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1.
Front Pharmacol ; 13: 837261, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35586042

RESUMO

Cancer immunotherapy often involves the use of engineered molecules to selectively bind and activate T cells located within tumour tissue. Fundamental to the success of such treatments is the presence or recruitment of T cells localised within the tumour microenvironment. Advanced organ-on-a-chip systems provide an in vitro setting in which to investigate how novel molecules influence the spatiotemporal dynamics of T cell infiltration into tissue, both in the context of anti-tumour efficacy and off-tumour toxicity. While highly promising, the complexity of these systems is such that mathematical modelling plays a crucial role in the quantitative evaluation of experimental results and maximising the mechanistic insight derived. We develop a mechanistic, mathematical model of a novel microphysiological in vitro platform that recapitulates T cell infiltration into epithelial tissue, which may be normal or transformed. The mathematical model describes the spatiotemporal dynamics of infiltrating T cells in response to chemotactic cytokine signalling. We integrate the model with dynamic imaging data to optimise key model parameters. The mathematical model demonstrates a good fit to the observed experimental data and accurately describes the distribution of infiltrating T cells. This model is designed to complement the in vitro system; with the potential to elucidate complex biological mechanisms, including the mode of action of novel therapies and the drivers of safety events, and, ultimately, improve the efficacy-safety profile of T cell-targeted cancer immunotherapies.

2.
Stem Cells ; 39(3): 296-305, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33438789

RESUMO

Skeletal progenitor/stem cells (SSCs) play a critical role in postnatal bone growth and maintenance. Telomerase (Tert) activity prevents cellular senescence and is required for maintenance of stem cells in self-renewing tissues. Here we investigated the role of mTert-expressing cells in postnatal mouse long bone and found that mTert expression is enriched at the time of adolescent bone growth. mTert-GFP+ cells were identified in regions known to house SSCs, including the metaphyseal stroma, growth plate, and the bone marrow. We also show that mTert-expressing cells are a distinct SSC population with enriched colony-forming capacity and contribute to multiple mesenchymal lineages, in vitro. In contrast, in vivo lineage-tracing studies identified mTert+ cells as osteochondral progenitors and contribute to the bone-forming cell pool during endochondral bone growth with a subset persisting into adulthood. Taken together, our results show that mTert expression is temporally regulated and marks SSCs during a discrete phase of transitional growth between rapid bone growth and maintenance.


Assuntos
Células Epiteliais/metabolismo , Células-Tronco/metabolismo , Telomerase/metabolismo , Animais , Medula Óssea/metabolismo , Ciclo Celular/fisiologia , Proliferação de Células/fisiologia , Senescência Celular/fisiologia , Camundongos
3.
Cell Mol Gastroenterol Hepatol ; 9(3): 507-526, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31778828

RESUMO

BACKGROUND & AIMS: The mucus layer in the human colon protects against commensal bacteria and pathogens, and defects in its unique bilayered structure contribute to intestinal disorders, such as ulcerative colitis. However, our understanding of colon physiology is limited by the lack of in vitro models that replicate human colonic mucus layer structure and function. Here, we investigated if combining organ-on-a-chip and organoid technologies can be leveraged to develop a human-relevant in vitro model of colon mucus physiology. METHODS: A human colon-on-a-chip (Colon Chip) microfluidic device lined by primary patient-derived colonic epithelial cells was used to recapitulate mucus bilayer formation, and to visualize mucus accumulation in living cultures noninvasively. RESULTS: The Colon Chip supports spontaneous goblet cell differentiation and accumulation of a mucus bilayer with impenetrable and penetrable layers, and a thickness similar to that observed in the human colon, while maintaining a subpopulation of proliferative epithelial cells. Live imaging of the mucus layer formation on-chip showed that stimulation of the colonic epithelium with prostaglandin E2, which is increased during inflammation, causes rapid mucus volume expansion via an Na-K-Cl cotransporter 1 ion channel-dependent increase in its hydration state, but no increase in de novo mucus secretion. CONCLUSIONS: This study shows the production of colonic mucus with a physiologically relevant bilayer structure in vitro, which can be analyzed in real time noninvasively. The Colon Chip may offer a new preclinical tool to analyze the role of mucus in human intestinal homeostasis as well as diseases, such as ulcerative colitis and cancer.


Assuntos
Colo/metabolismo , Mucosa Intestinal/metabolismo , Dispositivos Lab-On-A-Chip , Muco/metabolismo , Células Cultivadas , Dinoprostona/metabolismo , Células Caliciformes/fisiologia , Humanos , Organoides , Cultura Primária de Células/métodos , Membro 1 da Família 12 de Carreador de Soluto/metabolismo
4.
Nat Biomed Eng ; 3(7): 583, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213704

RESUMO

In the version of this Article originally published, the authors mistakenly cited Fig. 5d in the sentence beginning 'Importantly, the microbiome cultured in these primary Intestine Chips...'; the correct citation is Supplementary Table 2. This has now been amended.

5.
Nat Biomed Eng ; 3(7): 520-531, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31086325

RESUMO

The diverse bacterial populations that comprise the commensal microbiome of the human intestine play a central role in health and disease. A method that sustains complex microbial communities in direct contact with living human intestinal cells and their overlying mucus layer in vitro would thus enable the investigation of host-microbiome interactions. Here, we show the extended coculture of living human intestinal epithelium with stable communities of aerobic and anaerobic human gut microbiota, using a microfluidic intestine-on-a-chip that permits the control and real-time assessment of physiologically relevant oxygen gradients. When compared to aerobic coculture conditions, the establishment of a transluminal hypoxia gradient in the chip increased intestinal barrier function and sustained a physiologically relevant level of microbial diversity, consisting of over 200 unique operational taxonomic units from 11 different genera and an abundance of obligate anaerobic bacteria, with ratios of Firmicutes and Bacteroidetes similar to those observed in human faeces. The intestine-on-a-chip may serve as a discovery tool for the development of microbiome-related therapeutics, probiotics and nutraceuticals.


Assuntos
Técnicas de Cultura de Células/métodos , Microbioma Gastrointestinal/fisiologia , Mucosa Intestinal/microbiologia , Dispositivos Lab-On-A-Chip , Microbiota/fisiologia , Técnicas Analíticas Microfluídicas/métodos , Anaerobiose , Bactérias/classificação , Bactérias/crescimento & desenvolvimento , Bacteroidetes , Biodiversidade , Células CACO-2 , Células Epiteliais , Fezes/microbiologia , Firmicutes , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Hipóxia , Técnicas In Vitro , Muco , Oxigênio
6.
Microbiome ; 7(1): 43, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30890187

RESUMO

BACKGROUND: Species-specific differences in tolerance to infection are exemplified by the high susceptibility of humans to enterohemorrhagic Escherichia coli (EHEC) infection, whereas mice are relatively resistant to this pathogen. This intrinsic species-specific difference in EHEC infection limits the translation of murine research to human. Furthermore, studying the mechanisms underlying this differential susceptibility is a difficult problem due to complex in vivo interactions between the host, pathogen, and disparate commensal microbial communities. RESULTS: We utilize organ-on-a-chip (Organ Chip) microfluidic culture technology to model damage of the human colonic epithelium induced by EHEC infection, and show that epithelial injury is greater when exposed to metabolites derived from the human gut microbiome compared to mouse. Using a multi-omics approach, we discovered four human microbiome metabolites-4-methyl benzoic acid, 3,4-dimethylbenzoic acid, hexanoic acid, and heptanoic acid-that are sufficient to mediate this effect. The active human microbiome metabolites preferentially induce expression of flagellin, a bacterial protein associated with motility of EHEC and increased epithelial injury. Thus, the decreased tolerance to infection observed in humans versus other species may be due in part to the presence of compounds produced by the human intestinal microbiome that actively promote bacterial pathogenicity. CONCLUSION: Organ-on-chip technology allowed the identification of specific human microbiome metabolites modulating EHEC pathogenesis. These identified metabolites are sufficient to increase susceptibility to EHEC in our human Colon Chip model and they contribute to species-specific tolerance. This work suggests that higher concentrations of these metabolites could be the reason for higher susceptibility to EHEC infection in certain human populations, such as children. Furthermore, this research lays the foundation for therapeutic-modulation of microbe products in order to prevent and treat human bacterial infection.


Assuntos
Bactérias/metabolismo , Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/patologia , Intestinos/citologia , Técnicas de Cultura de Órgãos/métodos , Animais , Benzoatos/farmacologia , Caproatos/farmacologia , Células Cultivadas , Escherichia coli Êntero-Hemorrágica/metabolismo , Infecções por Escherichia coli/microbiologia , Feminino , Microbioma Gastrointestinal , Ácidos Heptanoicos/farmacologia , Humanos , Intestinos/microbiologia , Masculino , Camundongos , Procedimentos Analíticos em Microchip , Especificidade da Espécie
7.
Cell Mol Gastroenterol Hepatol ; 5(4): 659-668, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29713674

RESUMO

Microfluidic organ-on-a-chip models of human intestine have been developed and used to study intestinal physiology and pathophysiology. In this article, we review this field and describe how microfluidic Intestine Chips offer new capabilities not possible with conventional culture systems or organoid cultures, including the ability to analyze contributions of individual cellular, chemical, and physical control parameters one-at-a-time; to coculture human intestinal cells with commensal microbiome for extended times; and to create human-relevant disease models. We also discuss potential future applications of human Intestine Chips, including how they might be used for drug development and personalized medicine.

8.
PLoS Biol ; 16(3): e2002417, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29596476

RESUMO

Inflammatory bowel disease (IBD) is a chronic condition driven by loss of homeostasis between the mucosal immune system, the commensal gut microbiota, and the intestinal epithelium. Our goal is to understand how these components of the intestinal ecosystem cooperate to control homeostasis. By combining quantitative measures of epithelial hyperplasia and immune infiltration with multivariate analysis of inter- and intracellular signaling, we identified epithelial mammalian target of rapamycin (mTOR) signaling as a potential driver of inflammation in a mouse model of colitis. A kinetic analysis of mTOR inhibition revealed that the pathway regulates epithelial differentiation, which in turn controls the cytokine milieu of the colon. Consistent with our in vivo analysis, we found that cytokine expression of organoids grown ex vivo, in the absence of bacteria and immune cells, was dependent on differentiation state. Our study suggests that proper differentiation of epithelial cells is an important feature of colonic homeostasis because of its effect on the secretion of inflammatory cytokines.


Assuntos
Colite/metabolismo , Colo/imunologia , Citocinas/metabolismo , Animais , Autofagia , Comunicação Celular , Diferenciação Celular , Colo/metabolismo , Colo/patologia , Epitélio/imunologia , Epitélio/metabolismo , Microbioma Gastrointestinal , Homeostase , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/patologia , Cinética , Camundongos , Análise Multivariada , Fosforilação , Análise de Componente Principal , Transdução de Sinais , Sirolimo/farmacologia , Biologia de Sistemas , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo
9.
Sci Rep ; 8(1): 2871, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29440725

RESUMO

Here we describe a method for fabricating a primary human Small Intestine-on-a-Chip (Intestine Chip) containing epithelial cells isolated from healthy regions of intestinal biopsies. The primary epithelial cells are expanded as 3D organoids, dissociated, and cultured on a porous membrane within a microfluidic device with human intestinal microvascular endothelium cultured in a parallel microchannel under flow and cyclic deformation. In the Intestine Chip, the epithelium forms villi-like projections lined by polarized epithelial cells that undergo multi-lineage differentiation similar to that of intestinal organoids, however, these cells expose their apical surfaces to an open lumen and interface with endothelium. Transcriptomic analysis also indicates that the Intestine Chip more closely mimics whole human duodenum in vivo when compared to the duodenal organoids used to create the chips. Because fluids flowing through the lumen of the Intestine Chip can be collected continuously, sequential analysis of fluid samples can be used to quantify nutrient digestion, mucus secretion and establishment of intestinal barrier function over a period of multiple days in vitro. The Intestine Chip therefore may be useful as a research tool for applications where normal intestinal function is crucial, including studies of metabolism, nutrition, infection, and drug pharmacokinetics, as well as personalized medicine.


Assuntos
Intestino Delgado/citologia , Dispositivos Lab-On-A-Chip , Organoides/citologia , Biópsia , Proliferação de Células , Células Epiteliais/citologia , Humanos
10.
Stem Cell Reports ; 10(1): 17-26, 2018 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-29276155

RESUMO

The intestinal epithelium serves as an essential barrier to the outside world and is maintained by functionally distinct populations of rapidly cycling intestinal stem cells (CBC ISCs) and slowly cycling, reserve ISCs (r-ISCs). Because disruptions in the epithelial barrier can result from pathological activation of the immune system, we sought to investigate the impact of inflammation on ISC behavior during the regenerative response. In a murine model of αCD3 antibody-induced small-intestinal inflammation, r-ISCs proved highly resistant to injury, while CBC ISCs underwent apoptosis. Moreover, r-ISCs were induced to proliferate and functionally contribute to intestinal regeneration. Further analysis revealed that the inflammatory cytokines interferon gamma and tumor necrosis factor alpha led to r-ISC activation in enteroid culture, which could be blocked by the JAK/STAT inhibitor, tofacitinib. These results highlight an important role for r-ISCs in response to acute intestinal inflammation and show that JAK/STAT-1 signaling is required for the r-ISC regenerative response.


Assuntos
Enterite/metabolismo , Mucosa Intestinal/fisiologia , Intestino Delgado/metabolismo , Janus Quinases/metabolismo , Regeneração , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Enterite/induzido quimicamente , Enterite/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Mucosa Intestinal/patologia , Intestino Delgado/patologia , Janus Quinases/antagonistas & inibidores , Camundongos , Camundongos Transgênicos , Piperidinas/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Fator de Transcrição STAT1/antagonistas & inibidores , Células-Tronco/patologia
11.
Nature ; 538(7625): 350-355, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27680706

RESUMO

Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR-Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7-knockout mice, combined with knockdown of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7-knockout mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Colo/metabolismo , Epitélio/metabolismo , Receptores Frizzled/metabolismo , Animais , Antígenos/metabolismo , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Sítios de Ligação , Células CHO , Sistemas CRISPR-Cas , Linhagem Celular , Clostridioides difficile/patogenicidade , Cricetulus , Feminino , Receptores Frizzled/química , Receptores Frizzled/deficiência , Receptores Frizzled/genética , Técnicas de Inativação de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Organoides/metabolismo , Domínios Proteicos , Proteoglicanas/metabolismo , Fatores de Virulência/metabolismo , Proteínas Wnt/metabolismo
12.
Cell Stem Cell ; 18(3): 410-21, 2016 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-26908146

RESUMO

The gastrointestinal (GI) epithelium is a highly regenerative tissue with the potential to provide a renewable source of insulin(+) cells after undergoing cellular reprogramming. Here, we show that cells of the antral stomach have a previously unappreciated propensity for conversion into functional insulin-secreting cells. Native antral endocrine cells share a surprising degree of transcriptional similarity with pancreatic ß cells, and expression of ß cell reprogramming factors in vivo converts antral cells efficiently into insulin(+) cells with close molecular and functional similarity to ß cells. Induced GI insulin(+) cells can suppress hyperglycemia in a diabetic mouse model for at least 6 months and regenerate rapidly after ablation. Reprogramming of antral stomach cells assembled into bioengineered mini-organs in vitro yielded transplantable units that also suppressed hyperglycemia in diabetic mice, highlighting the potential for development of engineered stomach tissues as a renewable source of functional ß cells for glycemic control.


Assuntos
Técnicas de Reprogramação Celular , Reprogramação Celular , Mucosa Gástrica/metabolismo , Células Secretoras de Insulina/metabolismo , Animais , Mucosa Gástrica/citologia , Mucosa Gástrica/transplante , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/transplante , Camundongos
13.
Cell Rep ; 13(11): 2403-2411, 2015 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-26686631

RESUMO

The cellular and molecular mechanisms underlying adaptive changes to physiological stress within the intestinal epithelium remain poorly understood. Here, we show that PTEN, a negative regulator of the PI3K→AKT→mTORC1-signaling pathway, is an important regulator of dormant intestinal stem cells (d-ISCs). Acute nutrient deprivation leads to transient PTEN phosphorylation within d-ISCs and a corresponding increase in their number. This release of PTEN inhibition renders d-ISCs functionally poised to contribute to the regenerative response during re-feeding via cell-autonomous activation of the PI3K→AKT→mTORC1 pathway. Consistent with its role in mediating cell survival, PTEN is required for d-ISC maintenance at baseline, and intestines lacking PTEN have diminished regenerative capacity after irradiation. Our results highlight a PTEN-dependent mechanism for d-ISC maintenance and further demonstrate the role of d-ISCs in the intestinal response to stress.


Assuntos
Intestinos/citologia , Estado Nutricional , PTEN Fosfo-Hidrolase/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Proliferação de Células , Feminino , Genes Reporter , Intestinos/patologia , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Complexos Multiproteicos/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Telomerase/genética , Telomerase/metabolismo
14.
Stem Cell Reports ; 5(5): 673-681, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26489894

RESUMO

Lgr5-expressing intestinal stem cells (ISCs) renew the adult gut epithelium by producing mature villus cells (VCs); the transcriptional basis for ISC functions remains unclear. RNA sequencing analysis identified transcripts modulated during differentiation of Lgr5(+) ISCs into VCs, with high expression of the intestine-restricted transcription factor (TF) gene Cdx2 in both populations. Cdx2-deleted mouse ISCs showed impaired proliferation and long-term inability to produce mature lineages, revealing essential ISC functions. Chromatin immunoprecipitation sequencing analysis of CDX2 in Lgr5(+) ISCs, coupled with mRNA profiling of control and Cdx2(-/-) ISCs, identified features of CDX2 regulation distinct from VCs. Most CDX2 binding in ISCs occurs in anticipation of future gene expression, but whereas CDX2 primarily activates VC genes, direct ISC targets are activated and repressed. Diverse CDX2 requirements in stem and differentiated cells may reflect the versatility of TFs that specify a tissue in development and control the same tissue in adults.


Assuntos
Células-Tronco Adultas/metabolismo , Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Mucosa Intestinal/metabolismo , Fatores de Transcrição/metabolismo , Células-Tronco Adultas/citologia , Animais , Fator de Transcrição CDX2 , Proteínas de Homeodomínio/genética , Mucosa Intestinal/citologia , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/genética , Ativação Transcricional
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