Hans Clevers

Publications of Hans Clevers

• A CRITICAL ROLE FOR THE WNT EFFECTOR TCF4 IN ADULT INTESTINAL HOMEOSTATIC SELF-RENEWAL.

  Authors: Johan H van Es, Andrea Haegebarth, Pekka Kujala, Shalev Itzkovitz, Bon-Kyoung Koo, Sylvia F Boj, Jeroen Korving, Maaike van den Born, Alexander van Oudenaarden, Sylvie Robine, Hans Clevers

  Molecular and cellular biology. 03/2012;

  Throughout life, intestinal Lgr5(+) stem cells give rise to proliferating transient amplifying cells in crypts, which subsequently differentiate into one of the five main cell types and migrate alongThroughout life, intestinal Lgr5(+) stem cells give rise to proliferating transient amplifying cells in crypts, which subsequently differentiate into one of the five main cell types and migrate along the crypt-villus axis. These dynamic processes are coordinated by a relatively small number of evolutionarily conserved signaling pathways, which includes the Wnt signaling pathway. The DNA-binding proteins of the T-cell factor family, Tcf1/Tcf7, Lef, Tcf3/Tcf7l1 and Tcf4/Tcf7l2, constitute the downstream effectors of the Wnt signaling pathway. While Tcf4 is the major member active during embryogenesis, the role of these Wnt effectors in the homeostasis of the adult mouse intestinal epithelium is unresolved. Using Tcf1(-/-), Tcf3(flox) and novel Tcf4(flox) mice, we demonstrate an essential role for Tcf4 during homeostasis of the adult mouse intestine.

• Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling.

  Authors: Vivian W Y Wong, Daniel E Stange, Mahalia E Page, Simon Buczacki, Agnieszka Wabik, Satoshi Itami, Marc van de Wetering, Richard Poulsom, Nicholas A Wright, Matthew W B Trotter, Fiona M Watt, Doug J Winton, Hans Clevers, Kim B Jensen

  Nature cell biology. 03/2012; 14(4):401-8.

  Maintenance of adult tissues is carried out by stem cells and is sustained throughout life in a highly ordered manner. Homeostasis within the stem-cell compartment is governed by positive- andMaintenance of adult tissues is carried out by stem cells and is sustained throughout life in a highly ordered manner. Homeostasis within the stem-cell compartment is governed by positive- and negative-feedback regulation of instructive extrinsic and intrinsic signals. ErbB signalling is a prerequisite for maintenance of the intestinal epithelium following injury and tumour formation. As ErbB-family ligands and receptors are highly expressed within the stem-cell niche, we hypothesize that strong endogenous regulators must control the pathway in the stem-cell compartment. Here we show that Lrig1, a negative-feedback regulator of the ErbB receptor family, is highly expressed by intestinal stem cells and controls the size of the intestinal stem-cell niche by regulating the amplitude of growth-factor signalling. Intestinal stem-cell maintenance has so far been attributed to a combination of Wnt and Notch activation and Bmpr inhibition. Our findings reveal ErbB activation as a strong inductive signal for stem-cell proliferation. This has implications for our understanding of ErbB signalling in tissue development and maintenance and the progression of malignant disease.

• Wnt/β-Catenin and MAPK Signaling: Allies and Enemies in Different Battlefields.

  Authors: Daniele Guardavaccaro, Hans Clevers

  Science signaling. 01/2012; 5(219):pe15.

  Two papers published in Science Signaling reveal extensive crosstalk between Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling in cancer. Although both studies describe previouslyTwo papers published in Science Signaling reveal extensive crosstalk between Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling in cancer. Although both studies describe previously unknown links between these two signaling pathways, the relationship between Wnt/β-catenin and MAPK signaling depends on the specific cellular context. Indeed, in melanoma, hyperactivated MAPK signaling down-regulates the Wnt/β-catenin signal transduction cascade, thereby establishing a negative crosstalk between the two signaling pathways. In contrast, in colorectal cancer, stimulation of the Wnt/β-catenin pathway leads to activation of the MAPK pathway through Ras stabilization, representing an example of positive crosstalk. Moreover, activation of Wnt/β-catenin signaling has context-dependent functions that trigger opposing effects on tumor growth. In melanoma, aberrant activation of Wnt/β-catenin signaling may have anti-oncogenic functions by promoting programmed cell death; by contrast, in the intestine, Wnt/β-catenin signaling drives malignant transformation. Thus, there is no single correct way to target the Wnt/β-catenin pathway for all cancers.

• Cdx2 determines the fate of postnatal intestinal endoderm.

  Authors: Emma J Stringer, Isabelle Duluc, Thoueiba Saandi, Irwin Davidson, Monika Bialecka, Toshiro Sato, Nick Barker, Hans Clevers, Catrin A Pritchard, Doug J Winton, Nicholas A Wright, Jean-Noel Freund, Jacqueline Deschamps, Felix Beck

  Development (Cambridge, England). 12/2011; 139(3):465-74.

  Knock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in theKnock out of intestinal Cdx2 produces different effects depending upon the developmental stage at which this occurs. Early in development it produces histologically ordered stomach mucosa in the midgut. Conditional inactivation of Cdx2 in adult intestinal epithelium, as well as specifically in the Lgr5-positive stem cells, of adult mice allows long-term survival of the animals but fails to produce this phenotype. Instead, the endodermal cells exhibit cell-autonomous expression of gastric genes in an intestinal setting that is not accompanied by mesodermal expression of Barx1, which is necessary for gastric morphogenesis. Cdx2-negative endodermal cells also fail to express Sox2, a marker of gastric morphogenesis. Maturation of the stem cell niche thus appears to be associated with loss of ability to express positional information cues that are required for normal stomach development. Cdx2-negative intestinal crypts produce subsurface cystic vesicles, whereas untargeted crypts hypertrophy to later replace the surface epithelium. These observations are supported by studies involving inactivation of Cdx2 in intestinal crypts cultured in vitro. This abolishes their ability to form long-term growing intestinal organoids that differentiate into intestinal phenotypes. We conclude that expression of Cdx2 is essential for differentiation of gut stem cells into any of the intestinal cell types, but they maintain a degree of cell-autonomous plasticity that allows them to switch on a variety of gastric genes.

• Method for estimating the single molecular affinity.

  Authors: Richard B M Schasfoort, Wim de Lau, Alex van der Kooi, Hans Clevers, Gerard H M Engbers

  Analytical biochemistry. 12/2011; 421(2):794-6.

  Affinity constants (k(d), k(a), and K(D)) can be determined by methods that apply immobilized ligands such as immunoassays and label-free biosensor technologies. This article outlines a new surfaceAffinity constants (k(d), k(a), and K(D)) can be determined by methods that apply immobilized ligands such as immunoassays and label-free biosensor technologies. This article outlines a new surface plasmon resonance (SPR) array imaging method that yields affinity constants that can be considered as the best estimate of the affinity constant for single biomolecular interactions. Calculated rate (k(d) and k(a)) and dissociation equilibrium (K(D)) constants for various ligand densities and analyte concentrations are extrapolated to the K(D) at the zero response level (K(D)(R0)). By applying this method to an LGR5-exo-Fc-RSPO1-FH interaction couple, the K(D)(R0) was determined as 3.1nM.

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• Controlled gene expression in primary Lgr5 organoid cultures.

  Authors: Bon-Kyoung Koo, Daniel E Stange, Toshiro Sato, Wouter Karthaus, Henner F Farin, Meritxell Huch, Johan H van Es, Hans Clevers

  Nature methods. 12/2011; 9(1):81-3.

  The study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive andThe study of gene function in endodermal epithelia such as of stomach, small intestine and colon relies heavily on transgenic approaches. Establishing such animal models is laborious, expensive and time-consuming. We present here a method based on Cre recombinase-inducible retrovirus vectors that allows the conditional manipulation of gene expression in primary mouse organoid culture systems.

• Integrated genome-wide analysis of transcription factor occupancy, RNA polymerase II binding and steady-state RNA levels identify differentially regulated functional gene classes.

  Authors: Michal Mokry, Pantelis Hatzis, Jurian Schuijers, Nico Lansu, Frans-Paul Ruzius, Hans Clevers, Edwin Cuppen

  Nucleic acids research. 09/2011; 40(1):148-58.

  Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation.Routine methods for assaying steady-state mRNA levels such as RNA-seq and micro-arrays are commonly used as readouts to study the role of transcription factors (TFs) in gene expression regulation. However, cellular RNA levels do not solely depend on activity of TFs and subsequent transcription by RNA polymerase II (Pol II), but are also affected by RNA turnover rate. Here, we demonstrate that integrated analysis of genome-wide TF occupancy, Pol II binding and steady-state RNA levels provide important insights in gene regulatory mechanisms. Pol II occupancy, as detected by Pol II ChIP-seq, was found to correlate better with TF occupancy compared to steady-state RNA levels and is thus a more precise readout for the primary transcriptional mechanisms that are triggered by signal transduction. Furthermore, analysis of differential Pol II occupancy and RNA-seq levels identified genes with high Pol II occupancy and relatively low RNA levels and vice versa. These categories are strongly enriched for genes from different functional classes. Our results demonstrate a complementary value in Pol II chip-seq and RNA-seq approaches for better understanding of gene expression regulation.

• Isolation and in vitro expansion of human colonic stem cells.

  Authors: Peter Jung, Toshiro Sato, Anna Merlos-Suárez, Francisco M Barriga, Mar Iglesias, David Rossell, Herbert Auer, Mercedes Gallardo, Maria A Blasco, Elena Sancho, Hans Clevers, Eduard Batlle

  Nature medicine. 09/2011; 17(10):1225-7.

  Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell typesHere we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population.

• Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.

  Authors: Toshiro Sato, Daniel E Stange, Marc Ferrante, Robert G J Vries, Johan H Van Es, Stieneke Van den Brink, Winan J Van Houdt, Apollo Pronk, Joost Van Gorp, Peter D Siersema, Hans Clevers

  Gastroenterology. 09/2011; 141(5):1762-72.

  We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple cryptWe previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon. Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems. Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus. We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.

• Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.

  Authors: Wim de Lau, Nick Barker, Teck Y Low, Bon-Kyoung Koo, Vivian S W Li, Hans Teunissen, Pekka Kujala, Andrea Haegebarth, Peter J Peters, Marc van de Wetering, Daniel E Stange, Johan E van Es, Daniele Guardavaccaro, Richard B M Schasfoort, Yasuaki Mohri, Katsuhiko Nishimori, Shabaz Mohammed, Albert J R Heck, Hans Clevers

  Nature. 08/2011; 476(7360):293-7.

  The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs WntThe adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.

• Comparative proteomics of colon cancer stem cells and differentiated tumor cells identifies BIRC6 as a potential therapeutic target.

  Authors: W J Van Houdt, B L Emmink, T V Pham, S R Piersma, A Verheem, R G Vries, S A Fratantoni, A Pronk, H Clevers, I H M Borel Rinkes, C R Jimenez, O Kranenburg

  Molecular & cellular proteomics : MCP. 07/2011; 10(12):M111.011353.

  Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have beenPatients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.

• Stem cell self-renewal in intestinal crypt.

  Authors: Benjamin D Simons, Hans Clevers

  Experimental cell research. 07/2011; 317(19):2719-24.

  As a rapidly cycling tissue capable of fast repair and regeneration, the intestinal epithelium has emerged as a favored model system to explore the principles of adult stem cell biology. However,As a rapidly cycling tissue capable of fast repair and regeneration, the intestinal epithelium has emerged as a favored model system to explore the principles of adult stem cell biology. However, until recently, the identity and characteristics of the stem cell population in both the small intestine and colon has remained the subject of debate. Recent studies based on targeted lineage tracing strategies, combined with the development of an organotypic culture system, have identified the crypt base columnar cell as the intestinal stem cell, and have unveiled the strategy by which the balance between proliferation and differentiation is maintained. These results show that intestinal stem cells operate in a dynamic environment in which frequent and stochastic stem cell loss is compensated by the proliferation of neighboring stem cells. We review the basis of these experimental findings and the insights they offer into the mechanisms of homeostatic stem cell regulation.

• Highly sensitive proteome analysis of FACS-sorted adult colon stem cells.

  Authors: Serena Di Palma, Daniel Stange, Marc van de Wetering, Hans Clevers, Albert J R Heck, Shabaz Mohammed

  Journal of proteome research. 06/2011; 10(8):3814-9.

  In proteomics, multidimensional liquid chromatography combined with mass spectrometry has become a standard technique to reduce sample complexity and tackle the vast dynamic range. Such fractionationIn proteomics, multidimensional liquid chromatography combined with mass spectrometry has become a standard technique to reduce sample complexity and tackle the vast dynamic range. Such fractionation is necessary to obtain a comprehensive analysis of biological samples such as tissues and cell lines. However, extensive fractionation comes at the expense of sample losses, hampering the analysis of limited material. We previously described a highly sensitive multidimensional chromatographic strategy based on a combination of hydrophilic interaction liquid chromatography and reversed phase chromatography, which allows proteomic analysis with minimal sample losses. Here we apply this strategy to the analysis of a limited number of FACS-sorted colon stem cells extracted from mouse intestine, obtaining a proteome coverage comparable to current methods that generally require 100-fold more starting material. We propose that this alternative multidimensional chromatographic technology will find ample application such as in the analysis of distinct cellular populations obtained by laser microdissection.

• Actomyosin-mediated cellular tension drives increased tissue stiffness and β-catenin activation to induce epidermal hyperplasia and tumor growth.

  Authors: Michael S Samuel, Jose I Lopez, Ewan J McGhee, Daniel R Croft, David Strachan, Paul Timpson, June Munro, Ewald Schröder, Jing Zhou, Valerie G Brunton, Nick Barker, Hans Clevers, Owen J Sansom, Kurt I Anderson, Valerie M Weaver, Michael F Olson

  Cancer cell. 06/2011; 19(6):776-91.

  Tumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tensionTumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tension to re-establish force equilibrium. To determine how actomyosin tension affects tissue homeostasis and tumor development, we expressed conditionally active ROCK2 in mouse skin. ROCK activation elevated tissue stiffness via increased collagen. β-catenin, a key element of mechanotranscription pathways, was stabilized by ROCK activation leading to nuclear accumulation, transcriptional activation, and consequent hyperproliferation and skin thickening. Inhibiting actomyosin contractility by blocking LIMK or myosin ATPase attenuated these responses, as did FAK inhibition. Tumor number, growth, and progression were increased by ROCK activation, while ROCK blockade was inhibitory, implicating actomyosin-mediated cellular tension and consequent collagen deposition as significant tumor promoters.

• Strategies for homeostatic stem cell self-renewal in adult tissues.

  Authors: Benjamin D Simons, Hans Clevers

  Cell. 06/2011; 145(6):851-62.

  In adult tissues, an exquisite balance exists between stem cell proliferation and the generation of differentiated offspring. Classically, it has been argued that this balance is obtained at theIn adult tissues, an exquisite balance exists between stem cell proliferation and the generation of differentiated offspring. Classically, it has been argued that this balance is obtained at the level of a single stem cell, which divides strictly into a new stem cell and a progenitor. However, recent evidence suggests that balance can also be achieved at the level of the stem cell population. Some stem cells might be lost due to differentiation or damage, whereas others divide symmetrically to fill this gap. Here, we consider the general strategies for stem cell self-renewal and review the evidence for stochastic stem cell fate in adult tissues across a range of tissue types and organisms.

• The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish.

  Authors: Yme U van der Velden, Liqin Wang, John Zevenhoven, Ellen van Rooijen, Maarten van Lohuizen, Rachel H Giles, Hans Clevers, Anna-Pavlina G Haramis

  Proceedings of the National Academy of Sciences of the United States of America. 03/2011; 108(11):4358-63.

  Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz-Jeghers syndrome). LKB1 has many targets,Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz-Jeghers syndrome). LKB1 has many targets, including the AMP-activated protein kinase (AMPK) that is phosphorylated under low-energy conditions. AMPK phosphorylation in turn, affects several processes, including inhibition of the target of rapamycin (TOR) pathway, and leads to proliferation inhibition. To gain insight into how LKB1 mediates its effects during development, we generated zebrafish mutants in the single LKB1 ortholog. We show that in zebrafish lkb1 is dispensable for embryonic survival but becomes essential under conditions of energetic stress. After yolk absorption, lkb1 mutants rapidly exhaust their energy resources and die prematurely from starvation. Notably, intestinal epithelial cells were polarized properly in the lkb1 mutants. We show that attenuation of metabolic rate in lkb1 mutants, either by application of the TOR inhibitor rapamycin or by crossing with von Hippel-Lindau (vhl) mutant fish (in which constitutive hypoxia signaling results in reduced metabolic rate), suppresses key aspects of the lkb1 phenotype. Thus, we demonstrate a critical role for LKB1 in regulating energy homeostasis at the whole-organism level in a vertebrate. Zebrafish models of Lkb1 inactivation could provide a platform for chemical genetic screens to identify compounds that target accelerated metabolism, a key feature of tumor cells.

• Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium.

  Authors: François Gerbe, Johan H van Es, Leila Makrini, Bénédicte Brulin, Georg Mellitzer, Sylvie Robine, Béatrice Romagnolo, Noah F Shroyer, Jean-François Bourgaux, Christine Pignodel, Hans Clevers, Philippe Jay

  The Journal of cell biology. 03/2011; 192(5):767-80.

  The unique morphology of tuft cells was first revealed by electron microscopy analyses in several endoderm-derived epithelia. Here, we explore the relationship of these cells with the other cellThe unique morphology of tuft cells was first revealed by electron microscopy analyses in several endoderm-derived epithelia. Here, we explore the relationship of these cells with the other cell types of the intestinal epithelium and describe the first marker signature allowing their unambiguous identification. We demonstrate that although mature tuft cells express DCLK1, a putative marker of quiescent stem cells, they are post-mitotic, short lived, derive from Lgr5-expressing epithelial stem cells, and are found in mouse and human tumors. We show that whereas the ATOH1/MATH1 transcription factor is essential for their differentiation, Neurog3, SOX9, GFI1, and SPDEF are dispensable, which distinguishes these cells from enteroendocrine, Paneth, and goblet cells, and raises from three to four the number of secretory cell types in the intestinal epithelium. Moreover, we show that tuft cells are the main source of endogenous intestinal opioids and are the only epithelial cells that express cyclooxygenase enzymes, suggesting important roles for these cells in the intestinal epithelium physiopathology.

• The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse.

  Authors: Anna Merlos-Suárez, Francisco M Barriga, Peter Jung, Mar Iglesias, María Virtudes Céspedes, David Rossell, Marta Sevillano, Xavier Hernando-Momblona, Victoria da Silva-Diz, Purificación Muñoz, Hans Clevers, Elena Sancho, Ramón Mangues, Eduard Batlle

  Cell stem cell. 03/2011; 8(5):511-24.

  A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts diseaseA frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse.

• Differentiated human colorectal cancer cells protect tumor-initiating cells from irinotecan.

  Authors: Benjamin L Emmink, Winan J Van Houdt, Robert G Vries, Frederik J H Hoogwater, Klaas M Govaert, Andre Verheem, Maarten W Nijkamp, Ernst J A Steller, Connie R Jimenez, Hans Clevers, Inne H M Borel Rinkes, Onno Kranenburg

  Gastroenterology. 03/2011; 141(1):269-78.

  Stem cells of normal tissues have resistance mechanisms that allow them to survive genotoxic insults. The stem cell-like cells of tumors are defined by their tumor-initiating capacity and may haveStem cells of normal tissues have resistance mechanisms that allow them to survive genotoxic insults. The stem cell-like cells of tumors are defined by their tumor-initiating capacity and may have retained these resistance mechanisms, making them resistant to chemotherapy. We studied the relationship between resistance to the topoisomerase I inhibitor irinotecan and tumor-initiating potential in human colonosphere cultures and in mice with colorectal xenograft tumors. Colonosphere cultures were established from human colorectal tumor specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. Stem cell and differentiation markers were analyzed by immunoblotting and fluorescence-activated cell sorting. Clone- and tumor-initiating capacities were assessed by single-cell cloning and in immune-deficient mice. Sensitivity to irinotecan was assessed in vitro and in tumor-bearing mice. The relationship between drug resistance and tumor-initiating capacity was tested by fluorescence-activated cell sorting of colonosphere cells, based on expression of ABCB1 and aldehyde dehydrogenase (ALDH) activity. Colonosphere cultures had a high capacity to initiate tumors in mice and were resistant to irinotecan. Inhibition of the drug-efflux pump ABCB1 by PSC-833 allowed irinotecan to eradicate tumor-initiating cells. However, ABCB1 was expressed only by a subpopulation of differentiated tumor cells that did not form clones or tumors. Conversely, tumor-initiating cells were ABCB1-negative and were identified by high ALDH activity. Tumorigenic ALDHhigh/ABCB1negative cells generated nontumorigenic ALDHlow/ABCB1positive daughter cells in vitro and in tumor xenografts. PSC-833 increased the antitumor efficacy of irinotecan in mice. The resistance of colorectal tumors to irinotecan requires the cooperative action of tumor-initiating ALDHhigh/ABCB1negative cells and their differentiated, drug-expelling, ALDHlow/ABCB1positive daughter cells.

• Tracking adult stem cells.

  Authors: Hugo J Snippert, Hans Clevers

  EMBO reports. 02/2011; 12(2):113-22.

  The maintenance of stem-cell-driven tissue homeostasis requires a balance between the generation and loss of cell mass. Adult stem cells have a close relationship with the surrounding tissue--knownThe maintenance of stem-cell-driven tissue homeostasis requires a balance between the generation and loss of cell mass. Adult stem cells have a close relationship with the surrounding tissue--known as their niche--and thus, stem-cell studies should preferably be performed in a physiological context, rather than outside their natural environment. The mouse is an attractive model in which to study adult mammalian stem cells, as numerous experimental systems and genetic tools are available. In this review, we describe strategies commonly used to identify and functionally characterize adult stem cells in mice and discuss their potential, limitations and interpretations, as well as how they have informed our understanding of adult stem-cell biology. An accurate interpretation of physiologically relevant stem-cell assays is crucial to identify adult stem cells and elucidate how they self-renew and give rise to differentiated progeny.