271 results on '"Organ Cultures"'
Search Results
102. Circadian misalignment by environmental light/dark shifting causes circadian disruption in colon
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Faraz Bishehsari, Christopher B. Forsyth, Garth Swanson, Ali Keshavarzian, Maliha Shaikh, Lijuan Zhang, Dana M. Hayden, Robin M. Voigt, Laura Tran, Sherry Wilber, and Sarah B. Jochum
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Male ,Biochemistry ,Mice ,Medicine and Health Sciences ,Organ Cultures ,Luciferases ,Barrier function ,Chronobiology ,Multidisciplinary ,Animal Models ,Period Circadian Proteins ,Circadian Rhythm ,Cell biology ,Organoids ,Intestines ,PER2 ,Circadian Rhythms ,Circadian Oscillators ,Experimental Organism Systems ,Physical Sciences ,Medicine ,Suprachiasmatic Nucleus ,Biological Cultures ,Anatomy ,Research Article ,endocrine system ,Colon ,Science ,Photoperiod ,Period (gene) ,Materials Science ,Material Properties ,Mouse Models ,Motor Activity ,Biology ,Research and Analysis Methods ,Permeability ,Model Organisms ,In vivo ,Circadian Clocks ,Organoid ,Animals ,Humans ,Circadian rhythm ,Biology and Life Sciences ,Gastrointestinal Tract ,Mice, Inbred C57BL ,Animal Studies ,Digestive System ,Ex vivo - Abstract
Background Physiological circadian rhythms (CRs) are complex processes with 24-hour oscillations that regulate diverse biological functions. Chronic weekly light/dark (LD) shifting (CR disruption; CRD) in mice results in colonic hyperpermeability. However, the mechanisms behind this phenomenon are incompletely understood. One potential innovative in vitro method to study colonic CRs are colon organoids. The goals of this study were to utilize circadian clock gene Per2 luciferase reporter (Per2::Luc) mice to measure the effects of chronic LD shifting on colonic tissue circadian rhythmicity ex vivo and to determine if organoids made from shifted mice colons recapitulate the in vivo phenotype. Methods Non-shifted (NS) and shifted (S) BL6 Per2::Luc mice were compared after a 22-week experiment. NS mice had a standard 12h light/12h dark LD cycle throughout. S mice alternated 12h LD patterns weekly, with light from 6am-6pm one week followed by shifting light to 6pm-6am the next week for 22 weeks. Mice were tested for intestinal permeability while colon tissue and organoids were examined for CRs of bioluminescence and proteins of barrier function and cell fate. Results There was no absolute difference in NS vs. S 24h circadian period or phase. However, chronic LD shifting caused Per2::Luc S mice colon tissue to exhibit significantly greater variability in both the period and phase of Per2::Luc rhythms than NS mice colon tissue and organoids. Chronic LD shifting also resulted in increased colonic permeability of the Per2::Luc mice as well as decreased protein markers of intestinal permeability in colonic tissue and organoids from shifted Per2:Luc mice. Conclusions Our studies support a model in which chronic central circadian disruption by LD shifting alters the circadian phenotype of the colon tissue and results in colon leakiness and loss of colonic barrier function. These CRD-related changes are stably expressed in colon stem cell derived organoids from CRD mice.
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- 2021
103. GSK3ß inhibitor CHIR 99021 modulates cerebral organoid development through dose-dependent regulation of apoptosis, proliferation, differentiation and migration
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Vincent A. Pham, Hayley W. S. Tsang, Chloé Delépine, and Mriganka Sur
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0301 basic medicine ,Pyridines ,Organogenesis ,Apoptosis ,0302 clinical medicine ,Animal Cells ,Cell Movement ,Organ Cultures ,Induced pluripotent stem cell ,Cells, Cultured ,Multidisciplinary ,Cell Death ,Stem Cells ,Neurogenesis ,Brain ,Cell Differentiation ,Cell biology ,Organoids ,Neuroepithelial cell ,Cell Motility ,Cell Processes ,Medicine ,Biological Cultures ,Cellular Types ,Stem cell ,Neuronal Differentiation ,Research Article ,Cerebral organoid ,Cell Survival ,Science ,Induced Pluripotent Stem Cells ,Cell Migration ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Organoid ,Humans ,Neuron Migration ,Progenitor cell ,Cell Proliferation ,Glycogen Synthase Kinase 3 beta ,Brain Development ,Biology and Life Sciences ,Cell Biology ,Pyrimidines ,030104 developmental biology ,Organism Development ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Cerebral organoids generated from human pluripotent stem cells (hiPSCs) are unique in their ability to recapitulate human-specific neurodevelopmental events. They are capable of modeling the human brain and its cell composition, including human-specific progenitor cell types; ordered laminar compartments; and both cell-specific transcriptional signatures and the broader telencephalic transcriptional landscape. The serine/threonine kinase, GSK3β, plays a critical role in neurodevelopment, controlling processes as varied as neurogenesis, morphological changes, polarization, and migration. In the generation of cerebral organoids, inhibition of GSK3β at low doses has been used to increase organoid size and decrease necrotic core. However, little is known of the effects of GSK3β inhibition on organoid development. Here, we demonstrate that while low dose of GSK3β inhibitor CHIR 99021 increases organoid size, higher dose actually reduces organoid size; with the highest dose arresting organoid growth. To examine the mechanisms that may contribute to the phenotypic size differences observed in these treatment groups, we show that low dose of CHIR 99021 increases cell survival, neural progenitor cell proliferation and neuronal migration. A higher dose, however, decreases not only apoptosis but also proliferation, and arrests neural differentiation, enriching the pool of neuroepithelial cells, and decreasing the pools of early neuronal progenitors and neurons. These results reveal new mechanisms of the pleiotropic effects of GSK3β during organoid development, providing essential information for the improvement of organoid production and ultimately shedding light on the mechanisms of embryonic brain development.
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- 2021
104. Analysis of genetically engineered oncolytic herpes simplex viruses in human prostate cancer organotypic cultures.
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Passer, B. J., Wu, C-l., Wu, S., Rabkin, S. D., and Martuza, R. L.
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PROSTATE cancer , *HERPES simplex virus , *EPITHELIAL cells , *CANCER cells , *EXTRACELLULAR space - Abstract
Oncolytic herpes simplex viruses type 1 (oHSVs) such as G47Δ and G207 are genetically engineered for selective replication competence in cancer cells. Several factors can influence the overall effectiveness of oHSV tropism, including HSV-1 receptor expression, extracellular matrix milieu and cellular permissiveness. We have taken advantage of human prostate organ cultures derived from radical prostatectomies to investigate oHSV tropism. In this study, we show that both G47Δ and G207 specifically replicate in epithelial cells of the prostatic glands but not in the surrounding stroma. In contrast, both the epithelial and stromal cell compartments were readily infected by wild-type HSV-1. Analysis of oHSV replication in prostate surgical specimens 3 days post infection showed that G47Δ generated ∼30-fold more viral progeny than did G207. This correlated with the enhanced expression of G47Δ-derived glycoprotein gB protein levels as compared with G207. In benign prostate tissues, G207 and G47Δ titers were notably reduced, whereas strain F titers were maintained at similar levels compared with prostate cancer specimens. Overall, our results show that these oncolytic herpes vectors show both target specificity and replication competence in human prostate cancer specimens and point to the utility of using human prostate organ cultures in assessing oHSV tropism and cellular specificity. [ABSTRACT FROM AUTHOR]
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- 2009
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105. A novel system to study adenovirus tropism to normal and malignant colon tissues
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Kolodkin-Gal, D., Zamir, G., Pikarski, E., Pikarski, A., Shimony, N., Wu, H., Haviv, Y.S., and Panet, A.
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ADENOVIRUS diseases , *TROPISMS , *COLON cancer , *DIAGNOSTIC immunohistochemistry - Abstract
Abstract: We describe here a new organ culture system for the evaluation of viral tropism to colon carcinomas and normal colon tissues. Organ cultures of mouse and human colon retained viability for several days and thus facilitated studies of viral tropism. Two adenoviral vectors (AD) were compared in the study: AD5, that utilizes the CAR receptor, demonstrated poor infectivity to both normal and carcinoma tissues, while a capsid-modified-AD, recognizing haparan-sulfate receptor, demonstrated efficient infectivity of both tissues. Immunohistochemistry analysis demonstrated different viral tropism; while AD5 infected only the colon epithelia, the capsid-modified-adeno infected both the epithelia and mesothelial layers. To investigate other determinants in the tissue that influence viral tropism, human cancer tissues were pretreated with collagenase and infected with the AD viruses. Increased infectivity and altered tropism were noted in the treated tumor tissue. Taken together, this ex vivo system indicated that receptor utilization and extracellular-matrix components influence AD viral tropism in solid tissues. [Copyright &y& Elsevier]
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- 2007
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106. Nitrosomethylurea disturbs differentiation of mouse embryonic lungs in organ cultures.
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Popova, N. and Rossi, L.
- Abstract
We have studied the effect of nitrosomethylurea (NMU) on the differentiation of early rudiments of mouse embryonic lungs (12th day of embryogenesis) explanted into an organ culture. We have demonstrated that nontoxic doses of NMU are capable of accelerating normal lung differentiation both at the early (increase in the number of epithelial buds) and at the late (increase in the number of explants with regions of well-developed alveoli) stages of cultivation. However, NMU induces disturbances of differentiation, which appear as polycystic structures and hyperplastic nodules generally absent in the control. [ABSTRACT FROM AUTHOR]
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- 2000
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107. Pregnane X receptor activation constrains mucosal NF-κB activity in active inflammatory bowel disease
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Sunrui Chen, Meng Li, J. Jasper Deuring, Wenshi Wang, Colin de Haar, C. Janneke van der Woude, Wanlu Cao, Maikel P. Peppelenbosch, and Gastroenterology & Hepatology
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0301 basic medicine ,Biopsy ,Cell Lines ,Gene Expression ,Pathology and Laboratory Medicine ,Inflammatory bowel disease ,chemistry.chemical_compound ,0302 clinical medicine ,Gene expression ,Medicine and Health Sciences ,Organ Cultures ,Intestinal Mucosa ,Immune Response ,Pregnane X receptor ,Multidisciplinary ,medicine.diagnostic_test ,NF-kappa B ,Pregnane X Receptor ,Organoids ,Medicine ,Cytokines ,030211 gastroenterology & hepatology ,Biological Cultures ,Signal transduction ,Anatomy ,Rifampin ,Research Article ,Signal Transduction ,Colon ,Science ,Immunology ,Surgical and Invasive Medical Procedures ,Gastroenterology and Hepatology ,Research and Analysis Methods ,digestive system ,03 medical and health sciences ,Signs and Symptoms ,Western blot ,Diagnostic Medicine ,Cell Line, Tumor ,medicine ,Genetics ,Humans ,Inflammation ,business.industry ,Inflammatory Bowel Disease ,Biology and Life Sciences ,NF-κB ,medicine.disease ,Inflammatory Bowel Diseases ,digestive system diseases ,Gastrointestinal Tract ,030104 developmental biology ,chemistry ,Caco-2 ,Cell culture ,Cancer research ,Caco-2 Cells ,business ,Digestive System - Abstract
Background The Pregnane X Receptor (PXR) is a principal signal transducer in mucosal responses to xenobiotic stress. It is well-recognized that inflammatory bowel disease is accompanied by xenobiotic stress, but the importance of the PXR in limiting inflammatory responses in inflammatory bowel disease remains obscure at best. Methods We stimulate a total of 106 colonic biopsies from 19 Crohn’s disease patients with active disease, 36 colonic biopsies from 8 control patients, colonic organoids and various cell culture models (either proficient or genetically deficient with respect to PXR) in vitro with the PXR ligand rifampicin or vehicle. Effects on NF-κB activity are assessed by measuring interleukin-8 (IL-8) and interleukin-1s (IL-1s) mRNA levels by qPCR and in cell culture models by NF-κB reporter-driven luciferase activity and Western blot for signal transduction elements. Results We observe a strict inverse correlation between colonic epithelial PXR levels and NF-κB target gene expression in colonic biopsies from Crohn’s disease patients. PXR, activated by rifampicin, is rate-limiting for mucosal NF-κB activation in IBD. The correlation between colonic epithelial PXR levels and NF-κB target gene expression was also observed in intestinal organoids system. Furthermore, in preclinical in vitro models of intestinal inflammation, including intestinal organoids, genetic inactivation of PXR unleashes NF-κB-dependent signal transduction whereas conversely NF-κB signaling reduces levels of PXR expression. Conclusions Our data indicate that the PXR is a major and clinically relevant antagonist of NF-κB activity in the intestinal epithelial compartment during inflammatory bowel disease.
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- 2019
108. Shiga toxin sub-type 2a increases the efficiency of Escherichia coli O157 transmission between animals and restricts epithelial regeneration in bovine enteroids
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Javier Palarea-Albaladejo, Rachel Young, Nur Indah Ahmad, Stephen F. Fitzgerald, David L. Gally, Sharif Shaaban, Neil A. Mabbott, James L. Bono, Liam J. Morrison, Jason K. Morgan, Sean P. McAteer, Tom N. McNeilly, and Amy E. Beckett
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Male ,Physiology ,Molting ,medicine.disease_cause ,Toxicology ,Pathology and Laboratory Medicine ,Shiga Toxin 2 ,Gene expression ,Medicine and Health Sciences ,Toxins ,Bacteriophages ,Biology (General) ,Organ Cultures ,Escherichia coli Infections ,Mammals ,0303 health sciences ,Gastrointestinal tract ,Virulence ,Eukaryota ,Shiga toxin ,Ruminants ,Organoids ,Vertebrates ,Viruses ,Biological Cultures ,Anatomy ,Research Article ,Lysis (Medicine) ,QH301-705.5 ,Immunology ,Toxic Agents ,Excretion ,Cattle Diseases ,Biology ,Research and Analysis Methods ,Escherichia coli O157 ,Microbiology ,Gastrointestinal epithelium ,03 medical and health sciences ,Bovines ,Ileum ,Virology ,Tissue Repair ,Genetics ,medicine ,Animals ,Molecular Biology ,Escherichia coli ,Prophage ,030304 developmental biology ,030306 microbiology ,Toxin ,Organisms ,Biology and Life Sciences ,Epithelial Cells ,RC581-607 ,Gastrointestinal Tract ,Amniotes ,biology.protein ,Parasitology ,Cattle ,Immunologic diseases. Allergy ,Physiological Processes ,Digestive System - Abstract
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., Author summary Enterohaemorrhagic E. coli (EHEC) O157 strains are found in cattle where they are asymptomatic, while human exposure can lead to severe symptoms including bloody diarrhoea and kidney damage due to the activity of Shiga toxin (Stx). The most serious symptoms in humans are associated with isolates that encode Stx subtype 2a. The advantage of these toxins in the animal reservoir is still not clear, however there is experimental evidence implicating Stx with increased bacterial adherence, immune modulation and suppression of predatory protozoa. In this study, the hypothesis that Stx2a is important for super-shedding and calf-to-calf transmission was tested by comparing excretion and transmission dynamics of E. coli O157 strains with and without Stx2a. While Stx2a did not alter excretion levels when calfs were orally challenge, it enabled colonisation of more in contact ‘sentinel’ animals in our transmission model. We show that Stx2a is generally induced more rapidly than Stx2c, resulting in increased levels of Stx2a expression. Both Stx2a and Stx2c were able to restrict cellular proliferation of epithelial cells in cultured bovine enteroids. Taken together, we propose that rapid production of Stx2a and its role in establishing E. coli O157 colonisation in the bovine gastrointestinal tract facilitate effective transmission and have led to its expansion in the cattle E. coli O157 population.
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- 2019
109. The cat is out the bag about Toxoplasma host range
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Ursula Hofer
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Artificial Gene Amplification and Extension ,Biochemistry ,Polymerase Chain Reaction ,Toxoplasma Gondii ,Medicine and Health Sciences ,Morphogenesis ,Organ Cultures ,Genetics ,Protozoans ,0303 health sciences ,Sexual Differentiation ,Fatty Acids ,Eukaryota ,Lipids ,Intestines ,Organoids ,Infectious Diseases ,Biological Cultures ,Anatomy ,Toxoplasma ,Research Article ,Biology ,Research and Analysis Methods ,Microbiology ,Linoleoyl-CoA Desaturase ,Host Specificity ,Host-Parasite Interactions ,Linoleic Acid ,03 medical and health sciences ,parasitic diseases ,Parasite Groups ,Dietary Fatty Acid ,Animals ,Molecular Biology Techniques ,Molecular Biology ,General Immunology and Microbiology ,030306 microbiology ,Host (biology) ,Oocysts ,Organisms ,Toxoplasma gondii ,Biology and Life Sciences ,biology.organism_classification ,Parasitic Protozoans ,Sexual reproduction ,Gastrointestinal Tract ,Cats ,Parasitology ,Apicomplexa ,Digestive System ,Developmental Biology - Abstract
Many eukaryotic microbes have complex life cycles that include both sexual and asexual phases with strict species specificity. Whereas the asexual cycle of the protistan parasite Toxoplasma gondii can occur in any warm-blooded mammal, the sexual cycle is restricted to the feline intestine. The molecular determinants that identify cats as the definitive host for T. gondii are unknown. Here, we defined the mechanism of species specificity for T. gondii sexual development and break the species barrier to allow the sexual cycle to occur in mice. We determined that T. gondii sexual development occurs when cultured feline intestinal epithelial cells are supplemented with linoleic acid. Felines are the only mammals that lack delta-6-desaturase activity in their intestines, which is required for linoleic acid metabolism, resulting in systemic excess of linoleic acid. We found that inhibition of murine delta-6-desaturase and supplementation of their diet with linoleic acid allowed T. gondii sexual development in mice. This mechanism of species specificity is the first defined for a parasite sexual cycle. This work highlights how host diet and metabolism shape coevolution with microbes. The key to unlocking the species boundaries for other eukaryotic microbes may also rely on the lipid composition of their environments as we see increasing evidence for the importance of host lipid metabolism during parasitic lifecycles. Pregnant women are advised against handling cat litter, as maternal infection with T. gondii can be transmitted to the fetus with potentially lethal outcomes. Knowing the molecular components that create a conducive environment for T. gondii sexual reproduction will allow for development of therapeutics that prevent shedding of T. gondii parasites. Finally, given the current reliance on companion animals to study T. gondii sexual development, this work will allow the T. gondii field to use of alternative models in future studies., The sexual cycle of Toxoplasma gondii is restricted to cats, the only mammals to lack delta-6-desaturase activity, with consequent high levels of linoleic acid. This study shows that inhibition of delta-6-desaturase and diet supplementation with linoleic acid allows Toxoplasma sexual development in mice, potentially opening up alternative model hosts.
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- 2019
110. Morphophenotypic classification of tumor organoids as an indicator of drug exposure and penetration potential
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Sharan Poonja, Katarzyna A. Rejniak, and Aleksandra Karolak
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0301 basic medicine ,Cell division ,Cell ,Cancer Treatment ,0302 clinical medicine ,Neoplasms ,Medicine and Health Sciences ,Tumor Cells, Cultured ,Tumor Microenvironment ,Cell Cycle and Cell Division ,Biology (General) ,Organ Cultures ,Materials ,Ecology ,Pharmaceutics ,Simulation and Modeling ,Phenotype ,3. Good health ,Cell biology ,Organoids ,medicine.anatomical_structure ,Computational Theory and Mathematics ,Oncology ,Cell Processes ,Modeling and Simulation ,Physical Sciences ,Disease Progression ,Biological Cultures ,Research Article ,QH301-705.5 ,Surface Properties ,Materials Science ,Geometry ,Antineoplastic Agents ,Biology ,Research and Analysis Methods ,Models, Biological ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,Imaging, Three-Dimensional ,Drug Therapy ,In vivo ,Adhesives ,Genetics ,Organoid ,medicine ,Humans ,Computer Simulation ,Epigenetics ,Molecular Biology ,Cell Cycle Inhibitors ,Ecology, Evolution, Behavior and Systematics ,Mathematical Modeling ,Contact inhibition ,Biology and Life Sciences ,Computational Biology ,Cell Biology ,030104 developmental biology ,Radii ,Tumor progression ,Drug Screening Assays, Antitumor ,030217 neurology & neurosurgery ,Mathematics - Abstract
The dynamics of tumor progression is driven by multiple factors, which can be exogenous to the tumor (microenvironment) or intrinsic (genetic, epigenetic or due to intercellular interactions). While tumor heterogeneity has been extensively studied on the level of cell genetic profiles or cellular composition, tumor morphological diversity has not been given as much attention. The limited analysis of tumor morphophenotypes may be attributed to the lack of accurate models, both experimental and computational, capable of capturing changes in tumor morphology with fine levels of spatial detail. Using a three-dimensional, agent-based, lattice-free computational model, we generated a library of multicellular tumor organoids, the experimental analogues of in vivo tumors. By varying three biologically relevant parameters—cell radius, cell division age and cell sensitivity to contact inhibition, we showed that tumor organoids with similar growth dynamics can express distinct morphologies and possess diverse cellular compositions. Taking advantage of the high-resolution of computational modeling, we applied the quantitative measures of compactness and accessible surface area, concepts that originated from the structural biology of proteins. Based on these analyses, we demonstrated that tumor organoids with similar sizes may differ in features associated with drug effectiveness, such as potential exposure to the drug or the extent of drug penetration. Both these characteristics might lead to major differences in tumor organoid’s response to therapy. This indicates that therapeutic protocols should not be based solely on tumor size, but take into account additional tumor features, such as their morphology or cellular packing density., Author summary Primary tumors and tumor metastases grow as three-dimensional (3D) masses of cells. Depending on the surrounding stroma, they may acquire various shapes, more or less irregular. Tumor organoids are the 3D experimental cultures that mimic growth of in vivo tumors, as well as their response to treatments. However, it is difficult to assess experimentally in a reproducible and quantitative way, how tumor morphology influences treatment efficacy. Here, we used mathematical modeling and computer simulations to analyze the structure of the simulated organoids and to classify them with regards to two quantitative features: the tumor accessible surface area (ASA) describing organoid exposure to the drug and the extent of drug penetration through the tumor tissue (organoid compactness). We showed that organoids of similar sizes and growth dynamics can, in fact, be characterized by distinct values of compactness and ASA, and thus may respond differently to the drug treatment. We suggest that these tumor features should be taken into consideration in addition to tumor size, when the therapeutic interventions are designed.
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- 2019
111. Oct1/Pou2f1 is selectively required for colon regeneration and regulates colon malignancy
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Jared Rutter, Claire L. Bensard, Xinjian Chen, Dean Tantin, John C. Schell, Eric R. Swanson, and Karina Vázquez-Arreguín
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Cancer Research ,Colorectal cancer ,Carcinogenesis ,Gene Expression ,QH426-470 ,Epithelium ,Receptors, G-Protein-Coupled ,Mice ,0302 clinical medicine ,Intestine, Small ,Medicine and Health Sciences ,Organ Cultures ,Genetics (clinical) ,Regulation of gene expression ,Mice, Knockout ,0303 health sciences ,Dextran Sulfate ,3. Good health ,Organoids ,Gene Expression Regulation, Neoplastic ,Oncology ,Colonic Neoplasms ,Neoplastic Stem Cells ,Small Intestine ,Female ,Biological Cultures ,Stem cell ,Anatomy ,Research Article ,Signal Transduction ,Tumor suppressor gene ,Colon ,Azoxymethane ,Biology ,Malignancy ,Research and Analysis Methods ,03 medical and health sciences ,medicine ,Genetics ,Animals ,Humans ,Regeneration ,Molecular Biology ,Transcription factor ,Mitosis ,Immunohistochemistry Techniques ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Colorectal Cancer ,Integrases ,Gene Expression Profiling ,Biology and Life Sciences ,Cancers and Neoplasms ,medicine.disease ,HCT116 Cells ,Survival Analysis ,Gene expression profiling ,Gastrointestinal Tract ,Histochemistry and Cytochemistry Techniques ,Disease Models, Animal ,Tamoxifen ,Biological Tissue ,Cancer research ,Immunologic Techniques ,Digestive System ,030217 neurology & neurosurgery ,Octamer Transcription Factor-1 - Abstract
The transcription factor Oct1/Pou2f1 promotes poised gene expression states, mitotic stability, glycolytic metabolism and other characteristics of stem cell potency. To determine the effect of Oct1 loss on stem cell maintenance and malignancy, we deleted Oct1 in two different mouse gut stem cell compartments. Oct1 deletion preserved homeostasis in vivo and the ability to establish organoids in vitro, but blocked the ability to recover from treatment with dextran sodium sulfate, and the ability to maintain organoids after passage. In a chemical model of colon cancer, loss of Oct1 in the colon severely restricted tumorigenicity. In contrast, loss of one or both Oct1 alleles progressively increased tumor burden in a colon cancer model driven by loss-of-heterozygosity of the tumor suppressor gene Apc. The different outcomes are consistent with prior findings that Oct1 promotes mitotic stability, and consistent with differentially expressed genes between the two models. Oct1 ChIPseq using HCT116 colon carcinoma cells identifies target genes associated with mitotic stability, metabolism, stress response and malignancy. This set of gene targets overlaps significantly with genes differentially expressed in the two tumor models. These results reveal that Oct1 is selectively required for recovery after colon damage, and that Oct1 has potent effects in colon malignancy, with outcome (pro-oncogenic or tumor suppressive) dictated by tumor etiology., Author summary Colorectal cancer is the second leading cause of cancer death in the United States. Approximately 35% of diagnosed patients eventually succumb to disease. The high incidence and mortality due to colon cancer demand a better understanding of factors controlling the physiology and pathophysiology of the gastrointestinal tract. Previously, we and others showed that the widely expressed transcription factor Oct1 is expressed at higher protein levels in stem cells, including intestinal stem cells. Here we use deletion of a conditional mouse Oct1 (Pou2f1) allele in two different intestinal stem cell compartments to study gut homeostasis. We then proceed to investigate the effect of Oct1 loss in colon regeneration and malignancy. The results indicate that Oct1 loss is dispensable for maintenance of the mouse gut, but required for recovery after damage to the colon epithelium. We also find that Oct1 loss has opposing effects in two different mouse colon cancer models, and further that the two models are associated with different gene expression signatures. The differentially expressed genes are enriched for Oct1 targets, suggesting that differential gene control by Oct1 is one mechanism underlying the different outcomes.
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- 2019
112. Thyroxine (T4) may promote re-epithelialisation and angiogenesis in wounded human skin ex vivo
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Frank Siemers, Wolfgang Funk, Guo You Zhang, Ralf Paus, N. Meier, and Ewan A. Langan
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0301 basic medicine ,Keratinocytes ,Male ,Lydia Becker Institute ,Fibroblast Growth Factor ,Angiogenesis ,Physiology ,Human skin ,Fibroblast growth factor ,Cardiovascular Physiology ,Biochemistry ,Epithelium ,Neovascularization ,Tissue Culture Techniques ,030207 dermatology & venereal diseases ,0302 clinical medicine ,Endocrinology ,Re-Epithelialization ,Animal Cells ,Keratin ,Medicine and Health Sciences ,Organ Cultures ,Skin ,chemistry.chemical_classification ,Multidisciplinary ,integumentary system ,Middle Aged ,Medicine ,Keratins ,Female ,Biological Cultures ,medicine.symptom ,Anatomy ,Integumentary System ,Cellular Types ,Research Article ,Adult ,Science ,Neovascularization, Physiologic ,Research and Analysis Methods ,03 medical and health sciences ,In vivo ,ResearchInstitutes_Networks_Beacons/lydia_becker_institute_of_immunology_and_inflammation ,Growth Factors ,Tissue Repair ,medicine ,Humans ,Aged ,Wound Healing ,Endocrine Physiology ,business.industry ,Biology and Life Sciences ,Proteins ,Epithelial Cells ,Cell Biology ,Fibroblasts ,Cytoskeletal Proteins ,Thyroxine ,030104 developmental biology ,Biological Tissue ,chemistry ,Gene Expression Regulation ,Cancer research ,Epidermis ,Wound healing ,business ,Physiological Processes ,Energy Metabolism ,Ex vivo ,Developmental Biology - Abstract
There is a pressing need for improved preclinical model systems in which to study human skin wound healing. Here, we report the development and application of a serum-free full thickness human skin wound healing model. Not only can re-epithelialization (epidermal repair) and angiogenesis be studied in this simple and instructive model, but the model can also be used to identify clinically relevant wound-healing promoting agents, and to dissect underlying candidate mechanisms of action in the target tissue. We present preliminary ex vivo data to suggest that Thyroxine (T4), which reportedly promotes skin wound healing in rodents in vivo, may promote key features of human skin wound healing. Namely, T4 stimulates re-epithelialisation and angiogenesis, and modulates both wound healing-associated epidermal keratin expression and energy metabolism in experimentally wound human skin. Functionally, the wound healing-promoting effects of T4 are at least partially mediated via fibroblast growth factor/fibroblast growth factor receptor-mediated signalling, since they could be significantly antagonized by bFGF-neutralizing antibody. Thus, this pragmatic, easy-to-use full-thickness human skin wound healing model provides a useful preclinical research tool in the search for clinically relevant candidate wound healing-promoting agents. These ex vivo data encourage further pre-clinical testing of topical T4 as a cost-efficient, novel agent in the management of chronic human skin wounds.
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- 2019
113. 2-Cl-C.OXT-A Stimulates Contraction through the Suppression of Phosphodiesterase Activity in Human Induced Pluripotent Stem Cell-derived Cardiac Organoids
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Takahiro Kitsuka, Takahiro Nishida, Jun-ichi Oyama, Manabu Itoh, Koichi Node, Koichi Nakayama, Shigeki Morita, Kenichi Arai, Sojiro Amamoto, and Shuji Toda
- Subjects
0301 basic medicine ,Adenosine ,Contraction (grammar) ,Phosphodiesterase Inhibitors ,Physiology ,030204 cardiovascular system & hematology ,Pharmacology ,Cardiovascular Physiology ,Biochemistry ,Epithelium ,chemistry.chemical_compound ,0302 clinical medicine ,Animal Cells ,Medicine and Health Sciences ,Myocyte ,Myocytes, Cardiac ,Organ Cultures ,Receptor ,Connective Tissue Cells ,Multidisciplinary ,Forskolin ,Drugs ,Phosphodiesterase ,Heart ,Troponin ,Organoids ,Connective Tissue ,Medicine ,Biological Cultures ,Anatomy ,Cellular Types ,Research Article ,medicine.drug ,Science ,Suramin ,Induced Pluripotent Stem Cells ,Muscle Tissue ,Research and Analysis Methods ,Cell Line ,03 medical and health sciences ,Adenosine A1 receptor ,medicine ,Organoid ,Humans ,Muscle Cells ,Phosphoric Diester Hydrolases ,Isoproterenol ,Biology and Life Sciences ,Endothelial Cells ,Proteins ,Epithelial Cells ,Cell Biology ,Fibroblasts ,Myocardial Contraction ,Cytoskeletal Proteins ,Biological Tissue ,030104 developmental biology ,chemistry ,Cardiovascular Anatomy ,Angiogenesis ,Developmental Biology - Abstract
Background2-Cl-C.OXT-A (COA-Cl) is a novel synthesized adenosine analog that activates S1P1 receptor (S1P1R) and combines with adenosine A1 receptor (A1R) in G proteins and was shown to enhance angiogenesis and improve the brain function in rat stroke models. However, the role of COA-Cl in hearts remains unclear. COA-Cl, which has a similar structure to xanthine derivatives, has the potential to suppress phosphodiesterase (PDE), which is an important factor involved in the beating of heart muscle.Methods and resultsCardiac organoids with fibroblasts, human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs), and hiPSC-derived endothelial cells (hiPSC-ECs) were cultured until they started beating. The beating and contraction of organoids were observed before and after the application of COA-Cl. COA-Cl significantly increased the beating rate and fractional area change in organoids. To elucidate the mechanism underlying these effects of COA-Cl on cardiac myocytes, pure hiPSC-CM spheroids were evaluated in the presence/absence of Suramin (antagonist of A1R). The effects of COA-Cl, SEW2871 (direct stimulator of S1P1R), two positive inotropes (Isoproterenol [ISO] and Forskolin [FSK]), and negative inotrope (Propranolol [PRP]) on spheroids were assessed based on the beating rates and cAMP levels. COA-Cl stimulated the beating rates about 1.5-fold compared with ISO and FSK, while PRP suppressed the beating rate. However, no marked changes were observed with SEW2871. COA-Cl, ISO, and FSK increased the cAMP level. In contrast, the level of cAMP did not change with PRP or SEW2871 treatment. The results were the same in the presence of Suramin as absence. Furthermore, an enzyme analysis showed that COA-Cl suppressed the PDE activity by half.ConclusionsCOA-Cl, which has neovascularization effects, suppressed PDE and increased the contraction of cardiac organoids, independent of S1P1R and A1R. These findings suggest that COA-Cl may be useful as an inotropic agent for promoting angiogenesis in the future.
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- 2019
114. Effects of insulin signaling on mouse taste cell proliferation
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Yuzo Ninomiya, Robert F. Margolskee, Yu Watanabe, Ikiru Atsuta, Kiyoshi Koyano, Keisuke Sanematsu, Shingo Takai, Noriatsu Shigemura, Peihua Jiang, and Ryusuke Yoshida
- Subjects
Male ,0301 basic medicine ,Taste ,Sensory Receptors ,Physiology ,Cellular differentiation ,Social Sciences ,Gene Expression ,Biochemistry ,Mice ,Endocrinology ,0302 clinical medicine ,TAS1R3 ,Animal Cells ,Medicine and Health Sciences ,Insulin ,Psychology ,Organ Cultures ,Multidisciplinary ,biology ,Chemistry ,TOR Serine-Threonine Kinases ,Stem Cells ,Cell Differentiation ,Taste Buds ,Immunohistochemistry ,Cell biology ,Organoids ,medicine.anatomical_structure ,Medicine ,Female ,Sensory Perception ,Biological Cultures ,Cellular Types ,Signal Transduction ,Research Article ,Science ,Research and Analysis Methods ,03 medical and health sciences ,Taste bud ,Genetics ,medicine ,Animals ,Mechanistic target of rapamycin ,PI3K/AKT/mTOR pathway ,Cell Proliferation ,Diabetic Endocrinology ,Endocrine Physiology ,Insulin Signaling ,Biology and Life Sciences ,Cell Biology ,Gustducin ,Receptor, Insulin ,Hormones ,Insulin receptor ,030104 developmental biology ,biology.protein ,Biomarkers ,030217 neurology & neurosurgery ,Neuroscience ,Developmental Biology - Abstract
Expression of insulin and its receptor (IR) in rodent taste cells has been proposed, but exactly which types of taste cells express IR and the function of insulin signaling in taste organ have yet to be determined. In this study, we analyzed expression of IR mRNA and protein in mouse taste bud cells in vivo and explored its function ex vivo in organoids, using RT-PCR, immunohistochemistry, and quantitative PCR. In mouse taste tissue, IR was expressed broadly in taste buds, including in type II and III taste cells. With using 3-D taste bud organoids, we found insulin in the culture medium significantly decreased the number of taste cell and mRNA expression levels of many taste cell genes, including nucleoside triphosphate diphosphohydrolase-2 (NTPDase2), Tas1R3 (T1R3), gustducin, carbonic anhydrase 4 (CA4), glucose transporter-8 (GLUT8), and sodium-glucose cotransporter-1 (SGLT1) in a concentration-dependent manner. Rapamycin, an inhibitor of mechanistic target of rapamycin (mTOR) signaling, diminished insulin’s effects and increase taste cell generation. Altogether, circulating insulin might be an important regulator of taste cell growth and/or proliferation via activation of the mTOR pathway.
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- 2019
115. A more physiological approach to lipid metabolism alterations in cancer: CRC-like organoids assessment
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Ana Ramírez de Molina, Daniel E. Stange, Ruth Sánchez-Martínez, Silvia Cruz-Gil, Sebastian Schölch, Kristin Werner, and Sonia Wagner-Reguero
- Subjects
Colorectal cancer ,Cancer Treatment ,Gene Expression ,Stem cell marker ,Biochemistry ,Drug Metabolism ,Medicine and Health Sciences ,Enzyme assays ,Colorimetric assays ,Organ Cultures ,Bioassays and physiological analysis ,MTT assay ,Multidisciplinary ,LGR5 ,Warburg effect ,Metformin ,Organoids ,Nucleic acids ,Oncology ,Medicine ,Biological Cultures ,Anatomy ,Research Article ,medicine.drug ,Science ,Research and Analysis Methods ,microRNA ,Genetics ,medicine ,Pharmacokinetics ,Non-coding RNA ,Colorectal Cancer ,Pharmacology ,Natural antisense transcripts ,Biology and life sciences ,business.industry ,Cancers and Neoplasms ,Cancer ,medicine.disease ,digestive system diseases ,Gene regulation ,Gastrointestinal Tract ,MicroRNAs ,Biochemical analysis ,Cancer cell ,Cancer research ,RNA ,business ,Digestive System - Abstract
Precision medicine might be the response to the recent questioning of the use of metformin as an anticancer drug in colorectal cancer (CRC). Thus, in order to establish properly its benefits, metformin application needs to be assayed on the different progression stages of CRC. In this way, intestinal organoids imply a more physiological tool, representing a new therapeutic opportunity for CRC personalized treatment to assay tumor stage-dependent drugs. The previously reported lipid metabolism-related axis, Acyl-CoA synthetases/ Stearoyl-CoA desaturase (ACSLs/SCD), stimulates colon cancer progression and metformin is able to rescue the invasive and migratory phenotype conferred to cancer cells upon this axis overexpression. Therefore, we checked ACSL/SCD axis status, its regulatory miRNAs and the effect of metformin treatment in intestinal organoids with the most common acquired mutations in a sporadic CRC (CRC-like organoids) as a model for specific and personalized treatment. Despite ACSL4 expression is upregulated progressively in CRC-like organoids, metformin is able to downregulate its expression, especially in the first two stages (I, II). Besides, organoids are clearly more sensitive in the first stage (Apc mutated) to metformin than current chemotherapeutic drugs such as fluorouracil (5-FU). Metformin performs an independent “Warburg effect” blockade to cancer progression and is able to reduce crypt stem cell markers expression such as LGR5+. These results suggest a putative increased efficiency of the use of metformin in early stages of CRC than in advanced disease.
- Published
- 2018
116. Differential induction of interferon stimulated genes between type I and type III interferons is independent of interferon receptor abundance
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Megan L. Stanifer, Steeve Boulant, John McLauchlan, K. Christopher Garcia, Thomas Höfer, Connor G. G. Bamford, Kalliopi Pervolaraki, Soheil Rastgou Talemi, Felix Bormann, Dorothee Albrecht, and Juan L. Mendoza
- Subjects
0301 basic medicine ,RNA viruses ,Gene Expression ,Transcript profiling ,Pathology and Laboratory Medicine ,Biochemistry ,Interferon Lambda ,0302 clinical medicine ,Interferon ,Gene expression ,Medicine and Health Sciences ,Biology (General) ,Intestinal Mucosa ,Organ Cultures ,Receptor ,Receptors, Interferon ,Interferon receptor ,biology ,Chemistry ,3. Good health ,Cell biology ,Enzymes ,Organoids ,Vesicular stomatitis virus ,Vesicular Stomatitis Virus ,Medical Microbiology ,030220 oncology & carcinogenesis ,Viral Pathogens ,Interferon Type I ,Viruses ,Biological Cultures ,Signal transduction ,Anatomy ,Pathogens ,Oxidoreductases ,Luciferase ,medicine.drug ,Signal Transduction ,Research Article ,QH301-705.5 ,Colon ,Immunology ,Research and Analysis Methods ,Antiviral Agents ,Microbiology ,Rhabdoviruses ,Cell Line ,03 medical and health sciences ,Virology ,Extracellular ,medicine ,Genetics ,Humans ,Molecular Biology ,Gene ,Microbial Pathogens ,Cell specific ,Interleukins ,Organisms ,Biology and Life Sciences ,Proteins ,Epithelial Cells ,RC581-607 ,biology.organism_classification ,Viral Replication ,Gastrointestinal Tract ,030104 developmental biology ,Viral replication ,Cell culture ,Enzymology ,Parasitology ,Interferons ,Immunologic diseases. Allergy ,Digestive System - Abstract
It is currently believed that type I and III interferons (IFNs) have redundant functions. However, the preferential distribution of type III IFN receptor on epithelial cells suggests functional differences at epithelial surfaces. Here, using human intestinal epithelial cells we could show that although both type I and type III IFNs confer an antiviral state to the cells, they do so with distinct kinetics. Type I IFN signaling is characterized by an acute strong induction of interferon stimulated genes (ISGs) and confers fast antiviral protection. On the contrary, the slow acting type III IFN mediated antiviral protection is characterized by a weaker induction of ISGs in a delayed manner compared to type I IFN. Moreover, while transcript profiling revealed that both IFNs induced a similar set of ISGs, their temporal expression strictly depended on the IFNs, thereby leading to unique antiviral environments. Using a combination of data-driven mathematical modeling and experimental validation, we addressed the molecular reason for this differential kinetic of ISG expression. We could demonstrate that these kinetic differences are intrinsic to each signaling pathway and not due to different expression levels of the corresponding IFN receptors. We report that type III IFN is specifically tailored to act in specific cell types not only due to the restriction of its receptor but also by providing target cells with a distinct antiviral environment compared to type I IFN. We propose that this specific environment is key at surfaces that are often challenged with the extracellular environment., Author summary The human intestinal tract plays two important roles in the body: first it is responsible for nutrient absorption and second it is the primary barrier which protects the human body from the outside environment. This complex tissue is constantly exposed to commensal bacteria and is often exposed to both bacterial and viral pathogens. To protect itself, the gut produces, among others, secreted agents called interferons which help to fight against pathogen attacks. There are several varieties (type I, II, and III) of interferons and our work aims at understanding how type I and III interferon act to protect human intestinal epithelial cells (hIECs) during viral infection. In this study, we confirmed that both interferons can protect hIECs against viral infection but with different kinetics. We determined that type I confer an antiviral state to hIECs faster than type III interferons. We uncovered that these differences were intrinsic to each pathway and not the result of differential abundance of the respective interferon receptors. The results of this study suggest that type III interferon may provide a different antiviral environment to the epithelium target cells which is likely critical for maintaining gut homeostasis. Our findings will also help us to design therapies to aid in controlling and eliminating viral infections of the gut.
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- 2018
117. OrganoidTracker: Efficient cell tracking using machine learning and manual error correction
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Laetitia Hebert, Guizela Huelsz-Prince, Katarzyna Bozek, Sander J. Tans, Yvonne J. Goos, Xuan Zheng, Jeroen S. van Zon, Greg J. Stephens, Rutger N.U. Kok, Physics of Living Systems, and LaserLaB - Molecular Biophysics
- Subjects
0301 basic medicine ,Computer science ,Tracking (particle physics) ,computer.software_genre ,Convolutional neural network ,Machine Learning ,Automation ,Cognition ,Learning and Memory ,0302 clinical medicine ,Software ,Microscopy ,Medicine and Health Sciences ,Cell Cycle and Cell Division ,Organ Cultures ,Multidisciplinary ,Artificial neural network ,Software Engineering ,Cell Differentiation ,Division (mathematics) ,Organoids ,Cell Tracking ,Cell Processes ,Memory Recall ,Medicine ,Engineering and Technology ,Biological Cultures ,Anatomy ,Algorithms ,Research Article ,Computer and Information Sciences ,Neural Networks ,Imaging Techniques ,Science ,Research and Analysis Methods ,Machine learning ,Computer Software ,03 medical and health sciences ,Memory ,Fluorescence Imaging ,Organoid ,Humans ,business.industry ,Volume (computing) ,Biology and Life Sciences ,Cell Biology ,Gastrointestinal Tract ,030104 developmental biology ,Cognitive Science ,Neural Networks, Computer ,Artificial intelligence ,business ,Error detection and correction ,Digestive System ,computer ,030217 neurology & neurosurgery ,Neuroscience ,Developmental Biology - Abstract
Time-lapse microscopy is routinely used to follow cells within organoids, allowing direct study of division and differentiation patterns. There is an increasing interest in cell tracking in organoids, which makes it possible to study their growth and homeostasis at the single-cell level. As tracking these cells by hand is prohibitively time consuming, automation using a computer program is required. Unfortunately, organoids have a high cell density and fast cell movement, which makes automated cell tracking difficult. In this work, a semi-automated cell tracker has been developed. To detect the nuclei, we use a machine learning approach based on a convolutional neural network. To form cell trajectories, we link detections at different time points together using a min-cost flow solver. The tracker raises warnings for situations with likely errors. Rapid changes in nucleus volume and position are reported for manual review, as well as cases where nuclei divide, appear and disappear. When the warning system is adjusted such that virtually error-free lineage trees can be obtained, still less than 2% of all detected nuclei positions are marked for manual analysis. This provides an enormous speed boost over manual cell tracking, while still providing tracking data of the same quality as manual tracking.
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- 2020
118. Herpes simplex virus type 1 infection leads to neurodevelopmental disorder-associated neuropathological changes
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Haowen Qiao, Jia Shang, Bin Li, Pu Chen, Nian Liu, Ying Zhou, Moujian Guo, Ying Wu, Zhenyan Wang, and Wen Zhao
- Subjects
Organogenesis ,Herpesvirus 1, Human ,Pathology and Laboratory Medicine ,medicine.disease_cause ,Biochemistry ,Nestin ,Medical Conditions ,Neurodevelopmental disorder ,Neural Stem Cells ,Animal Cells ,Medicine and Health Sciences ,Medicine ,Organ Cultures ,Biology (General) ,Induced pluripotent stem cell ,Neurons ,0303 health sciences ,030302 biochemistry & molecular biology ,Neurogenesis ,Brain ,Cell Differentiation ,Neural stem cell ,Organoids ,Neuroepithelial cell ,Neurology ,Medical Microbiology ,Viral Pathogens ,Viruses ,Herpes Simplex Virus-1 ,Biological Cultures ,Pathogens ,Cellular Types ,Neuronal Differentiation ,Research Article ,Cerebral organoid ,Herpesviruses ,QH301-705.5 ,Induced Pluripotent Stem Cells ,Immunology ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Developmental Neuroscience ,Virology ,Genetics ,Organoid ,Humans ,Microbial Pathogens ,Molecular Biology ,030304 developmental biology ,business.industry ,Organisms ,Brain Development ,Biology and Life Sciences ,Proteins ,Herpes Simplex ,Cell Biology ,RC581-607 ,medicine.disease ,Herpes Simplex Virus ,Cytoskeletal Proteins ,Herpes simplex virus ,Neurodevelopmental Disorders ,Cellular Neuroscience ,Parasitology ,Immunologic diseases. Allergy ,DNA viruses ,business ,Organism Development ,Neuroscience ,Developmental Biology - Abstract
Neonatal herpes simplex virus type 1 (HSV-1) infections contribute to various neurodevelopmental disabilities and the subsequent long-term neurological sequelae into the adulthood. However, further understanding of fetal brain development and the potential neuropathological effects of the HSV-1 infection are hampered by the limitations of existing neurodevelopmental models due to the dramatic differences between humans and other mammalians. Here we generated in vitro neurodevelopmental disorder models including human induced pluripotent stem cell (hiPSC)-based monolayer neuronal differentiation, three-dimensional (3D) neuroepithelial bud, and 3D cerebral organoid to study fetal brain development and the potential neuropathological effects induced by the HSV-1 infections. Our results revealed that the HSV-1-infected neural stem cells (NSCs) exhibited impaired neural differentiation. HSV-1 infection led to dysregulated neurogenesis in the fetal neurodevelopment. The HSV-1-infected brain organoids modelled the pathological features of the neurodevelopmental disorders in the human fetal brain, including the impaired neuronal differentiation, and the dysregulated cortical layer and brain regionalization. Furthermore, the 3D cerebral organoid model showed that HSV-1 infection promoted the abnormal microglial activation, accompanied by the induction of inflammatory factors, such as TNF-α, IL-6, IL-10, and IL-4. Overall, our in vitro neurodevelopmental disorder models reconstituted the neuropathological features associated with HSV-1 infection in human fetal brain development, providing the causal relationships that link HSV biology with the neurodevelopmental disorder pathogen hypothesis., Author summary HSV-1 is one of the most prevalent human pathogens that can spread into the fetal central nervous system by maternal-fetal transmission, and thus resulting in long-term neurological sequelae in adult, including cognitive dysfunction and learning disabilities. However, there is a very limited progress in understanding the role of HSV-1 on human fetal brain development due to limited access to fetal human brain tissue as well as the limitations of existing neurodevelopmental and infection models. Here, we generated the in vitro neurodevelopmental disorder models including hiPSC-based monolayer neuronal differentiation, three-dimensional (3D) neuroepithelial bud, and 3D cerebral organoid to study the neurodevelopmental disorder-associated neuropathological changes with HSV-1 infection in human fetal brain development. Our results revealed that HSV-1 infection led to impaired neural differentiation and dysregulated neurogenesis in the fetal neurodevelopment. Additionally, HSV-1 infection impaired neuronal differentiation and dysregulated brain regionalization in our cerebral organoid model. Furthermore, the cerebral organoid model showed that HSV-1 infection led to the abnormal microglial proliferation and activation, accompanied by the induction of inflammatory factors including TNF-α, IL-6, IL-10, and IL-4. Taken together, our study provides novel evidence that HSV-1 infection impaired human brain development and contributed to neurodevelopmental disorder pathogen hypothesis, and would have implications for raising the therapeutic opportunities for targeting of viral reservoirs relevant to neurodevelopmental disorder.
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- 2020
119. Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes
- Author
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Manti Guha, Prasanna M. Chandramouleeswaran, Andres J. Klein-Szanto, James Garifallou, Clementina Mesaros, Hiroshi Nakagawa, Renata Pellegrino da Silva, Ian A. Blair, Noah Engel, Eishi Noguchi, Joseph A. Baur, Masataka Shimonosono, Hakon Hakonarson, Uma M. Sachdeva, Gordon Ruthel, Michael Gonzalez, Shinya Ohashi, Hisatsugu Maekawa, Kelly A. Whelan, and Sarmistha Mukherjee
- Subjects
Keratinocytes ,Male ,0301 basic medicine ,Mitochondrion ,medicine.disease_cause ,Biochemistry ,Epithelium ,Mice ,Spectrum Analysis Techniques ,0302 clinical medicine ,AMP-Activated Protein Kinase Kinases ,AMP-activated protein kinase ,Animal Cells ,Superoxides ,Medicine and Health Sciences ,Organ Cultures ,Energy-Producing Organelles ,Cells, Cultured ,Membrane Potential, Mitochondrial ,Alcohol Consumption ,Multidisciplinary ,Cell Death ,biology ,Chemistry ,TOR Serine-Threonine Kinases ,Oxides ,Flow Cytometry ,Cytoprotection ,Mitochondria ,Cell biology ,Organoids ,Cell Processes ,Spectrophotometry ,030220 oncology & carcinogenesis ,Physical Sciences ,Medicine ,Female ,Biological Cultures ,Cytophotometry ,Cellular Structures and Organelles ,Cellular Types ,Anatomy ,Microtubule-Associated Proteins ,Research Article ,Signal Transduction ,Science ,Autophagic Cell Death ,Bioenergetics ,Research and Analysis Methods ,Cell Line ,03 medical and health sciences ,Esophagus ,Autophagy ,medicine ,Animals ,Viability assay ,Mechanistic target of rapamycin ,Nutrition ,Ethanol ,Chemical Compounds ,Biology and Life Sciences ,AMPK ,Epithelial Cells ,Cell Biology ,Diet ,Mice, Inbred C57BL ,Repressor Proteins ,Oxidative Stress ,Biological Tissue ,030104 developmental biology ,biology.protein ,Protein Kinases ,Oxidative stress - Abstract
During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.
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- 2020
120. Outgrowth of erlotinib-resistant subpopulations recapitulated in patient-derived lung tumor spheroids and organoids
- Author
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Masahiro Inoue, Karen L. McKim, Malathi Banda, Meagan B. Myers, and Barbara L. Parsons
- Subjects
Male ,0301 basic medicine ,Cellular pathology ,Lung Neoplasms ,Molecular biology ,Mutant ,Cancer Treatment ,Apoptosis ,medicine.disease_cause ,Lung and Intrathoracic Tumors ,0302 clinical medicine ,Medicine and Health Sciences ,Tumor Cells, Cultured ,Epidermal growth factor receptor ,Organ Cultures ,Multidisciplinary ,Middle Aged ,Organoids ,Oncology ,030220 oncology & carcinogenesis ,Medicine ,Adenocarcinoma ,Female ,Biological Cultures ,KRAS ,Erlotinib ,Research Article ,medicine.drug ,Science ,Antineoplastic Agents ,Biology ,Research and Analysis Methods ,Biomolecular isolation ,Erlotinib Hydrochloride ,03 medical and health sciences ,Malignant Tumors ,Spheroids, Cellular ,Biomarkers, Tumor ,medicine ,Humans ,Lung cancer ,Aged ,Cell Proliferation ,Colorectal Cancer ,Biology and life sciences ,Carcinoma ,Spheroid ,Cancers and Neoplasms ,medicine.disease ,DNA isolation ,Molecular biology techniques ,030104 developmental biology ,Drug Resistance, Neoplasm ,Mutation ,Cancer research ,biology.protein - Abstract
A model that recapitulates development of acquired therapeutic resistance is needed to improve oncology drug development and patient outcomes. To achieve this end, we established methods for the preparation and growth of spheroids from primary human lung adenocarcinomas, including methods to culture, passage, monitor growth, and evaluate changes in mutational profile over time. Primary lung tumor spheroids were cultured in Matrigel® with varying concentrations of erlotinib, a small molecule kinase inhibitor of epidermal growth factor receptor (EGFR) that is ineffective against KRAS mutant cells. Subtle changes in spheroid size and number were observed within the first two weeks of culture. Spheroids were cultured for up to 24 weeks, during which time interactions between different cell types, movement, and assembly into heterogeneous organoid structures were documented. Allele-specific competitive blocker PCR (ACB-PCR) was used to quantify low frequency BRAF V600E, KRAS G12D, KRAS G12V, and PIK3CA H1047R mutant subpopulations in tumor tissue residue (TR) samples and cultured spheroids. Mutant subpopulations, including multiple mutant subpopulations, were quite prevalent. Twelve examples of mutant enrichment were found in eight of the 14 tumors analyzed, based on the criteria that a statistically-significant increase in mutant fraction was observed relative to both the TR and the no-erlotinib control. Of the mutants quantified in erlotinib-treated cultures, PIK3CA H1047 mutant subpopulations increased most often (5/14 tumors), which is consistent with clinical observations. Thus, this ex vivo lung tumor spheroid model replicates the cellular and mutational tumor heterogeneity of human lung adenocarcinomas and can be used to assess the outgrowth of mutant subpopulations. Spheroid cultures with characterized mutant subpopulations could be used to investigate the efficacy of lung cancer combination therapies.
- Published
- 2020
121. Room temperature shipment does not affect the biological activity of pluripotent stem cell-derived retinal organoids
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Lewin, Alfred S, Georgiou, Maria, Chichagova, Valeria, Hilgen, Gerrit, Dorgau, Birthe, Sernagor, Evelyne, Armstrong, Lyle, and Lako, Majlinda
- Subjects
Photoreceptors ,Retinal Ganglion Cells ,0301 basic medicine ,Atmospheric Science ,Sensory Receptors ,Light ,Cellular differentiation ,Cell ,Drug Evaluation, Preclinical ,Social Sciences ,Apoptosis ,chemistry.chemical_compound ,0302 clinical medicine ,Animal Cells ,Psychology ,Organ Cultures ,Induced pluripotent stem cell ,Neurons ,Staining ,Multidisciplinary ,Cell Death ,C100 ,Temperature ,Cell Differentiation ,Biological activity ,C500 ,C700 ,Cell biology ,Organoids ,Neuroepithelial cell ,Chemistry ,medicine.anatomical_structure ,Cell Processes ,Physical Sciences ,Medicine ,Sensory Perception ,Biological Cultures ,Cellular Types ,Cellular Structures and Organelles ,Research Article ,Signal Transduction ,Ganglion Cells ,Science ,Induced Pluripotent Stem Cells ,Biology ,Research and Analysis Methods ,Retina ,Cell Line ,Greenhouse Gases ,03 medical and health sciences ,Retinal Diseases ,medicine ,Organoid ,Humans ,Environmental Chemistry ,Postal Service ,Cilia ,Ecology and Environmental Sciences ,Chemical Compounds ,Biology and Life Sciences ,Afferent Neurons ,Retinal ,Cell Biology ,Carbon Dioxide ,C400 ,Nuclear Staining ,030104 developmental biology ,chemistry ,Specimen Preparation and Treatment ,Cell culture ,Cellular Neuroscience ,Atmospheric Chemistry ,Earth Sciences ,030221 ophthalmology & optometry ,Neuroscience - Abstract
The generation of laminated and light responsive retinal organoids from induced pluripotent stem cells (iPSCs) provides a powerful tool for the study of retinal diseases and drug discovery and a robust platform for cell-based therapies. The aim of this study is to investigate whether retinal organoids can retain their morphological and functional characteristics upon storage at room temperature (RT) conditions and shipment by air using a commercially available container that maintains the environment at ambient temperature. Morphological analysis and measurements of neuroepithelial thickness revealed no differences between control, RT incubated and shipped organoids. Similarly immunohistochemical analysis showed no differences in cell type composition and position within the laminated retinal structure. All groups showed a similar response to light, suggesting that the biological function of retinal organoids was not affected by RT storage or shipment. These findings provide an advance in transport of ready-made retinal organoids, increasing their availability to many research and pharma labs worldwide and facilitating cross-collaborative research.
- Published
- 2020
122. Folic acid supplementation alleviates reduced ureteric branching, nephrogenesis, and global DNA methylation induced by maternal nutrient restriction in rat embryonic kidney
- Author
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Mariko Hida and Midori Awazu
- Subjects
B Vitamins ,0301 basic medicine ,Methyltransferase ,Physiology ,Organogenesis ,Kidney development ,030204 cardiovascular system & hematology ,Kidney ,Biochemistry ,Rats, Sprague-Dawley ,chemistry.chemical_compound ,0302 clinical medicine ,Medicine and Health Sciences ,Choline ,Organ Cultures ,DNA methylation ,Multidisciplinary ,Organic Compounds ,Vitamins ,Chromatin ,Nucleic acids ,Chemistry ,medicine.anatomical_structure ,Physiological Parameters ,Physical Sciences ,Medicine ,Epigenetics ,Biological Cultures ,Anatomy ,DNA modification ,Chromatin modification ,Research Article ,Chromosome biology ,Cell biology ,Offspring ,Science ,Biology ,Research and Analysis Methods ,Kidney Development ,Andrology ,03 medical and health sciences ,Folic Acid ,Genetics ,medicine ,Animals ,Nutrition ,Methionine ,Biology and life sciences ,urogenital system ,Organic Chemistry ,Body Weight ,Chemical Compounds ,Kidneys ,DNA ,Renal System ,Nephrons ,Nutrients ,Embryo, Mammalian ,Rats ,030104 developmental biology ,chemistry ,DNMT1 ,Gene expression ,Ureter ,Food Deprivation ,Organism Development ,Developmental Biology - Abstract
We previously reported that maternal nutrient restriction (NR) inhibited ureteric branching, metanephric growth, and nephrogenesis in the rat. Here we examined whether folic acid, a methyl-group donor, rescues the inhibition of kidney development induced by NR and whether DNA methylation is involved in it. The offspring of dams given food ad libitum (CON) and those subjected to 50% food restriction (NR) were examined. NR significantly reduced ureteric tip number at embryonic day 14, which was attenuated by folic acid supplementation to nutrient restricted dams. At embryonic day 18, glomerular number, kidney weight, and global DNA methylation were reduced by NR, and maternal folic acid supplementation again alleviated them. Among DNA methyltransferases (DNMTs), DNMT1 was strongly expressed at embryonic day 15 in CON but was reduced in NR. In organ culture, an inhibitor of DNA methylation 5-aza-2 '-deoxycytidine as well as medium lacking methyl donors folic acid, choline, and methionine, significantly decreased ureteric tip number and kidney size mimicking the effect of NR. In conclusion, global DNA methylation is necessary for normal kidney development. Folic acid supplementation to nutrient restricted dams alleviated the impaired kidney development and DNA methylation in the offspring.
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- 2020
123. Gastrointestinal organoid technology advances studies of enteric virus biology
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Kolawole, Abimbola O. and Wobus, Christiane E.
- Subjects
Viral Diseases ,Cellular differentiation ,Cell Culture Techniques ,Enteroendocrine cell ,Pearls ,Epithelium ,Medicine and Health Sciences ,Astrovirus Infection ,Gastrointestinal Infections ,Organ Cultures ,Biology (General) ,Induced pluripotent stem cell ,Immune Response ,Enterovirus ,Microfold cell ,0303 health sciences ,030302 biochemistry & molecular biology ,Cell Differentiation ,Intestinal epithelium ,3. Good health ,Cell biology ,Organoids ,Infectious Diseases ,Biological Cultures ,Anatomy ,Stem cell ,Cell type ,QH301-705.5 ,Immunology ,Gastroenterology and Hepatology ,Biology ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Virology ,Enterovirus Infections ,Genetics ,Humans ,Progenitor cell ,Molecular Biology ,Rotavirus Infection ,030304 developmental biology ,Biology and Life Sciences ,RC581-607 ,Immunity, Innate ,Gastrointestinal Tract ,Biological Tissue ,Parasitology ,Immunologic diseases. Allergy ,Digestive System ,Developmental Biology - Abstract
The historical lack of models recapitulating the complexity of the human intestinal epithelium has hindered studies into many aspects of human enteric virus biology. Immortalized and transformed cell lines are typically limited by the presence of only one cell type, whereas susceptibility in animal models often requires infection routes that differ from humans or necessitates modification of immune system components [1, 2]. In addition, comparing animals from different species remains a confounding factor when trying to infer how findings may apply to human health. Thus, the development of human gastrointestinal organoids to study virus–host interactions marks a significant advance. They provide a physiologically relevant ex vivo platform in which human enteric microorganisms can be studied interacting with the human intestinal epithelium. Furthermore, their intermediate complexity, falling between cell lines and animal models, adds an additional tool to better understand these viruses. Here, we highlight how gastrointestinal organoids have provided new insights into the biology of human enteric viruses and the potential of this technology for advancing the field. The different flavors of gastrointestinal organoids Gastrointestinal organoids are three-dimensional (3D) structures derived from primary tissues (i.e., patient biopsy) containing intestinal stem/progenitor cells or from human pluripotent stem cells (hPSCs) [3]. They contain multiple intestinal epithelial cell types that perform critical functions that are also observed in the human intestine (e.g., absorption, barrier function, differentiation). Induced pluripotent stem cell–derived human intestinal organoids (HIOs) most closely resemble the human fetal intestine [4] and may encompass an epithelium alone [5] or also contain a mesenchyme [6]. However, most infectious disease laboratories work with human intestinal enteroids (HIEs), patient-derived, 3D epithelium-only structures that can be transitioned to a 2D monolayer on plates or transwells. HIEs maintain the physiological and genetic characteristics of their sources for long periods [7–10]. They can be differentiated from the crypt-like state into villus-like state by withdrawing growth factors required to maintain stem cells (i.e., WNT3A) from the culture media [11–13]. Cellular differentiation of specific cell lineages can be further achieved by pharmacologic or genetic means. For example, secretory cells are enriched following dibenzazepine (DBZ) treatment to block NOTCH signaling [12] and enteroendocrine cells by overexpression of NEUROGENIN-3 [14]. In addition, receptor activator of NF-κB ligand (RANKL) treatment of HIE with and without tumor necrosis factor alpha (TNF-α) addition has been used to drive microfold cell development [15, 16]. However, HIE and HIO do not completely mimic the intestinal epithelium in vivo. For example, they do not entirely reproduce the cellularity observed in vivo as few Paneth cells and no Tuft cells are present [17]. Furthermore, the lack of villi and genetic or pharmacologic skewing of differentiation may also affect the proportionality of individual cell lineages.
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- 2020
124. Modeling the effect of prolonged ethanol exposure on global gene expression and chromatin accessibility in normal 3D colon organoids
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Matthew Devall, Steven M. Powell, Graham Casey, Lucas T. Jennelle, Jennifer Bryant, Stephanie A. Bien, and Ulrike Peters
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Male ,0301 basic medicine ,Molecular biology ,Colorectal cancer ,Gene Expression ,Transcriptome ,0302 clinical medicine ,Gene expression ,Medicine and Health Sciences ,Organ Cultures ,Cells, Cultured ,Multidisciplinary ,Organic Compounds ,Chromosome Biology ,Genomics ,Middle Aged ,Chromatin ,3. Good health ,Organoids ,Chemistry ,RNA isolation ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,Physical Sciences ,Medicine ,Epigenetics ,Biological Cultures ,RNA extraction ,Anatomy ,Research Article ,Adult ,Colon ,Science ,Biology ,Research and Analysis Methods ,Biomolecular isolation ,03 medical and health sciences ,Cell Line, Tumor ,Genetics ,medicine ,Organoid ,Humans ,Colorectal Cancer ,Ethanol ,Organic Chemistry ,Chemical Compounds ,Biology and Life Sciences ,Cancers and Neoplasms ,Computational Biology ,Cell Biology ,Genome Analysis ,Genomic Libraries ,Chromatin Assembly and Disassembly ,medicine.disease ,Epithelium ,Gastrointestinal Tract ,Molecular biology techniques ,030104 developmental biology ,Cell culture ,Alcohols ,Cancer research ,Digestive System - Abstract
In this study we aimed to explore the potential biological effect of ethanol exposure on healthy colon epithelial cells using normal human colon 3D organoid "mini-gut" cultures. In numerous published studies ethanol use has been shown to be an environmental risk factor for colorectal cancer (CRC) development; however, the influence of ethanol exposure on normal colon epithelial cell biology remains poorly understood. We investigated the potential molecular effects of ethanol exposure in normal colon 3D organoids in a small pilot study (n = 3) using RNA-seq and ATAC-seq. We identify 1965 differentially expressed genes and 2217 differentially accessible regions of chromatin in response to ethanol treatment. Further, by cross-referencing our results with previously published analysis in colorectal cancer cell lines, we have not only validated a number of reported differentially expressed genes, but also identified several novel candidates for future investigation. In summary, our data highlights the potential importance for the use of normal colon 3D organoid models as a novel tool for the investigation of the relationship between the effects of environmental risk factors associated with colorectal cancer and the molecular mechanisms through which they confer this risk.
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- 2020
125. Surrogate R-spondins for tissue-specific potentiation of Wnt Signaling
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Yi Miao, K. Christopher Garcia, Xingnan Li, Calvin Kuo, Vincent C. Luca, and Michael J. Hollander
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0301 basic medicine ,medicine.medical_treatment ,Cell ,Yeast and Fungal Models ,0302 clinical medicine ,Cell Signaling ,Medicine and Health Sciences ,Organ Cultures ,Receptor ,Wnt Signaling Pathway ,WNT Signaling Cascade ,Tissue homeostasis ,0303 health sciences ,Multidisciplinary ,Chemistry ,Wnt signaling pathway ,Eukaryota ,Signaling Cascades ,3. Good health ,Cell biology ,Organoids ,Cytokine ,medicine.anatomical_structure ,Experimental Organism Systems ,Organ Specificity ,Medicine ,Engineering and Technology ,Saccharomyces Cerevisiae ,Biological Cultures ,Anatomy ,Stem cell ,Research Article ,Signal Transduction ,Cell signaling ,Cell type ,Colon ,Science ,Ubiquitin-Protein Ligases ,Research and Analysis Methods ,Saccharomyces ,03 medical and health sciences ,Organ Culture Techniques ,Model Organisms ,medicine ,Humans ,030304 developmental biology ,Binding Sites ,HEK 293 cells ,Interleukin-2 Receptor alpha Subunit ,Organisms ,Fungi ,Biology and Life Sciences ,Cell Biology ,Yeast ,Gastrointestinal Tract ,HEK293 Cells ,030104 developmental biology ,Signal Processing ,Animal Studies ,Thrombospondins ,Digestive System ,Function (biology) ,030217 neurology & neurosurgery ,Single-Chain Antibodies - Abstract
Secreted R-spondin1-4 proteins (RSPO1-4) orchestrate stem cell renewal and tissue homeostasis by potentiating Wnt/β-catenin signaling. RSPOs induce the turnover of negative Wnt regulators RNF43 and ZNRF3 through a process that requires RSPO interactions with Leucine-rich repeat-containing G-protein coupled receptors (LGRs), or through an LGR-independent mechanism that is enhanced by RSPO binding to heparin sulfate proteoglycans (HSPGs). Here, we describe the engineering of 'surrogate RSPOs' that function independently of LGRs to potentiate Wnt signaling on cell types expressing a target surface marker. These bispecific proteins were generated by fusing an RNF43- or ZNRF3-specific single chain antibody variable fragment (scFv) to the immune cytokine IL-2. Surrogate RSPOs mimic the function of natural RSPOs by crosslinking the extracellular domain (ECD) of RNF43 or ZNRF3 to the ECD of the IL-2 receptor CD25, which sequesters the complex and results in highly selective amplification of Wnt signaling on CD25+ cells. Furthermore, surrogate RSPOs were able substitute for wild type RSPO in a colon organoid growth assay when intestinal stem cells were transduced to express CD25. Our results provide proof-of-concept for a technology that may be adapted for use on a broad range of cell- or tissue-types and will open new avenues for the development of Wnt-based therapeutics for regenerative medicine.
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- 2020
126. Activation of metastatic potential in african green monkey kidney cell lines by prolonged in vitro culture.
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Contreras, G., Bather, R., and Furesz, J.
- Abstract
Studies on the tumorigenicity of Vero kidney cells of Cercopithecus aethiops monkey origin were extended to various passage levels of BSC-1 aneuploid cells and to low passage CV-1 diploid cells (derived also from C. aethiops monkey kidney). It was found that BSC-1 cells-like Vero cells-showed increased tumorigenicity with increasing passage level in antitymocyte globulins (ATG) treated newborn rats and in nude mice. Cells passaged over 250 times in cultures formed invasive adenocarcinomas in newborn rats. Their malignant tumor growth was further demonstrated around the 500 passage level when tumor metastases were detected in the lungs of four of the 14 inoculated rats. Vero cells induced such lung metastases in rats already at passage 227. CV-1 diploid cells at low passage level produced small nodules of epithelioid cells in newborn rats at 6th day after inoculation that had disappeared by the 21st day, and caused no local invasion nor lung metastasis. In vitro tumorigenicity tests on BSC-1 and CV-1 cells, using chick embryo skin, human muscle and colony formation in agarose, confirmed the animal test results. The results of this study indicate that BSC-1 and Vero cell lines at low and high passage levels may prove to be useful tools to study the molecular basis of malignancy. [ABSTRACT FROM AUTHOR]
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- 1985
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127. In vitro modulation of differentiation by calcium in organ cultures of human and murine epithelial tissue.
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Sacks, P., Parnes, S., Price, J., Risemberg, H., Goldstein, J., Marko, M., and Parsons, D.
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The differentiation of epithelial tissue in organ cultures of murine buccal mucosa, various human oral mucosa, and human newborn foreskin was found to be dependent on the calcium concentration of the culture media. In low calcium medium (≤0.07 m M) epithelial differentiation was inhibited. The original stratifying layers separate and can be removed, producing a destratified explant. Histologically such an explant consits of a dorsal epithelial layer of basal keratinocytes resting on an intact basal lamina with subjacent stroma. At 0.01 m M calcium, the epithelial layer was one to two cells thick whereas at 0.07 m M it could be three or more layers in thickness with the most superficial cells being spread over the underlying cells. In addition to differentiation, keratinocyte migration over the sides of the explant (epiboly) and epithelial proliferation as determined by [H]thymidine autoradiography were reduced by culture in low calcium medium. Redifferentiation occurs upon return to normal calcium levels (1.8 m M); addition of hydrocortisone to low calcium media was found to facilitate this redifferentiation. [ABSTRACT FROM AUTHOR]
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- 1985
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128. Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed to Mycoplasma pneumoniae cells or membranes.
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Gabridge, Michael and Polisky, Robert
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The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10 to 10 CFU of virulent Mycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 μg per ml of purified M. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures. [ABSTRACT FROM AUTHOR]
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- 1977
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129. Exposure of organ cultures from human tracheal epithelium to chemical carcinogens and subsequent long-term co-cultivation with autologous isotopic fibroblasts.
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Haas, I., Koldovsky, P., and Ganzer, U.
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As a continuation of previous experiments introducing an extracorporeal model for transformation of human respiratory epithelium that might be able to mimic a spontaneously occurring malignant tumor, we prepared organ cultures from tracheal specimens and exposed them repeatedly to chemical carcinogens, using benzo(a)pyrene and methylnitronitrosoguanine for 6 weeks. We then tried to select possibly initiated cells by subsequent co-cultivation with autologous isotopic fibroblasts for 2 years. Nontreated controls were maintained from the same specimens and cultured in the same manner. By this technique we selected from specimen La24 three long-living cell lines with varying morphology and an antigenic pattern indicating dedifferentiation. The cells expressed simultaneously a panel of cytokeratins, vimentin and neuroectodermal antigens. Transplantation of these cell lines under the subrenal capsule of athymic mice resulted in tumor-like nodules of limited size. Success rate was dependent on time of previous in vitro culture and carcinogen treatment. None of the lines produced invasive or metastasizing tumors. [ABSTRACT FROM AUTHOR]
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- 1996
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130. Tankyrases maintain homeostasis of intestinal epithelium by preventing cell death
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Pan Ye, Ye-Guang Chen, Zhen Qi, Yuan Liu, Shan Wang, Y. Jeffrey Chiang, Xintong Li, and Yehua Li
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0301 basic medicine ,Male ,Cancer Research ,Physiology ,Cell Culture Techniques ,Apoptosis ,Epithelium ,Receptors, G-Protein-Coupled ,Mice ,Poly ADP Ribosylation ,Cell Signaling ,Tankyrases ,Medicine and Health Sciences ,Homeostasis ,Organ Cultures ,Intestinal Mucosa ,Wnt Signaling Pathway ,Genetics (clinical) ,WNT Signaling Cascade ,Cells, Cultured ,Cell Death ,Microfilament Proteins ,Wnt signaling pathway ,LGR5 ,Intestinal epithelium ,Signaling Cascades ,Cell biology ,Organoids ,Adult Stem Cells ,medicine.anatomical_structure ,Cell Processes ,Small Intestine ,Female ,Biological Cultures ,Stem cell ,Anatomy ,Heterocyclic Compounds, 3-Ring ,Research Article ,Signal Transduction ,Programmed cell death ,lcsh:QH426-470 ,Colon ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Genetics ,medicine ,Animals ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Cell Proliferation ,Biology and Life Sciences ,Cell Biology ,Gastrointestinal Tract ,Mice, Inbred C57BL ,lcsh:Genetics ,030104 developmental biology ,Biological Tissue ,Physiological Processes ,Digestive System - Abstract
Lgr5+ intestinal stem cells are crucial for fast homeostatic renewal of intestinal epithelium and Wnt/β-catenin signaling plays an essential role in this process by sustaining stem cell self-renewal. The poly(ADP-ribose) polymerases tankyrases (TNKSs) mediate protein poly-ADP-ribosylation and are involved in multiple cellular processes such as Wnt signaling regulation, mitotic progression and telomere maintenance. However, little is known about the physiological function of TNKSs in epithelium homeostasis regulation. Here, using Villin-creERT2;Tnks1-/-;Tnks2fl/fl (DKO) mice, we observed that loss of TNKSs causes a rapid decrease of Lgr5+ intestinal stem cells and magnified apoptosis in small intestinal crypts, leading to intestine degeneration and increased mouse mortality. Consistently, deletion of Tnks or blockage of TNKS activity with the inhibitor XAV939 significantly inhibits the growth of intestinal organoids. We further showed that the Wnt signaling agonist CHIR99021 sustains the growth of DKO organoids, and XAV939 does not cause growth retardation of Apc-/- organoids. Consistent with the promoting function of TNKSs in Wnt signaling, Wnt/β-catenin signaling is significantly decreased with stabilized Axin in DKO crypts. Together, our findings unravel the essential role of TNKSs-mediated protein parsylation in small intestinal homeostasis by modulating Wnt/β-catenin signaling., Author summary Although tankyrases have been indicated to play important roles in telomere maintenance, mitosis and Wnt signaling regulation, little is known about their physiological function in intestinal epithelium. Using Villin-creERT2;Tnks1-/-;Tnks2fl/fl mice, which harbored conventional Tnks1 deletion and inducible intestinal epithelium-specific Tnks2 knockout, we show that tankyrases regulate adult intestinal Lgr5+ stem cells and epithelium homeostasis by preventing cell death and promoting cell proliferation.
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- 2018
131. Canonical PRC2 function is essential for mammary gland development and affects chromatin compaction in mammary organoids
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Michael J. G. Milevskiy, Bhupinder Pal, Caleb A. Dawson, Paul R. Jamieson, Ewa M. Michalak, Geoffrey J. Lindeman, Stuart H. Orkin, Johanna F. Dekkers, Yifang Hu, Gordon K. Smyth, Rachel Joyce, Warren S. Alexander, and Jane E. Visvader
- Subjects
0301 basic medicine ,Euchromatin ,Molecular biology ,Polycomb-Group Proteins ,Gene Expression ,Biochemistry ,Histones ,Mice ,Sequencing techniques ,Heterochromatin ,Breast Tumors ,Medicine and Health Sciences ,Histone code ,Organ Cultures ,Biology (General) ,biology ,Chromosome Biology ,General Neuroscience ,Polycomb Repressive Complex 2 ,RNA sequencing ,Animal Models ,Mammary Glands ,Chromatin ,Cell biology ,Histone Code ,Organoids ,Histone ,Experimental Organism Systems ,Oncology ,Female ,Epigenetics ,Biological Cultures ,Anatomy ,General Agricultural and Biological Sciences ,PRC2 ,Research Article ,QH301-705.5 ,Primary Cell Culture ,Mouse Models ,macromolecular substances ,Research and Analysis Methods ,Methylation ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Mammary Glands, Animal ,Exocrine Glands ,Model Organisms ,DNA-binding proteins ,Breast Cancer ,Organoid ,Polycomb-group proteins ,Genetics ,Animals ,Enhancer of Zeste Homolog 2 Protein ,General Immunology and Microbiology ,Lysine ,Reproductive System ,Biology and Life Sciences ,Proteins ,Cancers and Neoplasms ,Cell Biology ,030104 developmental biology ,Molecular biology techniques ,biology.protein ,Protein Processing, Post-Translational ,Breast Tissue - Abstract
Distinct transcriptional states are maintained through organization of chromatin, resulting from the sum of numerous repressive and active histone modifications, into tightly packaged heterochromatin versus more accessible euchromatin. Polycomb repressive complex 2 (PRC2) is the main mammalian complex responsible for histone 3 lysine 27 trimethylation (H3K27me3) and is integral to chromatin organization. Using in vitro and in vivo studies, we show that deletion of Suz12, a core component of all PRC2 complexes, results in loss of H3K27me3 and H3K27 dimethylation (H3K27me2), completely blocks normal mammary gland development, and profoundly curtails progenitor activity in 3D organoid cultures. Through the application of mammary organoids to bypass the severe phenotype associated with Suz12 loss in vivo, we have explored gene expression and chromatin structure in wild-type and Suz12-deleted basal-derived organoids. Analysis of organoids led to the identification of lineage-specific changes in gene expression and chromatin structure, inferring cell type–specific PRC2-mediated gene silencing of the chromatin state. These expression changes were accompanied by cell cycle arrest but not lineage infidelity. Together, these data indicate that canonical PRC2 function is essential for development of the mammary gland through the repression of alternate transcription programs and maintenance of chromatin states., Author summary The formation of mammary glands requires the tight regulation of many genes that govern cell fate decisions in the cells that form them. However, most of these genes remain undefined. The Polycomb repressive complex 2 (PRC2) has a role in gene silencing, and it is comprised of several subunits, which include either Enhancer of Zeste homolog 2 (Ezh2) or Ezh1 in combination with Suppressor of Zeste 12 protein homolog (Suz12) and embryonic ectoderm development (EED). Dysregulation of these subunits can lead to breast cancer. Although previous studies have analyzed the contribution of these complexes in mammary epithelium, failure to inactivate all canonical PRC2 complexes has made this task difficult. Deletion of Suz12 resulted in nonfunctional PRC2 and led to growth defects in mammary epithelial cells in vivo and in vitro. Here, we have used a 3D mammary organoid system to circumvent the lethality associated with Suz12 loss and studied chromatin dynamics and gene expression of PRC2 complexes. Our results suggest that loss of all canonical PRC2 complexes results in failure to repress transcriptional programs associated with early commitment and differentiation of mammary epithelial cells.
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- 2018
132. Loss of adenomatous polyposis coli function renders intestinal epithelial cells resistant to the cytokine IL-22
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Chen, Yu, Vandereyken, Maud, Newton, Ian P., Moraga, Ignacio, Näthke, Inke S., and Swamy, Mahima
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Male ,0301 basic medicine ,Carcinogenesis ,medicine.medical_treatment ,Gene Expression ,medicine.disease_cause ,Biochemistry ,Epithelium ,Interleukin 22 ,Mice ,0302 clinical medicine ,Animal Cells ,Cellular stress response ,Intestine, Small ,Gene expression ,Medicine and Health Sciences ,Organ Cultures ,Post-Translational Modification ,Phosphorylation ,Intestinal Mucosa ,Biology (General) ,STAT3 ,Staining ,0303 health sciences ,biology ,General Neuroscience ,Cell Staining ,Animal Models ,Intestinal epithelium ,3. Good health ,Organoids ,Intestines ,Cytokine ,Experimental Organism Systems ,Oncology ,Adenomatous Polyposis Coli ,030220 oncology & carcinogenesis ,Cytokines ,Female ,Biological Cultures ,Anatomy ,Cellular Types ,Colorectal Neoplasms ,General Agricultural and Biological Sciences ,Research Article ,Signal Transduction ,STAT3 Transcription Factor ,QH301-705.5 ,Adenomatous polyposis coli ,Mouse Models ,Research and Analysis Methods ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Model Organisms ,Downregulation and upregulation ,Genetics ,medicine ,Animals ,030304 developmental biology ,General Immunology and Microbiology ,Sequence Analysis, RNA ,Interleukins ,Biology and Life Sciences ,Proteins ,Epithelial Cells ,Cell Biology ,Gastrointestinal Tract ,Mice, Inbred C57BL ,Biological Tissue ,030104 developmental biology ,Specimen Preparation and Treatment ,Animal Studies ,biology.protein ,STAT protein ,Cancer research ,Wound healing ,Digestive System ,030217 neurology & neurosurgery - Abstract
Interleukin-22 (IL-22) is a critical immune defence cytokine that maintains intestinal homeostasis and promotes wound healing and tissue regeneration, which can support the growth of colorectal tumours. Mutations in the adenomatous polyposis coli gene (Apc) are a major driver of familial colorectal cancers (CRCs). How IL-22 contributes to APC-mediated tumorigenesis is poorly understood. To investigate IL-22 signalling in wild-type (WT) and APC-mutant cells, we performed RNA sequencing (RNAseq) of IL-22–treated murine small intestinal epithelial organoids. In WT epithelia, antimicrobial defence and cellular stress response pathways were most strongly induced by IL-22. Surprisingly, although IL-22 activates signal transducer and activator of transcription 3 (STAT3) in APC-mutant cells, STAT3 target genes were not induced. Our analyses revealed that ApcMin/Min cells are resistant to IL-22 due to reduced expression of the IL-22 receptor, and increased expression of inhibitors of STAT3, particularly histone deacetylases (HDACs). We further show that IL-22 increases DNA damage and genomic instability, which can accelerate cellular transition from heterozygosity (ApcMin/+) to homozygosity (ApcMin/Min) to drive tumour formation. Our data reveal an unexpected role for IL-22 in promoting early tumorigenesis while excluding a function for IL-22 in transformed epithelial cells., The adenomatous polyposis coli (APC) gene is mutated in 85% of colorectal cancers. This study shows that when APC is mutated in murine intestinal epithelial cells, they no longer respond to IL-22, a cytokine that is considered important for colorectal cancer progression; this has implications for IL-22 as a therapeutic target for cancer treatment.
- Published
- 2019
133. Ex vivo live cell tracking in kidney organoids using light sheet fluorescence microscopy
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Marie Held, Raphaël Lévy, Patricia Murray, Bettina Wilm, and Ilaria Santeramo
- Subjects
0301 basic medicine ,Fluorescence-lifetime imaging microscopy ,lcsh:Medicine ,Kidney ,Basement Membrane ,Mice ,Tissue culture ,Fluorescence Microscopy ,Medicine and Health Sciences ,Fluorescence microscope ,Organ Cultures ,lcsh:Science ,Cells, Cultured ,Microscopy ,Multidisciplinary ,Chemistry ,Physics ,Light Microscopy ,Extracellular Matrix ,Cell biology ,Organoids ,Physical Sciences ,Focal Planes ,Female ,Biological Cultures ,Single-Cell Analysis ,Anatomy ,Cellular Structures and Organelles ,Research Article ,Materials by Structure ,Imaging Techniques ,Amorphous Solids ,Materials Science ,Research and Analysis Methods ,03 medical and health sciences ,In vivo ,Fluorescence Imaging ,Organoid ,Animals ,lcsh:R ,Biology and Life Sciences ,Kidneys ,Optics ,Renal System ,Cell Biology ,Embryonic stem cell ,030104 developmental biology ,Microscopy, Fluorescence ,Mixtures ,Light sheet fluorescence microscopy ,lcsh:Q ,Gels ,Ex vivo - Abstract
Screening cells for their differentiation potential requires a combination of tissue culture models and imaging methods that allow for long-term tracking of the location and function of cells. Embryonic kidney re-aggregation in vitro assays have been established which allow for the monitoring of organotypic cell behaviour in re-aggregated and chimeric renal organoids. However, evaluation of cell integration is hampered by the high photonic load of standard fluorescence microscopy which poses challenges for imaging three-dimensional systems in real-time over a time course. Therefore, we employed light sheet microscopy, a technique that vastly reduces photobleaching and phototoxic effects. We have also developed a new method for culturing the re-aggregates which involves immersed culture, generating organoids which more closely reflect development in vivo. To facilitate imaging from various angles, we embedded the organoids in a freely rotatable hydrogel cylinder. Endpoint fixing and staining were performed to provide additional biomolecular information. We succeeded in imaging labelled cells within re-aggregated kidney organoids over 15 hours and tracking their fate while simultaneously monitoring the development of organotypic morphological structures. Our results show that Wt1-expressing embryonic kidney cells obtained from transgenic mice could integrate into re-aggregated chimeric kidney organoids and contribute to developing nephrons. Furthermore, the nascent proximal tubules that formed in the re-aggregated tissues using the new culture method displayed secretory function, as evidenced by their ability to secrete an organic anion mimic into the tubular lumen.
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- 2018
134. Generation of hepatocyte- and endocrine pancreatic-like cells from human induced endodermal progenitor cells
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Nicky Helsen, Philip Roelandt, Ana Rita Mestre Rosa, Paul de Vos, Eric Kalo, Satish Khurana, Manoj Kumar, Marijke M. Faas, Catherine M. Verfaillie, Veerle Vanslembrouck, Sumitava Dastidar, Conny Gysemans, Manmohan Bajaj, Jos Laureys, Renate Akkerman, Rangarajan Sambathkumar, Reproductive Origins of Adult Health and Disease (ROAHD), Translational Immunology Groningen (TRIGR), Man, Biomaterials and Microbes (MBM), Basic (bio-) Medical Sciences, Division of Gene Therapy & Regenerative Medicine, and Faculty of Medicine and Pharmacy
- Subjects
0301 basic medicine ,Embryology ,Cellular differentiation ,lcsh:Medicine ,Gene Expression ,Immunostaining ,Biochemistry ,VIVO ,Animal Cells ,Insulin-Secreting Cells ,SECRETING CELLS ,HUMAN FIBROBLASTS ,Medicine and Health Sciences ,EFFICIENT DIFFERENTIATION ,Cellular Reprogramming Techniques ,Organ Cultures ,Induced pluripotent stem cell ,lcsh:Science ,Staining ,Multidisciplinary ,Agricultural and Biological Sciences(all) ,Endoderm ,Cell Differentiation ,Cell biology ,Multidisciplinary Sciences ,Organoids ,Liver ,embryonic structures ,Science & Technology - Other Topics ,Biological Cultures ,Stem cell ,Cellular Types ,Anatomy ,Reprogramming ,Adult stem cell ,Research Article ,EXPRESSION ,Induced Pluripotent Stem Cells ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Albumins ,HUMAN LIVER ,Genetics ,Humans ,INSULIN-PRODUCING CELLS ,Progenitor cell ,Science & Technology ,Biochemistry, Genetics and Molecular Biology(all) ,lcsh:R ,Biology and Life Sciences ,Proteins ,Kidneys ,Cell Biology ,Renal System ,IN-VITRO ,Embryonic stem cell ,030104 developmental biology ,Specimen Preparation and Treatment ,Hepatocytes ,lcsh:Q ,DEFINED FACTORS ,EMBRYONIC STEM-CELLS ,Developmental Biology - Abstract
Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or β-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14 transcription factors (TFs) in reprogramming MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic-like cells. We demonstrated that 14 TF-iENDO cells can be expanded for at least 20 passages, differentiate spontaneously to hepatocyte-, endocrine pancreatic-, gut tube-like cells as well as endodermal tumor formation when grafted in immunodeficient mice. Furthermore, iENDO cells can be differentiated in vitro into hepatocyte- and endocrine pancreatic-like cells. However, the pluripotency TF OCT4, which is not silenced in iENDO cells, may contribute to the incomplete differentiation to mature cells in vitro and to endodermal tumor formation in vivo. Nevertheless, the studies presented here provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiate to multiple endodermal cell types, might be a valid alternative for the use of PSCs for creation of endodermal cell types. ispartof: PLOS ONE vol:13 issue:5 ispartof: location:United States status: published
- Published
- 2018
135. miR-122 inhibition in a human liver organoid model leads to liver inflammation, necrosis, steatofibrosis and dysregulated insulin signaling
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Marjan Mehrab-Mohseni, Anthony Atala, Hossein Sendi, Colin E. Bishop, Herbert L. Bonkovsky, Ivy Mead, Meimei Wan, and Kenneth L. Koch
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0301 basic medicine ,RNA viruses ,Liver cytology ,Physiology ,lcsh:Medicine ,Pathology and Laboratory Medicine ,Endocrinology ,Non-alcoholic Fatty Liver Disease ,Animal Cells ,MiR-122 ,Medicine and Health Sciences ,Insulin ,Organ Cultures ,lcsh:Science ,Immune Response ,Chemokine CCL2 ,Chemokine CCL3 ,Multidisciplinary ,Glucose Transporter Type 4 ,biology ,Chemistry ,Liver Diseases ,Fatty liver ,3. Good health ,Organoids ,Matrix Metalloproteinase 8 ,Liver ,Matrix Metalloproteinase 9 ,Medical Microbiology ,Viral Pathogens ,Viruses ,Liver Fibrosis ,Biological Cultures ,Cellular Types ,Anatomy ,Pathogens ,Signal Transduction ,Research Article ,Kupffer Cells ,Immunology ,Gastroenterology and Hepatology ,Research and Analysis Methods ,Microbiology ,Proinflammatory cytokine ,03 medical and health sciences ,Necrosis ,Signs and Symptoms ,Diagnostic Medicine ,Retroviruses ,Organoid ,medicine ,Humans ,Microbial Pathogens ,Inflammation ,Endocrine Physiology ,Interleukin-6 ,lcsh:R ,Lentivirus ,Insulin Signaling ,Organisms ,Biology and Life Sciences ,Cell Biology ,medicine.disease ,Fibrosis ,Fatty Liver ,Insulin receptor ,MicroRNAs ,030104 developmental biology ,Hepatic stellate cell ,Cancer research ,biology.protein ,Hepatocytes ,Insulin Receptor Substrate Proteins ,lcsh:Q ,Liver function ,Developmental Biology - Abstract
To investigate the role of miR-122 in the development and regression of non-alcoholic fatty liver disease (NAFLD) in vitro, we used multicellular 3D human liver organoids developed in our laboratory. These organoids consist of primary human hepatocytes, Kupffer cells, quiescent stellate cells and liver sinusoidal endothelial cells. They remain viable and functional for 4 weeks expressing typical markers of liver function such as synthesis of albumin, urea, and alpha-1 p450 drug metabolism. Before mixing, hepatic cells were transduced with lentivirus to inhibit miR122 expression (ABM, CA). Immediately after the organoids were fully formed (day 4) or after 1 or 2 weeks of additional incubation (days 11 or 18), the organoids were analyzed using fluorescent live/dead staining and ATP production; total RNA was extracted for qPCR gene expression profiling. Our results show that miR-122 inhibition in liver organoids leads to inflammation, necrosis, steatosis and fibrosis. This was associated with increase in inflammatory cytokines (IL6, TNF), chemokines (CCL2, CCL3) and increase in a subset of Matrix Metaloproteinases (MMP8, MMP9). An altered expression of key genes in lipid metabolism (i.e LPL, LDLR) and insulin signaling (i.e GLUT4, IRS1) was also identified. Conclusion: Our results highlight the role of miR-122 inhibition in liver inflammation, steatofibrosis and dysregulation of insulin signaling. Patients with NAFLD are known to have altered levels of miR-122, therefore we suggest that miR-122 mimics could play a useful role in reversing liver steatofibrosis and insulin resistance seen in patients with NAFLD.
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- 2018
136. A20 upregulation during treated HIV disease is associated with intestinal epithelial cell recovery and function
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Montha Pao, Averil Ma, Luis J. Montaner, Mohamed Abdel-Mohsen, Steven G. Deeks, Yenny Y. Rosli, Joseph M. McCune, Michael G. Kattah, Monika Deswal, Charles C. Kim, Ma Somsouk, Peter W. Hunt, Avantika S. Chitre, and Douek, Daniel C
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0301 basic medicine ,RNA viruses ,Male ,Physiology ,medicine.medical_treatment ,Biopsy ,Gene Expression ,HIV Infections ,Pathology and Laboratory Medicine ,Biochemistry ,Mice ,0302 clinical medicine ,Immunodeficiency Viruses ,Immune Physiology ,Antiretroviral Therapy, Highly Active ,Medicine and Health Sciences ,030212 general & internal medicine ,Biology (General) ,Organ Cultures ,Intestinal Mucosa ,Innate Immune System ,Middle Aged ,Viral Load ,3. Good health ,Organoids ,Intestines ,Cytokine ,Medical Microbiology ,Viral Pathogens ,Viruses ,Cytokines ,Infectious diseases ,Female ,Biological Cultures ,medicine.symptom ,Anatomy ,Pathogens ,Viral load ,Research Article ,Adult ,Programmed cell death ,QH301-705.5 ,Immunology ,Antiretroviral Therapy ,Viremia ,Inflammation ,Surgical and Invasive Medical Procedures ,Viral diseases ,Research and Analysis Methods ,Peripheral blood mononuclear cell ,Microbiology ,03 medical and health sciences ,Downregulation and upregulation ,Virology ,Retroviruses ,medicine ,Organoid ,Genetics ,Animals ,Humans ,Highly Active ,Molecular Biology ,Microbial Pathogens ,Tumor Necrosis Factor alpha-Induced Protein 3 ,business.industry ,Lentivirus ,Organisms ,Biology and Life Sciences ,HIV ,Proteins ,Epithelial Cells ,RC581-607 ,Molecular Development ,medicine.disease ,Gastrointestinal Tract ,Good Health and Well Being ,030104 developmental biology ,Immune System ,HIV-1 ,Parasitology ,Interferons ,Immunologic diseases. Allergy ,business ,Digestive System ,Developmental Biology - Abstract
Untreated Human Immunodeficiency Virus (HIV) infection is characterized by intestinal epithelial barrier dysfunction and chronic inflammation, related features that are attenuated to variable degrees by suppressive antiretroviral therapy (ART). Specific mediators of intestinal epithelial cell (IEC) dysfunction and restoration during HIV disease and treatment have yet to be identified. We studied IECs isolated from intestinal biopsies by RNAseq and found that mRNA levels for the ubiquitin-modifying enzyme, A20, are upregulated in ART-treated individuals and are positively correlated with markers of epithelial function (e.g., CTNNB, CLDN4, and TJP1). In a murine intestinal organoid model, A20 expression was suppressed by interferon-alpha (IFNα), which is highly expressed during HIV viremia and induces IFN-mediated signaling. Notably, A20 deletion rendered intestinal organoids more susceptible to cell death and inhibition of barrier-related genes mediated by interferon-gamma (IFNγ), a cytokine also present at elevated levels during untreated infection. Furthermore, A20 specifically restricted expression of IL-17A-induced inflammatory genes in organoids. Finally, ART-suppressed chronically infected individuals treated with pegylated IFNα2a for five weeks demonstrated reduced expression of A20 in peripheral blood mononuclear cells. Our results are thus consistent with a model in which enhanced type I interferons suppress A20 levels, leading to IFNγ-mediated dysfunction. As such, variation in A20 expression during the course of HIV infection could underlie both the development of epithelial dysfunction before the initiation of ART and the recovery of intestinal epithelial integrity thereafter. Trial registration ClinicalTrials.gov Clinical Trial NCT00594880, Author summary Though the advent of antiretroviral therapy (ART) has significantly improved the lives of individuals infected with the Human Immunodeficiency Virus (HIV), those on therapy still suffer from an enhanced risk of morbidities and mortalities that is caused, at least in part by, overactivation of the immune system. Disruption in the intestinal epithelial barrier, which when intact cordons off the immune system from components in the gut that can drive an inflammatory response, is thought to contribute significantly to this process. How HIV causes this disruption has not been identified. In this study, we analyzed the expression profile of intestinal epithelial cells from HIV-infected individuals, on and off therapy, compared to uninfected controls and observed an increase in expression of an anti-inflammatory regulator, A20, in treated participants but not in those without treatment. In a culture system that parallels features of the intact gut barrier, we saw that a cytokine induced by HIV suppressed the expression of A20. In addition, treatment of ART-suppressed HIV-infected individuals with this same cytokine led to reductions in A20 expression. Furthermore, experimental deletion of A20 in our culture system and treatment with HIV-associated cytokines led to changes consistent with a defective intestinal barrier. As such, we suggest A20 is central to epithelial dynamics during HIV disease and could protect the epithelium from damage associated with HIV-induced cytokine changes.
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- 2018
137. In vitro mouse spermatogenesis with an organ culture method in chemically defined medium
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Yuta Asayama, Tatsuma Yao, Noriaki Arakawa, Hiroyuki Sanjo, Takeru Abe, Takuya Sato, Hiroyuki Yamanaka, Masahiro Yao, Hisashi Hirano, Mitsuru Komeya, Takehiko Ogawa, Kumiko Katagiri, Yoko Ino, and Akio Matsuhisa
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0301 basic medicine ,Male ,Physiology ,Retinoic acid ,lcsh:Medicine ,Biochemistry ,chemistry.chemical_compound ,Follicle-stimulating hormone ,Mice ,Reproductive Physiology ,Animal Cells ,Spermatocytes ,Testis ,Medicine and Health Sciences ,Testosterone ,Cell Cycle and Cell Division ,Organ Cultures ,lcsh:Science ,Mice, Inbred ICR ,Multidisciplinary ,Chromosome Biology ,Organic Compounds ,Age Factors ,Serum Albumin, Bovine ,Lipids ,Spermatids ,Meiosis ,Chemistry ,medicine.anatomical_structure ,Cell Processes ,Physical Sciences ,Triiodothyronine ,Biological Cultures ,Cellular Types ,Luteinizing hormone ,Research Article ,Signal Transduction ,Mice, Transgenic ,Tretinoin ,Biology ,In Vitro Techniques ,Organ culture ,Research and Analysis Methods ,Andrology ,03 medical and health sciences ,Organ Culture Techniques ,Albumins ,medicine ,Animals ,Spermatogenesis ,Spermatid ,Ethanol ,lcsh:R ,Organic Chemistry ,Chemical Compounds ,Biology and Life Sciences ,Proteins ,Cell Biology ,Luteinizing Hormone ,Sperm ,Spermatogonia ,Culture Media ,Mice, Inbred C57BL ,Chemically defined medium ,030104 developmental biology ,Germ Cells ,chemistry ,Alcohols ,lcsh:Q ,Cattle ,Follicle Stimulating Hormone - Abstract
We previously reported the successful induction and completion of mouse spermatogenesis by culturing neonatal testis tissues. The culture medium consisted of α-minimum essential medium (α-MEM), supplemented with Knockout serum replacement (KSR) or AlbuMAX, neither of which were defined chemically. In this study, we formulated a chemically defined medium (CDM) that can induce mouse spermatogenesis under organ culture conditions. It was found that bovine serum albumin (BSA) purified through three different procedures had different effects on spermatogenesis. We also confirmed that retinoic acid (RA) played crucial roles in the onset of spermatogonial differentiation and meiotic initiation. The added lipids exhibited weak promoting effects on spermatogenesis. Lastly, luteinizing hormone (LH), follicle stimulating hormone (FSH), triiodothyronine (T3), and testosterone (T) combined together promoted spermatogenesis until round spermatid production. The CDM, however, was not able to produce elongated spermatids. It was also unable to induce spermatogenesis from the very early neonatal period, before 2 days postpartum, leaving certain factors necessary for spermatogenic induction in mice unidentified. Nonetheless, the present study provided important basic information on testis organ culture and spermatogenesis in vitro.
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- 2018
138. Colon organoid formation and cryptogenesis are stimulated by growth factors secreted from myofibroblasts
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Chin Wee Tan, Hon Yan Kelvin Yip, Antony W. Burgess, and Yumiko Hirokawa
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0301 basic medicine ,Cell signaling ,lcsh:Medicine ,Signal transduction ,Biochemistry ,Animal Cells ,Medicine and Health Sciences ,Organ Cultures ,Myofibroblasts ,lcsh:Science ,Connective Tissue Cells ,Notch Signaling ,Multidisciplinary ,Microvilli ,Receptors, Notch ,biology ,Chemistry ,Stem Cells ,Wnt signaling pathway ,Signaling cascades ,Proteases ,Enzymes ,Cell biology ,Organoids ,Bone morphogenetic protein 4 ,Connective Tissue ,Intercellular Signaling Peptides and Proteins ,Biological Cultures ,Anatomy ,Cellular Types ,Stem cell ,Research Article ,BMP signaling ,Colon ,Cell Survival ,Research and Analysis Methods ,Cell Line ,03 medical and health sciences ,Spheroids, Cellular ,TGF beta signaling pathway ,Organoid ,Animals ,Mesenchymal stem cell ,lcsh:R ,Biology and Life Sciences ,Proteins ,Feeder Cells ,Transforming growth factor beta ,Fibroblasts ,digestive system diseases ,Gastrointestinal Tract ,Mice, Inbred C57BL ,Wnt Proteins ,Biological Tissue ,030104 developmental biology ,TGF-beta signaling cascade ,Cell culture ,Culture Media, Conditioned ,Enzymology ,biology.protein ,lcsh:Q ,Serine Proteases ,Digestive System ,Biomarkers - Abstract
Although small intestinal epithelial stem cells form crypts when using intestinal culture conditions, colon stem cells usually form colonospheres. Colon mesenchymal cell feeder layers can stimulate colon crypts to form organoids and produce crypts. We have investigated whether conditioned medium from colon mesenchymal cells can also stimulate colonosphere and organoid cryptogenesis. We prepared conditioned medium (CM) from WEHI-YH2 cells (mouse colon myofibroblasts); the CM stimulated both colonosphere formation and organoid cryptogenesis in vitro. The colon organoid-stimulating factors in WEHI-YH2 CM are inactivated by heating and trypsin digestion and proteins can be concentrated by ultrafiltration. Both the colonosphere- and organoid cryptogenesis- stimulatory effects of the CM are independent of canonical Wnt and Notch signaling. In contrast, bone morphogenetic protein 4 (BMP4) abolishes colonosphere formation and organoid cryptogenesis. The Transforming Growth Factor beta (TGFβ) Type I receptor kinase inhibitor (A83-01) stimulates colonosphere formation, whereas the Epidermal Growth Factor receptor (EGFR) kinase inhibitor (AG1478) reduces the formation of colonospheres, but in the presence of EGF, a "just-right" concentration of AG1478 increases colon organoid cryptogenesis.
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- 2018
139. Effects of six common dietary nutrients on murine intestinal organoid growth
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Tenson Cai, Qun Wang, Yijun Qi, Michael J. Wannemuehler, Terrence A. Barrett, and Albert E. Jergens
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0301 basic medicine ,Organic chemistry ,lcsh:Medicine ,Ascorbic Acid ,Biochemistry ,Epithelium ,chemistry.chemical_compound ,Mice ,Animal Cells ,Sodium Glutamate ,Caffeic acid ,Medicine and Health Sciences ,Vitamin C ,Organ Cultures ,lcsh:Science ,Gastrointestinal tract ,Multidisciplinary ,Stem Cells ,Neurochemistry ,Vitamins ,Neurotransmitters ,Intestinal epithelium ,Cell biology ,Organoids ,Physical sciences ,Chemistry ,medicine.anatomical_structure ,Biological Cultures ,Anatomy ,Cellular Types ,Glutamate ,Chlorogenic Acid ,Research Article ,Curcumin ,Coumaric Acids ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Chemical compounds ,Caffeic Acids ,Organic compounds ,Organoid ,medicine ,Animals ,Nutrition ,Ethanol ,Parietal Cells ,lcsh:R ,Biology and Life Sciences ,Epithelial Cells ,Nutrients ,Cell Biology ,Diet ,Gastrointestinal Tract ,030104 developmental biology ,Biological Tissue ,chemistry ,Alcohols ,lcsh:Q ,Digestive System ,Ex vivo ,Neuroscience - Abstract
The intestinal epithelium of the gastrointestinal (GI) tract constantly renews itself to absorb nutrients and provide protection for the body from the outside world. Since the intestinal epithelium is constantly exposed to various chemicals and dietary components, it is critical to determine which constituents promote or inhibit intestinal epithelium health and growth rate. Intestinal organoids, three-dimensional miniature models of the intestines, represent an ex vivo tool to investigate intestinal physiology and growth patterns. In this study, we measured the growth rates of murine intestinal organoids exposed to various concentrations of different dietary constituents. Results indicate that caffeic acid inhibited organoid growth in a concentration-dependent manner, curcumin exhibited variable effectiveness, and vitamin C had no effect on organoid growth.
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- 2018
140. Assessment of p.Phe508del-CFTR functional restoration in pediatric primary cystic fibrosis airway epithelial cells
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Amelia K. Scaffidi, David F. Fischer, Kevin Looi, Stephen M. Stick, Clara J. Foo, Luke W. Garratt, Erika N. Sutanto, Michela Tessari, Richard A.J. Janssen, and Anthony Kicic
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0301 basic medicine ,Yellow fluorescent protein ,Cystic Fibrosis ,Pulmonology ,Cytotoxicity ,Cystic Fibrosis Transmembrane Conductance Regulator ,lcsh:Medicine ,Toxicology ,Pathology and Laboratory Medicine ,Biochemistry ,Pediatrics ,Cystic fibrosis ,Epithelium ,Transduction (genetics) ,Transduction, Genetic ,Animal Cells ,Medicine and Health Sciences ,Organ Cultures ,Child ,lcsh:Science ,Cells, Cultured ,Multidisciplinary ,biology ,Chemistry ,respiratory system ,Transmembrane protein ,Transport protein ,Cell biology ,Trachea ,Organoids ,Protein Transport ,Genetic Diseases ,Physical Sciences ,Biological Cultures ,Cellular Types ,Anatomy ,Research Article ,congenital, hereditary, and neonatal diseases and abnormalities ,Yellow Fluorescent Protein ,Phenylalanine ,High-throughput screening ,Genetic Vectors ,Bronchi ,Research and Analysis Methods ,Adenoviridae ,03 medical and health sciences ,Autosomal Recessive Diseases ,medicine ,Humans ,Molecular Biology Techniques ,Molecular Biology ,Clinical Genetics ,Molecular Biology Assays and Analysis Techniques ,Reporter gene ,Ussing chamber ,lcsh:R ,Chemical Compounds ,Biology and Life Sciences ,Proteins ,Epithelial Cells ,Cell Biology ,Iodides ,medicine.disease ,Fibrosis ,High Throughput Screening ,respiratory tract diseases ,Luminescent Proteins ,Biological Tissue ,030104 developmental biology ,biology.protein ,lcsh:Q ,Developmental Biology - Abstract
Background Mutations in the cystic fibrosis transmembrane regulator (CFTR) gene can reduce function of the CFTR ion channel activity and impair cellular chloride secretion. The gold standard method to assess CFTR function of ion transport using the Ussing chamber requires a high number of airway epithelial cells grown at air-liquid interface, limiting the application of this method for high throughput screening of potential therapeutic compounds in primary airway epithelial cells (pAECs) featuring less common CFTR mutations. This study assessed an alternative approach, using a small scale halide assay that can be adapted for a personalized high throughput setting to analyze CFTR function of pAEC. Methods Pediatric pAECs derived from children with CF (pAECCF) were established and expanded as monolayer cultures, before seeding into 96-well plates for the halide assay. Cells were then transduced with an adenoviral construct containing yellow fluorescent protein (eYFP) reporter gene, alone or in combination with either wild-type CFTR (WT-CFTR) or p.Phe508del CFTR. Four days post transduction, cells were stimulated with forskolin and genistein, and assessed for quenching of the eYFP signal following injection of iodide solution into the assay media. Results Data showed that pAECCF can express eYFP at high efficiency following transduction with the eYFP construct. The halide assay was able to discriminate functional restoration of CFTR in pAECCF treated with either WT-CFTR construct or the positive controls syntaxin 8 and B-cell receptor-associated protein 31 shRNAs. Significance The current study demonstrates that the halide assay can be adapted for pediatric pAECCF to evaluate restoration of CFTR function. With the ongoing development of small molecules to modulate the folding and/or activity of various mutated CFTR proteins, this halide assay presents a small-scale personalized screening platform that could assess therapeutic potential of molecules across a broad range of CFTR mutations.
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- 2018
141. Intestinal manipulation affects mucosal antimicrobial defense in a mouse model of postoperative ileus
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Lena Hieggelke, Joerg C. Kalff, Judith Kikhney, Mariola Lysson, Sven Wehner, Jan Wehkamp, Kathy Stein, Sabine Nuding, Annette Moter, Bianca Schneiker, and Burkhard Stoffels
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0301 basic medicine ,Male ,Gene Expression ,lcsh:Medicine ,Mucin 2 ,Biochemistry ,Mice ,Postoperative Complications ,Cryptdin ,Medicine and Health Sciences ,Mesenteric lymph nodes ,Intestinal Mucosa ,Organ Cultures ,lcsh:Science ,Multidisciplinary ,Antimicrobial ,Organoids ,medicine.anatomical_structure ,Jejunum ,Anaerobic bacteria ,Biological Cultures ,Anatomy ,Signal Transduction ,Research Article ,Antimicrobial peptides ,Ileum ,Anaerobic Bacteria ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Ileus ,medicine ,Genetics ,Animals ,Receptors, Interleukin-1 Type I ,Bacteria ,business.industry ,Mucin ,lcsh:R ,Organisms ,Biology and Life Sciences ,Proteins ,Gastrointestinal Tract ,Disease Models, Animal ,030104 developmental biology ,lcsh:Q ,business ,Digestive System - Abstract
Aim To explore the effects of abdominal surgery and interleukin-1 signaling on antimicrobial defense in a model of postoperative ileus. Methods C57BL/6 and Interleukin-1 receptor type I (IL-1R1) deficient mice underwent intestinal manipulation to induce POI. Expression of mucosal IL-1α, IL-1β and IL-1R1 and several antimicrobial peptides and enzymes were measured by quantitative PCR or ELISA, western blotting or immunohistochemistry. Bacterial overgrowth was determined by fluorescent in-situ hybridization and counting of jejunal luminal bacteria. Translocation of aerobic and anaerobic bacteria into the intestinal wall, mesenteric lymph nodes, liver and spleen was determined by counting bacterial colonies on agar plates 48h after plating of tissue homogenates. Antimicrobial activity against E. coli and B. vulgatus was analyzed in total and cationic fractions of small bowel mucosal tissue homogenates by a flow cytometry-based bacterial depolarization assay. Results Jejunal bacterial overgrowth was detected 24h after surgery. At the same time point, but not in the early phase 3h after surgery, bacterial translocation into the liver and mesenteric lymph nodes was observed. Increased antimicrobial activity against E. coli was induced within early phase of POI. Basal antimicrobial peptide and enzyme gene expression was higher in the ileal compared to the jejunal mucosa. The expression of lysozyme 1, cryptdin 1, cryptdin 4 and mucin 2 were reduced 24h after surgery in the ileal mucosa and mucin 2 was also reduced in the jejunum. Postoperative IL-1α and IL-1β were increased in the postoperative mucosa. Deficiency of IL-1R1 affected the expression of antimicrobial peptides during homeostasis and POI. Conclusion Small bowel antimicrobial capacity is disturbed during POI which is accompanied by bacterial overgrowth and translocation. IL-1R1 is partially involved in the gene expression of mucosal antimicrobial peptides. Altered small bowel antimicrobial activity may contribute also to POI development and manifestation in patients undergoing abdominal surgery.
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- 2018
142. A G542X cystic fibrosis mouse model for examining nonsense mutation directed therapies
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Ronald A. Conlon, Calvin U. Cotton, Rachel J. Mann, Mitchell L. Drumm, David F. LePage, Dana M. Valerio, Alexander Miron, M. Steele, Craig A. Hodges, and Daniel R. McHugh
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0301 basic medicine ,Male ,Cystic Fibrosis ,Pulmonology ,Cystic Fibrosis Transmembrane Conductance Regulator ,lcsh:Medicine ,Artificial Gene Amplification and Extension ,Cystic fibrosis ,Polymerase Chain Reaction ,Tissue Culture Techniques ,Genome editing ,Medicine and Health Sciences ,CRISPR ,Organ Cultures ,lcsh:Science ,Cells, Cultured ,Multidisciplinary ,Insertion Mutation ,Translational readthrough ,Nonsense Mutation ,Animal Models ,respiratory system ,Organoids ,Intestines ,Experimental Organism Systems ,Genetic Diseases ,Codon, Nonsense ,Female ,Biological Cultures ,Anatomy ,Research Article ,congenital, hereditary, and neonatal diseases and abnormalities ,Nonsense mutation ,Mouse Models ,Mice, Transgenic ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Model Organisms ,Autosomal Recessive Diseases ,medicine ,Genetics ,Animals ,Insertion ,RNA, Messenger ,Molecular Biology Techniques ,Molecular Biology ,Clinical Genetics ,Messenger RNA ,Cas9 ,lcsh:R ,Biology and Life Sciences ,Genetic Therapy ,medicine.disease ,Fibrosis ,Gastrointestinal Tract ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,Mutation ,Cancer research ,lcsh:Q ,CRISPR-Cas Systems ,Digestive System ,Developmental Biology - Abstract
Nonsense mutations are present in 10% of patients with CF, produce a premature termination codon in CFTR mRNA causing early termination of translation, and lead to lack of CFTR function. There are no currently available animal models which contain a nonsense mutation in the endogenous Cftr locus that can be utilized to test nonsense mutation therapies. In this study, we create a CF mouse model carrying the G542X nonsense mutation in Cftr using CRISPR/Cas9 gene editing. The G542X mouse model has reduced Cftr mRNA levels, demonstrates absence of CFTR function, and displays characteristic manifestations of CF mice such as reduced growth and intestinal obstruction. Importantly, CFTR restoration is observed in G542X intestinal organoids treated with G418, an aminoglycoside with translational readthrough capabilities. The G542X mouse model provides an invaluable resource for the identification of potential therapies of CF nonsense mutations as well as the assessment of in vivo effectiveness of these potential therapies targeting nonsense mutations.
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- 2018
143. Different culture media modulate growth, heterogeneity, and senescence in human mammary epithelial cell cultures
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Martha R. Stampfer, James C. Garbe, Jessica Bloom, Jonathan K. Lee, Mark A. LaBarge, Arantzazu Zubeldia-Plazaola, and Chalmers, Jeffrey
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0301 basic medicine ,Mammaplasty ,Cellular differentiation ,Mammary gland ,lcsh:Medicine ,Epithelium ,Spectrum Analysis Techniques ,Animal Cells ,Medicine and Health Sciences ,Organ Cultures ,lcsh:Science ,Cells, Cultured ,Cellular Senescence ,Staining ,education.field_of_study ,Cultured ,Multidisciplinary ,Ecology ,medicine.diagnostic_test ,Cell Staining ,Cell Differentiation ,Middle Aged ,Flow Cytometry ,Mammary Glands ,Phenotype ,Cell biology ,Organoids ,Laboratory Equipment ,medicine.anatomical_structure ,Spectrophotometry ,Engineering and Technology ,Female ,Biological Cultures ,Cytophotometry ,Cellular Types ,Anatomy ,Human ,Research Article ,Adult ,Senescence ,Ecological Metrics ,General Science & Technology ,Cells ,1.1 Normal biological development and functioning ,Population ,Equipment ,Biology ,Research and Analysis Methods ,Flow cytometry ,Young Adult ,03 medical and health sciences ,Underpinning research ,MD Multidisciplinary ,medicine ,Humans ,Cell Lineage ,Mammary Glands, Human ,education ,Ecology and Environmental Sciences ,lcsh:R ,Myoepithelial cell ,Biology and Life Sciences ,Species Diversity ,Epithelial Cells ,Cell Biology ,Cell Cultures ,Culture Media ,Biological Tissue ,030104 developmental biology ,Specimen Preparation and Treatment ,Cell culture ,lcsh:Q ,Developmental Biology - Abstract
© 2018 Lee et al. The ability to culture normal human mammary epithelial cells (HMEC) greatly facilitates experiments that seek to understand both normal mammary cell biology and the many differences between normal and abnormal human mammary epithelia. To maximize in vivo relevance, the primary cell culture conditions should maintain cells in states that resemble in vivo as much as possible. Towards this goal, we compared the properties of HMEC strains from two different reduction mammoplasty tissues that were grown in parallel using different media and culture conditions. Epithelial organoids were initiated into three different media: two commonly used serum-free-media, MCDB 170-type (e.g. MEGM) and WIT-P, and a low stress media, M87A. Growth, lineage heterogeneity, p16 protein expression, and population doublings to senescence were measured for each culture condition. MCDB 170 caused rapid senescence and loss of heterogeneity within 2 to 3 passages, but some cultures went through the 1 to 2 month process of selection to generate clonal finite post-selection poststasis cells. WIT-P caused impressive expansion of luminal cells in 2nd passage followed by their near complete disappearance by passage 4 and senescence shortly thereafter. M87A supported as much as twice the number of population doublings compared to either serumfree medium, and luminal and myoepithelial cells were present for as many as 8 passages. Thus, of the three media compared, WIT-P and MCDB 170 imposed rapid senescence and loss of lineage heterogeneity, phenotypes consistent with cells maintained in high-stress conditions, while M87A supported cultures that maintained multiple lineages and robust growth for up to 60 population doublings. In conjunction with previous studies examining the molecular properties of cultures grown in these media, we conclude that M87A medium is most able to support long-term culture of multiple lineages similar to in vivo conditions, thereby facilitating investigations of normal HMEC biology relevant to the mammary gland in situ.
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- 2018
144. Modeling APC mutagenesis and familial adenomatous polyposis using human iPS cells
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Gustavo Mostoslavsky, Ignacio S. Caballero, F.J. Molina-Estevez, Andreia Gianotti-Sommer, Nicholas Skvir, Cesar Sommer, Amalia Capilla, and Sanjib Chowdhury
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0301 basic medicine ,Cellular differentiation ,Gene Expression ,lcsh:Medicine ,medicine.disease_cause ,Loss of heterozygosity ,Animal Cells ,Medicine and Health Sciences ,Organ Cultures ,Induced pluripotent stem cell ,lcsh:Science ,Cells, Cultured ,Connective Tissue Cells ,Staining ,Mutation ,Multidisciplinary ,biology ,Stem Cells ,Gene Expression Regulation, Developmental ,Cell Staining ,Cell Differentiation ,3. Good health ,Intestines ,Organoids ,Cell Transformation, Neoplastic ,Adenomatous Polyposis Coli ,Connective Tissue ,Biological Cultures ,Cellular Types ,Anatomy ,Signal Transduction ,Research Article ,Adenomatous polyposis coli ,Immune Cells ,Adenomatous Polyposis Coli Protein ,Induced Pluripotent Stem Cells ,Immunology ,Antigen-Presenting Cells ,Mutagenesis (molecular biology technique) ,Research and Analysis Methods ,Familial adenomatous polyposis ,03 medical and health sciences ,Genetics ,medicine ,Humans ,lcsh:R ,Biology and Life Sciences ,Cell Biology ,Fibroblasts ,medicine.disease ,Gastrointestinal Tract ,HEK293 Cells ,Biological Tissue ,030104 developmental biology ,Mutagenesis ,Specimen Preparation and Treatment ,Cancer research ,biology.protein ,lcsh:Q ,Carcinogenesis ,Digestive System ,Developmental Biology - Abstract
Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.
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- 2018
145. Experimental Study of Tuberculosis: From Animal Models to Complex Cell Systems and Organoids
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Margarida Saraiva, Kaori L. Fonseca, Pedro Rodrigues, I. Anna S. Olsson, and Instituto de Investigação e Inovação em Saúde
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0301 basic medicine ,Bacterial Diseases ,Lung Development ,Organogenesis ,3d model ,Disease ,Review ,Medicine and Health Sciences ,Biology (General) ,Organ Cultures ,Animal Models ,3. Good health ,Vaccination ,Actinobacteria ,Organoids ,Infectious Diseases ,Experimental Organism Systems ,Biological Cultures ,medicine.medical_specialty ,Tuberculosis ,QH301-705.5 ,Immunology ,Mouse Models ,Biology ,Research and Analysis Methods ,Microbiology ,Models, Biological ,Mycobacterium tuberculosis ,03 medical and health sciences ,Model Organisms ,Virology ,Genetics ,medicine ,Animals ,Humans ,Animal Models of Disease ,Intensive care medicine ,Molecular Biology ,Bacteria ,Public health ,Organisms ,Biology and Life Sciences ,RC581-607 ,medicine.disease ,biology.organism_classification ,Tropical Diseases ,Animal Models of Infection ,Disease Models, Animal ,030104 developmental biology ,Infectious disease (medical specialty) ,Animal Studies ,Parasitology ,Immunologic diseases. Allergy ,Organism Development ,Mycobacterium Tuberculosis ,Developmental Biology - Abstract
Tuberculosis (TB) is a devastating disease to mankind that has killed more people than any other infectious disease. Despite many efforts and successes from the scientific and health communities, the prospect of TB elimination remains distant. On the one hand, sustainable public health programs with affordable and broad implementation of anti-TB measures are needed. On the other hand, achieving TB elimination requires critical advances in three areas: vaccination, diagnosis, and treatment. It is also well accepted that succeeding in advancing these areas requires a deeper knowledge of host—pathogen interactions during infection, and for that, better experimental models are needed. Here, we review the potential and limitations of different experimental approaches used in TB research, focusing on animal and human-based cell culture models. We highlight the most recent advances in developing in vitro 3D models and introduce the potential of lung organoids as a new tool to study Mycobacterium tuberculosis infection., Author summary Tuberculosis (TB) is the number 1 killer in the world due to a bacterial infection. The study of this disease through clinical and epidemiological data and through the use of different experimental models has provided important knowledge on the role of the immune response generated during infection. This is critical for the development of novel vaccines and therapeutic strategies. However, in spite of the advances made, it is well accepted that better models are needed to study TB. This review discusses the different models used to study TB, highlighting the advantages and disadvantages of the available animal and cellular models and introducing recently developed state-of-the-art approaches based on human-based cell culture systems. These new advances are integrated in a road map for future study of TB, converging for the potential of lung organoids in TB research.
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- 2017
146. Metabolic Imaging of Head and Neck Cancer Organoids
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Melissa C. Skala, Amy T. Shah, and Tiffany M. Heaster
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0301 basic medicine ,Receptor, ErbB-2 ,medicine.medical_treatment ,Cancer Treatment ,lcsh:Medicine ,Cetuximab ,Mice ,0302 clinical medicine ,Drug Metabolism ,Drug Discovery ,Medicine and Health Sciences ,Electrochemistry ,Image Processing, Computer-Assisted ,Tumor Cells, Cultured ,Organ Cultures ,lcsh:Science ,Multidisciplinary ,Cell Death ,Optical Imaging ,Chemical Reactions ,Primary tumor ,3. Good health ,Tumor Burden ,Organoids ,Chemistry ,Treatment Outcome ,Oncology ,Cell Processes ,Head and Neck Neoplasms ,030220 oncology & carcinogenesis ,Physical Sciences ,Carcinoma, Squamous Cell ,Biological Cultures ,medicine.drug ,Research Article ,Cell Physiology ,Combination therapy ,Imaging Techniques ,Mice, Nude ,Antineoplastic Agents ,Research and Analysis Methods ,03 medical and health sciences ,In vivo ,Fluorescence Imaging ,medicine ,Organoid ,Animals ,Humans ,Pharmacokinetics ,Cell Proliferation ,Fluorescent Dyes ,Pharmacology ,Chemotherapy ,business.industry ,Cell growth ,lcsh:R ,Head and neck cancer ,Biology and Life Sciences ,Cell Biology ,medicine.disease ,Cell Metabolism ,High-Throughput Screening Assays ,030104 developmental biology ,Otorhinolaryngology ,Head and Neck Cancers ,Cancer research ,lcsh:Q ,business ,Oxidation-Reduction Reactions - Abstract
Head and neck cancer patients suffer from toxicities, morbidities, and mortalities, and these ailments could be minimized through improved therapies. Drug discovery is a long, expensive, and complex process, so optimized assays can improve the success rate of drug candidates. This study applies optical imaging of cell metabolism to three-dimensional in vitro cultures of head and neck cancer grown from primary tumor tissue (organoids). This technique is advantageous because it measures cell metabolism using intrinsic fluorescence from NAD(P)H and FAD on a single cell level for a three-dimensional in vitro model. Head and neck cancer organoids are characterized alone and after treatment with standard therapies, including an antibody therapy, a chemotherapy, and combination therapy. Additionally, organoid cellular heterogeneity is analyzed quantitatively and qualitatively. Gold standard measures of treatment response, including cell proliferation, cell death, and in vivo tumor volume, validate therapeutic efficacy for each treatment group in a parallel study. Results indicate that optical metabolic imaging is sensitive to therapeutic response in organoids after 1 day of treatment (p
- Published
- 2017
147. Identification of the SUMO E3 ligase PIAS1 as a potential survival biomarker in breast cancer
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Donald G. Morris, Lili Deng, Elizabeth N. Kornaga, Shirin Bonni, Ayan Chanda, Emeka K. Enwere, and Angela Chan
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Proteomics ,0301 basic medicine ,Oncology ,SUMO protein ,lcsh:Medicine ,Biochemistry ,Metastasis ,Cohort Studies ,0302 clinical medicine ,Transforming Growth Factor beta ,Breast Tumors ,Basic Cancer Research ,Medicine and Health Sciences ,Transcriptional regulation ,Organ Cultures ,skin and connective tissue diseases ,lcsh:Science ,Aged, 80 and over ,Multidisciplinary ,Protein Stability ,Middle Aged ,Protein Inhibitors of Activated STAT ,3. Good health ,Ubiquitin ligase ,Organoids ,Protein Transport ,030220 oncology & carcinogenesis ,Small Ubiquitin-Related Modifier Proteins ,Biomarker (medicine) ,Female ,Biological Cultures ,Post-translational modification ,Research Article ,Adult ,medicine.medical_specialty ,Immunoblotting ,Molecular Probe Techniques ,Breast Neoplasms ,Biology ,Research and Analysis Methods ,03 medical and health sciences ,Breast cancer ,Cell Line, Tumor ,Internal medicine ,Breast Cancer ,Biomarkers, Tumor ,medicine ,Humans ,Neoplasm Invasiveness ,Molecular Biology Techniques ,Molecular Biology ,Immunohistochemistry Techniques ,Aged ,Cell Proliferation ,Cell Nucleus ,Biology and life sciences ,lcsh:R ,Sumoylation ,Cancers and Neoplasms ,Proteins ,Cancer ,medicine.disease ,Survival Analysis ,Sumoylation Pathway ,Histochemistry and Cytochemistry Techniques ,HEK293 Cells ,030104 developmental biology ,Tissue Array Analysis ,Immunologic Techniques ,biology.protein ,Cancer research ,lcsh:Q ,Biomarkers ,Protein Abundance - Abstract
Metastasis is the ultimate cause of breast cancer related mortality. Epithelial-mesenchymal transition (EMT) is thought to play a crucial role in the metastatic potential of breast cancer. Growing evidence has implicated the SUMO E3 ligase PIAS1 in the regulation of EMT in mammary epithelial cells and breast cancer metastasis. However, the relevance of PIAS1 in human cancer and mechanisms by which PIAS1 might regulate breast cancer metastasis remain to be elucidated. Using tissue-microarray analysis (TMA), we report that the protein abundance and subcellular localization of PIAS1 correlate with disease specific overall survival of a cohort of breast cancer patients. In mechanistic studies, we find that PIAS1 acts via sumoylation of the transcriptional regulator SnoN to suppress invasive growth of MDA-MB-231 human breast cancer cell-derived organoids. Our studies thus identify the SUMO E3 ligase PIAS1 as a prognostic biomarker in breast cancer, and suggest a potential role for the PIAS1-SnoN sumoylation pathway in controlling breast cancer metastasis.
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- 2017
148. Cytokeratin-14 contributes to collective invasion of salivary adenoid cystic carcinoma
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Ya-ling Tang, Min-xin Cao, Ya-ping Jiang, Xiao Cen, Xiao-lei Gao, Ya-Jie Tang, Xin-hua Liang, Jia-Shun Wu, Qianming Chen, Sha-sha Wang, and Shi-yu Gao
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0301 basic medicine ,Male ,lcsh:Medicine ,Biochemistry ,Salivary Glands ,Metastasis ,Tissue Culture Techniques ,0302 clinical medicine ,Basic Cancer Research ,Medicine and Health Sciences ,Small interfering RNAs ,Organ Cultures ,lcsh:Science ,Multidisciplinary ,Cell migration ,Middle Aged ,Prognosis ,Salivary Gland Neoplasms ,Head and Neck Tumors ,Carcinoma, Adenoid Cystic ,Organoids ,Nucleic acids ,Cell Motility ,Oncology ,030220 oncology & carcinogenesis ,Biomarker (medicine) ,Female ,Biological Cultures ,Anatomy ,Research Article ,Adenoid cystic carcinoma ,Cell Migration ,Biology ,Research and Analysis Methods ,Carcinomas ,03 medical and health sciences ,Cytokeratin ,Immune system ,Exocrine Glands ,Diagnostic Medicine ,Carcinoma ,medicine ,Genetics ,Humans ,Neoplasm Invasiveness ,Salivary Gland Tumors ,Non-coding RNA ,lcsh:R ,Keratin-14 ,Biology and Life Sciences ,Cancers and Neoplasms ,Cell Biology ,medicine.disease ,Gene regulation ,stomatognathic diseases ,030104 developmental biology ,Cancer research ,RNA ,lcsh:Q ,Gene expression ,Wound healing ,Digestive System ,Developmental Biology - Abstract
Collective invasion of cells plays a fundamental role in tissue growth, wound healing, immune response and cancer metastasis. This paper aimed to investigate cytokeratin-14 (CK14) expression and analyze its association with collective invasion in the invasive front of salivary adenoid cystic carcinoma (SACC) to uncover the role of collective invasion in SACC. Here, in the clinical data of 121 patients with SACC, the positive expression of CK14 was observed in 35/121(28.93%) of the invasive front of SACC. CK14 expression in the invasive front, local regional recurrence and distant metastasis were independent and significant prognostic factors in SACC patients. Then, we found that in an ex vivo 3D culture assay, CK14 siRNA receded the collective invasion, and in 2D monolayer culture, CK14 overexpression induced a collective SACC cell migration. These data indicated that the presence of characterized CK14+ cells in the invasive front of SACC promoted collective cell invasion of SACC and may be a biomarker of SACC with a worse prognosis.
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- 2017
149. The comprehensive role of E-cadherin in maintaining prostatic epithelial integrity during oncogenic transformation and tumor progression
- Author
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Won Kyung Kim, Joseph Aldahl, Robert D. Cardiff, Joseph Geradts, Erika Hooker, Yongfeng He, Adam Olson, Eun Jeong Yu, Zijie Sun, Dong Hong Lee, Vien Le, and Barsh, Gregory S
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Male ,Aging ,Cancer Research ,Carcinogenesis ,Apoptosis ,Tumor initiation ,QH426-470 ,Cell Transformation ,Epithelium ,Transgenic ,CDH1 ,Mice ,Prostate cancer ,0302 clinical medicine ,Animal Cells ,Medicine and Health Sciences ,2.1 Biological and endogenous factors ,RNA, Small Interfering ,Organ Cultures ,Aetiology ,beta Catenin ,Genetics (clinical) ,Cancer ,Staining ,Prostatic Intraepithelial Neoplasia ,0303 health sciences ,Tumor ,Cell Death ,Prostate Cancer ,Prostate Diseases ,Prostate ,Cell Staining ,Animal Models ,Cadherins ,CD ,Organoids ,Cell Transformation, Neoplastic ,medicine.anatomical_structure ,Experimental Organism Systems ,Oncology ,Cell Processes ,Disease Progression ,Biological Cultures ,Anatomy ,Cellular Types ,Research Article ,Urologic Diseases ,Urology ,Primary Cell Culture ,Mice, Transgenic ,Mouse Models ,Biology ,Research and Analysis Methods ,Small Interfering ,Cell Line ,03 medical and health sciences ,Exocrine Glands ,Model Organisms ,Antigens, CD ,Cell Line, Tumor ,Genetics ,medicine ,Animals ,Humans ,Antigens ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Cell Proliferation ,030304 developmental biology ,Neoplastic ,Goblet cell ,Animal ,Cadherin ,PTEN Phosphohydrolase ,Biology and Life Sciences ,Cancers and Neoplasms ,Prostatic Neoplasms ,Epithelial Cells ,Cell Biology ,medicine.disease ,Actin cytoskeleton ,Disease Models, Animal ,Genitourinary Tract Tumors ,Biological Tissue ,HEK293 Cells ,Specimen Preparation and Treatment ,Tumor progression ,Catenin ,Disease Models ,Animal Studies ,Cancer research ,biology.protein ,RNA ,Prostate Gland ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
E-cadherin complexes with the actin cytoskeleton via cytoplasmic catenins and maintains the functional characteristics and integrity of the epithelia in normal epithelial tissues. Lost expression of E-cadherin disrupts this complex resulting in loss of cell polarity, epithelial denudation and increased epithelial permeability in a variety of tissues. Decreased expression of E-cadherin has also been observed in invasive and metastatic human tumors. In this study, we investigated the effect of E-cadherin loss in prostatic epithelium using newly developed genetically engineered mouse models. Deletion of E-cadherin in prostatic luminal epithelial cells with modified probasin promoter driven Cre (PB-Cre4) induced the development of mouse prostatic intraepithelial neoplasia (PIN). An increase in levels of cytoplasmic and nuclear β-catenin appeared in E-cadherin deleted atypical cells within PIN lesions. Using various experimental approaches, we further demonstrated that the knockdown of E-cadherin expression elevated free cytoplasmic and nuclear β-catenin and enhanced androgen-induced transcription and cell growth. Intriguingly, pathological changes representing prostatic epithelial cell denudation and increased apoptosis accompanied the above PIN lesions. The essential role of E-cadherin in maintaining prostatic epithelial integrity and organization was further demonstrated using organoid culture approaches. To directly assess the role of loss of E-cadherin in prostate tumor progression, we generated a new mouse model with bigenic Cdh1 and Pten deletion in prostate epithelium. Early onset, aggressive tumor phenotypes presented in the compound mice. Strikingly, goblet cell metaplasia was observed, intermixed within prostatic tumor lesions of the compound mice. This study provides multiple lines of novel evidence demonstrating a comprehensive role of E-cadherin in maintaining epithelial integrity during the course of prostate oncogenic transformation, tumor initiation and progression., Author summary The biological significance of E-cadherin in maintaining prostatic epithelial integrity and related molecular mechanisms are still unclear. In this study, using mouse genetic tools, we directly address this important and unresolved question. Conditional deletion of E-cadherin in mouse prostatic epithelia resulted in prostatic intraepithelial neoplasia (PIN) development but no prostatic tumor formation. Both in vivo and in vitro data showed that loss of E-cadherin modulates the cellular localization of β-catenin, elevates its cytoplasmic and nuclear levels, and enhances its activity in transcription and cell proliferation. Intriguingly, in addition to PIN lesions, increased epithelial denudation and cell apoptosis also appeared within PIN lesions. This implicates that although lost E-cadherin is sufficient to introduce oncogenic transformation in prostatic epithelia, it also induces cell apoptosis and disrupts epithelial structure, preventing atypical PIN cells from progressing to tumor cells. Simultaneous deletion of Pten, a tumor suppressor, and E-cadherin in prostatic epithelia resulted in early onset, invasive prostatic tumors with admixture of goblet cells. These results demonstrate a critical role of E-cadherin in promoting prostatic tumor transdifferentiation and progression. This study further elucidates the dynamic role of E-cadherin in maintaining prostatic epithelial integrity during tumor initiation and progression.
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- 2019
150. Recombinant human PRG4 (rhPRG4) suppresses breast cancer cell invasion by inhibiting TGFβ-Hyaluronan-CD44 signalling pathway
- Author
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Shirin Bonni, Kunal Karve, Suresh C. Regmi, Gregory D. Jay, Lili Deng, Frank R. Jirik, Anusi Sarkar, Tannin A. Schmidt, and Ayan Chanda
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Proteomics ,0301 basic medicine ,Smad Proteins ,Biochemistry ,Metastasis ,Extracellular matrix ,Contractile Proteins ,0302 clinical medicine ,Cell Movement ,Transforming Growth Factor beta ,Breast Tumors ,Medicine and Health Sciences ,Hyaluronic Acid ,Organ Cultures ,Microscopy ,Multidisciplinary ,biology ,Light Microscopy ,Metastatic breast cancer ,Recombinant Proteins ,Cancer Cell Migration ,Enzymes ,3. Good health ,Organoids ,Cell Motility ,Hyaluronan Receptors ,Oncology ,030220 oncology & carcinogenesis ,Medicine ,Female ,Proteoglycans ,Biological Cultures ,Oxidoreductases ,Luciferase ,Signal Transduction ,Research Article ,Science ,Immunoblotting ,Molecular Probe Techniques ,Breast Neoplasms ,Cell Migration ,Research and Analysis Methods ,03 medical and health sciences ,Proteoglycan 4 ,Cell Line, Tumor ,Breast Cancer ,medicine ,Humans ,Neoplasm Invasiveness ,Molecular Biology Techniques ,Molecular Biology ,Cell Proliferation ,CD44 ,Cancers and Neoplasms ,Biology and Life Sciences ,Proteins ,Cancer ,Cell Biology ,Transforming growth factor beta ,medicine.disease ,Actins ,Molecular Weight ,Cytoskeletal Proteins ,030104 developmental biology ,Cancer cell ,Enzymology ,biology.protein ,Cancer research ,Protein Abundance ,Developmental Biology - Abstract
Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an anti-invasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFβ) to induce phenotypic disruption of three-dimensional human breast cancer cell-derived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFβ-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFβ-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFβ has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFβ has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFβ actions. Finally, these findings point to rhPRG4's potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer.
- Published
- 2019
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