifen. The ribbon of blue cells extending from the crypt (outlined) to the tip of the villus demonstrates that Lgr5-positive CBC cells are capable of generating the entire villus epithelium. Similar observations at much later time points (60 days postinduction) prove that the Lgr5-positive CBC cells are multipotent and long lived, allowing us to conclude that they are the true stem cells of the intestine. Crossing the Lgr5-EGFP mice with APC min mice allowed us to examine Lgr5 expression within the premalignant adenomas, which spontaneously arise in their intestine as a result of chronic activation of the Wnt signalling pathway. 22,23 In contrast with other Wnt target genes, which typically exhibit a uniform high-level expression throughout these tumors, Lgr5-GFP expression was restricted to a small number of cells within large adenomas. It is therefore tempting to speculate that Lgr5 marks not only the normal intestinal stem cells, but also the limited population of tumor-initiating/propagating cells thought to exist within colon cancers the so-called cancer stem cells. Conclusions The observations on Lgr5 confirm that each crypt in the small intestine and colon contains approximately 6 long-lived stem cells. It is somewhat unexpected that these stem cells are not quiescent, but that they complete a cell cycle every day. The CBC cells likely persist for the entire lifetime of a mouse. This implies that these cells have to go through many hundreds of cell divisions, without apparent stem cell exhaustion, telomere collapse, loss of essential genetic information, or excessive malignant transformation. The Lgr5 marker allows identification of adult stem cells based on a single parameter. This phenomenon may not be 1758

5 restricted to the intestine; we observe highly restricted Lgr5 expression in a variety of other tissues. In the hair follicle, mammary gland, and stomach epithelium, ongoing lineage tracing experiments support the notion that Lgr5 ve cells represent stem cells (Barker N and Clevers H, 2007, unpublished data). Lgr5 may thus represent a more general marker of adult stem cells. We first identified Lgr5 as a gene expressed in colon cancer cells. It is expressed in other cancers and, as described in our study, also in premalignant mouse adenomas. Based on these observations, we speculate that Lgr5 may mark cancer stem cells ( tumor-initiating cells ) in solid tumors. Of note, the Lgr5 gene encodes a surface receptor. If Lgr5-positive cells in human tumors indeed can be recognized by monoclonal antibodies, it will become possible to visualize, isolate, and even eradicate Lgr5-bearing cancer stem cells. One application of such antibodies could be in the evaluation of established or experimental cancer treatment regimens as to their effectiveness toward eradication of cancer stem cells. The specific expression of GFP driven from the Lgr5 locus allows the visualization of live stem cells in their niche. With this tool, it should become possible to study stem cell niche interactions, asymmetric cell divisions, and so on. An important outstanding question in cancer biology is that of the identity of the cell that sustains the first oncogenic mutation. Is the cell that initiates a tumor always a stem cell, or can it also be a transit-amplifying cell, or even a terminally differentiated cell? The inducible Cre enzyme knocked into the Lgr5 locus allows us to specifically introduce oncogenic mutations in stem cells and compare the effect with the same mutation introduced in any other cell type. The molecular function of Lgr5 is currently unknown. It is predicted to be a receptor for a peptide ligand of unknown nature. Given the close association of Lgr5 expression with stemness, it is tempting to speculate that the ligand controls stem cell behavior. It may thus present a pharmacological promise in its own right. References 1. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 1997;3: Dalerba P, Dylla SJ, Park IK, et al. Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA 2007;104: O Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007;445: Prince ME, Sivanandan R, Kaczorowski A, et al. Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci U S A 2007;104: Singh SK, Hawkins C, Clarke ID, et al. 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Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am J Anat 1974;141: Cheng H, Leblond CP. 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: Bjerknes M, Cheng H. Clonal analysis of mouse intestinal epithelial progenitors. Gastroenterology 1999;116: Bjerknes 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: Bjerknes 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: Hoffman J, Kuhnert F, Davis CR, et al. Wnts as essential growth factors for the adult small intestine and colon. Cell Cycle 2004; 3: Korinek V, Barker N, Moerer P, et al. 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6 lular carcinomas with beta-catenin mutations. Hepatology 2003; 37: Barker N, van Es JH, Kuipers J, et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 2007; 449: Morita H, Mazerbourg S, Bouley DM, et al. Neonatal lethality of LGR5 null mice is associated with ankyloglossia and gastrointestinal distension. Mol Cell Biol 2004;24: Soriano P. Generalized lacz expression with the ROSA26 Cre reporter strain. Nat Genet 1999;21: Luo CW, Hsueh AJ. Genomic analyses of the evolution of LGR genes. Chang Gung Med J 2006;29:2 8. Address requests for reprints to: Nick Barker, PhD, Hubrecht Institute for Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584CT Utrecht, The Netherlands. n.barker@niob.knaw.nl; fax: (31) H. Clevers is an inventor on several patents involving Wnt target genes, as is N. Barker. Supported by the following sources: Genmab B.V., Koninklijke Nederlandse Akademie van Wetenschappen (KNAW), Koningin Wilhelmina Fonds (KWF), Maag Lever en Darm Stichting (MLDS), European Molecular Biology Organization (EMBO), SenterNovem BSIK, Louis Jeantet Foundation, and the European Union (EU:FOOD- CT ). Ramstedt of the Ramstedt Pyloromyotomy Conrad Ramstedt ( ) was born in the Prussian village of Hamersleben, the son of a local physician. After preparatory courses at the gymnasium in Magdeburg, he proceeded to study medicine at Heidelberg, Berlin, and then Halle, where he received his medical degree. After working as an assistant in the surgical clinic at Halle, he served with distinction as a medical officer in the German army during World War I. On discharge in 1919, he was appointed chief surgeon to the Rafaelsklinik at Munster where he remained for the rest of his professional career. Congenital pyloric stenosis in infants was first described by the Danish pediatrician Harald Hirschsprung in Attempts to correct the defect by medical or surgical means were almost uniformly fatal. In 1912, while at Halle, Ramstedt described a safe and effective method of incising the pyloric sphincter, then leaving the cut open, a procedure adapted shortly thereafter by Ernst Heller at a tight lower esophageal sphincter to relieve achalasia. Ramstedt was a quiet and humble man. His 1912 paper was the only one he ever published on the subject. Contributed by WILLIAM S. HAUBRICH, MD The Scripps Clinic, La Jolla, California Conrad Ramstedt, MD. Reprinted with permission (Photo source: ramstedt.htm right). 1760