METHOD FOR IDENTIFYING, EXPANDING, AND REMOVING ADULT STEM CELLS AND CANCER STEM CELLS

Abstract

The invention relates to the fields of biochemistry, pharmacy and oncology. The invention particularly relates to the use of novel stem cell markers for the isolation of stem cells. The invention further relates to the obtained stem cells and their use in for example research or treatment, for example, for the preparation of a medicament for the treatment of damaged or diseased tissue.

In one of the embodiments, the invention provides a method for obtaining (or isolating) stem cells comprising optionally preparing a cell suspension from a tissue or organ sample, contacting said cell suspension with an Lgr 6 or 5 binding compound, identify the cells bound to said binding compound, and optionally isolating the stem cells from said binding compound.

The invention further relates to means suitable for cancer treatment and even more specific for the treatment of cancer by eradicating cancer stem cells.

Claims

1-58. (canceled)

59. An in vitro or ex vivo method of maintaining or culturing tissue or organ stem cells comprising providing the tissue or organ stem cells with an Lgr5 and/or Lgr6 binding compound.

60. The method of claim 59, wherein binding compound is an antibody or an antibody derivative or an antibody fragment capable of binding to Lgr5 and/or Lgr6.

61. The method of claim 60, wherein the antibody is a monoclonal antibody.

62. The method of claim 60, wherein the antibody fragment is scFv, Fab, or (Fab)2 fragment.

63. The method of claim 60, wherein the antibody derivative is a chimeric antibody, nanobody, bifunctional antibody, or humanized antibody.

64. The method of claim 59, wherein the binding compound is an Lgr5 or Lgr6 ligand.

65. The method of claim 59, wherein the binding compound is a small molecule agonist of Lgr5 or Lgr6.

66. The method of claim 59, wherein the stem cells are adult stem cells.

67. The method of claim 59, wherein the stem cells are isolated stem cells.

68. The method of claim 67, further comprising isolating the stem cells from a cell suspension by: providing a cell suspension; contacting said cell suspension with an antibody specific for Lgr5 so as to bind the antibody specific for Lgr5 to Lgr5 positive cells in the cell suspension; and at least partially purifying cells bound to the antibody specific for Lgr5 from cells not bound to the antibody specific for Lgr5 from the cell suspension so as to obtain the stem cells.

69. The method of claim 59, wherein the stem cells are intestine, brain, lung, heart, skin, liver, retina, stomach, pancreas, ovary, adrenal medulla, bladder, bone, connective tissue, ear, muscle, prostate, placenta, uterus, or breast stem cells.

70. The method of claim 59, wherein the stem cells are cancer stem cells.

71. The method of claim 70, wherein the cancer stem cells are obtained from a tumor and the method further comprises determining the cancer stem cell content of the tumor.

72. The method of claim 70, wherein the cancer stem cells are comprised in a tumor and the method further comprises determining the cancer stem cell content of the tumor.

73. The method of claim 71, wherein the cancer stem cells are comprised in a tumor and the method further comprises determining the cancer stem cell content of the tumor.

74. The method of claim 72, wherein determining the cancer stem cell content of the tumor comprises contacting the tumor with the Lgr5 and/or Lgr6 binding compound or a second Lgr5 and/or Lgr6 binding compound, removing unbound Lgr5 and/or Lgr6 binding compound and determining whether any bound binding compound is present in the tumour.

75. The method of claim 73, wherein determining the cancer stem cell content of the tumor comprises contacting the tumor with the Lgr5 and/or Lgr6 binding compound or a second Lgr5 and/or Lgr6 binding compound, removing unbound Lgr5 and/or Lgr6 binding compound and determining whether any bound binding compound is present in the tumour.

Description

FIGURE LEGENDS

[0322] FIGS. 1A-1C. Gpr49/Lgr5 is a Wnt target gene in a human colon cancer cell line and is expressed in mouse crypts. FIG. 1A: Northern blot analysis (upper panel); ethidium bromide-stained gel (lower panel). Lane 1: Control Ls174T-L8 cells. Lane 2: Ls174T cells after 24 hours doxycycline induced Wnt pathway inhibition as in 6 (References 2). Note the strong downregulation of the 4.4 kb Grp49 mRNA upon Wnt pathway inhibition. Lane 3: RNA extracted from isolated mouse small intestinal crypts, which unavoidably suffers from limited degradation resulting in some smearing. Lane 4: RNA extracted from isolated mouse villi. Note the specific expression of Grp49 in mouse crypts. FIGS. 1B-1C: Two overlapping images of an in-situ hybridization performed on small intestines of an APC min mouse, illustrating the ubiquitous expression of Grp49 at crypt bottoms (examples marked with white arrows) and the expression in the adenoma in the left panel (marked by a broken line).

[0323] FIGS. 2A-2I. Gpr49/Lgr5 expression in cycling Crypt Base Columnar (CBC) cells of the small intestine. FIGS. 2A-2C: In-Situ hybridization was performed with probes specific for 3 Tcf target genes demonstrating non-overlapping expression patterns on the crypt epithelium. FIG. 2A: Cryptdin specifically marks Paneth cells at the crypt base; FIG. 2B: KIAA0007 marks the TA cells located above the Paneth cells FIG. 2C: Gpr49/Lgr5 is specifically expressed in 4-8 cells intermingled with the Paneth cells at the crypt base. All sense controls were negative (not shown). FIG. 2D: CBC cells (circled) are only poorly visible on heamatoxylin/eosin stained sections. FIG. 2E: CBC cells (circled) are K167+ FIG. 2F: Some CBC cells express the M-phase marker phospho-histidine H3 (circled). FIG. 2G: BrdU incorporation in CBC cells 4 hours after a single dose of BrdU (circled). FIG. 2H: BrdU incorporation in CBC cells after 24 hour continuous BrdU labeling (circled). Black bars: Numbers of BrdU-positive CBC cells per crypt section after 4 hours or 24 hours. White bar: Total number of CBC cells per crypt section assessed by counting LacZ-positive cells in Gpr49-LacZ mice.

[0324] FIGS. 3A-3I. Restricted expression of a GPR49-LacZ reporter gene in adult mice FIG. 3A: Generation of mice carrying lacZ integrated into the last exon of the Gpr49 gene, removing all transmembrane regions of the encoded Gpr49 protein. FIGS. 3B-3H, Expression of GPR491acZ in selected adult mouse tissues. FIGS. 3B-3C: In the small intestine expression is restricted to 6-8 slender cells intermingled with the Paneth cells at the crypt base. FIGS. 3D-3E: In the colon, expression is confined to a few cells located at the crypt base. FIGS. 3F-3G: Expression in the stomach is limited to the base of the glands. FIG. 3H: In the mammary glands, expression was evident only in smaller, actively proliferating glands, where it was restricted to basal epithelial cells. FIG. 3I: In the skin, expression occurs in the outer root sheath of the hair follicles in a domain extending from the bulge to the dermal papilla.

[0325] FIGS. 4A-4H. EGFP expression in a GPR49-EGFP-Ires-CreERT2 knock-in mouse faithfully reproduces the GPR49lacZ expression pattern in the intestinal tract. FIG. 4A: Generation of mice expressing EGFP and CreERT2 from a single bicistronic message by gene knock-in into the first exon of Gpr49. FIGS. 4B,4C,4E: Confocal GFP imaging counterstained with the red DNA dye ToPro-3 confirms that Gpr49 expression is restricted to the 6-8 slender cells sandwiched between the Paneth cells at the crypt base of the small intestine. FIG. 4B: Entire crypt-villus unit; FIG. 4C: enlargement of crypt regions; FIG. 4D: Immunohistochemical analysis of EGFP expression in intestinal crypts. FIG. 4E: 2D image of 3D reconstruction supplied as supplemental movie in FIGS. 7A-7B. FIG. 4F. Confocal imaging of EGFP expression in the colon confirms Gpr49 expression is restricted to a few cells located at the crypt base. FIG. 4G: CryoEM section of crypt stained for GFP with immunogold (scale bar=1000 nm). Quantification of specificity of labeling: Gold particles were counted over 255 μm2 of CBC cell cytosol (1113 particles), 261 μm2 of Paneth cell cytosol (305 particles) and 257 μm2 of fibroblast cytosol (263 particles) outside the crypt. Thus CBC cytoplasm had 4.36 gold particles/μμm2 compared to the Paneth cells 1.17 gold particles/μm2 and to the fibroblast control 1.02 gold particles/μm2. C=Crypt lumen; P=Paneth cells; CBC=Crypt Base Columnar cells. FIG. 4H: Unlabeled CryoEM section (scale bar=2000 nm), underscoring the ultrastructural characteristics of CBC cells and their positioning relative to Paneth cells.

[0326] FIGS. 5A-5K. Lineage tracing in the small intestine and colon. FIG. 5A: GPR49-EGFP-Ires-CreERT2 knock-in mouse crossed with Rosa26-LacZ reporter mice 12 hours after Tamoxifen injection FIG. 5B: frequency at which the blue cells appeared at specific positions relative to the crypt bottom, according to the scheme in the inset of FIG. 5B. The large majority of the Cre+ LacZ-labeled CBC cells occurred at positions between the Paneth cells, while only 10% of these cells were observed at the +4 position directly above the cells (blue line). Quantitative data on the position of long term DNA label-retaining cells obtained in adult mice post-irradiation (marking the “+4” intestinal stem cell) were published recently by Potten and colleagues 17. Comparison of these data (red line) with the position of CBC cells carrying activated Cre. FIGS. 5C-5E: Histological analysis of LacZ activity in small intestine 1 day post-induction (FIG. 5C), 5 days post-induction (FIG. 5D) and 60 days post-induction (FIG. 5E). FIGS. 5F-5H: Double-labelling of LacZ-stained intestine using PAS demonstrates the presence of Goblet cells (FIG. 5F; white arrows) and Paneth Cells (FIG. 5G; blue arrows) in induced blue clones. Double-labelling with Synaptophysin demonstrates the presence of enteroendocrine cells within the induced blue clones (FIG. 5H; black arrows). FIGS. 5I-5K: Histological analysis of LacZ activity in colon 1 day post-induction (FIG. 5I), 5 days post-induction (FIG. 5J) and 60 days post-induction (FIG. 5K).

[0327] FIGS. 6A-6C. Strategy for EGFP-ires-CreERT2 cassette knock-in into the Gpr49 locus

[0328] FIG. 6A: Schematic structure of the mouse Gpr49 gene

[0329] FIG. 6B: Southern blotting strategy to screen ES cells transfected with a knock-in construct targeting the ATG translational start in Exon I.

[0330] FIG. 6C: Four ES cell clones out of a total of 500 scored positive for the recombined BamHI band running at 4.3 kb. After re-screening of these 4 ES clones, the first two (asterisks) were selected for blastocyst injections.

[0331] FIGS. 7A-7B. Relative radiation sensitivity of CBC cells, +4 cells, and TA cells. Adult mice were irradiated with 1 Gy or 10 Gy and subsequently sacrificed 6 hours later, at the peak of apoptosis. FIG. 7A: Active Caspase-3-positive cells were visualized by immunohistochemistry (Upper panel—black arrows highlighting positive +4 cells following 1 Gy irradiation: Lower panel-white arrows highlighting positive CBC cells following 10 Gy irradiation). FIG. 7B: The frequency of positive cells per crypt was determined by counting three classes: CBC cells (located between the Paneth cells), +4 cells (located directly above the Paneth cells) and TA cells: located at position 5-15. Maximal apoptosis at +4 is already reached at 1 Gy while 10 Gy causes significantly more apoptosis than 1 Gy irradiation in CBC cells.

[0332] FIGS. 8A-8C. Whole mount analysis of LacZ expression in small intestine of GPR49-EGFP-Ires-CreERT2 knock-in mice crossed with Rosa26-LacZ reporter mice at the indicated time points following Tamoxifen injection FIG. 8A: 1 day post-induction. FIG. 8B: 5 days post-induction. FIG. 8C: 60 days post-induction.

[0333] FIG. 9. Colocalisation of proliferation marker Ki67 and GFP-positive CBC cells in the intestinal crypts of GPR49-EGFP-CreERT2 mice (serial sections).

[0334] FIGS. 10A-10B. Sequences of the human, mouse and rat receptors.

[0335] FIG. 11. Predicted structure of Lgr4, 5 and 6.

[0336] FIGS. 12A-12D. Restricted expression of a GPR49-LacZ reporter gene in adult mice. Expression of GPR49lacZ in selected adult mouse tissues. LGR5 is restricted to rare cell populations in the brain (glomeruli of the olfactory bulb and several other poorly defined regions) (FIG. 12A), the eye (inner nuclear layer of the retina) (FIG. 12B), liver (cells surrounding the portal triads) (FIG. 12C) and adrenal gland (FIG. 12D).

[0337] FIGS. 13A-13B. Lineage tracing in the stomach Lgr5-EGFP-CreERT2 mice were crossed with Rosa26R reporter mice and Cre enzyme activity induced in the LGR5+ve cells by IP injection of Tamoxifen. LacZ reporter gene activity is initially restricted to the LGR5 cells (FIG. 13A), but rapidly expands to include the entire epithelium in the Stomach over time (FIG. 13B). This “lineage tracing” is maintained over long periods of time (FIG. 13B). This demonstrates that all epithelial cells are derived from the LGR5+ve population in this tissue, proving that they are stem cells.

[0338] FIGS. 14A-14B. Lineage tracing in the mammary gland Lgr5-EGFP-CreERT2 mice were crossed with Rosa26R reporter mice and Cre enzyme activity induced in the LGR5+ve cells by IP injection of Tamoxifen. LacZ reporter gene activity is initially restricted to the LGR5 cells (FIG. 14A), but expands to include the myoepithelium of newly-formed milk glands in lactating females (FIG. 14B), indicating that LGR5 is specifically marking myoepithelial stem cells in this organ.

[0339] FIGS. 15A-15B. Lineage tracing in the adrenal gland. Lgr5-EGFP-CreERT2 mice were crossed with Rosa26R reporter mice and Cre enzyme activity induced in the LGR5+ve cells by IP injection of Tamoxifen. LacZ reporter gene activity is initially restricted to the LGR5 cells (FIG. 15A), but expands to include the medulla of the adrenal gland (FIG. 15B), indicating that LGR5 is specifically marking adrenal medulla stem cells.

[0340] FIGS. 16A-16C. Lgr6 is expressed in cells of the upper bulge area of the mouse hair follicle and in basal cells of the epidermis. Skin sections of appr. 26 days old Lgr6-EGFP-Ires-CreERT2 mice (early anagen) were obtained and stained for nuclear DNA (Topro) and EGFP visualized using confocal microscopy (FIGS. 16A-C). During early anagen Lgr6 is expressed in the upper bulge (FIGS. 16A, 16C) and the basal epidermis (FIGS. 16A, 16B).

[0341] FIGS. 17A-17F. The progeny of Lgr6+ cells contribute to all structures of the hair follicles (HF), interfollicular epidermis (IFE) and sebaceous glands (SG). To trace the progeny of Lgr6+ cells Lgr6-EGFP-Ires-CreERT2/ROSA26-LacZ mice were injected with tamoxifen (TM) at P20 when HFs are in telogen (FIG. 17A). At P23 a first staining in the IFE and HFs was detected (FIG. 17B). Analysis of LacZ staining progeny at P38 (1st anagen, FIGS. 17C, 17D) and P52 (2nd telogen, FIGS. 17E, 17F) revealed contribution to all parts of the HFs, IFEs and SGs.

[0342] FIGS. 18A-18B. The progeny of Lgr6+ cells contribute to the myoepithelium of the lung. To trace the progeny of Lgr6+ cells Lgr6-EGFP-Ires-CreERT2/ROSA26-LacZ mice were injected with tamoxifen (TM) at P20. Analysis of LacZ staining progeny at P38 (FIG. 18A, 10×, 20× and 40× magnification from left to right) and P52 (FIG. 18B, 10×, 20× and 40× magnification from left to right) revealed contribution to the myoepithelium underlying the bronchioles of the lung.

[0343] FIGS. 19A-19C. Low-dose oral induction with β-NF does not induce Cre-mediated deletion in stem cells of AHCre mice. Intestinal whole-mounts stained for β-galactosidase from AhCre+ Rosa26R+ mice. FIG. 19A: No activation of the Rosa-lacZ reporter gene is observed in intestines from non-induced AhCre+ Rosa26R+ mice. FIG. 19B: Readily visible expression of lacZ throughout the intestine 2 days after a single gavage of 1 mg/kg β-napthoflavone, indicating efficient Cre-mediated activation of the lacZ reporter. No lacZ expression is visible at the crypt base (lower panel) demonstrating the absence of Cre-mediated recombination at the crypt base. FIG. 19C: No lacZ-positive crypt/villus units are visible on whole-mount intestines 100 days post-induction, indicating that this dosing regime very rarely causes recombination within the intestinal stem cells.

[0344] FIGS. 20A-20H. Transformation of non-stem cells through loss of APC does not efficiently drive adenoma formation over extended time-periods. FIGS. 20A-20C: β-catenin IHC performed on intestinal sections from AhCre+ Rosa26R+ Apcfl/fl 3 days following a single gavage of 1.0 mg/kg β-napthoflavone. Clusters of transformed cells with nuclear β-catenin were frequently observed on the villus (FIG. 20A) and upper regions of the crypt (FIG. 20B). β-cateninhigh clusters were only very rarely observed at the crypt base (FIG. 20C). These clusters are highlighted with black arrows. FIG. 20D: Quantification of the location of the β-cateninhigh cell clusters on intestinal sections from AhCre+ Rosa26R+ Apcfl/fl 4 days following a single gavage of 1.0 mg/kg β-napthoflavone. Box-plots showing numbers of foci observed at the crypt base, the upper crypt and the villus in 1600 crypt-villus units. Significantly more clusters were seen at the upper regions of the crypt than any other region (p=0.04, Mann Whitney, n=3). Nuclear β-catenin foci were observed only very rarely at the crypt base. FIG. 20E: □-β-catenin IHC performed on intestinal section from AhCre+ Rosa26R+ Apcfl/fl 24 days following a single gavage of 1.0 mg/kg β-napthoflavone. Here, nuclear β-catenin is seen in a small lesion 24 days after cre induction. FIGS. 20F-20G: β-catenin IHC performed on intestinal section from AhCre+ Rosa26R+ Apcfl/fl 167 days following a single gavage of 1.0 mg/kg β-napthoflavone showing a microadenoma (FIG. 20F) and small adenoma (FIG. 20G) with nuclear β-catenin. FIG. 20H: Quantification of adenoma formation over extended time-periods in AhCre+ Rosa26R+ Apcfl/fl following a single gavage of 1.0 mg/kg β-napthoflavone. Lesion size was scored on intestinal whole-mounts from AhCre+ Rosa26R+ Apcfl/fl mice that had been stained for lacZ to help visualise the small lesions (at least 3 mice were used for each time-point). No adenomas were seen in mice up to and including day 24 and there was only the very rare microadenoma in mice at day 24. The occasional adenoma was observed in AhCre+ Rosa26R+ Apcfl/fl at 100 days (plus), however the majority of lesion remained microscopic showing that most lesions were not progressing to adenoma despite a long latency period.

[0345] FIGS. 21A-21I. Lgr5+ve intestinal stem cells transformed following loss of APC persist and fuel the rapid formation of β-cateninhigh microadenomas.

[0346] FIGS. 21A-21I: The consequences of Lgr5+ve intestinal stem cell transformation and their subsequent fate was tracked over an eight day period using β-catenin and GFP as markers of transformed cells and Lgr5+ve stem cells respectively. FIGS. 21A-21C: Accumulation of the Wnt effector, β-catenin is first observed in scattered Lgr5+ve stem cells 3 days after Cre induction in Lgr5-EGFP-Ires-CreERT2/APCfl/fl intestines. Representative examples of β-cateninhigh Lgr5+ve stem cells are circled. FIG. 21D-21F: Five days post-induction the transformed Lgr5-GFP+ve stem cells remain (FIGS. 21E-21F black arrows) and are associated with clusters of transformed (β-cateninhigh) cells within the TA compartment. FIGS. 21G-21H: Eight days post-induction the clusters of transformed cells have expanded to fill the TA compartment (FIG. 21H: red circle). The transformed Lgr5-GFP+ve stem cells at the crypt base persist (FIGS. 21H-21I: black arrows), but their transformed progeny within the TA compartment are Lgr5-GFP-ve. (FIGS. 21H-21I: red circles).

[0347] FIGS. 22A-22H. Selective transformation of Lgr5+ve stem cells following loss of APC efficiently drives adenoma formation throughout the small intestine. FIGS. 22A-22H: The appearance and development of intestinal adenomas and the expression of the Lgr5-GFP stem cell marker within these adenomas was tracked over a 36 day period using GFP (FIG. 22F) and β-catenin (all others) IHC. FIGS. 22A-22B: Multiple small adenomas are readily visible throughout the intestine 14 days after Lgr5+ve stem cell transformation. FIGS. 22C-22F: Multiple macroscopic adenomas (>100) are present after 24 days. Lgr5-GFP expression in adenomas is restricted to rare scattered cells (FIG. 22F; circled). FIGS. 22G-22H: At 36 days, a large proportion of the intestine is filled with macroscopic adenomas. FIGS. 23A-23B. Presence of Lgr5+ stem cells in intestinal adenomas. Intestinal adenomas express high levels of β-catenin as a result of chronic activation of the Wnt pathway (FIG. 23A). In contrast to other Wnt target genes which are highly expressed throughout the adenoma (not shown), expression of the intestinal stem cell marker Lgr5-GFP is restricted to scattered cells with characteristic stem cell morphology: slender, comma-shaped cells; indicated with black arrow (FIG. 23B). We speculate that these Lgr5+ve cells within the adenoma are stem cells dedicated to maintaining the growth of the adenoma (so-called cancer stem cells).

[0348] FIG. 24. FACS analyses of LGR5 expression in L8 cells, which are clonal derivatives of LS174T cells, which express dominant negative Tcf4 (DNTcf4) upon Doxycycline (DOX). DNTcf4 turns off constitutive active Wnt pathway. After 48 hrs of DOX induction, a reduction in hLgr5 protein levels is observed. Rat IgG is used as negative isotype control. 9G5 is a rat monoclonal derived antibody directed against hLgr5.

[0349] FIGS. 25A-25B. Comparison of Lgr5+ stem cells and their direct progeny GFP-positive epithelial cells from cell suspensions prepared from freshly isolated crypts of Lgr5-EGFP-ires-CreERT2 mice. FACS analysis distinguished a GFP-high (GFPhi) and a GFP-low (GFPlo) population, which we tentatively identified as CBC cells and their immediate transit-amplifying daughters, respectively (FIG. 25A). An example of a Wnt-responsive gene, Sox9, which shows high level expression in CBC cells, but TA cells directly above the Paneth cells also express this gene in in situ hybridizations, albeit at a much lower level (FIG. 25B).

[0350] FIG. 26. Endogenous hLgr5 staining of a human colon cancer cell line (L8) using several Lgr5-specific monoclonal antibodies. L8 cells are a clonal derivative of the parental LS1 74Tcell-line. Following Doxycycline (DOX) induction the L8 cells express a dominant-negative form of Tcf-4 (DNTcf4). DNTcf4 efficiently blocks the constitutive Wnt pathway activity in these cells and consequently switches off Tcf target genes. After 48 hrs of DOX induction a major reduction in hLgr5 protein levels is observed. Rat lgG is used as negative isotype control.

[0351] FIG. 27. Light chain+ heavy chain sequences analyzed using KABAT method. CDR regions are in bold and in italics.

EXAMPLES

Example 1

[0352] Experimental Part

[0353] Northern blotting and induced Wnt pathway inhibition in LS174T clone L8: As in van de Wetering, M. et al. The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111, 241-50 (2002). The probe spanned the entire reading frame of mouse Gpr49. Crypt and villus epithelial preparations for RNA isolation were generated from 0.5 cm lengths of intestine by 4 successive rounds of incubation in pre-warmed 30 mM EDTA at 37° C. for 10 minutes, followed by vigorous shaking (10×) in ice-cold PBS. Fractions 1 and 4, comprising predominantly villi and crypts respectively were used for RNA isolation.

[0354] Mice: GPR49-LacZ mice were generated by homologous recombination in ES cells targeting an Ires-LacZ cassette to the 5′ end of the last exon, essentially removing the region containing all TM regions and creating a null allele (Lexicon). GPR49-EGFP-Ires-CreERT2 mice were generated by homologous recombination in ES cells targeting an EGFP-Ires-CreERT2 cassette to the ATG of GPR49. Rosa26-lacZ Cre reporter mice were obtained from Jackson Labs.

[0355] Tamoxifen induction: Mice of at least 8 weeks of age were injected once intraperitoneally with 200 μl of Tamoxifen in sunflower oil at 10 mg/ml.

[0356] BrdU injection: Mice were injected intraperitoneally at four hour intervals with 200 μl of a BrdU solution in PBS at 5 mg/ml.

[0357] Immuno Electron Microscopy: Intestines were dissected and perfuse-fixed in 4% PFA in 0.2 M PHEM-buffer, embedded in gelatine, cryosectioned with a Leica FCS cryoultratome and immunolabelled against GFP with polyclonal rabbit anti-GFP antibody. Samples were trimmed using a diamond Cryotrim 90 knife at −100° C. (Diatome, Switzerland) and ultrathin sections of 70 nm were cut at −120° C. using a Cryoimmuno knife (Diatome, Switzerland). For the low magnification EM images the 15 nm protein A-gold particles (UMCU, Utrecht, The Netherlands) were briefly silver enhanced with R-GENT SE-EM (Aurion, The Netherlands) according to the manufacturers instructions. Aspecific binding to Paneth cell granules was diminished by applying Blocking solution (Aurion, The Netherlands) prior to the primary antibody.

[0358] Tissue sample preparation for immunohistochemistry, in-situ hybridization and LacZ expression analysis: All performed as previously described in Muncan, V. et al. Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc. Mol Cell Biol 26, 8418-26 (2006). In-situ probes comprising a 1 kb N-terminal fragment of mGPR49 were generated from sequence-verified Image Clone 30873333. Ki67 antibodies were purchased from Monosan (The Netherlands), Phospho-histone H3 from Campro Scientific (The Netherlands), anti-synaptophysin from Dako, anti BrdU from Roche. Polyclonal rabbit anti-GFP was provided by Edwin Cuppen, Hubrecht Institute.

[0359] Generation of Suspension of Human (Tumor) Tissue Cells.

[0360] Using a razor blade, mince freshly isolated human (tumor) tissue as much as possible. Do this in serum-free media. Draw minced tumor into a 25 ml pipette. Place the solution into a 50 ml conical tube. Incubate at 37 C for 30-60 min after adding collagenase IV (200 units/m1) (Sigma). The final concentration should be 200 units/ml. Pipette up and down a few times every 10 min (approx). Pass the solution through a filter (45 micrometer pore size; Becton Dickinson). Wash the filter with 4-5 ml of serum-free medium. Centrifuge the solution 1500 rpm for 10 min (4° C.) Resuspend the pellet in hypotonic ammonium chloride (approx. 5 ml). Leave 10 min at room temperature (this will lyse red blood cells). Then add equal volume of serum-free media and centrifuge again. Resuspend pellet in serum free medium. If clumpy then pass through another filter. Count with trypan blue to see the percent dead cells.

[0361] Cancer Stem Cell Assay by Xenografting in Immunodeficient Mice

[0362] The mice are sublethally irradiated with 320 Rad. The experimental procedure involves injecting human (colon) cancer cell suspensions under the renal capsule of NOD/SCID mice. The mice are handled using sterile techniques and anaesthetized using inhalational anaesthesia: isoflurane. The mice are placed on a heating pad during the procedure.

[0363] A clipper is used to shave the abdomen, which is then prepped sequentially with: (1) iodine based solution and (2) 70% ethanol solution. The area is then dabbed with a gauze. The mouse is placed on its side (left side up). A 1 cm (approximately) flank incision is made with scissors, just below the costal margin on the left side. Deliver the kidney into the wound. The cell suspension to be assayed for cancer stem cell activity is mixed 1:1 (medium:Matrigel) on ice. Utilizing a tuberculin syringe, inject 25 microliter of the cell suspension under the renal capsule. Deliver the kidney back into the abdomen. If cancer stem cell activity is present in the cell suspension, a tumor will grow out in the subsequent weeks/months which is analysed by histology and should resemble the original human tumor.

[0364] The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. Current models state that 4-6 crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined. Gpr49/Lgr5 was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Two knock-in alleles revealed exclusive expression of Gpr49 in cycling, columnar cells at the crypt base. In addition, Gpr49 was expressed in rare cells in several other tissues including stomach, breast and hair follicle. Using an inducible Cre knock-in allele and the Rosa26-LacZ reporter strain, lineage tracing experiments were performed in adult mice. The Gpr49.sup.+ve crypt base columnar cell (CBC) generated all epithelial lineages over a 60-day period, implying that it represents the stem cell of the small intestine and colon. The expression pattern of Gpr49 shows that it marks stem cells in multiple adult tissues and cancers.

[0365] The absorptive epithelium of the small intestine is ordered into crypts and (References 2). In the mouse, the small intestinal epithelium turns over every 3-5 days. The massive rate of cell production in the crypts is balanced by apoptosis at the tips of the villi. To date, intestinal stem cells have not been functionally identified, due to the lack of unique markers and the absence of stem cell assays. The analysis of mouse chimeras and mutagen-induced somatic clones.sup.2, 3 (References 2) and the study of regeneration upon injury have allowed an operational definition of stem cell characteristics. Stem cells are believed to cycle steadily to produce the rapidly proliferating transit amplifying (TA) cells capable of differentiating towards all lineages. Stem cells self-renew throughout life, and regenerate the epithelium following injury. The estimated number of stem cells is between 4 and 6 per crypt.sup.2 (References 2). Long-term DNA label retention has tentatively located stem cells at “position +4” directly above the Paneth cells.sup.4 (References 2). Three differentiated cell types (enterocytes, goblet cells and enteroendocrine cells) form from TA cells at the crypt-villus junction and continue their migration in coherent bands stretching along the crypt-villus axis. While crypts are monoclonal, each villus receives cells from multiple different crypts and is therefore polyclonal. The fourth major differentiated cell-type, the Paneth cell, resides at the crypt bottom. The colon epithelium contains crypts, but has a flat surface rather than carrying villi. This epithelium comprises two major differentiated cell types: the absorptive colonocytes and the goblet cells.sup.1 (References 2). To date, no stem cells have been identified in the colon.

[0366] Since Wnt signals constitute the major driving force behind the biology of the crypt.sup.5 (References 2), we hypothesized that one or more Wnt/Tcf4(Tcf712) target genes may be specifically expressed in the stem cells. We have previously described the Wnt/Tcf4 target gene program in colorectal cancer cells and found that it is physiologically expressed in intestinal crypts.sup.6, 7 (References 2). When we studied the expression of approximately 80 selected Tcf4 target genes.sup.7, the overwhelming majority was expressed either in Paneth cells or TA cells. The Gpr49/Lgr5 gene, however, was expressed in a unique fashion. The Gpr49 gene behaved as a Wnt target gene, as its expression was extinguished upon the induced inhibition of Wnt pathway activity by dominant-negative TCF4 in the human colorectal cancer cell line LS174T, a cell system described earlier.sup.6 (References 2) (FIG. 1a, lane 1 vs. 2). Accordingly, the gene was expressed in the crypts, but not the villi, of mouse small intestine (FIG. 1a, lane 3 vs. 4). In situ hybridization revealed expression in a limited number of cells located at all crypt bottoms as well as in adenomas in the small intestine of an APC.sup.min mouse (FIGS. 1b and c). This expression pattern, enlarged in FIG. 2c, clearly differed from that obtained with a Paneth cell-specific gene (FIG. 2a) or a TA-specific gene (FIG. 2b). The Gpr49 gene appeared to mark small cells interspersed between Paneth cells, the cycling Crypt Base Columnar (CBC) cells (FIG. 2d-h; see below).

[0367] Gpr49 encodes an orphan G protein-coupled receptor (GPCR), characterized by a large leucine-rich extracellular domain. It is closely related to GPCRs with glycoprotein ligands, such as the TSH-, FSH- and LH-receptors.sup.8 (References 2). Gpr49 was on our original list of Tcf4 targets in colorectal cancer.sup.6, but has since been observed to be overexpressed also in ovarian and hepatocellular carcinomas.sup.9, 10 (References 2). In order to study its expression in detail we obtained a knock-in allele, in which LacZ, preceded by an internal ribosome entry site (ices), is integrated just N-terminal to the first transmembrane domain essentially creating a null allele (FIG. 3a).

[0368] While our study was in progress. Morita et al published the Gpr49.sup.−/− phenotype.sup.11 (References 2). A malformation of the tongue and lower jaw causes newborn mutants to swallow large amounts of air leading to their demise soon after birth. We observed the same phenotype in our mice. Of note, crypts and intestinal stem cells are first established several weeks after birth.sup.12 (References 2). The heterozygous Gpr49-LacZ mice allowed us to detail the expression of Gpr49. Before birth, a dynamic and complex expression pattern was observed (Barker et al. in preparation). Around birth, Gpr49 expression subsided in virtually all tissues. Expression in adult mice was restricted to rare, scattered cells in the eye, brain, hair follicle, mammary gland, reproductive organs, stomach and intestinal tract (FIG. 3, and not shown). In the small intestine, Gpr49 expression was observed in slender cells located between the Paneth cells in the small intestine (FIGS. 3b and c) and in a similar number of cells at the bottom of colon crypts (FIGS. 3d and e). Counting of blue cells in small intestinal crypts sectioned through the lumen revealed the presence of approximately 3.5 of such cells per sectioned crypt (FIG. 2i, white bar). More than 30 years ago, Leblond and Cheng noted the presence of cycling cells between the Paneth cells and have coined the term “Crypt Base Columnar” (CBC) cells.sup.13 (References 2). Based on their position and their presence in long-term mutant epithelial clones, Cheng and Bjerknes.sup.2, 14 (References 2) and Gordon and colleagues.sup.15 (References 2) have proposed that these cells may harbor stem cell activity

[0369] By morphology, the slender Gpr49.sup.+ve CBC cells with their scant cytoplasm and flat, wedge-shaped nuclei pointing towards the crypt lumen were readily distinguishable from the adjacent Paneth cells. Occasionally (once in approximately every ten crypts), these cells also expressed the M phase marker phospho-histone H3, indicating that the cells are in cycle (FIG. 2f). Indeed, a 4 hour pulse of BrdU labeled approximately 1 of these cells per crypt (FIGS. 2G and 2I, left black bar), while a 24 hour continuous BrdU labeling resulted in more than 3 positive cells per crypt (FIGS. 2h and i, right black bar), close to the total number of CBC cells per crypt (FIG. 2i, white bar). This observation implied that the average cycling time of CBC cells is in the order of 1 day. Direct colocalization of the proliferation marker Ki67 with GPR49-LacZ further confirmed that the LacZ positive CBC cells are typically cycling (FIG. 2e and FIG. 9).

[0370] In order to be able to visualize live CBC cells and to study their potential “sternness”, we generated another knock-in allele, in which we integrated an EGFP-ires-CreERT2 cassette at the first ATG codon of Gpr49 (FIG. 4a and FIG. 6). Heterozygous mice carrying this allele were healthy and fertile. The GFP pattern observed in adult tissues faithfully recapitulated the pattern previously seen with the Gpr49-LacZ allele in eye, brain, hair follicle, mammary gland, reproductive organs, stomach and intestinal tract (not shown, and FIG. 4). Confocal imaging allowed the visualization of the Gpr49.sup.+ve cells by GFP fluorescence in small intestine (FIG. 4b,c,e) and colon (FIG. 4f). Immuno-Electron Microscopy using immunogold labeling of the GFP-positive CBC cells and of neighbouring Paneth cells and fibroblasts illustrated the unique ultrastructural anatomy of the CBC cells (FIGS. 4g and h). Typically, the CBC cells were relatively broad at their base, contained a flat wedge-shaped nucleus and scarce organelles. A slender extension of apical cytoplasm was squeezed inbetween neighboring endoplasmic reticulum- and granule-rich Paneth cells, extended to the crypt lumen and carried some apical microvilli.

[0371] We then crossed the EGFP-ires-CreERT2 knock-in allele with the Cre-activatable Rosa26-LacZ reporter.sup.16 (See FIG. 4a for experimental strategy). Injection of Tamoxifen activates the CreERT2 fusion enzyme in Gpr49-expressing cells. Cre-mediated excision of the roadblock sequence in the Rosa26-LacZ reporter should then irreversibly mark the Gpr49.sup.+ve cells. Moreover, while potential progeny of these cells will no longer express GFP, the activated LacZ reporter should act as a genetic mark, facilitating lineage tracing.

[0372] LacZ expression was not observed in non-induced mice (not shown). To quantify the total number of CBC cells per crypt in which the latent Cre enzyme could be activated by Tamoxifen, we treated 2-3 months-old mice with Tamoxifen and sacrificed the mice 12 hours later. As evident in FIG. 5a, blue LacZ signals appeared at the typical CBC positions. We determined the frequency at which the blue cells appeared at specific positions relative to the crypt bottom, according to the scheme in FIG. 5b. The large majority of the Cre.sup.+ve, LacZ-labelled CBC cells occurred at positions between the Paneth cells, while only 10% of these cells were observed at the +4 position directly above the cells (FIG. 5b, blue line). Quantitative data on the position of long term DNA label-retaining cells obtained in adult mice post-irradiation (marking the “+4” intestinal stem cell) were published recently by Potten and colleagues.sup.17. Comparison of these data (FIG. 5b, red line) with the position of CBC cells with activatable Cre revealed that the two markers identified largely non-overlapping cell populations.

[0373] Another defining characteristic of the +4 cell is their exquisite sensitivity to low dose (<1 Gy) radiation.sup.4. To compare relative radiation sensitivity between CBC cells and +4 cells, adult mice were irradiated with 1 Gy or 10 Gy and subsequently sacrificed 6 hours later, at the peak of apoptosis. Active Caspase-3-positive cells were visualized by immunohistochemistry (FIG. 7a). The frequency of positive cells per crypt was determined by counting apoptotic cells in three classes: CBC cells (defined by their location between the Paneth cells), +4 cells (located directly above the Paneth cells) and TA cells: located at position 5-15 (FIG. 7b). Maximal apoptosis at the +4 position was already reached at 1 Gy (a: upper panel, black arrows) in concordance with.sup.4 (References 2), while 10 Gy caused significantly more apoptosis than 1 Gy irradiation in CBC (a: lower panel) and TA cells, confirming the different identities of the CBC and +4 cells.

[0374] Adult mice were then subjected to a Tamoxifen pulse and were sacrificed at 1, 5, 12 (not shown) and 60 days post-induction. One day post-induction, occasional CBC cells in the crypts of small intestine and colon were observed to express LacZ (FIGS. 5c and 5i respectively). As is demonstrated for whole-mount small intestine in FIG. 8, parallel ribbons of cells emanated from the crypt bottoms and ran up the side of adjacent villi at later time points. The kinetics of stripe formation was not uniform. Some stripes already reached the villus tips 5 days post-induction, while blue staining in occasional crypts was still restricted to crypts. At 5 days post-induction (FIG. 5d), such crypt-restricted expression was very rarely seen. The CBC cells were capable of long-term maintenance of the self-renewing epithelium, since in 60-day intestines (FIG. 5e and FIG. 8) the frequency of blue crypts and ribbons was essentially identical to that seen at 5-12 days post-induction.

[0375] Double-labeling of 60 day-induced intestine demonstrated the presence of PAS-positive goblet cells (FIG. 5f), PAS-positive Paneth cells (FIG. 5g) and synaptophysin-positive enteroendocrine cells (FIG. 5h) in the LacZ-stained clones originating from the GPR49.sup.+ve CBC cells. Using mutational marking, Cheng and Bjerknes have reported the existence of different types of long-lived epithelial clones, i.e. columnar (enterocyte) clones, mucous (goblet) clones and mixed clones.sup.2. The clones observed in our study were exclusively of the mixed variety. In blue clones, the frequency of goblet cells (114 out of 2043 total cells counted), enterocytes (1846/2043) and Paneth cells (83/2043) was comparable to the frequency of goblet cells (127 out of 3691 total cells counted), enterocytes (3345/3691) and Paneth cells (127/3691) in unmarked adjacent epithelium. As noted.sup.2 (References 2), the third secretory cell type, the enteroendocrine cell, was too rare to allow accurate enumeration. Taken together, we conclude that the Gpr49.sup.+ve CBC cells represent the genuine stem cells of the small intestine.

[0376] Analysis of the colon yielded essentially identical observations. The Gpr49.sup.+ve cells yielded blue clones emanating from the crypt bottom (FIG. 5i). These clones contained colonocytes as well as goblet cells, and essentially remained unchanged during the 60 days of chase (FIG. 5j,k). One significant difference with the situation in the small intestine involved the kinetics of clone-formation. At 5 days, blue staining in most crypts was still restricted to the bottom and entirely blue crypts were only rarely observed, implying that the colon stem cells were more often quiescent than their small intestinal counterparts. At later days, the relative number of entirely blue crypts increased. We concluded that the Gpr49.sup.+ve colon cells fulfilled the stem cell requirements in being pluripotent and capable of maintaining epithelial self-renewal over long periods of time.

[0377] Our observations provide the definitive characterization of the intestinal stem cell by lineage tracing using the expression of a single marker gene, Gpr49. The small intestinal Gpr49.sup.+ve cells are generally not quiescent, but are rapidly cycling, as evidenced by the expression of Ki67 and phospho-histone H3, the incorporation of BrdU, and by the kinetics of ribbon formation. Gpr49.sup.+ve cells of the small intestine appear more actively dividing than their colonic counterparts, likely reflecting differences in the rate of epithelial turnover between the two organs. It appears somewhat counterintuitive that stem cells cycle. This is, however, not unprecedented. Germ stem cells in the Drosophila testis and ovary of the fly, arguably the best understood adult stem cells in animals, cycle throughout the lifetime of the adult fly.sup.18 (References 2). Similarly, a recent elegant study demonstrated that adult stem cells of mammalian skin are continuously cycling.sup.19 (References 2).

[0378] The cycling +4 cells have previously been proposed by Potten and colleagues to represent the small intestinal stem cells.sup.4 (References 2), a notion not confirmed here. The notion was based on the observation that a DNA label incorporated during periods of high stem cell activity was specifically retained in cells at the +4 position. Long-term label retention is often used as an indirect strategy to identify stem cells.sup.12 (References 2). It should be noted, however, that terminally differentiating cells will also retain DNA labels and that label retention should therefore be interpreted with caution. Previous studies have proposed other markers for intestinal stem cells. Musashim.sup.20, 21 (References 2) and CD133.sup.22 (References 2) in our hands stain up to 30-50 cells per crypt (not shown), which appear to encompass CBC cells as well as early transit amplifying cells. Li and colleagues have described several molecular markers for the +4 cells, including phospho-PTEN, phospho-AKT and 14-3ζ.sup.23 (References 2). Our current study implies that the validity of these putative stem cell markers should be reconsidered.

[0379] It appears rather unique that adult stem cells can be identified based on the expression of a single gene. This phenomenon may not be restricted to the intestine, since we observe highly restricted Gpr49 expression in a variety of other tissues. In the anagen hair follicle, the Gpr49 gene is expressed in the bulge area as well as in the outer root sheath (FIG. 3i). While quiescent LTR stem cells reside exclusively in the bulge, activated stem cells migrate down through the outer root sheath towards the basal papilla.sup.24-26 (References 2). Indeed, Gpr49 was recently reported to be the second most highly upregulated gene as assessed by differential expression arraying on isolated hair follicle stem cells.sup.27 (References 2). Moreover, preliminary lineage tracing experiments in the hair follicle support the notion that Gpr49.sup.+ve cells represent stem cells (Barker, Clevers and Toftgard, unpublished). While patterns of proliferation in stomach glands have indicated that the epithelial stem cells reside at the isthmus, halfway between the gland base and epithelial surface.sup.28 (References 2) we find Gpr49 expressed at gland bottoms (FIG. 3f,g). Ongoing lineage tracing experiments imply that the entire glands derive from these cells (Barker and Clevers, unpublished). In the mammary gland, stem cells reside in the basal epithelial layer.sup.29 (References 2), where we observe Gpr49 expression (FIG. 3h). Gpr49 may thus represent a more general marker of adult stem cells. If true, the mouse models developed in the course of this study will allow the isolation as well as specific genetic modification of live adult stem cells in a variety of organs. We first identified Gpr49 as a gene expressed in colon cancer cells.sup.6 (References 2). It is expressed in other cancers.sup.9, 10 (References 2) and, as described in the current study, also in premalignant mouse adenomas. Based on the observations reported here, we now know that Gpr49 may mark cancer stem cells (“tumor-initiating cells”) in colorectal adenocarcinomas.

Example 2 Lgr5 Tissue Expression and Evidence for Lgr5.SUP.+ Stem Cells in these Tissues

[0380] Materials and Methods

[0381] For experimental details, we refer to materials and methods as used in example 1

[0382] Results and Discussion

[0383] Lgr5 Expression Also Detected in Brain, Retina, Liver and Adrenal Gland

[0384] We studied the Lgr5 expression in multiple other tissues in the mice carrying lacZ integrated into the last exon of the Gpr49 gene, removing all transmembrane regions of the encoded Gpr49 protein. We determined that, analogous to colon and small intestine, Lgr5.sup.+ cells were detected in brain, retina, liver and adrenal gland (FIG. 12). In adult mice, LGR5 is restricted to rare cell populations in the brain (glomeruli of the olfactory bulb and several other poorly defined regions), the eye (inner nuclear layer of the retina), liver (cells surrounding the portal triads) and adrenal gland.

[0385] Lineage Tracing in the Stomach, Mammary Gland and Adrenal Gland Proves that Lgr5 is Marking Stem Cell Populations in these Tissues

[0386] We used the LGR5KI/Rosa26-lacZ mice.sup.16 (See example 1 for experimental strategy) to study the presence of Lgr5.sup.+ stem cells in multiple other tissues. Injection of Tamoxifen activates the CreERT2 fusion enzyme in Gpr49.sup.− expressing cells. Cre-mediated excision of the roadblock sequence in the Rosa26-LacZ reporter should then irreversibly mark the Gpr49.sup.+ve cells. Moreover, while potential progeny of these cells will no longer express GFP, the activated LacZ reporter should act as a permanent genetic mark, which will be passed on to any descendents of the LGR5+ve cells, allowing us to track their appearance and fate in-vivo.

[0387] Lineage tracing was initiated in young LGR5KI/Rosa26-lacZ mice and the stomach epithelium analyzed for LacZ activity after 6 months. LGR5-lacZ positive cells are initially restricted to the base of the glands (FIG. 13a). After 6 months, multiple entirely lacZ-positive glands are visible throughout the stomach (FIG. 13b), demonstrating that the LGR5+ve cells are capable of generating all cell-types on the glandular epithelium over long periods of time.

[0388] Similar lineage tracing experiments were performed and the mammary gland epithelium analyzed for LacZ activity over a 3 month period. LGR5-lacZ positive cells are initially restricted to rare basal epithelial cells on virgin glands (FIG. 14a). Following pregnancy, LacZ-positive cells are visible in the basal epithelium of the newly-formed milk glands (FIG. 14b). This demonstrates that LGR5.sup.+ cells in the mammary gland are myoepithelial stem cells.

[0389] Lineage tracing in the adrenal glands analyzed for LacZ activity over a 3 month period. LGR5-lacZ positive cells are initially restricted to the periphery of the adrenal gland 5 days after induction (FIG. 15a). After 3 months the majority of the adrenal medulla is LacZ positive (FIG. 15b). This remains positive over a 14 month period (not shown). This demonstrates that the LGR5.sup.+ cells are the stem cells of the adrenal medulla.

Example 3 Lgr6 Tissue Expression and Lgr6 Expression in Related Stem Cells

[0390] Material and Methods

[0391] Transgenic Mice and Treatments.

[0392] Lgr6-EGFP-Ires-CreERT2 mice were generated by homologous recombination in embryonic stem cells targeting an EGFP-Ires-CreERT2 cassette to the ATG of Lgr6. Rosa26-LacZ reporter mice were obtained from the Jackson laboratory. Mice were fed ad libitum. The Cre recombinase was activated in Lgr6-EGFP-Ices-CreERT2/Rosa26-LacZ mice by injecting 200 μl of tamoxifen (10 mg/ml dissolved in sunflower oil) intraperitoneally.

[0393] Confocal Analysis of EGFP Expression

[0394] For confocal imaging the skin samples were fixed in formalin for 15 minutes at RT and embedded in 4% low melting agarose. Longitudinal sections between 100 and 200 μm thick were prepared using a vibratome. Sections were then permeabilized in PBS supplemented with 1% BSA+1% DMSO+0.1% TritonX, stained for 30 minutes with TO-PRO 1:1000 dilution (Molecular Probes) and embedded using Vectashield (Vector Labs). Sections were imaged with a Sp2 confocal microscope (Leica) and processed using Volocity and Photoshop CS2 software.

[0395] Detection of Beta-Galactosidase Activity

[0396] Freshly obtained tissues were fixed for 2 hours in 1% Formaldehyde/0.2% glutaraldehyde/0.02% NP40 in PBS0 solution at 4° C. on a rolling platform. Samples were washed 3 times for 20 min with rinse buffer (2 mM MgCl.sub.2/0.02% NP40/PBS0) and stained for 36-48 h in a solution consisting of 1 mg/ml X-gal, 5 mM ferrothiocyanide, 5 mM ferrithiocyanide, 0.1% sodium deoxycholate in rinse buffer. The substrate was removed and the samples washed twice in PBS0 for 20 min at room temperature on a rolling platform. The tissues were then fixed overnight in 4% PFA in PBS0 at 4° C. in the dark on a rolling platform. The PFA was removed and the tissues washed twice in PBS0 for 20 min at room temperature. The samples were embedded in paraffin, sectioned (4 μm) and counterstained with neutral red.

[0397] Results and Discussion

[0398] To characterize the expression of Lgr6 in the skin we utilized a knock-in mouse, where the Lgr6 promoter controls the expression of EGFP and the CreERT2 fusion protein, termed Lgr6-EGFP-Ires-CreERT2. At P25 when the hair follicles (HFs) are in the growing (anagen) phase, the GFP-positive cells were localized to cells of the upper bulge/isthmus area of the HF (FIG. 16A, C) and basal cells of the interfollicular epidermis (IFE, FIG. 16A, B). This expression pattern suggests that Lgr6 expression marks a SC/early progenitor cell population of the hair follicle and the epidermis.

[0399] To address the question whether the Lgr6.sup.+ cells of the anagen HF and IFE represent functional stem cells 20 day-old Lgr6-EGFP-Ires-CreERT2/Rosa26-LacZ mice were injected with tamoxifen. At P20 Lgr6 is expressed in the upper bulge/isthmus area of the HF and basal cells of the IFE (data not shown). Three days post tamoxifen injection a scattered pattern of labeled cells could be seen in the HFs and the IFE (FIG. 17B). At 18 days post-injection the progeny of Lgr6.sup.+ cells could be seen in the anagen HFs (FIG. 17C, D) as well as in the IFE and the sebaceous glands (SG) (FIG. 17C, D). In the next telogen labeled cells were found in the bulge and isthmus of the HFs (FIG. 17E, F) and the IFE and SGs (FIG. 17E, F). This observation strongly suggests that Lgr6.sup.+ cells located in the bulge/isthmus area of the HF and the basal IFE exhibit stem cell properties. In particular, Lgr6.sup.+ cells can contribute to all the appendages of the skin, i.e. the growing HFs, the IFE and the SG.

[0400] It seems rather unique that adult stem cells can be identified on the basis of expression of a single gene, in this case Lgr6. This phenomenon may not be restricted to the skin, because we observe highly restricted expression of Lgr6 in a variety of other tissues. To address the question whether the Lgr6.sup.+ cells represent functional stem cells in any other tissues 20 day-old Lgr6-EGFP-Ires-CreERT2/Rosa26-LacZ mice were injected with tamoxifen. LacZ staining was performed on 18 and 32 days post tamoxifen injection to assess for lineage tracing in a variety of tissues. Interestingly, LacZ positive cells were present in the myoepithelium underlying the bronchioles of the lung at both timepoints (FIG. 18). Thus, Lgr6.sup.+ cells contribute to the myoepithelium of the lung strongly suggesting that Lgr6.sup.+ cells located in the lung exhibit stem cell properties as well.

Example 4 the Role of Lgr5.SUP.+ Cancer Stem Cells in Adenoma

[0401] The anatomy of the intestinal crypt is uniquely suited to study adult stem cells in their niche. The epithelium of the murine small intestine renews every five days.sup.1, 2 (references 5). Vigorous proliferation occurs within the crypt compartment. We have recently identified slender, undifferentiated cells expressing the Lgr5 gene located at crypt bottoms as the stem cells of the small intestine and colon. Each small intestinal crypt contains approximately 6 independent, long-lived stem cells that are intermingled with Paneth cells in the small intestine and with goblet cells in the colon. Counter-intuitively, these cells are not quiescent, but complete a cell cycle every day.sup.3 (references 5). Leblond and colleagues have originally named these cells morphologically Crypt Base Columnar (CBC) cells.sup.4, 5 (references 5). Their daughter cells constitute the readily distinguishable transit amplifying (TA) crypt compartment. TA cells divide every 12-16 hours, generating some 300 cells per crypt every day.sup.6 (references 5). Newly-formed TA cells reside within crypts for approximately 48-72 hours, undergoing up to 6 rounds of cell division while migrating upwards.sup.6, (references 5). When the committed TA cells reach the crypt-villus junction, they rapidly and irreversibly differentiate. The proliferation is balanced by apoptosis at the other end of the epithelial conveyor belt, the tip of the villus. Only Paneth cells escape this flow; they have a residence time of 3-6 weeks at the crypt base.sup.7-9 (references 5). Initiating mutation in intestinal malignancies in mouse and man target components of the Wnt pathway, most frequently the negative Wnt regulator APC.sup.10, 11 (references 5). This results in the constitutive activation of a Wnt target gene program that drives the formation of benign adenomas or polyps.sup.12-15 (references 5). However, it remains unclear which cell type sustains the cancer-initiating mutation.

[0402] The Cytochrome P450-promoter-driven AH-Cre mouse allows conditional deletion of floxed alleles in the intestinal epithelium following administration of the inducing agent, β-Napthoflavone (β-NF). Importantly, the AH-Cre allele is highly active in all cell types of the epithelium, including the stem cells.sup.16 (references 5). We have previously employed a floxed allele of APC.sup.17 (references 5) in combination with the AH-Cre mouse line to demonstrate that acute loss of APC throughout the adult intestinal epithelium following IP injection of β-NF leads to an immediate quantitative transformation of the epithelium.sup.16 (references 5), a process almost entirely dependent on the downstream Wnt target gene c-Myc.sup.18 (references 5). High-dose oral 6-NF induces more stochastic deletion of APC, resulting in rapid adenoma formation throughout the intestine within 3 weeks.sup.19 (references 5). Both these high-dose induction protocols effect deletion in all compartments of the epithelium, including the stem cells at the crypt base.

[0403] Having validated the AHCre/APC.sup.flox/flox mouse as an inducible model of intestinal cancer, we sought to dissect the mechanism of adenoma formation by identifying its cell-of-origin. We reasoned that oral administration of low-dose @-NF would restrict its range of action to cells on the villi and the upper regions of the crypts. Careful titration of the required dosage revealed that following oral administration of 1 mg/kg β-NF, the efficiency of Cre activation in the stem cells at the crypt base was extremely low, as measured by the negligible frequency of long-term lineage tracing initiated in AHCre/R26R mice receiving this dose. This dose was still very efficient in inducing Cre activity in the TA compartment and villus epithelium, as detected using the Rosa26-LacZ mouse.sup.20 (references 5) as a Cre reporter (FIG. 19a, b). In a typical experiment over 70% of villi contained blue cells 2 days after induction, but at day 7 blue staining could no longer be detected. In line with this, no crypt/villus ribbons were detected at day 100 post-oral induction (FIG. 19c).

[0404] Using this dosing regime on AHCre/APC.sup.flox/flox/R26R mice, multiple β-catenin.sup.high foci/lesions rapidly became visible throughout the upper crypt and villus epithelium. Representative pictures taken at day 3 post-induction are given in FIG. 20. Mutant APC foci evident by high β-catenin levels occurred predominantly at crypt-villus junctions, but were also seen on the villi (FIG. 19d). Very infrequently these lesions were also seen near the crypt base.

[0405] The majority of the APC-deficient cells present on the villus epithelium were lost after 4-5 days, presumably by shedding. The remaining APC-deficient lacZ-positive lesions/foci present within the crypts failed to expand over a 24 day period. A typical example of such a lesion is given in FIG. 20e. No macroscopic adenomas were visible at this stage. Strikingly, these small lesions persisted over a 180 day period (FIG. 20g), and only very rarely progressed to small adenomas, which did not expand beyond 2-3 villi (FIG. 20f, h). This was in stark contrast to the high frequency formation of large adenomas initiated in the AHcre/APC.sup.flox/flox mice following high-dose β-NF induction. This suggested that the vast majority of adenomas in the latter mice resulted from loss of APC in stem cells.

[0406] In order to formally prove that transformation of intestinal stem cells is the major route to adenoma formation, we employed our Lgr5-EGFP-ires-CreERT2 knock-in mice as a stem cell-specific Cre line to inducibly delete the floxed APC. To this end, Lgr5-EGFP-ires-CreERT2×APC.sup.flox/flox mice were generated. In these mice, the stem cell-specific Cre enzyme was activated with a single IP injection of Tamoxifen (FIG. 21a). Subsequent phenotypic changes in the intestine were tracked over a 2 month period. Accumulation of the Wnt-effector protein β-catenin was first observed in isolated CBC cells at the crypt base after 3 days (FIG. 21a). These transformed cells were GFP-positive, confirming the targeted deletion of APC in the intestinal stem cells (FIG. 21b). After 5 days, multiple crypts throughout the intestine were observed to harbor transformed (i.e. β-catenin.sup.high) stem cells in association with highly proliferative clusters/pockets of β-catenin.sup.high cells within the transit-amplifying (TA) compartment (FIG. 21c, d). This indicated that the Wnt-transformed stem cells remain viable and rapidly generate an expanding population of transformed progeny higher up the crypts. Eight days after inducing APC deletion in the stem cells, the “pockets” of transformed cells had continued to expand within the crypts and outpockets/evaginations of the crypt epithelium and small microadenomas within the associated villus stroma became evident (FIG. 21e). Cells with accumulated β-catenin were never present on the villus epithelium in these mice, demonstrating that the expanding transformed population were restricted to the intestinal crypts. These observations are strikingly reminiscent of a model of adenoma formation, in which Wnt-transformed cells expressing high levels of the Wnt target gene EphB2 and -B3 expand within the crypt until they come into contact with the Ephrin-positive villus epithelium.sup.21, 22 (references 5). The resulting repulsive forces consequently dictate that the microadenoma can only continue to expand by invading the stroma of the neighbouring villus where it is shielded from the Ephrin-positive villus epithelium.

[0407] The “outpockets” and microadenomas present in the 8 day induced Lgr5KI/APC.sup.flox/flox mice continued their aggressive expansion, as evidenced by the presence of multiple large adenomas throughout the intestine 36 days after initiating stem cell transformation (FIG. 21f).

[0408] To further investigate the hierarchy that exists between the APC-deficient stem cells and their transformed progeny, we examined expression of the stem cell marker protein Lgr5-EGFP during the various stages of adenoma formation in our model. In non-transformed stem cells, Lgr5-EGFP expression was restricted to the Crypt Base Columnar (CBC) cells (FIG. 22a). Expression of this stem cell marker was maintained following the initial transformation of the stem cells after 3 days (FIG. 22b) and was also clearly evident in the “pockets” of recently expanded transformed progeny within the crypts after 8 days (FIG. 22c), indicating that at least some aspect of “stemness” was conferred to these cells. However, there was a marked down-regulation of Lgr5-EGFP expression on the larger adenomas present in the intestines of 36-day induced mice, despite uniformly high 6-catenin levels throughout the tumor (FIG. 22d). Lgr5-EGFP expression was limited to a few scattered cells within the tumor mass (FIG. 23). These GFP-positive cells retained the slender, wedge-shaped morphology characteristic of the CBC intestinal stem cells. It is therefore tempting to speculate that the Lgr5 expression in larger adenomas is marking a rare population of stem cells responsible for fueling their continued growth. Taken together, these data demonstrate that transformation of stem cells through loss of Apc is an extremely efficient route towards initiating intestinal adenoma formation. The kinetics of this process suggest that no further mutations are required once both Ape alleles are lost in intestinal epithelium, which is in accordance with the tissue-tropism of Apc's tumor suppressor activity.

Example 5 Generation and Use of Antibodies Directed Against LGR5 and LGR6

[0409] Materials and Methods

[0410] Monoclonal rat antibodies were generated by Genovac (Freiburg, Germany) by intramuscular injection of rats with an expression plasmid expressing either human Lgr5 or Lgr6. Rat B-cells were fused with mouse myeloma cells. The resulting hybridomas were screened on HEK293 cells that were transfected with human or Mouse Lgr5 or Lgr6 expression plasmids.

[0411] L8 (DNTcf4-LS174T) cells were cultured with and without Doxycycline for 48 hrs. L8 cells are clonal derivatives of LS174T cells. Upon Doxycycline (DOX) induction the L8 cells express a dominant negative form of T-cell Factor 4 (DNTcf4; see Roose et al., 1999, Science 285: 1923-1926). DNTcf4 turns off constitutive active Wnt pathway. Rat IgG was used as negative isotype control. After 48 hrs cells are washed with ice cold PBS and brought into suspension using 5 mM EDTA. All the following steps are done at 4° C. Cells were blocked for 30 min in PBS containing 2% BSA. Primary (1st) and Secondary (2nd) antibody reagent were incubated subsequently for 1 hr, and washed with ice cold PBS/2% BSA. For the primary antibody staining we used undiluted hybridoma supernatant, 2nd antibody staining was done using Qdot® 655 goat F(ab′)2 anti-rat IgG conjugates (H+L) (Molecular Probes/Invitrogen). Prior to analysis propidium iodine was added to exclude dead cells in the analysis.

[0412] Results

[0413] The specificity of some of the isolated antibodies is shown in Tables 4 and 5, as tested by FACS analysis. 9G5 is a rat monoclonal antibody directed against hLgr5. The analysis of endogenous Lgr5 expression was determined in L8 cells. L8 cells are clonal derivatives of LS174T cells. Upon Doxycycline (DOX) induction, L8 cells express dominant negative Tcf4 (DNTcf4). DNTcf4 turns off constitutive active Wnt pathway. This is reflected in FIG. 24, showing FACS staining of L8 cells with 9G5 antibody or IgG control antibody. After 48 hrs of DOX induction, indeed a reduction in endogenous hLgr5 protein levels was observed, as also becomes clear from the reduction in the fluorescent means of the peak for the L8 cells treated with doxycycline.

[0414] This experiment was also performed with LGRS-specific antibodies 2F10, 10C1 and 6C10. As shown in FIG. 26, similar results are obtained with any of these LGR5-specific antibodies.

TABLE-US-00004 TABLE 4 Specificity of Lgr5 antibodies. 9G5 recognize both mouse and human Lgr5. The colon cancer cell lines; DLD1 and SW480, LIM1863 do not show specific staining for Lgr5. These antibodies were tested negative for cross reactivity against mouse Lgr4, 6 and human Lgr4, and 6. mLgr5-293T hLgr5-293T L8 overexpression overexpression LS174 (DNTcf4-LS174) NR 1D9 − + + + NR 2F10 − + + + NR 4D11 − + + + NR 6C10 − + + + NR 9B3 − + + + NR 3A4 − + + + NR 5A7 − + + + NR 6G2 − + + + NR 9G5 ++ ++ ++ ++ NR 2B8 − ++ ++ ++ NR 3B9 − ++ ++ ++ NR 5C8 − + + + NR 7B11 − + + + NR10C1 − + + + NR 4D6 − + + + NR 5E9 − + + + NR 8F2 − ++ ++ ++ NR10F7 − + + +

TABLE-US-00005 TABLE 5 Specificity of Lgr6 antibodies. Antibodies 1d8 and 3d8 recognize mouse Lgr6 and hLgr5 in addition to human Lgr6. The colon cancer cell lines; LS174, DLD1 and SW480 do not show specific staining for Lgr6. These antibodies were tested negative for cross reactivity against mouse Lgr4, 5 and human Lgr4. Clone number 1d8 3d8 6d8 2f4 2h10 5e10 hLgr5-293T + + − − − − overexpression mLgr6-293T + + − − − − overexpression hLgr6-293T ++ ++ ++ ++ + ++ overexpression LS174 − − − − − − L8 (LS174-DNTcf4) − − − − − −

Example 6 Expression Analysis of Colon and Small Intestine Derived Stem Cells Compared to their Direct Progeny

[0415] Materials and Methods

[0416] Isolation of GFP Positive Epithelial Cells

[0417] Freshly isolated small intestines or colons were incised along their length and villi (in case of small intestine) were removed by scraping. The tissue was then incubated in PBS/5 mM EDTA for 5 minutes. Gentle shaking removed remaining villi and the intestinal tissue was subsequently incubated in PBS/EDTA for 30 minutes at 4° C. Vigorous shaking yielded free crypts which were incubated in PBS supplemented with Trypsine (10 mg/ml) and DNAse (0.8 u/μl) for 30 minutes at 37° C. After incubation, cells were spun down, resuspended in SMEM (Invitrogen) and filtered through a 40 μM mesh. GFP-expressing cells were isolated using a MoFlo cell sorter (DAKO).

[0418] Microarray Analysis

[0419] RNA was isolated from the GFP.sup.hi and GFP.sup.lo cell fractions of intestines from Lgr5-EGFP-ires-CreERT2 mice. 250 ng of total RNA was labeled using low RNA Input Linear Amp kit (Agilent Technologies, Pato Alto, Calif., USA). Labeling, hybridization, and washing protocols were done according to guidelines (Agilent Technologies, Santa Clara, Calif., USA). Differentially labelled cRNA from GFP.sup.hi and GFP.sup.lo cells from two different sorts (each combining three different mice) were combined and hybridised on 4X44K Agilent Whole Mouse Genome dual colour Microarrays (G4122F) in two dye swap experiments, resulting in four individual arrays. All data analyses were performed using ArrayAssist (Stratagene Inc, La Jolla, Calif., USA) and Microsoft Excel (Microsoft Corporation, Redmond, Wash., USA). Raw signal intensities were corrected by subtracting local background. Negative values were changed by a positive value close to zero (standard deviation of the local background) in order to allow calculation of rations between intensities for features only present in one sample (GFP.sup.hi or GFP.sup.lo). Data were filtered if both (GFP.sup.hi or GFP.sup.lo) intensities were changed or if both intensities were less than two time the background signal and normalized by a Loess algorithm. Statistical analysis was performed by running an Excel version of SAM (Significant Analysis of Microarrays) using an Excel plug-in of the software (Tusher PNAS 2001, References 6) and “one class” as the response value. Genes were considered to be significantly enriched in GFP.sup.hi cells if they had a q-value of <0.1 and where present in at least 3 out of 4 arrays and the average of all four arrays exceeded a log 2 ration of 0.6.

[0420] Results

[0421] In order to define a gene expression profile for Lgr5.sup.+ intestinal stem cells, we established a protocol to sort GFP-positive epithelial cells from cell suspensions prepared from freshly isolated crypts of Lgr5-EGFP-ires-CreERT2 mice (see Methods). FACS analysis distinguished a GFP-high (GFP.sup.hi) and a GFP-low (GFP.sup.lo) population, which we tentatively identified as CBC cells and their immediate transit-amplifying daughters, respectively (FIG. 25). A single mouse intestine routinely yielded several hundred thousand GFP.sup.hi and GFP.sup.lo cells. An example of an almost pure population of Lgr5-expressing cells is provided in FIG. 25A. In order to identify novel stem cell genes, mRNA samples of the two populations were subjected to comparative gene expression profiling. The gene that was most highly enriched in the GFP.sup.hi cells was, satisfactorily, the Lgr5 gene itself. Multiple genes on the list (Table 6) were already identified as intestinal Wnt target genes previously, for instance in human colon cancer (van der Flier et al, 2007, Gastroenterology 132, 628-632), which further validated the gene list. While in situ hybridizations on these Wnt target genes typically confirmed high level expression in CSC cells, TA cells directly above the Paneth cells also expressed these genes, albeit at a much lower level. As an example, FIG. 25B shows the expression of Sox9, a Wnt-responsive gene (Blache et al., 2004) crucial for Paneth cell specification (Bastide et al., 2007; Mori-Akiyama et al., 2007).

Discussion

[0422] In the intestine, a long-lived pool of cycling stem cells is defined by Lgr5 expression, a Wnt responsive orphan G-coupled receptor (Barker et al., 2007, Nature 449, 1003-1007). These Lgr5.sup.+ cells have previously been observed by Leblond and colleagues, who named them Crypt Base Columnar (CBC) cells and already speculated that these CBC cells represent the stem cells of the intestinal epithelium (Cheng and Leblond, 1974, Am J Anat 141, 461-79). Here, we define a minimal gene expression profile for these CBC cells by exploiting the Lgr5-EGFP-ires-CreERT2 knock-in mice for sorting, based on GFP expression. We defined a set genes differently expressed between GFP.sup.hi and GFP.sup.lo fractions. Based on confocal images of isolated crypts, we tentatively identified these as CBC cells and their daughters, respectively. Lgr5 was found as the most differential gene in this set, implying that its expression is strongly restricted to CBC cells. Many other genes in the signature represented previously identified Wnt-dependent genes, e.g. Ascl2, CD44, Ephb3, Sox9 and Sp5 (van der Flier et al, 2007, Gastroenterology 132, 628-632). Given the intimate connection between Wnt signaling and the biology of stem cells in many tissues (Reya and Clevers, 2005, Nature 434, 843-850), this was not surprising.

[0423] Table 6 Expression Analysis of Stem Cells and their Direct Progeny

[0424] Small intestinal (Table 6a) and colon stem cell (Table 6b) signature based on Lgr5 expression. GFP-positive epithelial cells from pure crypt preparations of Lgr5-EGFP-ires-CreERT2 mice were isolated using FACS sorting. FACS analysis distinguished two populations GFP.sup.hi and GFP.sup.lo cells, corresponding to the CBC cells and their immediate transit-amplifying daughters respectively. In order to identify novel stem cell genes, mRNA samples of the two populations were subjected to comparative gene expression profiling using Agilent microarray analysis.

TABLE-US-00006 TABLE 6a Comparison of small intestinal stem cells with direct progeny avg log2 ratio.sup.1 Gene name (gfp.sup.high/gfp.sup.low) Lgr5 2.54 Ephb3 1.38 Cd44 1.15 Rnf43 1.14 Sox9 1.12 Slc12a2 0.87 Ets2 0.80 .sup.1Q value <0.15

TABLE-US-00007 TABLE 6b Comparison of colon stem cells with direct progeny avg log2 ratio.sup.1 Gene name (gfp.sup.high/gfp.sup.low) Lgr5 2.98 Cd44 1.47 Cdca7 1.23 Ephb3 1.11 Myb 0.81 Myc 0.77 .sup.1Q value <0.05

Example 7 Sequence Determination of Light Chain and Heavy Chains, Including CDR Regions, of LGR5-Specific/LGR6-Specific Antibodies

[0425] Materials and Methods:

[0426] Hybridoma Sequence

[0427] The hybridomas were produced as described in the materials and methods section of Example 5.

[0428] Hybridoma sequences were determined from Lgr5-specific and/or Lgr6-specific clonal hybridoma cell lines NR 2F10 (see Table 4) and 6d8 and 2f4 (see Table 5). Total RNA was isolated using Trizol reagent and cDNA generated using superscript reverse transcriptase (Promega). cDNA was amplified using PCR primers designed to amplify the IgG antibody Fv-DNA sequences in a ‘touch-down’ PCR. PCR fragments were cloned into either PJET1.2 (Fermentas) or PGEM-T (Promega) cloning vectors and subsequently sequenced using vector-specific primers on an ABI sequencer.

[0429] IgG Antibody Fe-DNA Sequence PCR Primers:

TABLE-US-00008 Kappa L-chain reverse primers; 25 individually synthesized oligos, pooled, representing 50 variants: MVK-1 GACATTGTTCTCACCCAGTCTCC MVK-2 GACATTGTGCTSACCCAGTCTCC MVK-3 GACATTGTGATGACTCAGTCTCC MVK-4 GACATTGTGCTMACTCAGTCTCC MVK-5 GACATTGTGYTRACACAGTCTCC MVK-6 GACATTGTRATGACACAGTCTCC MVK-7 GACATTMAGATRACCCAGTCTCC MVK-8 GACATTGCAGATGAMCCAGTCTCC MVK-9 GACATTCAGATGACDCAGTCTCC MVK-10 GACATTCAGATGACACAGACTAC MVK-11 GACATTCAGATGATTCAGTCTCC MVK-12 GACATTGTTCTCAWCCAGTCTCC MVK-13 GACATTGTTCTCTCCCAGTCTCC MVK-14 GACATTGWGCTSACCCAATCTCC MVK-15 GACATTSTGATGACCCARTCTC MVK-16 GACATTKTGATGACCCARACTCC MVK-17 GACATTGTGATGACTCAGGCTAC MVK-18 GACATTGTGATGACBCAGGCTGC MVK-19 GACATTGTGATAACYCAGGATG MVK-20 GACATTGTGATGACCCAGTTTGC MVK-21 GACATTGTGATGACACAACCTGC MVK-22 GACATTGTGATGACCCAGATTCC MVK-23 GACATTTTGCTGACTCAGTCTCC MVK-24 GACATTGTAATGACCCAATCTCC MVK-25 GACATTGTGATGACCCACACTCC Kappa L-chain forward primer: mck-1 ACACTCATTCCTGTTGAAGCTCTTGAC

TABLE-US-00009 H-chain variable region reverse primers, 25 individually synthesized oligos, pooled, representing 88 variants: MVH-1 GCCGGCCATGGCCGAGGTRMAGCTTCAGGAGTCAGGAC MVH-2 GCCGGCCATGGCCGAGGTSCAGCTKCAGCAGTCAGGAC MVH-3 GCCGGCCATGGCCCAGGTGCAGCTGAAGSASTCAGG MVH-4 GCCGGCCATGGCCGAGGTGCAGCTTCAGGAGTCSGGAC MVH-5 GCCGGCCATGGCCGARGTCCAGCTGCAACAGTCYGGAC MVH-6 GCCGGCCATGGCCCAGGTCCAGCTKCAGCAATCTGG MVH-7 GCCGGCCATGGCCCAGSTBCAGCTGCAGCAATCTGG MVH-8 GCCGGCCATGGCCCAGGTYCAGCTGCAGCAGTCTGGRC MVH-9 GCCGGCCATGGCCCAGGTYCAGCTYCAGCAGTCTGG MVH-10 GCCGGCCATGGCCGAGGTCCARCTGCAACAATCTGGACC MVH-11 GCCGGCCATGGCCCAGGTCCACGTGAAGCAGTCTGGG MVH-12 GCCGGCCATGGCCGAGGTGAASSTGGTGGAATCTG MVH-13 GCCGGCCATGGCCGAVGTGAAGYTGGTGGAGTCTG MVH-14 GCCGGCCATGGCCGAGGTGCAGSKGGTGGAGTCTGGGG MVH-15 GCCGGCCATGGCCGAKGTGCAMCTGGTGGAGTCTGGG MVH-16 GCCGGCCATGGCCGAGGTGAAGCTGATGGARTCTGG MVH-17 GCCGGCCATGGCCGAGGTGCARCTTGTTGAGTCTGGTG MVH-18 GCCGGCCATGGCCGARGTRAAGCTTCTAGAGTCTGGA MVH-19 GCCGGCCATGGCCGAAGTGAARSTTGAGGAGTCTGG MVH-20 GCCGGCCATGGCCGAAGTGATGCTGGTGGAGTCTGGG MVH-21 GCCGGCCATGGCCCAGGTTACTCTRAAAGWGTSTGGCC MVH-22 GCCGGCCATGGCCCAGGTCCAACTVCAGCARCCTGG MVH-23 GCCGGCCATGGCCCAGGTYCARCTGCAGCAGTCTG MVH-24 GCCGGCCATGGCCGATGTGAACTTGGAAGTGTCTGG MVH-25 GCCGGCCATGGCCGAGGTGAAGGTCATCGAGTCTGG H-chain forward primers: MJH-REV1&2 GGGGGTGTCGTTTIGGCTGAGGAGACGGTGACCGTGG MJH-REV2INT GGGGGTGTCGTTTTGGCTGAGGAGACGGTGACAGTGG MJH-REV3 GGGGGTGTCGTTTTGGCTGAGGAGACGGTGACCAGAG MJH-REV4 GGGGGTGTCGTTTTGGCTGAGGAGACGGTGACCGAGG Variable position key: R (A/G); M (A/C); Y (T/C); W (A/T; S (G/C); K (G/T); H (A/T/C); B (G/C/T); V (G/A/C); D (G/A/T); N (G/A/T/C)

[0430] In this experiment, mouse-specific oligos are used. For more reproducible results, rat-specific oligos can be used as well.

[0431] Results

[0432] The light chain sequence of LGR5-specific antibody NR 2F10 (see Table 4) and the heavy chain sequences of LGR6-specific antibodies 6d8 and 2f4 (see Table 5) are depicted in FIG. 27. The CDR regions are indicated in bold and in italics. The CDR sequences were determined according to Kabat (Kabat et al., “Sequences of Proteins of Immunological Interest,” U.S. Dept. of Health and Human Services, National Institute of Health, 1987). Antibodies or functional equivalents thereof comprising at least one of these CDR sequences constitute a high affinity binding compound with a high specificity for their target proteins LGR5 and/or LGR6.

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