Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Genome-wide mRNA profiling of adult human pancreatic beta and duct cells in comparison to other human tissues

ABSTRACT: Aims: establishment of reference samples to investigate gene expression selective for endocrine or ductal-exocrine cells within the adult human pancreas. To this end, human islet endocrine cells, FACS-enriched in insulin+ cells, (n=3) and human exocrine ductal cells (n=2) are compared on Affymetrix HG133A platform with duplicate hybridizations of a panel of other primary human tissues. The microarray analysis was performed on 3 pools of human beta cell-enriched cell fractions, isolated from 10 non-selected donor organs, and 2 pools of duct cell-enriched fractions obtained from 6 non-selected donor pancreases. The cells were suspension-cultured for 2-3 weeks along standard procedures and with no specific treatment prior to FACS-sorting and RNA extraction. The average composition of human beta cell-preparations was 55 ± 13% insulin+ cells, 13±8% glucagon+ cells and 21 ±7% non-granulated cells. Pancreatic duct cells-enriched preparations contained 85 ±7% cytokeratin 19+ cells with 4 ± 1% insulin+ cells and 6 ±4% glucagon+ cells and were isolated as described by Heimberg H. et al.( Diabetes 2001,49: 571-579 ). Pancreatic cell mRNA profiles were compared to those of a panel of other human primary tissues (n=2 biological replicates). This dataset is part of the TransQST collection.

ORGANISM(S): Homo sapiens

SUBMITTER: Geert Martens

PROVIDER: E-GEOD-30803 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Clusters of conserved beta cell marker genes for assessment of beta cell phenotype.

Martens Geert A GA Jiang Lei L Hellemans Karine H KH Stangé Geert G Heimberg Harry H Nielsen Finn C FC Sand Olivier O Van Helden Jacques J Van Lommel Leentje L Schuit Frans F Gorus Frans K FK Pipeleers Daniel G DG

PloS one 20110902 9

<h4>Background and methodology</h4>The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse ...[more]

PMID: 21912665

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Project description:Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. Results: Ngn3 stimulates duct cells to express a focused set of genes that are abundant in islet endocrine cells and/or neural tissues. This neuro-endocrine shift, however, covers a minor fraction (5%) of the estimated genome-wide transcriptome difference between duct and islet endocrine cells. Interestingly, transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes. We used Affymetrix HG133A and HG133B to get a comprehensive view on the reprogramming potential in vitro of human pancreatic duct cell cultures (n=3-4) at 3 and 14/20 days after ectopic adenoviral expression of murine neurogenin 3 as compared to GFP-expressing control vectors. The microarray analysis was performed on 3 independent samples that each contained RNA extracted from a pool of 3 independent donor pancreata. The total number of non-selected donor organs is 9. Transcripts were considered as differentially regulated by Ngn3 when 1.5 fold (LCB, unpaired P < 0.05) up- or down-regulated in AdGFP-Ngn3 versus AdGFP controls, at 3 and/or 14 dpi. Transcripts that showed differential expression between day 3 and day 14 in AdGFP-Ngn3 duct cells but not in AdGFP control cells, were also considered Ngn3-regulated

Project description:Human type 1 diabetes (T1D) arises through autoimmunity towards the insulin-producing pancreatic β cells and is modeled by the BioBreeding (BB) rat. Factors associated with islet autoimmunity are dilute and difficult to directly measure in the periphery. Therefore, we previously utilized microarray-based bioassay where human T1D sera were used to induce a disease-specific gene expression signature in unrelated, healthy peripheral blood mononuclear cells (PBMC). Here we report that sera of both spontaneously diabetic BB DRlyp/lyp and diabetes inducible BB DR+/+ rats induce inflammatory signatures in healthy PBMC of non-diabetic Brown Norway (BN) rats, while allogeneic BN sera does not. Consistent with their common T1D susceptibility, transcriptional signatures of both BB sub-strains share identity, including cytokines, immune receptors and signaling molecules. However, like the human T1D signature, the DRlyp/lyp signature, which is associated with progression to diabetes, is differentiated from that of the DR+/+ by induction of many IL-1 regulated genes. Addition of IL-1RA to cultures significantly ablates the DRlyp/lyp signature. Daily treatment of DRlyp/lyp rats with human recombinant IL-1RA significantly delays onset and sera of treated animals do not induce the characteristic IL-1 signature. Consistent with the presence of immune regulation in DR+/+ rats is induction of a signature showing negative regulation of the NFκB pathway. This study supports prior human investigations of serum that reflect disease processes associated with progression to T1D and add to a growing body of evidence implicating the balance of IL-1 and IL-1RA as key factors influencing the severity and outcome of inflammatory responses. Experiment Overall Design: The induction of gene expression was accomplished by culturing the PBMCs of healthy day180 BN donor rats for 6 h at 37°C in 5% CO2 with a) 20% autologous BN plasma(10 replicates), b) allogeneic BN plasma (15 replicates), c) allogeneic DRlyp/lyp plasma (day 60, 4 replicates), or d) DR+/+ plasma (day 60, 4 replicates). Healthy BN PBMCs were also cultured with d60 DRlyp/lyp serum supplemented with IL-1RA (4 replicates) as well as autologous BN serum supplemented with IL-1B (4 replicates), to respectively block or induce IL-1 mediated gene expression. To determine if IL-1RA-treatment of DRlyp/lyp rats would result in modulation of the DRlyp/lyp signature, additional DRlyp/lyp rats were treated with hIL-1RA (n=6) or PBS (n=6). Rats were treated for 10 days, beginning at d30, then serum was collected at d40 and used to induce gene expression in PBMCs isolated from healthy d180 BN rats. Thus, there are a total of 53 chips being the addition of all the sample conditions mentioned above.

Dataset Information

Genome-wide mRNA profiling of adult human pancreatic beta and duct cells in comparison to other human tissues

Publications

Clusters of conserved beta cell marker genes for assessment of beta cell phenotype.

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