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1.
EMBO J ; 38(12)2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31036555

RESUMO

Immunotherapy using chimeric antigen receptor (CAR)-engineered lymphocytes has shown impressive results in leukemia. However, for solid tumors such as colorectal cancer (CRC), new preclinical models are needed that allow to test CAR-mediated cytotoxicity in a tissue-like environment. Here, we developed a platform to study CAR cell cytotoxicity against 3-dimensional (3D) patient-derived colon organoids. Luciferase-based measurement served as a quantitative read-out for target cell viability. Additionally, we set up a confocal live imaging protocol to monitor effector cell recruitment and cytolytic activity at a single organoid level. As proof of principle, we demonstrated efficient targeting in diverse organoid models using CAR-engineered NK-92 cells directed toward a ubiquitous epithelial antigen (EPCAM). Tumor antigen-specific cytotoxicity was studied with CAR-NK-92 cells targeting organoids expressing EGFRvIII, a neoantigen found in several cancers. Finally, we tested a novel CAR strategy targeting FRIZZLED receptors that show increased expression in a subgroup of CRC tumors. Here, comparative killing assays with normal organoids failed to show tumor-specific activity. Taken together, we report a sensitive in vitro platform to evaluate CAR efficacy and tumor specificity in a personalized manner.

2.
Oncogene ; 38(24): 4788-4803, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30816345

RESUMO

A role of sphingolipids for inflammatory bowel disease and cancer is evident. However, the relative and separate contribution of sphingolipid deterioration in inflammation versus carcinogenesis for the pathophysiology of colitis-associated colon cancer (CAC) was unknown and therefore examined in this study. We performed isogenic bone marrow transplantation of inducible sphingosine-1-phosphate (S1P) lyase knockout mice to specifically modulate sphingolipids and associated genes and proteins in a compartment-specific way in a DSS/AOM mediated CAC model. 3D organoid cultures were used in vitro. S1P lyase (SGPL1) knockout in either immune cells or tissue, caused local sphingolipid accumulation leading to a dichotomic development of CAC: Immune cell SGPL1 knockout (I-SGPL-/-) augmented massive immune cell infiltration initiating colitis with lesions and calprotectin increase. Pathological crypt remodeling plus extracellular S1P-signaling caused delayed tumor formation characterized by S1P receptor 1, STAT3 mRNA increase, as well as programmed cell death ligand 1 expression, accompanied by a putatively counter regulatory STAT1S727 phosphorylation. In contrast, tissue SGPL1 knockout (T-SGPL-/-) provoked immediate occurrence of epithelial-driven tumors with upregulated sphingosine kinase 1, S1P receptor 2 and epidermal growth factor receptor. Here, progressing carcinogenesis was accompanied by an IL-12 to IL-23 shift with a consecutive development of a Th2/GATA3-driven, tumor-favoring microenvironment. Moreover, the knockout models showed distinct lymphopenia and neutrophilia, different from the full SGPL1 knockout. This study shows that depending on the initiating cellular S1P source, the pathophysiology of inflammation-induced cancer versus cancer-induced inflammation develops through separate, discernible molecular steps.

3.
J Exp Med ; 216(3): 704-720, 2019 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-30792186

RESUMO

Constitutive Wnt activation upon loss of Adenoma polyposis coli (APC) acts as main driver of colorectal cancer (CRC). Targeting Wnt signaling has proven difficult because the pathway is crucial for homeostasis and stem cell renewal. To distinguish oncogenic from physiological Wnt activity, we have performed transcriptome and proteome profiling in isogenic human colon organoids. Culture in the presence or absence of exogenous ligand allowed us to discriminate receptor-mediated signaling from the effects of CRISPR/Cas9-induced APC loss. We could catalog two nonoverlapping molecular signatures that were stable at distinct levels of stimulation. Newly identified markers for normal stem/progenitor cells and adenomas were validated by immunohistochemistry and flow cytometry. We found that oncogenic Wnt signals are associated with good prognosis in tumors of the consensus molecular subtype 2 (CMS2). In contrast, receptor-mediated signaling was linked to CMS4 tumors and poor prognosis. Together, our data represent a valuable resource for biomarkers that allow more precise stratification of Wnt responses in CRC.

4.
Leukemia ; 33(7): 1700-1712, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30635626

RESUMO

The transcriptional regulator far upstream element binding protein 1 (FUBP1) acts as an oncoprotein in solid tumor entities and plays a role in the maintenance of hematopoietic stem cells. However, its potential function in leukemia is unknown. In murine models of chronic (CML) and acute myeloid leukemia (AML) induced by BCR-ABL1 and MLL-AF9, respectively, knockdown of Fubp1 resulted in prolonged survival, decreased numbers of CML progenitor cells, decreased cell cycle activity and increased apoptosis. Knockdown of FUBP1 in CML and AML cell lines recapitulated these findings and revealed enhanced DNA damage compared to leukemia cells expressing wild type FUBP1 levels. FUBP1 was more highly expressed in human CML compared to normal bone marrow cells and its expression correlated with disease progression. In AML, higher FUBP1 expression in patient leukemia cells was observed with a trend toward correlation with shorter overall survival. Treatment of mice with AML with irinotecan, known to inhibit topoisomerase I and FUBP1, significantly prolonged survival alone or in combination with cytarabine. In summary, our data suggest that FUBP1 acts as cell cycle regulator and apoptosis inhibitor in leukemia. We demonstrated that FUBP1 might play a role in DNA repair, and its inhibition may improve outcome in leukemia patients.

5.
Cell Mol Gastroenterol Hepatol ; 6(4): 477-493.e1, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30364784

RESUMO

Background & Aims: Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID. Methods: Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions. Results: Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin-positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane. Conclusions: We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.

6.
Gastroenterology ; 2018 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-30267714

RESUMO

Caspase-8 (CASP8) is a protease that initiates apoptosis and regulates inflammation and immune responses. We identified germline mutations in CASP8 in 3 unrelated patients with infant-onset inflammatory bowel disease: 2 patients were homozygous for the mutation 710A>G, p.Q237R, which resulted in reduced protein expression, and 1 patient carried the mutation 793C>T, p.R265W. We isolated peripheral blood mononuclear cells from our index patient and observed defects in T- and B-cell maturation, proliferation, and/or activation. Macrophages from 1 patient with CASP8 deficiency and monocytic BLaER1 cells with knockout of CASP8 or overexpression of CASP8 with the 710A>G mutation had altered inflammasome activity on stimulation with lipopolysaccharide. Patient-derived intestinal organoids and colon carcinoma cells with knockout of CASP8 had defects in cell death processes that involved loss of TRAIL signaling and increased necroptosis. These findings indicate that CASP8 controls inflammation, innate and adaptive immunity, and intestinal barrier integrity in humans.

7.
Proc Natl Acad Sci U S A ; 114(7): 1643-1648, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28130546

RESUMO

Loss of tumor suppressor adenomatous polyposis coli (APC) activates ß-catenin to initiate colorectal tumorigenesis. However, ß-catenin (CTNNB1) activating mutations rarely occur in human colorectal cancer (CRC). We found that APC loss also results in up-regulation of IL-6 signal transducer (IL-6ST/gp130), thereby activating Src family kinases (SFKs), YAP, and STAT3, which are simultaneously up-regulated in the majority of human CRC. Although, initial YAP activation, which stimulates IL6ST gene transcription, may be caused by reduced serine phosphorylation, sustained YAP activation depends on tyrosine phosphorylation by SFKs, whose inhibition, along with STAT3-activating JAK kinases, causes regression of established colorectal tumors. These results explain why APC loss is a more potent initiating event than the mere activation of CTNNB1.


Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Receptor gp130 de Citocina/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Adulto , Idoso , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Receptor gp130 de Citocina/genética , Feminino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , beta Catenina/genética , beta Catenina/metabolismo
8.
Dev Cell ; 39(2): 239-253, 2016 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-27720610

RESUMO

Numerous signals drive the proliferative expansion of the distal endoderm and the underlying mesenchyme during lung branching morphogenesis, but little is known about how these signals are integrated. Here, we show by analysis of conditional double mutants that the two T-box transcription factor genes Tbx2 and Tbx3 act together in the lung mesenchyme to maintain branching morphogenesis. Expression of both genes depends on epithelially derived Shh signaling, with additional modulation by Bmp, Wnt, and Tgfß signaling. Genetic rescue experiments reveal that Tbx2 and Tbx3 function downstream of Shh to maintain pro-proliferative mesenchymal Wnt signaling, in part by direct repression of the Wnt antagonists Frzb and Shisa3. In combination with our previous finding that Tbx2 and Tbx3 repress the cell-cycle inhibitors Cdkn1a and Cdkn1b, we conclude that Tbx2 and Tbx3 maintain proliferation of the lung mesenchyme by way of at least two molecular mechanisms: regulating cell-cycle regulation and integrating the activity of multiple signaling pathways.


Assuntos
Proteínas Hedgehog/metabolismo , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Morfogênese , Proteínas com Domínio T/metabolismo , Via de Sinalização Wnt , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Proliferação de Células , Feminino , Glicoproteínas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/citologia , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Camundongos , Proteínas Repressoras/metabolismo , Fator de Crescimento Transformador beta/metabolismo
9.
PLoS One ; 11(6): e0156787, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27253890

RESUMO

The epicardium, the outermost layer of the heart, is an essential source of cells and signals for the formation of the cardiac fibrous skeleton and the coronary vasculature, and for the maturation of the myocardium during embryonic development. The molecular factors that control epicardial mobilization and differentiation, and direct the epicardial-myocardial cross-talk are, however, insufficiently understood. The T-box transcription factor gene Tbx18 is specifically expressed in the epicardium of vertebrate embryos. Loss of Tbx18 is dispensable for epicardial development, but may influence coronary vessel maturation. In contrast, over-expression of an activator version of TBX18 severely impairs epicardial development by premature differentiation of epicardial cells into SMCs indicating a potential redundancy of Tbx18 with other repressors of the T-box gene family. Here, we show that Tbx2 and Tbx20 are co-expressed with Tbx18 at different stages of epicardial development. Using a conditional gene targeting approach we find that neither the epicardial loss of Tbx2 nor the combined loss of Tbx2 and Tbx18 affects epicardial development. Similarly, we observed that the heterozygous loss of Tbx20 with and without additional loss of Tbx18 does not impact on epicardial integrity and mobilization in mouse embryos. Thus, Tbx18 does not function redundantly with Tbx2 or Tbx20 in epicardial development.


Assuntos
Epistasia Genética , Pericárdio/embriologia , Pericárdio/metabolismo , Proteínas com Domínio T/genética , Alelos , Animais , Transição Epitelial-Mesenquimal/genética , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Fenótipo , Proteínas com Domínio T/metabolismo
10.
J Mol Cell Cardiol ; 97: 140-9, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27180262

RESUMO

Initiation of cardiac excitation depends on a specialized group of cardiomyocytes at the venous pole of the heart, the sinoatrial node (SAN). The T-box transcription factor gene Tbx18 is expressed in the SAN myocardium and is required for formation of a large portion of the pacemaker. Previous studies suggested that Tbx18 is also sufficient to reprogram ventricular cardiomyocytes into SAN cells in rat, guinea-pig and pig hearts. To evaluate the consequences of misexpression of Tbx18 for imposing a nodal phenotype onto chamber myocardial cells in fetal mice, we used two independent conditional approaches with chamber-specific cre driver lines and an Hprt(Tbx18) misexpression allele. Myh6-Cre/+;Hprt(Tbx18/y) mice developed dilated atria with thickened walls, reduced right ventricles and septal defects that resulted in reduced embryonic and post-natal survival. Tagln-Cre/+;Hprt(Tbx18/y) mice exhibited slightly smaller hearts with rounded trabeculae that supported normal embryonic survival. Molecular analyses showed that the SAN gap junction and ion channel profile was not ectopically induced in chamber myocardium but the working myocardial gene program was partially inhibited in atria and ventricles of both misexpression models. Left atrial expression of Pitx2 was strongly repressed in Myh6-Cre/+;Hprt(Tbx18/y) embryos. We conclude that exclusion of Tbx18 expression from the developing atria and (right) ventricle is important to achieve normal cardiac left-right patterning and myocardial differentiation, and that Tbx18 is not sufficient to induce full SAN differentiation of chamber cardiomyocytes in fetal mice.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Miocárdio/metabolismo , Nó Sinoatrial/metabolismo , Proteínas com Domínio T/genética , Transcriptoma , Animais , Biomarcadores , Análise por Conglomerados , Feminino , Feto , Perfilação da Expressão Gênica , Genes Letais , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/patologia
11.
Nature ; 530(7590): 340-3, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26863187

RESUMO

Mammalian Wnt proteins are believed to act as short-range signals, yet have not been previously visualized in vivo. Self-renewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt. Wnt3 is produced specifically by Paneth cells. Here we have generated an epitope-tagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and 'plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.


Assuntos
Membrana Celular/metabolismo , Mucosa Intestinal/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt , Proteína Wnt3/metabolismo , Alelos , Animais , Adesão Celular , Divisão Celular , Difusão , Feminino , Receptores Frizzled/metabolismo , Técnicas de Introdução de Genes , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Wnt3/genética
12.
Cell Stem Cell ; 18(2): 203-13, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26831517

RESUMO

Intestinal crypts display robust regeneration upon injury. The relatively rare secretory precursors can replace lost stem cells, but it is unknown if the abundant enterocyte progenitors that express the Alkaline phosphate intestinal (Alpi) gene also have this capacity. We created an Alpi-IRES-CreERT2 (Alpi(CreER)) knockin allele for lineage tracing. Marked clones consist entirely of enterocytes and are all lost from villus tips within days. Genetic fate-mapping of Alpi(+) cells before or during targeted ablation of Lgr5-expressing stem cells generated numerous long-lived crypt-villus "ribbons," indicative of dedifferentiation of enterocyte precursors into Lgr5(+) stems. By single-cell analysis of dedifferentiating enterocytes, we observed the generation of Paneth-like cells and proliferative stem cells. We conclude that the highly proliferative, short-lived enterocyte precursors serve as a large reservoir of potential stem cells during crypt regeneration.


Assuntos
Linhagem da Célula , Enterócitos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Células-Tronco/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Biomarcadores/metabolismo , Desdiferenciação Celular , Linhagem Celular , Proliferação de Células , Enterócitos/patologia , Integrases/metabolismo , Neoplasias Intestinais/patologia , Camundongos , Mutação/genética , Organoides , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Regeneração/genética , Análise de Célula Única , beta-Galactosidase/metabolismo
13.
J Pathol ; 236(2): 155-64, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25712196

RESUMO

Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC.


Assuntos
Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/genética , Deleção de Genes , Mutação em Linhagem Germinativa/genética , Proteínas Supressoras de Tumor/genética , Polipose Adenomatosa do Colo/metabolismo , Adulto , Estudos de Casos e Controles , Cromossomos Humanos Par 9/genética , Neoplasias Colorretais/metabolismo , Variações do Número de Cópias de DNA/genética , Células Epiteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Fases de Leitura Aberta/genética , Proteínas Supressoras de Tumor/metabolismo
14.
Trends Cell Biol ; 25(2): 100-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25308311

RESUMO

Tissue homeostasis and regeneration are fueled by resident stem cells that have the capacity to self-renew, and to generate all the differentiated cell types that characterize a particular tissue. Classical models of such cellular hierarchies propose that commitment and differentiation occur unidirectionally, with the arrows 'pointing away' from the stem cell. Recent studies, all based on genetic lineage tracing, describe various strategies employed by epithelial stem cell hierarchies to replace damaged or lost cells. While transdifferentiation from one tissue type into another ('metaplasia') appears to be generally forbidden in nonpathological contexts, plasticity within an individual tissue stem cell hierarchy may be much more common than previously appreciated. In this review, we discuss recent examples of such plasticity in selected mammalian epithelia, highlighting the different modes of regeneration and their implications for our understanding of cellular hierarchy and tissue self-renewal.


Assuntos
Transdiferenciação Celular , Células Epiteliais/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Linhagem da Célula , Epigênese Genética , Humanos , Intestinos/citologia , Camundongos , Regeneração
15.
J Allergy Clin Immunol ; 134(6): 1354-1364.e6, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25174867

RESUMO

BACKGROUND: Inflammatory bowel disease (IBD) is one of the most common chronic gastrointestinal diseases, but the underlying molecular mechanisms remain largely unknown. Studies of monogenic diseases can provide insight into the pathogenesis of IBD. OBJECTIVE: We thought to determine the underlying molecular causes of IBD occurring in 2 unrelated families in association with an immune deficiency. METHODS: We performed genetic linkage analysis and candidate gene sequencing on 13 patients from a large consanguineous family affected by early-onset IBD, progressive immune deficiency, and, in some cases, autoimmunity and alopecia, a condition we named enteropathy-lymphocytopenia-alopecia. The candidate gene was also sequenced in an unrelated patient with a similar phenotype. We performed histologic analysis of patients' intestinal biopsy specimens and carried out functional assays on PBMCs. Gut organoids derived from a patient's biopsy specimen were analyzed. RESULTS: We identified biallelic missense mutations in tetratricopeptide repeat domain 7A (TTC7A) in all patients from both families. The resulting TTC7A depletion modified the proliferation, adhesion, and migratory capacities of lymphocytes through inappropriate activation of the RhoA signaling pathway. Normal function was restored by wild-type TTC7A expression or addition of a RhoA kinase inhibitor. The growth and polarity of gut epithelial organoids were also found to be dependent on the RhoA signaling pathway. CONCLUSIONS: We show that TTC7A regulates the actin cytoskeleton dynamics in lymphocytes through the RhoA signaling pathway and is required in both lymphocytes and epithelial cells for maintaining equilibrium between cell proliferation, migration, polarization, and cell death. Our study highlights variability in the phenotypic expression resulting from TTC7A deficiency and outlines that impairment of both epithelial cells and lymphocytes cooperatively causes IBD.


Assuntos
Alopecia , Doenças Inflamatórias Intestinais , Linfopenia , Proteínas/genética , Proteínas/imunologia , Adolescente , Adulto , Alopecia/genética , Alopecia/imunologia , Alopecia/patologia , Criança , Pré-Escolar , Colo/patologia , Duodeno/patologia , Feminino , Humanos , Lactente , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/patologia , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Antro Pilórico/patologia , Adulto Jovem , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/imunologia , Proteína rhoA de Ligação ao GTP/imunologia
16.
J Exp Med ; 211(7): 1393-405, 2014 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-24980747

RESUMO

Paneth cells (PCs) are terminally differentiated, highly specialized secretory cells located at the base of the crypts of Lieberkühn in the small intestine. Besides their antimicrobial function, PCs serve as a component of the intestinal stem cell niche. By secreting granules containing bactericidal proteins like defensins/cryptdins and lysozyme, PCs regulate the microbiome of the gut. Here we study the control of PC degranulation in primary epithelial organoids in culture. We show that PC degranulation does not directly occur upon stimulation with microbial antigens or bacteria. In contrast, the pro-inflammatory cytokine Interferon gamma (IFN-γ) induces rapid and complete loss of granules. Using live cell imaging, we show that degranulation is coupled to luminal extrusion and death of PCs. Transfer of supernatants from in vitro stimulated iNKT cells recapitulates degranulation in an IFN-γ-dependent manner. Furthermore, endogenous IFN-γ secretion induced by anti-CD3 antibody injection causes Paneth loss and release of goblet cell mucus. The identification of IFN-γ as a trigger for degranulation and extrusion of PCs establishes a novel effector mechanism by which immune responses may regulate epithelial status and the gut microbiome.


Assuntos
Degranulação Celular/imunologia , Defensinas/imunologia , Interferon gama/imunologia , Intestino Delgado/imunologia , Muramidase/imunologia , Células T Matadoras Naturais/imunologia , Celulas de Paneth/imunologia , Animais , Anticorpos/farmacologia , Complexo CD3/imunologia , Degranulação Celular/efeitos dos fármacos , Células Caliciformes/citologia , Células Caliciformes/imunologia , Intestino Delgado/citologia , Camundongos , Camundongos Knockout , Microbiota/imunologia , Muco/imunologia , Técnicas de Cultura de Órgãos , Celulas de Paneth/citologia
17.
Stem Cell Reports ; 2(6): 838-52, 2014 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-24936470

RESUMO

Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Perfilação da Expressão Gênica/métodos , Intestinos/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células Cultivadas , Humanos , Receptores Acoplados a Proteínas-G/metabolismo
18.
Nat Methods ; 11(1): 106-12, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24292484

RESUMO

Although Lgr5(+) intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5(+) intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5(+) cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types.


Assuntos
Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Colo/citologia , Intestino Delgado/citologia , Receptores Acoplados a Proteínas-G/metabolismo , Células-Tronco/citologia , Animais , Diferenciação Celular , Células Cultivadas , Cromossomos/ultraestrutura , Citometria de Fluxo , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Cariotipagem , Camundongos , Microscopia Confocal , Celulas de Paneth/citologia , Piridinas/química , Pirimidinas/química , Transdução de Sinais , Estômago/citologia , Ácido Valproico/química
19.
J Clin Invest ; 124(1): 328-37, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24292712

RESUMO

Multiple intestinal atresia (MIA) is a rare cause of bowel obstruction that is sometimes associated with a combined immunodeficiency (CID), leading to increased susceptibility to infections. The factors underlying this rare disease are poorly understood. We characterized the immunological and intestinal features of 6 unrelated MIA-CID patients. All patients displayed a profound, generalized lymphocytopenia, with few lymphocytes present in the lymph nodes. The thymus was hypoplastic and exhibited an abnormal distribution of epithelial cells. Patients also had profound disruption of the epithelial barrier along the entire gastrointestinal tract. Using linkage analysis and whole-exome sequencing, we identified 10 mutations in tetratricopeptide repeat domain­7A (TTC7A), all of which potentially abrogate TTC7A expression. Intestinal organoid cultures from patient biopsies displayed an inversion of apicobasal polarity of the epithelial cells that was normalized by pharmacological inhibition of Rho kinase. Our data indicate that TTC7A deficiency results in increased Rho kinase activity, which disrupts polarity, growth, and differentiation of intestinal epithelial cells, and which impairs immune cell homeostasis, thereby promoting MIA-CID development.


Assuntos
Atresia Intestinal/genética , Mucosa Intestinal/patologia , Proteínas/genética , Imunodeficiência Combinada Severa/genética , Sequência de Bases , Polaridade Celular , Células Cultivadas , Criança , Consanguinidade , Análise Mutacional de DNA , Células Epiteliais/fisiologia , Exoma , Feminino , Estudos de Associação Genética , Ligação Genética , Humanos , Lactente , Atresia Intestinal/imunologia , Atresia Intestinal/mortalidade , Atresia Intestinal/patologia , Linfonodos/patologia , Linfopenia/genética , Linfopenia/imunologia , Linfopenia/patologia , Masculino , Linhagem , Proteínas/metabolismo , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/mortalidade , Imunodeficiência Combinada Severa/patologia , Timo/anormalidades , Timo/patologia , Quinases Associadas a rho/metabolismo
20.
Diabetes ; 63(2): 410-20, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24130334

RESUMO

Upon a nutrient challenge, L cells produce glucagon-like peptide 1 (GLP-1), a powerful stimulant of insulin release. Strategies to augment endogenous GLP-1 production include promoting L-cell differentiation and increasing L-cell number. Here we present a novel in vitro platform to generate functional L cells from three-dimensional cultures of mouse and human intestinal crypts. We show that short-chain fatty acids selectively increase the number of L cells, resulting in an elevation of GLP-1 release. This is accompanied by the upregulation of transcription factors associated with the endocrine lineage of intestinal stem cell development. Thus, our platform allows us to study and modulate the development of L cells in mouse and human crypts as a potential basis for novel therapeutic strategies in patients with type 2 diabetes.


Assuntos
Células Enteroendócrinas/fisiologia , Intestino Delgado/citologia , Intestino Delgado/fisiologia , Organoides/citologia , Animais , Técnicas de Cultura de Células , Regulação da Expressão Gênica/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Tecidos
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