Pancreatic gene expression during recovery after pancreatitis reveals unique transcriptome profiles
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE99774 | GEO | 2018/01/23
REPOSITORIES: GEO
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Project description:BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing (scRNA-seq) studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We introduce FixNCut, a scRNA-seq approach where tissue is reversibly fixed with dithiobis(succinimidyl propionate) prior to dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate findings using GeoMx whole transcriptome atlas (WTA) profiling, and integrate our data with prior studies to benchmark our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition of challenging pancreatic cells including acinar and immune populations in homeostasis and acute pancreatitis, and identifies changes in all major cell types during injury and recovery. We define the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas towards type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.
2024-02-28 | GSE235874 | GEO
Project description:The present study was constructed to confirm previous findings that mice on a high fat diet (HFD) treated by subcutaneous injection with exenatide (EXE) at 3µg/kg once daily for 6 weeks develop exocrine pancreatic injury (Rouse et al. 2014). The present study included 12 weeks of EXE exposure at multiple concentrations (3, 10, or 30 μg/kg) with multiple endpoints (histopathology evaluations, immunoassay for cytokines, immunostaining of the pancreas, serum chemistries and measurement of trypsin, amylase, and, lipase, and gene expression profiles). Time- and dose-dependent exocrine pancreatic injury was observed in mice associated with EXE exposure in a HFD environment. The time- and dose-dependent morphological changes identified in the pancreas involved acinar cell injury and death (autophagy, apoptosis, necrosis, and atrophy), cell adaptations (hypertrophy and hyperplasia), and cell survival (regeneration) accompanied with varying degrees of inflammatory response leading to secondary injury in pancreatic blood vessels, ducts, and adipose tissues. Gene expression profiles supported the presence of increased signaling for cell survival and altered lipid metabolism. The potential for EXE to cause acute or early chronic pancreatic injury was identified in a HFD environment. In human disease, the influence of pancreatitis risk factors or pre-existing chronic pancreatitis on this injury potential requires further investigation.
2014-06-01 | GSE54774 | GEO
Project description:Analysis of CD4+ T cell gene expression changes after 4 and 24 weeks of Maraviroc administration relative to baseline in 25 immunodiscordant patients with viral suppression. Maraviroc elicited very modest changes in CD4+ T cell transcriptome. The goal of this study was to identify genes with a potential role in Maraviroc-induced CD4+ T cell recovery in immunodiscordant patients. The study measured gene expression in CD4+ T cells isolated from the whole blood of HIV-infected patients before and at two time points after addition of Maraviroc to the baseline antiretroviral therapy. Thirty two patients were recruited from 3 sites, University of California, San Diego (UCSD), University of California, Los Angeles (UCLA) and University of Southern California (USC), and peripheral blood samples were obtained at 3 visits, week 0 (baseline), week 4 and week 24 after initiation of Maraviroc therapy. Because of poor RNA quality and outliers on the microarrays, 7 patients were excluded from the study. Non-outlier week 0 and week 24 arrays from one of the patients (Patient ID 01-005) were used for array data normalization and processing, but were not included in the time course analysis since the week 4 sample was not available.
2014-04-16 | E-GEOD-56804 | biostudies-arrayexpress
Project description:The present study was constructed to confirm previous findings that mice on a high fat diet (HFD) treated by subcutaneous injection with exenatide (EXE) at 3µg/kg once daily for 6 weeks develop exocrine pancreatic injury (Rouse et al. 2014). The present study included 12 weeks of EXE exposure at multiple concentrations (3, 10, or 30 Œºg/kg) with multiple endpoints (histopathology evaluations, immunoassay for cytokines, immunostaining of the pancreas, serum chemistries and measurement of trypsin, amylase, and, lipase, and gene expression profiles). Time- and dose-dependent exocrine pancreatic injury was observed in mice associated with EXE exposure in a HFD environment. The time- and dose-dependent morphological changes identified in the pancreas involved acinar cell injury and death (autophagy, apoptosis, necrosis, and atrophy), cell adaptations (hypertrophy and hyperplasia), and cell survival (regeneration) accompanied with varying degrees of inflammatory response leading to secondary injury in pancreatic blood vessels, ducts, and adipose tissues. Gene expression profiles supported the presence of increased signaling for cell survival and altered lipid metabolism. The potential for EXE to cause acute or early chronic pancreatic injury was identified in a HFD environment. In human disease, the influence of pancreatitis risk factors or pre-existing chronic pancreatitis on this injury potential requires further investigation. Male C57BL/6 mice 6 to 8 weeks of age were purchased from Harlan Laboratories (Frederick, MD). Mice were housed individually in an environmentally controlled room (18C-21C, 40%-70% relative humidity) with a twelve-hour light/dark cycle. Mice were initially fed Certified Purina Rodent Chow #5002 (Ralston Purina Co., St. Louis, MO) providing 63% of calories through carbohydrates, 24% from protein, and 13% from fat. Per the experimental design, mice were subsequently placed on a Teklad Custom Research Diet, TD.06415 (Harlan, Frederick, MD), providing 45% of calories through fats (36% saturated, 47% monounsaturated, 17% polyunsaturated), 36% from carbohydrates, and 19% from protein. Water and food were available ad libitum. Thorough out the experiment all mice were weighed weekly and dosing adjusted accordingly for each mouse. A cohort of eighty 6 to 8 week-old male C57BL6 mice were received from Harlan Laboratories. The cohort represented experimental time point of 12 weeks. The cohort was further divided into 4 exenatide (EXE) treatment groups (0, 3, 10, or 30 ug/kg) of 20 mice each. Initially, all mice were placed on HFD for six weeks without additional treatment and were then maintained on HFD for an additional 12 weeks while receiving daily subcutaneous injections of saline or EXE (Creative Peptides, Shirley, NY; 3, 10, or 30 ug/kg). At the 12 week timepoint, twenty-four hours following their final dose, the mice from the treatment cohort were anesthetized with isoflurane and euthanized by exsanguination with collection of blood for serum harvest. The pancreas from 12 mice in each cohort was preserved in 10% formalin for histology processing. The pancreas from 5 mice in each cohort was placed in RNAlater (Life Technologies, Grand Island, NY) for subsequent RNA extraction and the pancreas from 3 mice in each cohort was immediately frozen in liquid nitrogen and then stored at -80C. Pancreas tissue was resected, preserved in RNALater, and stored at -80 C. After thawing, 2.5 mg of each sample were processed using miRNeasy Mini Kits (Cat # 217004) (Qiagen, Valencia, CA). Samples were homogenized in 700 uL of Qiazol Lysis Reagent (Qiagen), for 5 minutes at 50 hz in a TissueLyser LT (Qiagen), and processed using the automated purification of total RNA on a Qiacube (Qiagen), using the 'Purification of total RNA, including small RNAs, from animal tissues & cells (aqueous phase), version 2 (April 2010)' standard protocol, as described in the miRNeasy Mini Handbook (1073008 07/2012) & http://www.qiagen.com/QIAcube/Standard/ProtocolView.aspx?StandardProtocolID=847.Mean yield per 2.5 mg of pancreas tissue was approximately 35 ug, determined by NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Average RIN values were approximately 5.4 +/- 1.4, as assayed by a 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA), using an Agilent RNA 6000 Nano Kit (cat # 5067-1511). This RIN mean was consistent with our previous experiences in RNA extraction from murine pancreatic tissue. The isolated RNA was labeled and gene expression analysis performed by Expression Analysis, Inc. (Durham, NC). The 3 samples with the highest RIN from each treatment group were used to probe treatment effect on gene expression. Total RNA samples (100 ng) were converted into cDNA using Ovation WGA FFPE System (NuGEN, Part No. 6200). The second strand cDNA is then purified with Agencourt RNAClean beads and followed by SPIA amplification. Amplified SPIA cDNA product is purified using Agencourt RNAClean beads and quantitated using a NanoDrop ND-8000 spectrophotometer. Target preparation was performed using 4 ug of amplified cDNA and the NuGEN FL-Ovation cDNA Biotin Module V2. Fragmented and biotin-labeled target was hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 microarrays. Raw gene expression data were first analyzed using ArrayTrack bioinformatics tool with robust multichip average algorithm and quantile normalization. Differentially expressed genes were defined by p < 0.05 and fold change > 1.3 in Welch t-test comparing treatment and control groups. Expression data were then imported to Ingenuity Pathway Analysis (IPA) tool for further analyses of pathway, biofunction, and toxicity using general and pancreas-specific knowledge bases.
2014-06-01 | E-GEOD-54774 | biostudies-arrayexpress
Project description:T cells undergo autoimmunization following spinal cord injury (SCI) and play both protective and destructive roles during the recovery process. T-cell deficient athymic nude (AN) rats recover better than immunocompetent Sprague-Dawley (SD) rats following spinal cord transection. In the present study, we evaluated locomotor recovery in SD and AN rats following moderate spinal cord contusion. To explain variable locomotor outcome, we assessed whole-genome expression using RNA sequencing, in the acute (1 week post-injury) and chronic (8 weeks post-injury) phases of recovery. AN rats demonstrated greater locomotor function than SD rats only at 1 week post-injury, coinciding with peak T cell infiltration in immunocompetent rats. Genetic markers for T cells and helper T cells were acutely enriched in SD rats, while AN rats expressed genes for Th2 cells, cytotoxic T cells, NK cells, mast cells, IL-1a, and IL-6 at higher levels. Acute enrichment of cell death-related genes suggested that SD rats undergo secondary tissue damage from T cells. Additionally, SD rats exhibited increased acute expression of voltage-gated potassium (Kv) channel-related genes. However, AN rats demonstrated greater chronic expression of cell death-associated genes and less expression of axon-related genes. We put forth a model in which T cells facilitate early tissue damage, demyelination, and Kv channel dysregulation in SD rats following contusion SCI. However, compensatory features of the immune response in AN rats cause delayed tissue death and limit long-term recovery. T cell inhibition combined with other neuroprotective treatment may thus be a promising therapeutic avenue. 2x2 model with 4 groups and 12 total samples. 2 rat strains (athymic nude [AN] and Sprague-Dawley [SD]) and 2 time points (1 week post-injury [acute] and 8 weeks post-injury [chronic]). 3 samples per group, for a total of 12 samples. No technical replicates were performed. Acute SD group = rats 618, 619, and 620. Chronic SD group = rats 605, 606, and 608. Acute AN group = rats 714, 715, and 717. Chronic AN group = rats 707, 712, and 713.
2015-10-26 | E-GEOD-62760 | biostudies-arrayexpress
Project description:Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signaling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by Partial Duct Ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) which expand 5-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality We generated arrays from whole pancreas, islet, acinar and duct (Sox9+ sorted) cells and pancreas derived cultures maintained in our defined medium. For the clustering analysis we substracted the pancreas array to all arrays. Genes 2 fold differentially expressed in the ductal array were used for the analsyis. For the genes enriched in organoid cultures compared to pancreas we substracted the organoid culture arrays to the pancreas array. Genes >2-fold differentially expressed were used for the analysis.
2013-09-17 | E-GEOD-50103 | biostudies-arrayexpress
Project description:Glis3 is expressed in pancreatic beta and PP cells. To identify down stream target genes of Glis3, we performed microarray analysis using pancreas total RNAs from 1 week-old WT and Glis3KO2 mice. insulin and pancreatic polypeptide (Ppy) was significantly decreased together with several other β cell markers, Glut2 and MafA by microarray analysis. Immunohistochemistry, QRT-PCR, and transmission electron microscopy indicated that postnatal Glis3KO2 pancreas still contains a large population of β cells that express Pdx-1, Nkx6.1, and Isl-1; however, insulin production and secretory granules were greatly reduced in these cells. In addition, chromogranin A (ChgA) and Urocortin 3, which are associated with mature β cells, was dramatically decreased in Glis3KO2 pancreas. These observations suggest that Glis3 plays a critical role in the maturation of pancreatic β cell phenotype. Pancreatic total RNAs were purified from 4 WT and 4 Glis3KO2 at 1 week old age. Then the samples were applied to Agilent mouse genome chip.
2016-07-01 | E-GEOD-67238 | biostudies-arrayexpress
Project description:Spinal cord injury disrupts ascending and descending neural signals causing sensory and motor dysfunction below the injury. Neuromodulation with electrical stimulation is used in both clinical and research settings to induce neural plasticity and improve functional recovery following injury. However, the mechanisms by which electrical stimulation affects recovery remain unclear. In this study we examined the effects of cortical electrical stimulation following injury on transcription at several levels of the central nervous system. We performed a unilateral cervical spinal contusion injury in rats and delivered stimulation for one week to the contralesional motor cortex to activate a descending motor tract.RNA was purified from bilateral subcortical white matter, and 3 levels of the spinal cord. Here we provide the complete data set in the hope that it will be useful for researchers studying electrical stimulation as a therapy to improve recovery from the deficits associated with spinal cord injury.
2021-02-16 | GSE155610 | GEO
Project description:Expression data from pancreatic cancer cell lines and non-neoplastic pancreatic cell line HPDE To identify genes epigenetically silenced and regulated in pancreatic cancer We compared the gene expression profiles of 6 pancreatic cancer cell lines (panc215, A32-1, A38-5, panc2.5, panc2.8, and panc3.014), to the non-neoplastic pancreas cell line, HPDE. We also compared the baseline gene expression of the pancreatic cancer cell lines to expression patterns after treatment with 5-aza-dC alone, TSA alone, and to a combination of 5-aza-dC/TSA.
2010-04-14 | E-GEOD-21163 | biostudies-arrayexpress
Project description:The objective of this study was to understand the long-term effects of a chronic phase shift protocol on the pancreatic transcriptome. A total of 144 wild-type C57Bl/6J 4-6 week old mice were housed under normal circadian conditions or disrupted using a chronic phase shift protocol whereby the light-dark cycle is advanced 8 hour every 2-3 days for 4 weeks. 12 hours prior to sacrafice mice were normalized under normal circadian conditions. Starting at ZT 0, mice were serially sacraficed at 4-hour intervals for 48 hours and the pancreas extracted. Pancreatic RNA was isolated and subjected to RNA-seq.
2021-01-21 | GSE165198 | GEO