ABSTRACT
Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.
Subject(s)
Cell Culture Techniques/methods , Organoids/growth & development , Snake Venoms/metabolism , Adult Stem Cells/metabolism , Animals , Coral Snakes/metabolism , Gene Expression Profiling/methods , Organoids/metabolism , Salivary Glands/metabolism , Snake Venoms/genetics , Snakes/genetics , Snakes/growth & development , Stem Cells/metabolism , Toxins, Biological/genetics , Transcriptome/geneticsABSTRACT
Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.
Subject(s)
Enteroendocrine Cells/metabolism , RNA, Messenger/genetics , Cells, Cultured , Gastrointestinal Hormones/genetics , Gastrointestinal Tract/metabolism , Glucagon-Like Peptide 1/genetics , Humans , Organoids/metabolism , Transcription Factors/genetics , Transcriptome/geneticsABSTRACT
The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.
Subject(s)
Cell Proliferation , Hepatocytes/metabolism , Organoids/metabolism , Animals , Cell Culture Techniques , Cells, Cultured , Hepatocytes/cytology , Humans , Mice , Mice, Inbred BALB C , Mice, Knockout , Organoids/cytology , Stem Cells/cytology , Stem Cells/metabolism , Time FactorsABSTRACT
Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.
Subject(s)
Breast Neoplasms/pathology , Genetic Heterogeneity , Organoids/pathology , Tissue Banks , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Cells, Cultured , Drug Screening Assays, Antitumor/methods , Female , Humans , Mice , Mice, Nude , Organoids/drug effects , Precision Medicine/methodsABSTRACT
The prostate gland consists of basal and luminal cells arranged as pseudostratified epithelium. In tissue recombination models, only basal cells reconstitute a complete prostate gland, yet murine lineage-tracing experiments show that luminal cells generate basal cells. It has remained challenging to address the molecular details of these transitions and whether they apply to humans, due to the lack of culture conditions that recapitulate prostate gland architecture. Here, we describe a 3D culture system that supports long-term expansion of primary mouse and human prostate organoids, composed of fully differentiated CK5+ basal and CK8+ luminal cells. Organoids are genetically stable, reconstitute prostate glands in recombination assays, and can be experimentally manipulated. Single human luminal and basal cells give rise to organoids, yet luminal-cell-derived organoids more closely resemble prostate glands. These data support a luminal multilineage progenitor cell model for prostate tissue and establish a robust, scalable system for mechanistic studies.
Subject(s)
Organ Culture Techniques , Organoids , Prostate/cytology , Androgens/metabolism , Humans , Male , Stem Cells/cytology , Stem Cells/metabolismABSTRACT
FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.
Subject(s)
F-Box Proteins , Neoplasms , Humans , F-Box-WD Repeat-Containing Protein 7/genetics , F-Box-WD Repeat-Containing Protein 7/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Epidermal Growth Factor/genetics , Epidermal Growth Factor/pharmacology , Epidermal Growth Factor/metabolism , Proteomics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , F-Box Proteins/geneticsABSTRACT
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, TransgenicABSTRACT
The thyroid maintains systemic homeostasis by regulating serum thyroid hormone concentrations. Here we report the establishment of three-dimensional (3D) organoids from adult thyroid tissue representing murine and human thyroid follicular cells (TFCs). The TFC organoids (TFCOs) harbor the complete machinery of hormone production as visualized by the presence of colloid in the lumen and by the presence of essential transporters and enzymes in the polarized epithelial cells that surround a central lumen. Both the established murine as human thyroid organoids express canonical thyroid markers PAX8 and NKX2.1, while the thyroid hormone precursor thyroglobulin is expressed at comparable levels to tissue. Single-cell RNA sequencing and transmission electron microscopy confirm that TFCOs phenocopy primary thyroid tissue. Thyroid hormones are readily detectable in conditioned medium of human TFCOs. We show clinically relevant responses (increased proliferation and hormone secretion) of human TFCOs toward a panel of Graves' disease patient sera, demonstrating that organoids can model human autoimmune disease.
Subject(s)
Gene Expression Regulation/physiology , Graves Disease/metabolism , Organoids/metabolism , Thyroid Epithelial Cells/physiology , Animals , Culture Media , Humans , Mice , PAX8 Transcription Factor/genetics , PAX8 Transcription Factor/metabolism , Thyroglobulin/genetics , Thyroglobulin/metabolism , Thyroid Nuclear Factor 1/genetics , Thyroid Nuclear Factor 1/metabolismABSTRACT
Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
Subject(s)
Carcinoma, Non-Small-Cell Lung/pathology , Cystic Fibrosis/pathology , Epithelial Cells/pathology , Organ Culture Techniques/methods , Organoids/pathology , Respiratory Syncytial Virus Infections/pathology , Respiratory System/pathology , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/metabolism , Cells, Cultured , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Disease Models, Animal , Drug Screening Assays, Antitumor , Epithelial Cells/metabolism , Female , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred NOD , Mice, SCID , Organoids/metabolism , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/isolation & purification , Respiratory System/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Patient-derived human organoids can be used to model a variety of diseases. Recently, we described conditions for long-term expansion of human airway organoids (AOs) directly from healthy individuals and patients. Here, we first optimize differentiation of AOs towards ciliated cells. After differentiation of the AOs towards ciliated cells, these can be studied for weeks. When returned to expansion conditions, the organoids readily resume their growth. We apply this condition to AOs established from nasal inferior turbinate brush samples of patients suffering from primary ciliary dyskinesia (PCD), a pulmonary disease caused by dysfunction of the motile cilia in the airways. Patient-specific differences in ciliary beating are observed and are in agreement with the patients' genetic mutations. More detailed organoid ciliary phenotypes can thus be documented in addition to the standard diagnostic procedure. Additionally, using genetic editing tools, we show that a patient-specific mutation can be repaired. This study demonstrates the utility of organoid technology for investigating hereditary airway diseases such as PCD.
Subject(s)
Ciliary Motility Disorders , Organoids , Cilia , Ciliary Motility Disorders/genetics , Humans , Mutation , PhenotypeABSTRACT
Bladder cancer is a common malignancy that has a relatively poor outcome. Lack of culture models for the bladder epithelium (urothelium) hampers the development of new therapeutics. Here we present a long-term culture system of the normal mouse urothelium and an efficient culture system of human bladder cancer cells. These so-called bladder (cancer) organoids consist of 3D structures of epithelial cells that recapitulate many aspects of the urothelium. Mouse bladder organoids can be cultured efficiently and genetically manipulated with ease, which was exemplified by creating genetic knockouts in the tumor suppressors Trp53 and Stag2. Human bladder cancer organoids can be derived efficiently from both resected tumors and biopsies and cultured and passaged for prolonged periods. We used this feature of human bladder organoids to create a living biobank consisting of bladder cancer organoids derived from 53 patients. Resulting organoids were characterized histologically and functionally. Organoid lines contained both basal and luminal bladder cancer subtypes based on immunohistochemistry and gene expression analysis. Common bladder cancer mutations like TP53 and FGFR3 were found in organoids in the biobank. Finally, we performed limited drug testing on organoids in the bladder cancer biobank.
Subject(s)
Organoids/pathology , Urinary Bladder Neoplasms/pathology , Animals , Mice , Precision MedicineABSTRACT
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/metabolismABSTRACT
[This corrects the article DOI: 10.1371/journal.pbio.1000539.].
ABSTRACT
In the adenoma-carcinoma sequence, it is proposed that intestinal polyps evolve through a set of defined mutations toward metastatic colorectal cancer (CRC). Here, we dissect this adenoma-carcinoma sequence in vivo by using an orthotopic organoid transplantation model of human colon organoids engineered to harbor different CRC mutation combinations. We demonstrate that sequential accumulation of oncogenic mutations in Wnt, EGFR, P53, and TGF-ß signaling pathways facilitates efficient tumor growth, migration, and metastatic colonization. We show that reconstitution of specific niche signals can restore metastatic growth potential of tumor cells lacking one of the oncogenic mutations. Our findings imply that the ability to metastasize-i.e., to colonize distant sites-is the direct consequence of the loss of dependency on specific niche signals.
Subject(s)
Colorectal Neoplasms/genetics , Organoids/transplantation , Adult , Animals , Cell Movement , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Colorectal Neoplasms/physiopathology , Disease Models, Animal , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic , Genetic Engineering , Humans , Male , Mice , Mice, Inbred NOD , Middle Aged , Mutation , Neoplasm Metastasis/genetics , Neoplastic Processes , Organoids/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolismABSTRACT
Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.
Subject(s)
Colonic Neoplasms/etiology , Gene Expression Regulation , Integrases/genetics , Mice, Transgenic , Adenoma/etiology , Adenoma/metabolism , Adenoma/pathology , Animals , Biomarkers, Tumor , Carbonic Anhydrase I/genetics , Carbonic Anhydrase I/metabolism , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Disease Models, Animal , Disease Progression , Enzyme Activation , Gene Knock-In Techniques , Gene Targeting , Genes, APC , Genes, ras , Genetic Loci , Humans , Immunohistochemistry , Integrases/metabolism , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Mice , Mice, Knockout , Mutation , Organ Specificity/genetics , ResearchABSTRACT
Leucine-rich repeat-containing G-protein coupled receptor 5-positive (Lgr5(+)) stem cells reside at crypt bottoms of the small and large intestine. Small intestinal Paneth cells supply Wnt3, EGF, and Notch signals to neighboring Lgr5(+) stem cells. Whereas the colon lacks Paneth cells, deep crypt secretory (DCS) cells are intermingled with Lgr5(+) stem cells at crypt bottoms. Here, we report regenerating islet-derived family member 4 (Reg4) as a marker of DCS cells. To investigate a niche function, we eliminated DCS cells by using the diphtheria-toxin receptor gene knocked into the murine Reg4 locus. Ablation of DCS cells results in loss of stem cells from colonic crypts and disrupts gut homeostasis and colon organoid growth. In agreement, sorted Reg4(+) DCS cells promote organoid formation of single Lgr5(+) colon stem cells. DCS cells can be massively produced from Lgr5(+) colon stem cells in vitro by combined Notch inhibition and Wnt activation. We conclude that Reg4(+) DCS cells serve as Paneth cell equivalents in the colon crypt niche.
Subject(s)
Colonic Neoplasms/metabolism , Neoplasm Proteins/genetics , Receptors, G-Protein-Coupled/genetics , Stem Cells/metabolism , Animals , Colon/cytology , Colon/growth & development , Colon/metabolism , Colonic Neoplasms/pathology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Intestine, Small/cytology , Intestine, Small/metabolism , Mice , Neoplasm Proteins/metabolism , Organoids/growth & development , Organoids/metabolism , Pancreatitis-Associated Proteins , Paneth Cells/cytology , Paneth Cells/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Notch/genetics , Stem Cell Niche/genetics , Stem Cells/cytology , Wnt Signaling Pathway/geneticsABSTRACT
Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear. To address the specific role of MYO5B in the intestine, we generated an intestine-specific conditional Myo5b-deficient (Myo5bfl/fl;Vil-CreERT2) mouse model. We analyzed intestinal tissues and cultured organoids of Myo5bfl/fl;Vil-CreERT2 mice by electron microscopy, immunofluorescence, and immunohistochemistry. Our data showed that Myo5bfl/fl;Vil-CreERT2 mice developed severe diarrhea within 4 d after tamoxifen induction. Periodic Acid Schiff and alkaline phosphatase staining revealed subapical accumulation of intracellular vesicles in villus enterocytes. Analysis by electron microscopy confirmed an almost complete absence of apical microvilli, the appearance of microvillus inclusions, and enlarged intercellular spaces in induced Myo5bfl/fl;Vil-CreERT2 intestines. In addition, we determined that MYO5B is involved not only in apical but also basolateral trafficking of proteins. The analysis of the intestine during the early onset of the disease revealed that subapical accumulation of secretory granules precedes occurrence of microvillus inclusions, indicating involvement of MYO5B in early differentiation of epithelial cells. By comparing our data with a novel MVID patient, we conclude that our mouse model completely recapitulates the intestinal phenotype of human MVID. This includes severe diarrhea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules. Thus, loss of MYO5B disturbs both apical and basolateral trafficking of proteins and causes MVID in mice.
Subject(s)
Malabsorption Syndromes/metabolism , Microvilli/pathology , Mucolipidoses/metabolism , Myosin Type V/metabolism , Animals , Disease Models, Animal , Enterocytes/metabolism , Enterocytes/pathology , Enterocytes/ultrastructure , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial Cells/ultrastructure , Female , Humans , Immunohistochemistry , Intestinal Mucosa/metabolism , Intestines/pathology , Intestines/ultrastructure , Malabsorption Syndromes/chemically induced , Male , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Mice, Transgenic , Microscopy, Confocal , Microscopy, Electron, Transmission , Microvilli/metabolism , Microvilli/ultrastructure , Mucolipidoses/chemically induced , Myosin Type V/genetics , Organ Culture Techniques , Protein Transport/genetics , Protein Transport/physiology , TamoxifenABSTRACT
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/metabolismABSTRACT
BACKGROUND & AIMS: We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. METHODS: We generated organoids from surgical samples of human gastric corpus. Culture conditions were developed based on those for the mouse gastric and human intestinal systems. We used microinjection to infect the organoids with H pylori. Epithelial responses were measured using microarray and quantitative polymerase chain reaction analyses. RESULTS: Human gastric cells were expanded indefinitely in 3-dimensional cultures. We cultured cells from healthy gastric tissues, single-sorted stem cells, or tumor tissues. Organoids maintained many characteristics of their respective tissues based on their histology, expression of markers, and euploidy. Organoids from healthy tissue expressed markers of 4 lineages of the stomach and self-organized into gland and pit domains. They could be directed to specifically express either lineages of the gastric gland, or the gastric pit, by addition of nicotinamide and withdrawal of WNT. Although gastric pit lineages had only marginal reactions to bacterial infection, gastric gland lineages mounted a strong inflammatory response. CONCLUSIONS: We developed a system to culture human gastric organoids. This system can be used to study H pylori infection and other gastric pathologies.