RESUMEN
The intestinal epithelium is constantly exposed to microbes residing in the lumen. Traditionally, the response to microbial interactions has been studied in cell lines derived from cancerous tissues, e.g. Caco-2. It is, however, unclear how the responses in these cancer cell lines reflect the responses of a normal epithelium and whether there might be microbial strain-specific effects. To address these questions, we derived organoids from the small intestine from a cohort of healthy individuals. Culturing intestinal epithelium on a flat laminin matrix induced their differentiation, facilitating analysis of microbial responses via the apical membrane normally exposed to the luminal content. Here, it was evident that the healthy epithelium across multiple individuals (n = 9) demonstrates robust acute both common and strain-specific responses to a range of probiotic bacterial strains (BB-12â, LGGâ, DSM33361, and Bif195). Importantly, parallel experiments using the Caco-2 cell line provide no acute response. Collectively, we demonstrate that primary epithelial cells maintained as organoids represent a valuable resource for assessing interactions between the epithelium and luminal microbes across individuals, and that these models are likely to contribute to a better understanding of host microbe interactions.
Asunto(s)
Microbioma Gastrointestinal , Humanos , Células CACO-2 , Células Epiteliales/metabolismo , Organoides , Epitelio , Mucosa Intestinal/microbiologíaRESUMEN
During intestinal organogenesis, equipotent epithelial progenitors mature into phenotypically distinct stem cells that are responsible for lifelong maintenance of the tissue. While the morphological changes associated with the transition are well characterized, the molecular mechanisms underpinning the maturation process are not fully understood. Here, we leverage intestinal organoid cultures to profile transcriptional, chromatin accessibility, DNA methylation, and three-dimensional (3D) chromatin conformation landscapes in fetal and adult epithelial cells. We observed prominent differences in gene expression and enhancer activity, which are accompanied by local changes in 3D organization, DNA accessibility, and methylation between the two cellular states. Using integrative analyses, we identified sustained Yes-Associated Protein (YAP) transcriptional activity as a major gatekeeper of the immature fetal state. We found the YAP-associated transcriptional network to be regulated at various levels of chromatin organization and likely to be coordinated by changes in extracellular matrix composition. Together, our work highlights the value of unbiased profiling of regulatory landscapes for the identification of key mechanisms underlying tissue maturation.
Asunto(s)
Epigenómica , Mucosa Intestinal , Adulto , Humanos , Intestinos , Epitelio , Cromatina/genéticaRESUMEN
Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.
Asunto(s)
Proteínas Hedgehog/metabolismo , Mucosa Intestinal/metabolismo , Intestino Delgado/embriología , Células Madre Mesenquimatosas/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/genética , Mucosa Intestinal/citología , Mucosa Intestinal/embriología , Intestino Delgado/citología , Intestino Delgado/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Morfogénesis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Vía de Señalización Wnt/genéticaRESUMEN
Dietary patterns, microbiome dysbiosis, and gut microbial metabolites (GMMs) have a pivotal role in the homeostasis of intestinal epithelial cells and in disease progression, such as that of colorectal cancer (CRC). Although GMMs and microorganisms have crucial roles in many biological activities, models for deciphering diet-microbiome-host relationships are largely limited to animal models. Thus, intestinal organoids (IOs) have provided unprecedented opportunities for the generation of in vitro platforms with the sufficient level of complexity to model physiological and pathological diet-microbiome-host conditions. Overall, IO responses to GMM metabolites and microorganisms can provide new insights into the mechanisms by which those agents may prevent or trigger diseases, significantly extending our knowledge of diet-microbiome-host interactions.
Asunto(s)
Microbioma Gastrointestinal/fisiología , Organoides/microbiología , Animales , Humanos , Microbiota/fisiología , Análisis de la Célula IndividualRESUMEN
The processes involved in renewal of the epithelium that lines the mouse stomach remain unclear. Apart from the cells in the isthmus, several other populations located deeper in the gastric glands have been suggested to contribute to the maintenance of the gastric epithelium. Here, we reveal that Lrig1 is expressed in the basal layer of the forestomach and the lower part of glands in the corpus and pylorus. In the glandular epithelium of the stomach, Lrig1 marks a heterogeneous population comprising mainly non-proliferative cells. Yet, fate-mapping experiments using a knock-in mouse line expressing Cre specifically in Lrig1+ cells demonstrate that these cells are able to contribute to the long-term maintenance of the gastric epithelium. Moreover, when cultured in vitro, cells expressing high level of Lrig1 have much higher organoid forming potential than the corresponding cellular populations expressing lower levels of Lrig1. Taken together, these observations show that Lrig1 is expressed primarily by differentiated cells, but that these cells can be recruited to contribute to the maintenance of the gastric epithelium. This confirms previous observations that cells located in the lower segments of gastric glands can participate in tissue replenishment.
Asunto(s)
Biomarcadores , Proliferación Celular , Células Epiteliales/metabolismo , Mucosa Gástrica/metabolismo , Glicoproteínas de Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Biomarcadores/metabolismo , Desdiferenciación Celular/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/fisiología , Mucosa Gástrica/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estómago/citologíaRESUMEN
Inflammatory bowel disease (IBD) is a chronic intestinal disorder, with two main types: Crohn's disease (CD) and ulcerative colitis (UC), whose molecular pathology is not well understood. The majority of IBD-associated SNPs are located in non-coding regions and are hard to characterize since regulatory regions in IBD are not known. Here we profile transcription start sites (TSSs) and enhancers in the descending colon of 94 IBD patients and controls. IBD-upregulated promoters and enhancers are highly enriched for IBD-associated SNPs and are bound by the same transcription factors. IBD-specific TSSs are associated to genes with roles in both inflammatory cascades and gut epithelia while TSSs distinguishing UC and CD are associated to gut epithelia functions. We find that as few as 35 TSSs can distinguish active CD, UC, and controls with 85% accuracy in an independent cohort. Our data constitute a foundation for understanding the molecular pathology, gene regulation, and genetics of IBD.
Asunto(s)
Colitis Ulcerosa/genética , Enfermedad de Crohn/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Adulto , Biopsia , Estudios de Casos y Controles , Estudios de Cohortes , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/patología , Colon/diagnóstico por imagen , Colon/patología , Colonoscopía , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/patología , Femenino , Humanos , Mucosa Intestinal/diagnóstico por imagen , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ARN , Regulación hacia ArribaAsunto(s)
Dieta Alta en Grasa , Lípidos/análisis , Microbiota , Piel/química , Piel/microbiología , Adiposidad , Animales , HumanosRESUMEN
Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Reprogramación Celular , Matriz Extracelular/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Fosfoproteínas/metabolismo , Regeneración , Animales , Biomarcadores/metabolismo , Proteínas de Ciclo Celular , Feto/metabolismo , Humanos , Mecanotransducción Celular , Ratones Endogámicos C57BL , Transducción de Señal , Transcripción Genética , Activación Transcripcional/genética , Proteínas Señalizadoras YAPRESUMEN
Transgenic porcine cancer models bring novel possibilities for research. Their physical similarities with humans enable the use of surgical procedures and treatment approaches used for patients, which facilitates clinical translation. Here, we aimed to develop an inducible oncopig model of intestinal cancer. Transgenic (TG) minipigs were generated using somatic cell nuclear transfer by handmade cloning. The pigs encode two TG cassettes: (a) an Flp recombinase-inducible oncogene cassette containing KRAS-G12D, cMYC, SV40LT - which inhibits p53 - and pRB and (b) a 4-hydroxytamoxifen (4-OHT)-inducible Flp recombinase activator cassette controlled by the intestinal epithelium-specific villin promoter. Thirteen viable transgenic minipigs were born. The ability of 4-OHT to activate the oncogene cassette was confirmed in vitro in TG colonic organoids and ex vivo in tissue biopsies obtained by colonoscopy. In order to provide proof of principle that the oncogene cassette could also successfully be activated in vivo, three pigs were perorally treated with 400 mg tamoxifen for 2 × 5 days. After two months, one pig developed a duodenal neuroendocrine carcinoma with a lymph node metastasis. Molecular analysis of the carcinoma and metastasis confirmed activation of the oncogene cassette. No tumor formation was observed in untreated TG pigs or in the remaining two treated pigs. The latter indicates that tamoxifen delivery can probably be improved. In summary, we have generated a novel inducible oncopig model of intestinal cancer, which has the ability to form metastatic disease already two months after induction. The model may be helpful in bridging the gap between basic research and clinical usage. It opens new venues for longitudinal studies of tumor development and evolution, for preclinical assessment of new anticancer regimens, for pharmacology and toxicology assessments, as well as for studies into biological mechanisms of tumor formation and metastasis.
Asunto(s)
Animales Modificados Genéticamente/genética , Clonación de Organismos/métodos , Modelos Animales de Enfermedad , Neoplasias Intestinales/genética , Técnicas de Transferencia Nuclear , Porcinos Enanos/genética , Animales , Técnicas de Cultivo de Embriones/métodos , Transferencia de Embrión/métodos , Femenino , Humanos , Mucosa Intestinal/metabolismo , Neoplasias Intestinales/patología , Intestinos/patología , PorcinosRESUMEN
Reliable disease models are needed in order to improve quality of healthcare. This includes gaining better understanding of disease mechanisms, developing new therapeutic interventions and personalizing treatment. Up-to-date, the majority of our knowledge about disease states comes from in vivo animal models and in vitro cell culture systems. However, it has been exceedingly difficult to model disease at the tissue level. Since recently, the gap between cell line studies and in vivo modeling has been narrowing thanks to progress in biomaterials and stem cell research. Development of reliable 3D culture systems has enabled a rapid expansion of sophisticated in vitro models. Here we focus on some of the latest advances and future perspectives in 3D organoids for human disease modeling.
Asunto(s)
Enfermedad , Modelos Biológicos , Organoides/patología , Técnicas de Cultivo de Tejidos/métodos , Animales , HumanosRESUMEN
Thymidine kinase (TK) activity is a marker of biological activity that allows the indolent and aggressive forms of chronic lymphocytic leukemia (CLL) to be distinguished. The aims of the study were to determine the relationship between TK activity and clinical status and prognosis, as well as to compare its activity with that of other prognostic and predictive factors. TK activity was measured in patient sera at the time of diagnosis using the DiviTum method, with the mean value being 439 Du/L. A correlation was found between TK activity and risk of disease progression (p=0.045). The optimal discriminative value of TK activity in the prediction of CLL progression was found to be 600 Du/L. TK activity significantly differed between the patients who achieved complete remission and those who only partially responded to therapy. In 93% of patients without any response to treatment and 18 out of 20 patients with progressive disease, TK activity over 600 Du/L was noted. In addition, all of the 10 patients with 17p13 deletion displayed TK activity of over 600 Du/L (p=0.0004). High TK activity also correlated with elevated levels of LDH (p=0.001) and ß2microglobulin (p=0.03) in the study group. The results of the study indicated the importance of TK activity as a prognostic factor in patients with CLL.
Asunto(s)
Biomarcadores de Tumor/sangre , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/patología , Timidina Quinasa/sangre , Anciano , Biomarcadores , Pruebas Enzimáticas Clínicas , Progresión de la Enfermedad , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/sangre , Masculino , Persona de Mediana Edad , Estadificación de NeoplasiasRESUMEN
Activation of Wnt/ß-catenin signaling in adult mouse epidermis leads to expansion of the stem cell compartment and redirects keratinocytes in the interfollicular epidermis and sebaceous glands (SGs) to differentiate along the hair follicle (HF) lineages. Here we demonstrate that during epidermal development and homeostasis there is reciprocal activation of the androgen receptor (AR) and ß-catenin in cells of the HF bulb. AR activation reduced ß-catenin-dependent transcription, blocked ß-catenin-induced induction of HF growth, and prevented ß-catenin-mediated conversion of SGs into HFs. Conversely, AR inhibition enhanced the effects of ß-catenin activation, promoting HF proliferation and differentiation, culminating in the formation of benign HF tumors and a complete loss of SG identity. We conclude that AR signaling has a key role in epidermal stem cell fate selection by modulating responses to ß-catenin in adult mouse skin.
Asunto(s)
Células Epiteliales/metabolismo , Folículo Piloso/crecimiento & desarrollo , Receptores Androgénicos/metabolismo , Vía de Señalización Wnt/fisiología , beta Catenina/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Células Epiteliales/citología , Folículo Piloso/metabolismo , Humanos , Queratinocitos/metabolismo , Masculino , Ratones , Ratones Transgénicos , Modelos Animales , Reacción en Cadena en Tiempo Real de la Polimerasa , Glándulas Sebáceas/citología , Sensibilidad y Especificidad , Células Madre/metabolismoRESUMEN
Regeneration and homeostasis in the adult intestinal epithelium is driven by proliferative resident stem cells, whose functional properties during organismal development are largely unknown. Here, we show that human and mouse fetal intestine contains proliferative, immature progenitors, which can be expanded in vitro as Fetal Enterospheres (FEnS). A highly similar progenitor population can be established during intestinal differentiation of human induced pluripotent stem cells. Established cultures of mouse fetal intestinal progenitors express lower levels of Lgr5 than mature progenitors and propagate in the presence of the Wnt antagonist Dkk1, and new cultures can be induced to form mature intestinal organoids by exposure to Wnt3a. Following transplantation in a colonic injury model, FEnS contribute to regeneration of colonic epithelium by forming epithelial crypt-like structures expressing region-specific differentiation markers. This work provides insight into mechanisms underlying development of the mammalian intestine and points to future opportunities for patient-specific regeneration of the digestive tract.
Asunto(s)
Colon/lesiones , Colon/fisiología , Feto/citología , Intestinos/embriología , Regeneración , Trasplante de Células Madre , Células Madre/citología , Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Colon/citología , Colon/patología , Humanos , Mucosa Intestinal/patología , Intestinos/citología , Ratones , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Esferoides Celulares/patología , Células Madre/metabolismoRESUMEN
Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.
Asunto(s)
Caulobacter crescentus/genética , Replicación del ADN , Plásmidos , Ciclo Celular , ADN Bacteriano/metabolismo , Microscopía Fluorescente , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Although the sebaceous gland (SG) plays an important role in skin function, the mechanisms regulating SG differentiation and carcinoma formation are poorly understood. We previously reported that c-MYC overexpression stimulates SG differentiation. We now demonstrate roles for the androgen receptor (AR) and p53. MYC-induced SG differentiation was reduced in mice lacking a functional AR. High levels of MYC triggered a p53-dependent DNA damage response, leading to accumulation of proliferative SG progenitors and inhibition of AR signaling. Conversely, testosterone treatment or p53 deletion activated AR signaling and restored MYC-induced differentiation. Poorly differentiated human sebaceous carcinomas exhibited high p53 and low AR expression. Thus, the consequences of overactivating MYC in the SG depend on whether AR or p53 is activated, as they form a regulatory axis controlling proliferation and differentiation.