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1.
Cell ; 160(1-2): 299-312, 2015 Jan 15.
Article in English | MEDLINE | ID: mdl-25533785

ABSTRACT

Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.


Subject(s)
Liver/cytology , Organ Culture Techniques , Animals , Genomic Instability , Hepatocytes/cytology , Humans , Mice , Organoids/cytology
2.
Cell ; 161(4): 933-45, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25957691

ABSTRACT

In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.


Subject(s)
Biological Specimen Banks , Colorectal Neoplasms/pathology , Drug Screening Assays, Antitumor/methods , Organoids , Colorectal Neoplasms/drug therapy , DNA-Binding Proteins/metabolism , Humans , Oncogene Proteins/metabolism , Organ Culture Techniques , Organoids/drug effects , Precision Medicine , Ubiquitin-Protein Ligases
3.
Cell ; 155(2): 357-68, 2013 Oct 10.
Article in English | MEDLINE | ID: mdl-24120136

ABSTRACT

Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."


Subject(s)
Chief Cells, Gastric/cytology , Stem Cells/cytology , Stomach/cytology , Animals , Cell Lineage , Chief Cells, Gastric/chemistry , Gastric Mucosa/cytology , Mice , Organoids/cytology , Receptors, Tumor Necrosis Factor/analysis , Wnt Signaling Pathway
4.
Cell ; 136(5): 903-12, 2009 Mar 06.
Article in English | MEDLINE | ID: mdl-19269367

ABSTRACT

The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.


Subject(s)
Adult Stem Cells/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Intestine, Small/cytology , Animals , Cell Separation , Gene Deletion , Gene Expression Profiling , Mice , Mice, Transgenic
5.
Nature ; 556(7702): 457-462, 2018 04.
Article in English | MEDLINE | ID: mdl-29643510

ABSTRACT

Every cancer originates from a single cell. During expansion of the neoplastic cell population, individual cells acquire genetic and phenotypic differences from each other. Here, to investigate the nature and extent of intra-tumour diversification, we characterized organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells. Intra-tumour diversification of DNA methylation and transcriptome states also occurred; these alterations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. There were marked differences in responses to anticancer drugs between even closely related cells of the same tumour. The results indicate that colorectal cancer cells experience substantial increases in somatic mutation rate compared to normal colorectal cells, and that genetic diversification of each cancer is accompanied by pervasive, stable and inherited differences in the biological states of individual cancer cells.


Subject(s)
Antineoplastic Agents/pharmacology , Clone Cells/drug effects , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Evolution, Molecular , Mutation , Single-Cell Analysis , Cell Proliferation , Clone Cells/metabolism , Clone Cells/pathology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/metabolism , DNA Methylation , DNA Mutational Analysis , Gene Expression Regulation, Neoplastic , Humans , Intestinal Mucosa/metabolism , Intestines/cytology , Intestines/drug effects , Intestines/pathology , Mutation Rate , Organoids/cytology , Organoids/drug effects , Organoids/metabolism , Organoids/pathology , Transcriptome
6.
Proc Natl Acad Sci U S A ; 118(5)2021 02 02.
Article in English | MEDLINE | ID: mdl-33500353

ABSTRACT

Neuroblastoma is the most common extracranial solid tumor and accounts for ∼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor ("SCP") and the "neuroblast" cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.


Subject(s)
Adrenal Glands/pathology , Neuroblastoma/pathology , Schwann Cells/pathology , Single-Cell Analysis , Adrenal Medulla/pathology , Animals , Cell Aggregation , Gene Expression Regulation, Neoplastic , Humans , Mice, Inbred C57BL , Neoplasm Staging , Neural Stem Cells , Neuroblastoma/genetics , Phenotype
7.
EMBO J ; 38(15): e101654, 2019 08 01.
Article in English | MEDLINE | ID: mdl-31282586

ABSTRACT

Patient-derived tumour xenografts and tumour organoids have become important preclinical model systems for cancer research. Both models maintain key features from their parental tumours, such as genetic and phenotypic heterogeneity, which allows them to be used for a wide spectrum of applications. In contrast to patient-derived xenografts, organoids can be established and expanded with high efficiency from primary patient material. On the other hand, xenografts retain tumour-stroma interactions, which are known to contribute to tumorigenesis. In this review, we discuss recent advances in patient-derived tumour xenograft and tumour organoid model systems and compare their promises and challenges as preclinical models in cancer research.


Subject(s)
Neoplasms/pathology , Organoids/pathology , Animals , Cell Line, Tumor , Drug Evaluation, Preclinical , Humans , Models, Biological , Organoids/cytology , Xenograft Model Antitumor Assays
8.
Nature ; 538(7624): 260-264, 2016 Oct 13.
Article in English | MEDLINE | ID: mdl-27698416

ABSTRACT

The gradual accumulation of genetic mutations in human adult stem cells (ASCs) during life is associated with various age-related diseases, including cancer. Extreme variation in cancer risk across tissues was recently proposed to depend on the lifetime number of ASC divisions, owing to unavoidable random mutations that arise during DNA replication. However, the rates and patterns of mutations in normal ASCs remain unknown. Here we determine genome-wide mutation patterns in ASCs of the small intestine, colon and liver of human donors with ages ranging from 3 to 87 years by sequencing clonal organoid cultures derived from primary multipotent cells. Our results show that mutations accumulate steadily over time in all of the assessed tissue types, at a rate of approximately 40 novel mutations per year, despite the large variation in cancer incidence among these tissues. Liver ASCs, however, have different mutation spectra compared to those of the colon and small intestine. Mutational signature analysis reveals that this difference can be attributed to spontaneous deamination of methylated cytosine residues in the colon and small intestine, probably reflecting their high ASC division rate. In liver, a signature with an as-yet-unknown underlying mechanism is predominant. Mutation spectra of driver genes in cancer show high similarity to the tissue-specific ASC mutation spectra, suggesting that intrinsic mutational processes in ASCs can initiate tumorigenesis. Notably, the inter-individual variation in mutation rate and spectra are low, suggesting tissue-specific activity of common mutational processes throughout life.


Subject(s)
Adult Stem Cells/metabolism , Aging/genetics , Mutation Accumulation , Mutation Rate , Organ Specificity , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Child , Child, Preschool , Colon/metabolism , DNA Mutational Analysis , Female , Genes, Neoplasm/genetics , Humans , Incidence , Intestine, Small/metabolism , Liver/metabolism , Male , Mice , Middle Aged , Multipotent Stem Cells/metabolism , Neoplasms/epidemiology , Neoplasms/genetics , Organoids/metabolism , Point Mutation/genetics , Young Adult
9.
Proc Natl Acad Sci U S A ; 116(52): 26599-26605, 2019 Dec 26.
Article in English | MEDLINE | ID: mdl-31843916

ABSTRACT

Cycling intestinal Lgr5+ stem cells are intermingled with their terminally differentiated Paneth cell daughters at crypt bottoms. Paneth cells provide multiple secreted (e.g., Wnt, EGF) as well as surface-bound (Notch ligand) niche signals. Here we show that ablation of Paneth cells in mice, using a diphtheria toxin receptor gene inserted into the P-lysozyme locus, does not affect the maintenance of Lgr5+ stem cells. Flow cytometry, single-cell sequencing, and histological analysis showed that the ablated Paneth cells are replaced by enteroendocrine and tuft cells. As these cells physically occupy Paneth cell positions between Lgr5 stem cells, they serve as an alternative source of Notch signals, which are essential for Lgr5+ stem cell maintenance. Our combined in vivo results underscore the adaptive flexibility of the intestine in maintaining normal tissue homeostasis.

10.
Nature ; 521(7550): 43-7, 2015 May 07.
Article in English | MEDLINE | ID: mdl-25924068

ABSTRACT

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.


Subject(s)
Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Intestines/pathology , Mutation/genetics , Organoids/metabolism , Organoids/pathology , Stem Cells/pathology , Aneuploidy , Animals , CRISPR-Cas Systems , Child , Child, Preschool , Colorectal Neoplasms/metabolism , Female , Genes, APC , Genes, p53/genetics , Heterografts , Humans , Imidazoles , Intercellular Signaling Peptides and Proteins/metabolism , Intestinal Mucosa/metabolism , Mice , Middle Aged , Mutagenesis, Site-Directed , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Neoplasm Transplantation , Piperazines , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Smad4 Protein/deficiency , Stem Cell Niche/physiology , Stem Cells/metabolism
11.
Proc Natl Acad Sci U S A ; 115(4): E610-E619, 2018 01 23.
Article in English | MEDLINE | ID: mdl-29311336

ABSTRACT

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67iresCreER allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.


Subject(s)
Lateral Ventricles/cytology , Neural Stem Cells/physiology , Stem Cell Niche , Animals , Cell Lineage , Cell Proliferation , Mice , Neurogenesis , Receptors, Tumor Necrosis Factor/metabolism , Single-Cell Analysis , Transcriptome
12.
Nature ; 513(7518): 422-425, 2014 Sep 18.
Article in English | MEDLINE | ID: mdl-25043003

ABSTRACT

The somatic mutations present in the genome of a cell accumulate over the lifetime of a multicellular organism. These mutations can provide insights into the developmental lineage tree, the number of divisions that each cell has undergone and the mutational processes that have been operative. Here we describe whole genomes of clonal lines derived from multiple tissues of healthy mice. Using somatic base substitutions, we reconstructed the early cell divisions of each animal, demonstrating the contributions of embryonic cells to adult tissues. Differences were observed between tissues in the numbers and types of mutations accumulated by each cell, which likely reflect differences in the number of cell divisions they have undergone and varying contributions of different mutational processes. If somatic mutation rates are similar to those in mice, the results indicate that precise insights into development and mutagenesis of normal human cells will be possible.


Subject(s)
Cell Lineage/genetics , Clone Cells/cytology , Clone Cells/metabolism , Genome/genetics , Mutagenesis/genetics , Mutation/genetics , Animals , Biological Clocks/genetics , Cell Division , Cells, Cultured , Embryo, Mammalian/cytology , Humans , Male , Mice , Mice, Inbred C57BL , Mutation Rate , Organoids/cytology , Organoids/metabolism , Phylogeny , Sequence Analysis, DNA , Tail/cytology
13.
Nature ; 494(7436): 247-50, 2013 Feb 14.
Article in English | MEDLINE | ID: mdl-23354049

ABSTRACT

The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.


Subject(s)
Hepatocytes/cytology , Hepatocytes/metabolism , Receptors, G-Protein-Coupled/metabolism , Regeneration , Stem Cells/cytology , Stem Cells/metabolism , Wnt Signaling Pathway , Alleles , Animals , Bile Ducts/cytology , Bile Ducts/metabolism , Cell Lineage , Clone Cells/cytology , Clone Cells/metabolism , Culture Media/chemistry , Culture Media/metabolism , Disease Models, Animal , Female , Gene Knock-In Techniques , Hepatocytes/pathology , Hydrolases/deficiency , Hydrolases/genetics , Liver/cytology , Liver/metabolism , Liver/pathology , Liver Diseases/metabolism , Liver Diseases/pathology , Male , Mice , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Organoids/cytology , Organoids/transplantation , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Thrombospondins/deficiency , Thrombospondins/genetics , Thrombospondins/metabolism , Tyrosinemias/metabolism , Tyrosinemias/pathology
14.
Gastroenterology ; 153(4): 1040-1053.e4, 2017 10.
Article in English | MEDLINE | ID: mdl-28716720

ABSTRACT

BACKGROUND & AIMS: Resistance of metastatic human colorectal cancer cells to drugs that block epidermal growth factor (EGF) receptor signaling could be caused by aberrant activity of other receptor tyrosine kinases, activating overlapping signaling pathways. One of these receptor tyrosine kinases could be MET, the receptor for hepatocyte growth factor (HGF). We investigated how MET signaling, and its interaction with CD44 (a putative MET coreceptor regulated by Wnt signaling and highly expressed by intestinal stem cells [ISCs] and adenomas) affects intestinal homeostasis, regeneration, and adenoma formation in mini-gut organoids and mice. METHODS: We established organoid cultures from ISCs stimulated with HGF or EGF and assessed intestinal differentiation by immunohistochemistry. Mice with total epithelial disruption of MET (AhCre/Metfl/fl/LacZ) or ISC-specific disruption of MET (Lgr5Creert2/Metfl/fl/LacZ) and control mice (AhCre/Met+/+/LacZ, Lgr5Creert2/Met+/+/LacZ) were exposed to 10 Gy total body irradiation; intestinal tissues were collected, and homeostasis and regeneration were assessed by immunohistochemistry. We investigated adenoma organoid expansion stimulated by HGF or EGF using adenomas derived from Lgr5Creert2/Metfl/fl/Apcfl/fl and Lgr5Creert2/Met+/+/Apcfl/fl mice. The same mice were evaluated for adenoma prevalence and size. We also quantified adenomas in AhCre/Metfl/fl/Apcfl/+ mice compared with AhCre/Met+/+/Apcfl/+ control mice. We studied expansion of organoids generated from crypts and adenomas, stimulated by HGF or EGF, that were derived from mice expressing different CD44 splice variants (Cd44+/+, Cd44-/-, Cd44s/s, or Cd44v4-10/v4-10 mice). RESULTS: Crypts incubated with EGF or HGF expanded into self-organizing mini-guts with similar levels of efficacy and contained all differentiated cell lineages. MET-deficient mice did not have defects in intestinal homeostasis. Total body irradiation reduced numbers of proliferating crypts in AhCre/Metfl/fl/LacZ mice. Lgr5Creert2/Metfl/fl/LacZ mice had impaired regeneration of MET-deficient ISCs. Adenoma organoids stimulated with EGF or HGF expanded to almost twice the size of nonstimulated organoids. MET-deficient adenoma organoids did not respond to HGF stimulation, but did respond to EGF. ISC-specific disruption of Met (Lgr5Creert2/Metfl/fl/Apcfl/fl mice) caused a twofold increase in apoptosis in microadenomas, resulting in an approximately 50% reduction of microadenoma numbers and significantly reduced average adenoma size. Total epithelial disruption of Met (AhCre/Metfl/fl/Apcfl/+ mice) resulted in an approximate 50% reduction in (micro)adenoma numbers. Intestinal crypts from Cd44-/- mice did not expand to the same extent as crypts from Cd44+/+ mice on stimulation with HGF, but had the same response to EGF. The negative effect on HGF-mediated growth was overcome by expression of CD44v4-10, but not by CD44s. Similarly, HGF-mediated expansion of adenoma organoids required CD44v4-10. CONCLUSIONS: In studies of intestinal organoid cultures and mice with inducible deletion of MET, we found HGF receptor signaling to regulate intestinal homeostasis and regeneration, as well as adenoma formation. These activities of MET are promoted by the stem cell CD44 isoform CD44v4-10. Our findings provide rationale for targeting signaling via MET and CD44 during anti-EGF receptor therapy of patients with colorectal cancer or in patients resistant to EGF receptor inhibitors.


Subject(s)
Adenoma/metabolism , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Hyaluronan Receptors/metabolism , Intestinal Neoplasms/metabolism , Intestines/enzymology , Proto-Oncogene Proteins c-met/metabolism , Regeneration , Stem Cells/enzymology , Adenoma/genetics , Adenoma/pathology , Animals , Cell Differentiation , Cell Lineage , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Cells, Cultured , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Genotype , Hepatocyte Growth Factor/pharmacology , Homeostasis , Hyaluronan Receptors/genetics , Intestinal Neoplasms/genetics , Intestinal Neoplasms/pathology , Intestines/drug effects , Intestines/pathology , Intestines/radiation effects , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Proto-Oncogene Proteins c-met/genetics , Regeneration/drug effects , Regeneration/radiation effects , Signal Transduction , Stem Cells/drug effects , Stem Cells/pathology , Stem Cells/radiation effects , Time Factors , Tissue Culture Techniques , Tumor Burden
15.
Nature ; 488(7413): 665-9, 2012 Aug 30.
Article in English | MEDLINE | ID: mdl-22895187

ABSTRACT

LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5+ stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.


Subject(s)
DNA-Binding Proteins/metabolism , Endocytosis , Oncogene Proteins/metabolism , Receptors, Wnt/metabolism , Stem Cells/enzymology , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Wnt Signaling Pathway , Adenoma/metabolism , Adenoma/pathology , Animals , Cell Proliferation , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Frizzled Receptors/metabolism , HEK293 Cells , Humans , Lysosomes/metabolism , Mice , Oncogene Proteins/deficiency , Oncogene Proteins/genetics , Organoids/cytology , Organoids/metabolism , Organoids/pathology , Paneth Cells/metabolism , Paneth Cells/pathology , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, Wnt/antagonists & inhibitors , Stem Cells/cytology , Stem Cells/metabolism , Tumor Suppressor Proteins/deficiency , Tumor Suppressor Proteins/genetics , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Ubiquitination , Wnt Signaling Pathway/drug effects , beta Catenin/metabolism
16.
Proc Natl Acad Sci U S A ; 112(43): 13308-11, 2015 Oct 27.
Article in English | MEDLINE | ID: mdl-26460009

ABSTRACT

Tumor organoids are 3D cultures of cancer cells. They can be derived from the tumor of each individual patient, thereby providing an attractive ex vivo assay to tailor treatment. Using patient-derived tumor organoids for this purpose requires that organoids derived from biopsies maintain the genetic diversity of the in vivo tumor. In this study tumor biopsies were obtained from 14 patients with metastatic colorectal cancer (i) to test the feasibility of organoid culture from metastatic biopsy specimens and (ii) to compare the genetic diversity of patient-derived tumor organoids and the original tumor biopsy. Genetic analysis was performed using SOLiD sequencing for 1,977 cancer-relevant genes. Copy number profiles were generated from sequencing data using CopywriteR. Here we demonstrate that organoid cultures can be established from tumor biopsies of patients with metastatic colorectal cancer with a success rate of 71%. Genetic analysis showed that organoids reflect the metastasis from which they were derived. Ninety percent of somatic mutations were shared between organoids and biopsies from the same patient, and the DNA copy number profiles of organoids and the corresponding original tumor show a correlation of 0.89. Most importantly, none of the mutations that were found exclusively in either the tumor or organoid culture are in driver genes or genes amenable for drug targeting. These findings support further exploration of patient-derived organoids as an ex vivo platform to personalize anticancer treatment.


Subject(s)
Cell Culture Techniques/methods , Colorectal Neoplasms/genetics , Genetic Variation/genetics , Neoplasm Metastasis/genetics , Organoids/cytology , Organoids/growth & development , Antineoplastic Protocols/standards , Base Sequence , Colorectal Neoplasms/drug therapy , Genes, Neoplasm/genetics , Humans , Molecular Sequence Data , Organoids/chemistry , Precision Medicine/methods , Sequence Analysis, DNA
17.
EMBO J ; 32(20): 2708-21, 2013 Oct 16.
Article in English | MEDLINE | ID: mdl-24045232

ABSTRACT

Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.


Subject(s)
Adult Stem Cells/physiology , Cell Proliferation , Pancreas/cytology , Receptors, G-Protein-Coupled/physiology , Thrombospondins/physiology , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Animals , Cell Culture Techniques , Cells, Cultured , Embryo, Mammalian , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, SCID , Mice, Transgenic , Models, Biological , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Multipotent Stem Cells/physiology , Pancreas/metabolism , Rats , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/genetics , Thrombospondins/genetics , Thrombospondins/metabolism
18.
Nature ; 469(7330): 415-8, 2011 Jan 20.
Article in English | MEDLINE | ID: mdl-21113151

ABSTRACT

Homeostasis of self-renewing small intestinal crypts results from neutral competition between Lgr5 stem cells, which are small cycling cells located at crypt bottoms. Lgr5 stem cells are interspersed between terminally differentiated Paneth cells that are known to produce bactericidal products such as lysozyme and cryptdins/defensins. Single Lgr5-expressing stem cells can be cultured to form long-lived, self-organizing crypt-villus organoids in the absence of non-epithelial niche cells. Here we find a close physical association of Lgr5 stem cells with Paneth cells in mice, both in vivo and in vitro. CD24(+) Paneth cells express EGF, TGF-α, Wnt3 and the Notch ligand Dll4, all essential signals for stem-cell maintenance in culture. Co-culturing of sorted stem cells with Paneth cells markedly improves organoid formation. This Paneth cell requirement can be substituted by a pulse of exogenous Wnt. Genetic removal of Paneth cells in vivo results in the concomitant loss of Lgr5 stem cells. In colon crypts, CD24(+) cells residing between Lgr5 stem cells may represent the Paneth cell equivalents. We conclude that Lgr5 stem cells compete for essential niche signals provided by a specialized daughter cell, the Paneth cell.


Subject(s)
Intestines/cytology , Multipotent Stem Cells/cytology , Multipotent Stem Cells/metabolism , Paneth Cells/cytology , Receptors, G-Protein-Coupled/metabolism , Stem Cell Niche/cytology , Animals , CD24 Antigen/metabolism , Cell Count , Cell Proliferation , Coculture Techniques , Humans , Mice , Paneth Cells/metabolism , Stem Cell Niche/metabolism , Wnt Proteins/metabolism , Wnt3 Protein
19.
Nature ; 476(7360): 293-7, 2011 Jul 04.
Article in English | MEDLINE | ID: mdl-21727895

ABSTRACT

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 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.


Subject(s)
Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Thrombospondins/metabolism , Wnt Proteins/metabolism , Adult Stem Cells/metabolism , Animals , Cells, Cultured , Epithelial Cells/cytology , Epithelial Cells/metabolism , Frizzled Receptors/metabolism , Gene Deletion , HEK293 Cells , Humans , Mice , Protein Binding , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/deficiency , Receptors, G-Protein-Coupled/genetics , Regeneration , Signal Transduction/genetics , Wnt Proteins/genetics , Wnt3 Protein , Wnt3A Protein
20.
EMBO J ; 31(14): 3079-91, 2012 Jun 12.
Article in English | MEDLINE | ID: mdl-22692129

ABSTRACT

Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent '+4' cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5(+) CBC cells. Transcriptional profiling of FACS-sorted Lgr5(+) stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell-enriched genes was defined. Spatial expression patterns were further characterized by mRNA in-situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5(+)stem cells. Lineage tracing using a newly established knock-in mouse for one of the signature genes, Smoc2, confirmed its stem cell specificity. Using this resource, we find-and confirm by independent approaches-that the proposed quiescent/'+4' stem cell markers Bmi1, Tert, Hopx and Lrig1 are robustly expressed in CBC cells.


Subject(s)
Antigens, Differentiation/metabolism , Gene Expression Regulation , Intestinal Mucosa/metabolism , Receptors, G-Protein-Coupled/metabolism , Stem Cells/metabolism , Animals , Antigens, Differentiation/genetics , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Gene Expression Profiling , Intestines/cytology , Mice , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Receptors, G-Protein-Coupled/genetics , Stem Cells/cytology
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