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1.
Nature ; 585(7826): 574-578, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32939089

RESUMO

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology1-4. However, the approaches that are used at present to derive these organoids in three-dimensional matrices5,6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host-microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions.


Assuntos
Homeostase , Intestinos/embriologia , Morfogênese , Organoides/embriologia , Tecidos Suporte , Animais , Padronização Corporal , Diferenciação Celular , Linhagem da Célula , Cryptosporidium parvum/patogenicidade , Células-Tronco Embrionárias Humanas/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Intestinos/citologia , Intestinos/parasitologia , Intestinos/patologia , Camundongos , Modelos Biológicos , Organoides/citologia , Organoides/parasitologia , Organoides/patologia , Regeneração , Medicina Regenerativa , Células-Tronco , Técnicas de Cultura de Tecidos/métodos , Engenharia Tecidual
2.
Vet Microbiol ; 247: 108785, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32768229

RESUMO

Porcine deltacoronavirus (PDCoV) is a novel swine enteropathogenic coronavirus that causes watery diarrhea, vomiting and mortality in nursing piglets. Type III interferons (IFN-λs) are the major antiviral cytokines in intestinal epithelial cells, the target cells in vivo for PDCoV. In this study, we found that PDCoV infection remarkably inhibited Sendai virus-induced IFN-λ1 production by suppressing transcription factors IRF and NF-κB in IPI-2I cells, a line of porcine intestinal mucosal epithelial cells. We also confirmed that PDCoV infection impeded the activation of IFN-λ1 promoter stimulated by RIG-I, MDA5 and MAVS, but not by TBK1 and IRF1. Although the expression levels of IRF1 and MAVS were not changed, PDCoV infection resulted in reduction of the number of peroxisomes, the platform for MAVS to activate IRF1, and subsequent type III IFN production. Taken together, our study demonstrates that PDCoV suppresses type III IFN responses to circumvent the host's antiviral immunity.


Assuntos
Infecções por Coronavirus/veterinária , Células Epiteliais/imunologia , Células Epiteliais/virologia , Interações Hospedeiro-Patógeno/imunologia , Interferons/antagonistas & inibidores , Animais , Linhagem Celular , Coronavirus , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Fator Regulador 1 de Interferon/antagonistas & inibidores , Fator Regulador 1 de Interferon/imunologia , Interferons/imunologia , Intestinos/citologia , Intestinos/virologia , Rim/citologia , Rim/virologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Vírus Sendai/imunologia , Transdução de Sinais/imunologia , Suínos/virologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia
3.
Nat Commun ; 11(1): 3953, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769974

RESUMO

Many important cell types in adult vertebrates have a mesenchymal origin, including fibroblasts and vascular mural cells. Although their biological importance is undisputed, the level of mesenchymal cell heterogeneity within and between organs, while appreciated, has not been analyzed in detail. Here, we compare single-cell transcriptional profiles of fibroblasts and vascular mural cells across four murine muscular organs: heart, skeletal muscle, intestine and bladder. We reveal gene expression signatures that demarcate fibroblasts from mural cells and provide molecular signatures for cell subtype identification. We observe striking inter- and intra-organ heterogeneity amongst the fibroblasts, primarily reflecting differences in the expression of extracellular matrix components. Fibroblast subtypes localize to discrete anatomical positions offering novel predictions about physiological function(s) and regulatory signaling circuits. Our data shed new light on the diversity of poorly defined classes of cells and provide a foundation for improved understanding of their roles in physiological and pathological processes.


Assuntos
Diferenciação Celular , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miócitos de Músculo Liso/fisiologia , Pericitos/fisiologia , Animais , Separação Celular , Vasos Coronários/citologia , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Citometria de Fluxo , Intestinos/irrigação sanguínea , Intestinos/citologia , Masculino , Camundongos , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Liso Vascular/citologia , Miocárdio/citologia , Miócitos de Músculo Liso/citologia , Pericitos/citologia , RNA-Seq , Análise de Célula Única , Bexiga Urinária/irrigação sanguínea , Bexiga Urinária/citologia
4.
PLoS One ; 15(8): e0237357, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32780763

RESUMO

Fermented feeds contain abundant organic acids, amino acids, and small peptides, which improve the nutritional status as well as the morphology and microbiota composition of the intestine. Ginseng polysaccharides exhibit several biological activities and contribute to improving intestinal development. Here, Xuefeng black-bone chickens were fed a basal diet fermented by Bacillus subtilis, Saccharomyces cerevisiae, Lactobacillus plantarum, and Enterococcus faecium, with or without ginseng polysaccharides. The 100% microbially fermented feed (Fe) and 100% microbially fermented feed and ginseng polysaccharide (FP) groups showed significantly increased villus height and villus height to crypt depth ratio, and decreased crypt depth in the jejunum. In the 100% complete feed and ginseng polysaccharide (Po) group, the villus height to crypt depth ratio was significantly increased, crypt depth was reduced, and villus height remained unaffected. Next, we studied the intestinal microbial composition of 32 Xuefeng black-bone chickens. A total of 10 phyla and 442 genera were identified, among which Firmicutes, Proteobacteria, and Bacteroidetes were the most dominant phyla. At the genus level, Sutterella and Asteroleplasma abundance increased and decreased, respectively, in the FP and Po groups. Sutterella abundance was positively correlated to villus height and villus height to crypt depth ratio, and negatively correlated to crypt depth, and Asteroleplasma abundance was positively correlated to crypt depth and negatively correlated to villus height to crypt depth ratio. At the species level, the FP group showed significantly increased Bacteroides_vulgatus and Eubacterium_tortuosum and decreased Mycoplasma_gallinarum and Asteroleplasma_anaerobium abundance, and the Po group showed significantly increased Mycoplasma_gallinarum and Asteroleplasma_anaerobium abundance. Moreover, bacterial abundance was closely related to the jejunum histomorphology. Asteroleplasma_anaerobium abundance was positively correlated with crypt depth and negatively correlated with villus height to crypt depth ratio. Mycoplasma_gallinarum abundance was positively correlated to villus height, and Bacteroides_vulgatus and Eubacterium_tortuosum abundance was positively correlated with villus height to crypt depth ratio and negatively correlated with crypt depth. Therefore, fermented feeds with ginseng polysaccharides may be used as effective alternatives to antibiotics for improving intestinal morphology and microbial composition.


Assuntos
Ração Animal , Galinhas , Fermentação , Intestinos/efeitos dos fármacos , Microbiota/efeitos dos fármacos , Panax/química , Polissacarídeos/farmacologia , Animais , Biodiversidade , Intestinos/citologia , Intestinos/microbiologia
5.
Nat Commun ; 11(1): 3421, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32647184

RESUMO

The OX40-OX40L pathway provides crucial co-stimulatory signals for CD4 T cell responses, however the precise cellular interactions critical for OX40L provision in vivo and when these occur, remains unclear. Here, we demonstrate that provision of OX40L by dendritic cells (DCs), but not T cells, B cells nor group 3 innate lymphoid cells (ILC3s), is critical specifically for the effector Th1 response to an acute systemic infection with Listeria monocytogenes (Lm). OX40L expression by DCs is regulated by cross-talk with NK cells, with IFNγ signalling to the DC to enhance OX40L in a mechanism conserved in both mouse and human DCs. Strikingly, DC expression of OX40L is redundant in a chronic intestinal Th1 response and expression by ILC3s is necessary. Collectively these data reveal tissue specific compartmentalisation of the cellular provision of OX40L and define a mechanism controlling DC expression of OX40L in vivo.


Assuntos
Microambiente Celular , Ligante OX40/metabolismo , Células Th1/imunologia , Animais , Comunicação Celular , Sinais (Psicologia) , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Humanos , Interferon gama/biossíntese , Interleucina-12/farmacologia , Intestinos/citologia , Antígeno Ki-1/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Listeria monocytogenes/fisiologia , Camundongos Endogâmicos C57BL , Receptores CXCR5/metabolismo , Receptores OX40/metabolismo , Baço/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
Proc Natl Acad Sci U S A ; 117(29): 16969-16975, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32611816

RESUMO

Understanding to what extent stem cell potential is a cell-intrinsic property or an emergent behavior coming from global tissue dynamics and geometry is a key outstanding question of systems and stem cell biology. Here, we propose a theory of stem cell dynamics as a stochastic competition for access to a spatially localized niche, giving rise to a stochastic conveyor-belt model. Cell divisions produce a steady cellular stream which advects cells away from the niche, while random rearrangements enable cells away from the niche to be favorably repositioned. Importantly, even when assuming that all cells in a tissue are molecularly equivalent, we predict a common ("universal") functional dependence of the long-term clonal survival probability on distance from the niche, as well as the emergence of a well-defined number of functional stem cells, dependent only on the rate of random movements vs. mitosis-driven advection. We test the predictions of this theory on datasets of pubertal mammary gland tips and embryonic kidney tips, as well as homeostatic intestinal crypts. Importantly, we find good agreement for the predicted functional dependency of the competition as a function of position, and thus functional stem cell number in each organ. This argues for a key role of positional fluctuations in dictating stem cell number and dynamics, and we discuss the applicability of this theory to other settings.


Assuntos
Linhagem da Célula , Autorrenovação Celular , Nicho de Células-Tronco , Animais , Sobrevivência Celular , Feminino , Homeostase , Intestinos/citologia , Intestinos/crescimento & desenvolvimento , Rim/citologia , Rim/crescimento & desenvolvimento , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Modelos Teóricos , Razão Sinal-Ruído , Células-Tronco/citologia , Células-Tronco/fisiologia
7.
Nature ; 584(7821): 415-419, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641829

RESUMO

Sexual dimorphism arises from genetic differences between male and female cells, and from systemic hormonal differences1-3. How sex hormones affect non-reproductive organs is poorly understood, yet highly relevant to health given the sex-biased incidence of many diseases4. Here we report that steroid signalling in Drosophila from the ovaries to the gut promotes growth of the intestine specifically in mated females, and enhances their reproductive output. The active ovaries of the fly produce the steroid hormone ecdysone, which stimulates the division and expansion of intestinal stem cells in two distinct proliferative phases via the steroid receptors EcR and Usp and their downstream targets Broad, Eip75B and Hr3. Although ecdysone-dependent growth of the female gut augments fecundity, the more active and more numerous intestinal stem cells also increase female susceptibility to age-dependent gut dysplasia and tumorigenesis, thus potentially reducing lifespan. This work highlights the trade-offs in fitness traits that occur when inter-organ signalling alters stem-cell behaviour to optimize organ size.


Assuntos
Drosophila melanogaster/metabolismo , Fertilidade/fisiologia , Intestinos/crescimento & desenvolvimento , Longevidade/fisiologia , Tamanho do Órgão/fisiologia , Ovário/metabolismo , Esteroides/metabolismo , Envelhecimento , Animais , Carcinogênese , Proliferação de Células , Copulação/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/citologia , Drosophila melanogaster/fisiologia , Ecdisona/metabolismo , Feminino , Mucosa Intestinal/anatomia & histologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Intestinos/anatomia & histologia , Intestinos/citologia , Intestinos/patologia , Masculino , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo
8.
Am J Physiol Gastrointest Liver Physiol ; 319(2): G189-G196, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32628072

RESUMO

To fulfill the lifelong need to supply diverse epithelial cells, intestinal stem cells (ISCs) rely on executing accurate transcriptional programs. This review addresses the mechanisms that control those programs. Genes that define cell behaviors and identities are regulated principally through thousands of dispersed enhancers, each individually <1 kb long and positioned from a few to hundreds of kilobases away from transcription start sites, upstream or downstream from coding genes or within introns. Wnt, Notch, and other epithelial control signals feed into these cis-regulatory DNA elements, which are also common loci of polymorphisms and mutations that confer disease risk. Cell-specific gene activity requires promoters to interact with the correct combination of signal-responsive enhancers. We review the current state of knowledge in ISCs regarding active enhancers, the nucleosome modifications that may enable appropriate and hinder inappropriate enhancer-promoter contacts, and the roles of lineage-restricted transcription factors.


Assuntos
Diferenciação Celular/fisiologia , Epigênese Genética , Intestinos/citologia , Células-Tronco/fisiologia , Animais , Regulação da Expressão Gênica , Humanos
9.
J Steroid Biochem Mol Biol ; 202: 105720, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32565249

RESUMO

The synonymous single nucleotide polymorphism (SNP) rs731236, located in the vitamin D receptor (VDR) gene (Taq I) has been associated with both decreased levels of the protein in peripheral blood mononuclear cells and a fibrosis-related complication in Crohn´s disease (CD). Interactions between VDR and a protein-disulfide isomerase-associated 3 (PDIA3) in the regulation of extracellular matrix have been reported and we aim to analyze the relevance of the VDR genotypes and the effects of Vitamin D (VD) in the expression of VDR, PDIA3 and proliferation of intestinal fibroblasts. Human intestinal fibroblasts were isolated from the non-affected surgical resections of colorectal patients and classified according to the VDR genotype. In some cases, cells were transfected with specific PDIA3 siRNA. Basal and VD-stimulated expression of VDR, PDIA3 and Collagen 1A1 (COL1A1) as well as fibroblast migration/proliferation were analyzed. Our data show that intestinal fibroblasts homozygous for the C allele in the VDR gene exhibited lower VDR protein levels and higher proliferation than cells homozygous for the T allele. VD increased VDR and attenuated the accelerated proliferation of CC fibroblasts. The diminished VDR level detected in CC cells was associated with increased levels of both PDIA3 and COL1A1 expression and the transient silencing of PDIA3 significantly reduced COL1A1 expression. We conclude that intestinal fibroblasts homozygous for the C allele in the VDR gene exhibited: reduced VDR protein levels, increased proliferation and increased PDIA3/COL1A1 expression. Treatment with VD increased VDR and attenuated proliferation of these cells.


Assuntos
Fibroblastos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Adolescente , Adulto , Alelos , Proliferação de Células , Células Cultivadas , Feminino , Genótipo , Humanos , Intestinos/citologia , Masculino , Polimorfismo de Nucleotídeo Único , Adulto Jovem
10.
Nat Biomed Eng ; 4(9): 863-874, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32514094

RESUMO

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate. The absence of a solid matrix substantially reduces organoid heterogeneity, which we show for mouse and human gastrointestinal organoids. We use the devices to screen for anticancer drug candidates with patient-derived colorectal cancer organoids, and apply high-content image-based phenotypic analyses to reveal insights into mechanisms of drug action. The scalable organoid-culture technology should facilitate the use of organoids in drug development and diagnostics.


Assuntos
Técnicas de Cultura de Células/métodos , Organoides/citologia , Células-Tronco/citologia , Animais , Agregação Celular , Células Cultivadas , Dimetilpolisiloxanos/química , Avaliação Pré-Clínica de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Hidrogéis/química , Intestinos/citologia , Camundongos , Organogênese , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento
11.
Nature ; 585(7826): 591-596, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32526765

RESUMO

Recent clinical and experimental evidence has evoked the concept of the gut-brain axis to explain mutual interactions between the central nervous system and gut microbiota that are closely associated with the bidirectional effects of inflammatory bowel disease and central nervous system disorders1-4. Despite recent advances in our understanding of neuroimmune interactions, it remains unclear how the gut and brain communicate to maintain gut immune homeostasis, including in the induction and maintenance of peripheral regulatory T cells (pTreg cells), and what environmental cues prompt the host to protect itself from development of inflammatory bowel diseases. Here we report a liver-brain-gut neural arc that ensures the proper differentiation and maintenance of pTreg cells in the gut. The hepatic vagal sensory afferent nerves are responsible for indirectly sensing the gut microenvironment and relaying the sensory inputs to the nucleus tractus solitarius of the brainstem, and ultimately to the vagal parasympathetic nerves and enteric neurons. Surgical and chemical perturbation of the vagal sensory afferents at the hepatic afferent level reduced the abundance of colonic pTreg cells; this was attributed to decreased aldehyde dehydrogenase (ALDH) expression and retinoic acid synthesis by intestinal antigen-presenting cells. Activation of muscarinic acetylcholine receptors directly induced ALDH gene expression in both human and mouse colonic antigen-presenting cells, whereas genetic ablation of these receptors abolished the stimulation of antigen-presenting cells in vitro. Disruption of left vagal sensory afferents from the liver to the brainstem in mouse models of colitis reduced the colonic pTreg cell pool, resulting in increased susceptibility to colitis. These results demonstrate that the novel vago-vagal liver-brain-gut reflex arc controls the number of pTreg cells and maintains gut homeostasis. Intervention in this autonomic feedback feedforward system could help in the development of therapeutic strategies to treat or prevent immunological disorders of the gut.


Assuntos
Encéfalo/citologia , Intestinos/citologia , Intestinos/inervação , Fígado/citologia , Fígado/inervação , Neurônios/fisiologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Vias Aferentes , Animais , Células Apresentadoras de Antígenos/imunologia , Colite/imunologia , Colite/metabolismo , Colite/patologia , Homeostase , Humanos , Intestinos/imunologia , Masculino , Camundongos , Ratos , Receptores Muscarínicos/metabolismo , Baço/citologia , Baço/imunologia , Nervo Vago/fisiologia
12.
PLoS Negl Trop Dis ; 14(5): e0007942, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453724

RESUMO

Efforts to identify new drugs for therapeutic and preventive treatments against parasitic nematodes have gained increasing interest with expanding pathogen omics databases and drug databases from which new anthelmintic compounds might be identified. Here, a novel approach focused on integrating a pan-Nematoda multi-omics data targeted to a specific nematode organ system (the intestinal tract) with evidence-based filtering and chemogenomic screening was undertaken. Based on de novo computational target prioritization of the 3,564 conserved intestine genes in A. suum, exocytosis was identified as a high priority pathway, and predicted inhibitors of exocytosis were tested using the large roundworm (Ascaris suum larval stages), a filarial worm (Brugia pahangi adult and L3), a whipworm (Trichuris muris adult), and the non-parasitic nematode Caenorhabditis elegans. 10 of 13 inhibitors were found to cause rapid immotility in A. suum L3 larvae, and five inhibitors were effective against the three phylogenetically diverse parasitic nematode species, indicating potential for a broad spectrum anthelmintics. Several distinct pathologic phenotypes were resolved related to molting, motility, or intestinal cell and tissue damage using conventional and novel histologic methods. Pathologic profiles characteristic for each inhibitor will guide future research to uncover mechanisms of the anthelmintic effects and improve on drug designs. This progress firmly validates the focus on intestinal cell biology as a useful resource to develop novel anthelmintic strategies.


Assuntos
Anti-Helmínticos/farmacologia , Nematoides/efeitos dos fármacos , Animais , Células Cultivadas , Sistemas de Liberação de Medicamentos , Descoberta de Drogas , Intestinos/citologia , Intestinos/efeitos dos fármacos , Larva/efeitos dos fármacos
13.
Nat Commun ; 11(1): 2591, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444641

RESUMO

The intestine is a highly dynamic environment that requires tight control of the various inputs to maintain homeostasis and allow for proper responses to injury. It was recently found that the stem cell niche and epithelium is regenerated after injury by de-differentiated adult cells, through a process that gives rise to Sca1+ fetal-like cells and is driven by a transient population of Clu+ revival stem cells (revSCs). However, the molecular mechanisms that regulate this dynamic process have not been fully defined. Here we show that TNFAIP8 (also known as TIPE0) is a regulator of intestinal homeostasis that is vital for proper regeneration. TIPE0 functions through inhibiting basal Akt activation by the commensal microbiota via modulating membrane phospholipid abundance. Loss of TIPE0 in mice results in injury-resistant enterocytes, that are hyperproliferative, yet have regenerative deficits and are shifted towards a de-differentiated state. Tipe0-/- enterocytes show basal induction of the Clu+ regenerative program and a fetal gene expression signature marked by Sca1, but upon injury are unable to generate Sca-1+/Clu+ revSCs and could not regenerate the epithelium. This work demonstrates the role of TIPE0 in regulating the dynamic signaling that determines the injury response and enables intestinal epithelial cell regenerative plasticity.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Microbioma Gastrointestinal/fisiologia , Intestinos/citologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Ataxina-1/metabolismo , Diferenciação Celular , Colite/induzido quimicamente , Colite/patologia , Enterócitos/patologia , Feminino , Técnicas de Silenciamento de Genes , Homeostase , Intestinos/irrigação sanguínea , Intestinos/patologia , Intestinos/efeitos da radiação , Isquemia/genética , Isquemia/patologia , Antígenos Comuns de Leucócito/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/patologia , Regeneração/fisiologia , Transdução de Sinais , Nicho de Células-Tronco , Células-Tronco/metabolismo
14.
Nat Commun ; 11(1): 2493, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427826

RESUMO

Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. To assess the genetic risks induced by culturing, we determined all mutations in individual human stem cells by whole genome sequencing. Individual pluripotent, intestinal, and liver stem cells accumulate 3.5 ± 0.5, 7.2 ± 1.1 and 8.3 ± 3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of adult stem cells is nearly 40-fold higher than the in vivo mutation accumulation rate. Mutational signature analysis reveals that in vitro induced mutations are caused by oxidative stress. Reducing oxygen tension in culture lowers the mutational load. We use the mutation rates, spectra, and genomic distribution to model the accumulation of oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Our study provides empirically defined parameters to assess the mutational risk of stem cell based therapies.


Assuntos
Células-Tronco Adultas/metabolismo , Análise Mutacional de DNA/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Adulto , Células-Tronco Adultas/citologia , Algoritmos , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Intestinos/citologia , Fígado/citologia , Fígado/metabolismo , Modelos Genéticos , Acúmulo de Mutações , Taxa de Mutação , Medicina Regenerativa/métodos , Sequenciamento Completo do Genoma/métodos
15.
Tissue Cell ; 63: 101324, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32223952

RESUMO

Three-dimensional (3D) cell culture is more similar to in vivo studies and suitable for studies of interactions between cells and extracellular matrix. CD44 is a cell surface receptor that can relate with the extracellular matrix molecules. CD44 in gastric cancer (GC) is a metastatic and drug resistance marker. In this study the quantity of CD44+ cells in MKN-45 cell line in response to half maximal inhibitory concentration (IC50) dose of Docetaxel (DOC) was measured in 2D and 3D cultures. MKN-45 cell line was cultured in 2D and 3D environments. For 3D culture, rat gastric tissue was separated and decellularized and MKN-45 cells were injected and cultured in the prepared matrix. The frequency of CD44+ cells in 2D and 3D cultures were analyzed before and after treatment with IC50 of DOC by flow cytometry and immunohistochemistry. Despite different environmental conditions, The frequency of CD44+ cells increased significantly in 2D and 3D environments after treatment with IC50 of DOC (P < 0.05). Given the advantages of 3D, this environment seems more appropriate for study about CD44+ cells and drug resistance in GC.


Assuntos
Técnicas de Cultura de Células , Docetaxel/farmacologia , Receptores de Hialuronatos/genética , Neoplasias Gástricas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Matriz Extracelular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Ratos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia
16.
Food Chem ; 324: 126840, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32344339

RESUMO

Functional foods have created an open environment for the development of new solutions to health-related issues. In celiac disease, there is still no therapeutic alternative other than the observance of a gluten-free diet. In this context, we developed a wheat flour enriched in l-theanine aimed to be a potential alternative to the gluten-free diet. Through microbial transglutaminase-catalysed transamidation of gluten proteins using ethylamine as amine nucleophile, substantial amounts of glutamine residues were converted in theanine residues. Furthermore, using T-cell lines generated from intestinal biopsy specimens of celiac disease patients, this treatment showed the potential to strongly reduce the ability of gluten proteins to stimulate a T-cell-mediated immune response. From a rheological point of view, the functionality of gluten was retained. Considering L-theanine's evidence-based health benefits, a novel functional food is presented here and for celiac disease can be a path towards the development of an alternative to the gluten-free diet.


Assuntos
Doença Celíaca/imunologia , Farinha , Glutamatos/química , Glutens/química , Linfócitos T/imunologia , Doença Celíaca/dietoterapia , Dieta Livre de Glúten , Suplementos Nutricionais , Elasticidade , Etilaminas/metabolismo , Alimento Funcional , Glutens/metabolismo , Humanos , Intestinos/citologia , Intestinos/imunologia , Transglutaminases/metabolismo , Triticum
17.
Benef Microbes ; 11(2): 163-173, 2020 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-32131607

RESUMO

This study reports the probiotic attributes of Lactobacillus strains isolated from chicken faeces and mainly their capabilities to prevent infectious diseases and improve chicken production performance. Thus, 22 Lactobacillus strains were isolated from 50 chickens' faeces samples and assessed for their resistance to gastric acidity (pH 0.5, 1, 1.5, 2 and 2.5), tolerance to bile salts, adherence to broiler intestinal cells and antibacterial activity. These in vitro screening analyses revealed Lactobacillus plantarum S22 and L. plantarum S27 as the only strains capable to survive at pH 2.0 in MRS broth (log10 cfu/ml=5.02 and 8.46 log respectively), while the remaining strains were not resistant to pH≤2.0. Similarly, 21 strains were resistant to bile at 0.5% (log10 cfu/ml=0.09-3.32 log), but only Lactobacillus fermentum S26, L. plantarum S22 and L. plantarum S27 were able to grow in the presence of 0.1% (w/v) bile (8.23±0.15; 8.39±0.17 and 8.57±0.07 respectively). Most of these isolates (19/22) were active against Escherichia coli ATCC 25922, E. coli SL2016 and Salmonella enterica CIP 81-3. Lactic acid is likely the main antibacterial compound produced since the neutralised supernatant was devoid of any antibacterial activity. In vitro characterisation of these 22 novel strains, based on the aforementioned criteria revealed L. plantarum S27 as the most suitable strain for in vivo analyses. To this end, this strain was assessed for its sensitivity to different antibiotics and adhesion to poultry intestinal cells to ascertain it probiotic attributes. The administration of L. plantarum S27 to the chicks at 109 cfu/ml permitted to improve the animal food intake and weight. Taken together, data from in vitro and in vivo analyses indicated that L. plantarum S27 might be a worthy probiotic for chickens rather than adding antibiotics to animals feeding.


Assuntos
Fezes/microbiologia , Lactobacillus plantarum/isolamento & purificação , Lactobacillus/isolamento & purificação , Probióticos/administração & dosagem , Animais , Antibacterianos/farmacologia , Aderência Bacteriana , Ácidos e Sais Biliares/química , Galinhas/crescimento & desenvolvimento , Galinhas/microbiologia , Meios de Cultura/química , Células Epiteliais/microbiologia , Escherichia coli/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Intestinos/citologia , Ácido Láctico/metabolismo , Lactobacillus/classificação , Lactobacillus plantarum/efeitos dos fármacos , Lactobacillus plantarum/fisiologia , Masculino
18.
Anim Sci J ; 91(1): e13357, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32219956

RESUMO

Tight junctions (TJs) play an important role in intestinal barrier function. TJs in intestinal epithelial cells are composed of different junctional molecules, such as claudin and occludin, and regulate the paracellular permeability of water, ions, and macromolecules in adjacent cells. One of the most important roles of the TJ structure is to provide a physical barrier to luminal inflammatory molecules. Impaired integrity and structure of the TJ barrier result in a forcible activation of immune cells and chronic inflammation in different tissues. According to recent studies, the intestinal TJ barrier could be regulated, as a potential target, by dietary factors to prevent and reduce different inflammatory disorders, although the precise mechanisms underlying the dietary regulation remain unclear. This review summarizes currently available information on the regulation of the intestinal TJ barrier by food components.


Assuntos
Fenômenos Fisiológicos da Nutrição Animal , Alimentos , Intestinos/citologia , Intestinos/fisiologia , Nutrientes , Junções Íntimas/fisiologia , Animais , Água Corporal/metabolismo , Claudinas/metabolismo , Células Epiteliais/metabolismo , Inflamação , Intestinos/imunologia , Permeabilidade , Junções Íntimas/imunologia
19.
Parasit Vectors ; 13(1): 143, 2020 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-32188507

RESUMO

BACKGROUND: The porcine coccidium Cystoisospora suis is characterized by a complex life-cycle during which asexual multiplication is followed by sexual development with two morphologically distinct cell types, the micro- and macrogametes. Genes related to the sexual stages and cell cycle progression were previously identified in related Apicomplexa. Dynein light chain type 1 and male gamete fusion factor HAP2 are restricted to microgametes. Tyrosine-rich proteins and oocyst wall proteins are a part of the oocyst wall. The Rad51/Dmc1-like protein and Nima-related protein kinases are associated with the cell cycle and fertilization process. Here, the sexual stages of C. suis were characterized in vitro morphologically and for temporal expression changes of the mentioned genes to gain insight into this poorly known phase of coccidian development. METHODS: Sexual stages of C. suis developing in vitro in porcine intestinal epithelial cells were examined by light and electron microscopy. The transcriptional levels of genes related to merozoite multiplication and sexual development were evaluated by quantitative real-time PCR at different time points of cultivation. Transcription levels were compared for parasites in culture supernatants at 6-9 days of cultivation (doc) and intracellular parasites at 6-15 doc. RESULTS: Sexual stage of C. suis was detected during 8-11 doc in vitro. Microgamonts (16.8 ± 0.9 µm) and macrogamonts (16.6 ± 1.1 µm) are very similar in shape and size. Microgametes had a round body (3.5 ± 0.5 µm) and two flagella (11.2 ± 0.5 µm). Macrogametes were spherical with a diameter of 12.1 ± 0.5 µm. Merozoite gene transcription peaked on 10 doc and then declined. Genes related to the sexual stages and cell cycle showed an upregulation with a peak on 13 doc, after which they declined. CONCLUSIONS: The present study linked gene expression changes to the detailed morphological description of C. suis sexual development in vitro, including fertilization, meiosis and oocyst formation in this unique model for coccidian parasites. Following this process at the cellular and molecular level will elucidate details on potential bottlenecks of C. suis development (applicable for coccidian parasites in general) which could be exploited as a novel target for control.


Assuntos
Células Epiteliais/parasitologia , Merozoítos/crescimento & desenvolvimento , Merozoítos/genética , Sarcocystidae/crescimento & desenvolvimento , Sarcocystidae/genética , Animais , Células Cultivadas , Células Epiteliais/ultraestrutura , Feminino , Intestinos/citologia , Estágios do Ciclo de Vida , Masculino , Microscopia Eletrônica , Suínos
20.
Nat Cell Biol ; 22(3): 321-331, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123335

RESUMO

CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Técnicas de Introdução de Genes/métodos , Organoides/citologia , Hepatócitos/citologia , Hepatócitos/ultraestrutura , Humanos , Intestinos/citologia , Fígado/citologia , Organoides/ultraestrutura , Fuso Acromático/ultraestrutura , Proteína Supressora de Tumor p53/fisiologia
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