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2.
PLoS Pathog ; 15(10): e1008003, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31581229

RESUMO

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium.


Assuntos
Doenças dos Bovinos/transmissão , Células Epiteliais/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/efeitos dos fármacos , Íleo/microbiologia , Organoides/microbiologia , Toxina Shiga II/farmacologia , Animais , Bovinos , Doenças dos Bovinos/epidemiologia , Doenças dos Bovinos/microbiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/isolamento & purificação , Íleo/citologia , Íleo/metabolismo , Masculino , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Virulência
3.
Genes Dev ; 33(17-18): 1252-1264, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-31395740

RESUMO

Although MAX is regarded as an obligate dimerization partner for MYC, its function in normal development and neoplasia is poorly defined. We show that B-cell-specific deletion of Max has a modest effect on B-cell development but completely abrogates Eµ-Myc-driven lymphomagenesis. While Max loss affects only a few hundred genes in normal B cells, it leads to the global down-regulation of Myc-activated genes in premalignant Eµ-Myc cells. We show that the balance between MYC-MAX and MNT-MAX interactions in B cells shifts in premalignant B cells toward a MYC-driven transcriptional program. Moreover, we found that MAX loss leads to a significant reduction in MYC protein levels and down-regulation of direct transcriptional targets, including regulators of MYC stability. This phenomenon is also observed in multiple cell lines treated with MYC-MAX dimerization inhibitors. Our work uncovers a layer of Myc autoregulation critical for lymphomagenesis yet partly dispensable for normal development.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Carcinogênese/genética , Regulação Neoplásica da Expressão Gênica , Linfoma/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Carcinogênese/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Cinurenina/genética , Cinurenina/metabolismo , Linfoma/fisiopatologia , Camundongos , Organoides/crescimento & desenvolvimento , Organoides/fisiopatologia , Oximas/farmacologia , Sulfonamidas/farmacologia
4.
Nutrients ; 11(8)2019 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-31426533

RESUMO

Growth hormone (GH) and glutamine (Gln) stimulate the growth of the intestinal mucosa. GH activates the proliferation of intestinal stem cells (ISCs), enhances the formation of crypt organoids, increases ISC stemness markers in the intestinal organoids, and drives the differentiation of ISCs into Paneth cells and enterocytes. Gln enhances the proliferation of ISCs and increases crypt organoid formation; however, it mainly acts on the post-proliferation activity of ISCs to maintain the stability of crypt organoids and the intestinal mucosa, as well as to stimulate the differentiation of ISCs into goblet cells and possibly Paneth cells and enteroendocrine cells. Since GH and Gln have differential effects on ISCs. Their use in combination may have synergistic effects on ISCs. In this review, we summarize the evidence of the actions of GH and/or Gln on crypt cells and ISCs in the literature. Overall, most studies demonstrated that GH and Gln in combination exerted synergistic effects to activate the proliferation of crypt cells and ISCs and enhance crypt organoid formation and mucosal growth. This treatment influenced the proliferation of ISCs to a similar degree as GH treatment alone and the differentiation of ISCs to a similar degree as Gln treatment alone.


Assuntos
Glutamina/farmacologia , Hormônio do Crescimento/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Diferenciação Celular , Proliferação de Células , Sinergismo Farmacológico , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento , Células-Tronco/fisiologia
5.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31452509

RESUMO

Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.


Assuntos
Anticorpos/metabolismo , Receptores Frizzled/agonistas , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Linhagem Celular , Humanos , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/agonistas , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/agonistas , Camundongos , Organoides/efeitos dos fármacos , Organoides/crescimento & desenvolvimento , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/fisiologia , Ligação Proteica
6.
Elife ; 82019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31451149

RESUMO

The devastating effects and incurable nature of hereditary and sporadic retinal diseases such as Stargardt disease, age-related macular degeneration or retinitis pigmentosa urgently require the development of new therapeutic strategies. Additionally, a high prevalence of retinal toxicities is becoming more and more an issue of novel targeted therapeutic agents. Ophthalmologic drug development, to date, largely relies on animal models, which often do not provide results that are translatable to human patients. Hence, the establishment of sophisticated human tissue-based in vitro models is of upmost importance. The discovery of self-forming retinal organoids (ROs) derived from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) is a promising approach to model the complex stratified retinal tissue. Yet, ROs lack vascularization and cannot recapitulate the important physiological interactions of matured photoreceptors and the retinal pigment epithelium (RPE). In this study, we present the retina-on-a-chip (RoC), a novel microphysiological model of the human retina integrating more than seven different essential retinal cell types derived from hiPSCs. It provides vasculature-like perfusion and enables, for the first time, the recapitulation of the interaction of mature photoreceptor segments with RPE in vitro. We show that this interaction enhances the formation of outer segment-like structures and the establishment of in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics. In addition, we demonstrate the applicability of the RoC for drug testing, by reproducing the retinopathic side-effects of the anti-malaria drug chloroquine and the antibiotic gentamicin. The developed hiPSC-based RoC has the potential to promote drug development and provide new insights into the underlying pathology of retinal diseases.


Assuntos
Células-Tronco Pluripotentes Induzidas/fisiologia , Dispositivos Lab-On-A-Chip , Organoides/crescimento & desenvolvimento , Retina/fisiologia , Humanos
8.
Cells ; 8(5)2019 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035373

RESUMO

Human induced pluripotent stem cell (hiPSC)-derived organoids mimicking tissues and organs in vitro have advanced medical research, as they opened up new possibilities for in-depth basic research on human organ development as well as providing a human in vitro model for personalized therapeutic approaches. hiPSC-derived retinal organoids have proven to be of great value for modeling the human retina featuring a very similar cellular composition, layering, and functionality. The technically challenging imaging of three-dimensional structures such as retinal organoids has, however, raised the need for robust whole-organoid imaging techniques. To improve imaging of retinal organoids we optimized a passive clearing technique (PACT), which enables high-resolution visualization of fragile intra-tissue structures. Using cleared retinal organoids, we could greatly enhance the antibody labeling efficiency and depth of imaging at high resolution, thereby improving the three-dimensional microscopy output. In that course, we were able to identify the spatial morphological shape and organization of, e.g., photoreceptor cells and bipolar cell layers. Moreover, we used the synaptic protein CtBP2/Ribeye to visualize the interconnection points of photoreceptor and bipolar cells forming the retinal-specific ribbon synapses.


Assuntos
Células-Tronco Pluripotentes Induzidas/ultraestrutura , Organoides , Células Fotorreceptoras/ultraestrutura , Retina/ultraestrutura , Oxirredutases do Álcool/química , Técnicas de Cultura de Células/métodos , Proteínas Correpressoras/química , Humanos , Técnicas de Cultura de Órgãos/métodos , Organoides/crescimento & desenvolvimento , Organoides/ultraestrutura , Engenharia Tecidual/métodos
9.
Nat Biotechnol ; 37(4): 436-444, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30936566

RESUMO

Human brain organoids generated with current technologies recapitulate histological features of the human brain, but they lack a reproducible topographic organization. During development, spatial topography is determined by gradients of signaling molecules released from discrete signaling centers. We hypothesized that introduction of a signaling center into forebrain organoids would specify the positional identity of neural tissue in a distance-dependent manner. Here, we present a system to trigger a Sonic Hedgehog (SHH) protein gradient in developing forebrain organoids that enables ordered self-organization along dorso-ventral and antero-posterior positional axes. SHH-patterned forebrain organoids establish major forebrain subdivisions that are positioned with in vivo-like topography. Consistent with its behavior in vivo, SHH exhibits long-range signaling activity in organoids. Finally, we use SHH-patterned cerebral organoids as a tool to study the role of cholesterol metabolism in SHH signaling. Together, this work identifies inductive signaling as an effective organizing strategy to recapitulate in vivo-like topography in human brain organoids.


Assuntos
Proteínas Hedgehog/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Animais , Biotecnologia , Padronização Corporal , Diferenciação Celular , Colesterol/metabolismo , Humanos , Camundongos , Modelos Neurológicos , Células-Tronco Neurais/metabolismo , Neurogênese , Organoides/citologia , Células-Tronco Pluripotentes/metabolismo , Prosencéfalo/citologia , Transdução de Sinais
10.
Nature ; 569(7754): 66-72, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31019299

RESUMO

Intestinal organoids are complex three-dimensional structures that mimic the cell-type composition and tissue organization of the intestine by recapitulating the self-organizing ability of cell populations derived from a single intestinal stem cell. Crucial in this process is a first symmetry-breaking event, in which only a fraction of identical cells in a symmetrical sphere differentiate into Paneth cells, which generate the stem-cell niche and lead to asymmetric structures such as the crypts and villi. Here we combine single-cell quantitative genomic and imaging approaches to characterize the development of intestinal organoids from single cells. We show that their development follows a regeneration process that is driven by transient activation of the transcriptional regulator YAP1. Cell-to-cell variability in YAP1, emerging in symmetrical spheres, initiates Notch and DLL1 activation, and drives the symmetry-breaking event and formation of the first Paneth cell. Our findings reveal how single cells exposed to a uniform growth-promoting environment have the intrinsic ability to generate emergent, self-organized behaviour that results in the formation of complex multicellular asymmetric structures.


Assuntos
Intestinos/citologia , Organoides/citologia , Organoides/crescimento & desenvolvimento , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Proteínas de Ciclo Celular , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Organoides/metabolismo , Celulas de Paneth/citologia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Análise de Célula Única
11.
Biotechniques ; 66(3): 108-112, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30869546

RESUMO

Jenny Straiton explores the miniature world of organoids and discusses how these small models are making big changes in the world of neurological research.


Assuntos
Encéfalo/citologia , Células-Tronco/citologia , Encéfalo/crescimento & desenvolvimento , Humanos , Organoides/crescimento & desenvolvimento
12.
Nat Neurosci ; 22(4): 669-679, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30886407

RESUMO

Neural organoids have the potential to improve our understanding of human brain development and neurological disorders. However, it remains to be seen whether these tissues can model circuit formation with functional neuronal output. Here we have adapted air-liquid interface culture to cerebral organoids, leading to improved neuronal survival and axon outgrowth. The resulting thick axon tracts display various morphologies, including long-range projection within and away from the organoid, growth-cone turning, and decussation. Single-cell RNA sequencing reveals various cortical neuronal identities, and retrograde tracing demonstrates tract morphologies that match proper molecular identities. These cultures exhibit active neuronal networks, and subcortical projecting tracts can innervate mouse spinal cord explants and evoke contractions of adjacent muscle in a manner dependent on intact organoid-derived innervating tracts. Overall, these results reveal a remarkable self-organization of corticofugal and callosal tracts with a functional output, providing new opportunities to examine relevant aspects of human CNS development and disease.


Assuntos
Córtex Cerebral/crescimento & desenvolvimento , Neurônios/fisiologia , Organoides/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos/métodos , Axônios/fisiologia , Sobrevivência Celular , Córtex Cerebral/citologia , Feminino , Humanos , Masculino , Vias Neurais/citologia , Vias Neurais/fisiologia , Neurônios/citologia , Organoides/citologia , Células-Tronco Pluripotentes/fisiologia
13.
BMC Dev Biol ; 19(1): 3, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30841924

RESUMO

BACKGROUND: Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids has not been determined. RESULTS: Here we used computational fluid dynamics (CFD) to simulate two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the computed variables of the steering plates were closer to those of the spinning flask. CONCLUSION: Our protocol improves the initial steps of the standard brain organoid formation, and the produced organoids displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.


Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Técnicas de Cultura de Órgãos/métodos , Organoides/crescimento & desenvolvimento , Estresse Fisiológico/fisiologia , Linhagem Celular , Humanos , Hidrodinâmica , Organoides/citologia , Resistência ao Cisalhamento/fisiologia
14.
Cell Mol Gastroenterol Hepatol ; 7(3): 533-554, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30827941

RESUMO

BACKGROUND & AIMS: Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury. METHODS: Small intestine and enteroids from Defa4Cre;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4Cre;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4Cre;Rosa26reverse tetracycline transactivator-ires Enhanced Green Fluorescent Protein (EGFP);TetONICD), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4Cre;ADAM10flox/flox), and Adenomatous polyposis coli (APC) inactivation (Defa4Cre;APCflox/flox) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo. RESULTS: Defa4Cre-expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4Cre-marked cells showed clonogenic enteroid growth. Notch activation in Defa4Cre-expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4Cre-expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner. CONCLUSIONS: Our studies have shown that Defa4Cre-expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model.


Assuntos
Plasticidade Celular , Integrases/metabolismo , Intestinos/lesões , Intestinos/patologia , Celulas de Paneth/metabolismo , Receptores Notch/metabolismo , alfa-Defensinas/metabolismo , Proteína ADAM10/metabolismo , Adenoma/patologia , Proteína da Polipose Adenomatosa do Colo/metabolismo , Alelos , Animais , Desdiferenciação Celular , Linhagem da Célula , Células Clonais , Doxorrubicina , Deleção de Genes , Homeostase , Hiperplasia , Camundongos , Mitose , Células-Tronco Multipotentes/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/patologia , Regeneração
15.
Nat Neurosci ; 22(3): 362-373, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30718900

RESUMO

UTX is a chromatin modifier required for development and neural lineage specification, but how it controls these biological processes is unclear. To determine the molecular mechanisms of UTX, we identified novel UTX protein interaction partners. Here we show that UTX and 53BP1 directly interact and co-occupy promoters in human embryonic stem cells and differentiating neural progenitor cells. Human 53BP1 contains a UTX-binding site that diverges from its mouse homolog by 41%, and disruption of the 53BP1-UTX interaction abrogated human, but not mouse, neurogenesis in vitro. The 53BP1-UTX interaction is required to upregulate key neurodevelopmental genes during the differentiation of human embryonic stem cells into neurons or into cortical organoids. 53BP1 promotes UTX chromatin binding, and in turn H3K27 modifications and gene activation, at a subset of genomic regions, including neurogenic genes. Overall, our data suggest that the 53BP1-UTX interaction supports the activation of key genes required for human neurodevelopment.


Assuntos
Córtex Cerebral/metabolismo , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona Desmetilases/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Córtex Cerebral/crescimento & desenvolvimento , Feminino , Código das Histonas , Humanos , Masculino , Camundongos Endogâmicos C57BL , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Regiões Promotoras Genéticas
16.
Cell ; 176(4): 743-756.e17, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30735633

RESUMO

Direct comparisons of human and non-human primate brains can reveal molecular pathways underlying remarkable specializations of the human brain. However, chimpanzee tissue is inaccessible during neocortical neurogenesis when differences in brain size first appear. To identify human-specific features of cortical development, we leveraged recent innovations that permit generating pluripotent stem cell-derived cerebral organoids from chimpanzee. Despite metabolic differences, organoid models preserve gene regulatory networks related to primary cell types and developmental processes. We further identified 261 differentially expressed genes in human compared to both chimpanzee organoids and macaque cortex, enriched for recent gene duplications, and including multiple regulators of PI3K-AKT-mTOR signaling. We observed increased activation of this pathway in human radial glia, dependent on two receptors upregulated specifically in human: INSR and ITGB8. Our findings establish a platform for systematic analysis of molecular changes contributing to human brain development and evolution.


Assuntos
Córtex Cerebral/citologia , Organoides/metabolismo , Animais , Evolução Biológica , Encéfalo/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Córtex Cerebral/metabolismo , Redes Reguladoras de Genes/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Macaca , Neurogênese/genética , Organoides/crescimento & desenvolvimento , Pan troglodytes , Células-Tronco Pluripotentes/citologia , Análise de Célula Única , Especificidade da Espécie , Transcriptoma/genética
17.
Nat Methods ; 16(3): 255-262, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30742039

RESUMO

Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.


Assuntos
Rim/irrigação sanguínea , Organoides/crescimento & desenvolvimento , Células Cultivadas , Fibroblastos/citologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Dispositivos Lab-On-A-Chip , Técnicas de Cultura de Órgãos , Impressão Tridimensional , Engenharia Tecidual
18.
Cells ; 8(1)2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30634512

RESUMO

Human induced pluripotent stem cell (hiPSC)-derived three-dimensional retinal organoids are a new platform for studying the organoidogenesis. However, recurrent genomic aberration, acquired during generation of hiPSCs, limit its biomedical application and/or aberrant hiPSCs has not been evaluated for generation of differentiated derivatives, such as organoids and retinal pigment epithelium (RPE). In this study, we efficiently differentiated mosaic hiPSCs into retinal organoids containing mature photoreceptors. The feeder-free hiPSCs were generated from the human epidermal keratinocytes that were rapid in process with improved efficiency over several passages and maintained pluripotency. But, hiPSCs were cytogenetically mosaic with normal and abnormal karyotypes, while copy number variation analysis revealed the loss of chromosome 8q. Despite this abnormality, the stepwise differentiation of hiPSCs to form retinal organoids was autonomous and led to neuronal lamination. Furthermore, the use of a Notch inhibitor, DAPT, at an early timepoint from days 29⁻42 of culture improved the specification of the retinal neuron and the use of retinoic acid at days 70⁻120 led to the maturation of photoreceptors. hiPSC-derived retinal organoids acquired all subtypes of photoreceptors, such as RHODOPSIN, B-OPSIN and R/G-OPSIN. Additionally, the advanced maturation of photoreceptors was observed, revealing the development of specific sensory cilia and the formation of the outer-segment disc. This report is the first to show that hiPSCs with abnormal chromosomal content are permissive to the generation of three-dimensional retinal organoids.


Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas , Queratinócitos/citologia , Opsinas/metabolismo , Organoides/crescimento & desenvolvimento , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Organoides/citologia , Retina/citologia , Epitélio Pigmentado da Retina/citologia
19.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30642900

RESUMO

The enteric pathogen Shigella is one of the leading causes of moderate-to-severe diarrhea and death in young children in developing countries. Transformed cell lines and animal models have been widely used to study Shigella pathogenesis. In addition to altered physiology, transformed cell lines are composed of a single cell type that does not sufficiently represent the complex multicellular environment of the human colon. Most available animal models do not accurately mimic human disease. The human intestinal enteroid model, derived from LGR5+ stem cell-containing intestinal crypts from healthy subjects, represents a technological leap in human gastrointestinal system modeling and provides a more physiologically relevant system that includes multiple cell types and features of the human intestine. We established the utility of this model for studying basic aspects of Shigella pathogenesis and host responses. In this study, we show that Shigella flexneri is capable of infecting and replicating intracellularly in human enteroids derived from different segments of the intestine. Apical invasion by S. flexneri is very limited but increases ∼10-fold when enteroids are differentiated to include M cells. Invasion via the basolateral surface was at least 2-log10 units more efficient than apical infection. Increased secretion of interleukin-8 and higher expression levels of the mucin glycoprotein Muc2 were observed in the enteroids following S. flexneri infection. The human enteroid model promises to bridge some of the gaps between traditional cell culture, animal models, and human infection.


Assuntos
Disenteria Bacilar/microbiologia , Intestinos/citologia , Organoides/microbiologia , Shigella flexneri/fisiologia , Células Cultivadas , Humanos , Intestinos/microbiologia , Modelos Biológicos , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Shigella flexneri/genética , Shigella flexneri/crescimento & desenvolvimento , Shigella flexneri/patogenicidade , Células-Tronco/citologia , Células-Tronco/metabolismo , Virulência
20.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30642906

RESUMO

The enteric bacterium and intracellular human pathogen Shigella causes hundreds of millions of cases of the diarrheal disease shigellosis per year worldwide. Shigella is acquired by ingestion of contaminated food or water; upon reaching the colon, the bacteria invade colonic epithelial cells, replicate intracellularly, spread to adjacent cells, and provoke an intense inflammatory response. There is no animal model that faithfully recapitulates human disease; thus, cultured cells have been used to model Shigella pathogenesis. However, the use of transformed cells in culture does not provide the same environment to the bacteria as the normal human intestinal epithelium. Recent advances in tissue culture now enable the cultivation of human intestinal enteroids (HIEs), which are derived from human intestinal stem cells, grown ex vivo, and then differentiated into "mini-intestines." Here, we demonstrate that HIEs can be used to model Shigella pathogenesis. We show that Shigella flexneri invades polarized HIE monolayers preferentially via the basolateral surface. After S. flexneri invades HIE monolayers, S. flexneri replicates within HIE cells and forms actin tails. S. flexneri also increases the expression of HIE proinflammatory signals and the amino acid transporter SLC7A5. Finally, we demonstrate that disruption of HIE tight junctions enables S. flexneri invasion via the apical surface.


Assuntos
Disenteria Bacilar/microbiologia , Mucosa Intestinal/microbiologia , Modelos Biológicos , Organoides/microbiologia , Shigella flexneri/fisiologia , Técnicas de Cultura de Células , Disenteria Bacilar/genética , Disenteria Bacilar/metabolismo , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Transportador 1 de Aminoácidos Neutros Grandes/genética , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Shigella flexneri/patogenicidade , Células-Tronco/citologia , Células-Tronco/microbiologia , Virulência
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