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Genome-wide effect of islet isolation and in vitro culture on gene expression profile of pancreatic beta-cells enriched tissue.

ABSTRACT: It is known that the stresses encountered during islet isolation have deleterious effects on beta-cell physiology. The nature of these effects, however, is incompletely known, partly due to the heterogeneity of islet preparations. The objective was to assess the genome-wide effect of islet isolation and in-vitro culture on beta-cell transcriptome. Beta cells identified by their intrinsic autofluorescence, were captured from donor pancreas and isolated islets using Laser Capture Microdissection. Beta cells from donor pancreas serve as the control. Our results indicated induction of a strong inflammatory response induced by islet isolation which continues during in vitro culture manifested by upregulation of several cytokines, chemokines and their receptors. IL-8 was induced by 3.6-fold and 56-fold in fresh and 3-days cultured islets respectively. Concordantly, several pancreatic progenitor cell-specific transcription factors like were upregulated in cultured islets, suggesting progressive transformation of mature beta-cell phenotype toward an immature endocrine cell phenotype.

ORGANISM(S): Homo sapiens

PROVIDER: GSE29113 | GEO | 2012/02/12

SECONDARY ACCESSION(S): PRJNA140379

REPOSITORIES: GEO

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Genome-wide effect of islet isolation and in vitro culture on gene expression profile of pancreatic beta-cells enriched tissue.

Project description:It is known that the stresses encountered during islet isolation have deleterious effects on beta-cell physiology. The nature of these effects, however, is incompletely known, partly due to the heterogeneity of islet preparations. The objective was to assess the genome-wide effect of islet isolation and in-vitro culture on beta-cell transcriptome. Beta cells identified by their intrinsic autofluorescence, were captured from donor pancreas and isolated islets using Laser Capture Microdissection. Beta cells from donor pancreas serve as the control. Our results indicated induction of a strong inflammatory response induced by islet isolation which continues during in vitro culture manifested by upregulation of several cytokines, chemokines and their receptors. IL-8 was induced by 3.6-fold and 56-fold in fresh and 3-days cultured islets respectively. Concordantly, several pancreatic progenitor cell-specific transcription factors like were upregulated in cultured islets, suggesting progressive transformation of mature beta-cell phenotype toward an immature endocrine cell phenotype. There are three sample types in our experiment; 1) beta-cells captured from the frozen sections of donor pancreas n=8, 2) beta cells extracted from islets frozen immediately after isolation (d0 islets) n=8 and, 3) beta cells extracted from isolated islets frozen after culturing for 3 days (d3 islets) n=5. For the analysis, beta cells from pancreas were considered as the reference point.

2012-02-12 | E-GEOD-29113 | biostudies-arrayexpress

microRNA expression profile in isolated human pancreatic islets and in human pancreatic islet-derived extracellular vesicles -EVs-

Project description:Human pancreatic islets were isolated from pancreas of deceased donors by Ricordi's procedure and cultured in CMRL 1066 medium additioned with human albumin. EVs were isolated from conditioned medium derived from islet culture after isolation. Once isolated, RNA of islets and islet-derived EVs was extracted and analyzed for microRNA expression within 48 hours after isolation.

2012-07-30 | E-MEXP-3691 | biostudies-arrayexpress

Transcription profiling of cultured normal human islet tissue, islets treated with interferon alpha 2 beta, islet-depleted exocrine pancreas, pancreas, liver and kidney tissue

Project description:The purpose of this study was to identify mRNA transcripts encoding tissue restricted cell surface proteins on human islet tissue that, in turn, might serve as markers for determination of healthy, transplanted and/or diseased islet cell mass. Using oligonucleotide microarrays and clinical grade human islets, we obtained the gene expression profiles of cultured normal islet tissue and compared them to profiles of islets treated with interferon alpha 2 beta, islet-depleted exocrine pancreas, pooled whole pancreas, pooled liver and pooled kidney tissue. Data set filtering and comparisons of gene expression patterns revealed a small set of genes corresponding to transmembrane or membrane associated proteins with limited tissue distributions (present in Islets of Langerhans and, often, central or peripheral nervous system tissues, but absent from exocrine pancreas, liver, kidney and other tissues) with possible utility as novel islet markers. Under the influence of IFN alpha, some of these transcripts show differential expression as confirmed by real time PCR. In addition, we found significant differential expression of two sets of transcripts expressed in islets but with broad tissue distributions, non classical MHC class I b (HLA-G and F) and MHC Class II locus (e.g. HLA-DR alpha and HLA-DQ alpha and beta).

2007-04-06 | E-CBIL-27 | biostudies-arrayexpress

LCM human islet beta cells

Project description:Fresh frozen sections of islets obtained by surgery were laser capture microdissected using autofluorescence to guide selection of beta cell areas of the islet. RNA was extracted and amplified with 2 rounds of T7 linear amplification. Two technical replicates were hybridized to Affymetrix U95Av2 arrays.

2014-10-28 | E-MTAB-3042 | biostudies-arrayexpress

Differences in gene expression in islets isolated from pancreata donated after Cardiac Death relative to Brain Death

Project description:The decreasing numbers of Donation after Brain Death (DBD) donors necessitates the comprehensive evaluation of Donation after Cardiac Death Donors (DCD) as a source of pancreata. The aim of this study was to characterize pancreata and islets from DCD and DBD donors with respect to markers of cellular stress that may indicate compromised islet quality. Immunohistochemical staining of pre-isolation pancreas biopsies found increased numbers of caspase 3 positive islets in DBD, while markers of oxidative stress (nitrotyrosine, CML, and HNE) were elevated in DCD. Assessment of islet quality by standard (yield, morphology, fluorescence microscopy, and glucose stimulated insulin secretion) and novel methods (flow cytometry, HPLC quantification of ATP) did not reveal significant differences. However, the post culture loss of DCD islets was increased compared to DBD, and DCD islets showed delayed functional potency when transplanted into diabetic NOD.scid mice. Microarray analysis of cultured islets showed increased expression of multiple stress pathway related genes in DCD compared to DBD. Together these data indicate that the current standard donor management, pancreas recovery and preservation practices are insufficient to quench the oxidative stress injury suffered by DCD islets which leads to loss in culture and may complicate their use in clinical transplant. Keywords: cell type comparison

2010-02-09 | GSE14839 | GEO

Transcriptional profiling of stress-response in cultured porcine islets

Project description:An increasing demand for cell-based diabetes therapy could be met through xenotransplantation of adult porcine islets. Use of islet xenotranplantation on a large scale would require rigorous safety and quality control measures to maximize transplant success. Development of molecular tools to monitor porcine islet cellular responses to ischemic, osmotic, mechanical and oxidative stresses during islet cell processing and post-isolation culturing would aid the rational design of cytoprotective strategies aimed at improving transplant outcomes. In addition, gene expression signatures informative for islet quality could serve as an adjunct to physiological testing to establish the suitability of islet products for transplantation. Nine adult Landrace sows (2-3.5 y old, 249Â±27 kg) were sacrificed, the pancreases were dissected, and islet cells isolated as previously described [8]. Islet preparation purity was assessed by light microscopy after staining with diphenylthiocarbazone and ranged between 90-95% for the preparations used. Islet yield, based on islet equivalents (IEQ, number of islets standardized to 150 Âµm diameter), was estimated at 1930Â±520 per gram of pancreas tissue.Profiles of islet cells cultured under standard conditions were compared to islet cells cultured under stress conditions with elevated glucose (16.7 mM) or addition of inflammatory cytokines (IL-1ï¢, TNF-ï¡, and IFN-ï§), or both, for 48 hours.

2010-07-01 | E-GEOD-4744 | biostudies-arrayexpress

Expression data from of FACS separated acinar and duct cell at day 4 of suspension cultured human pancreatic exocrine cells

Project description:Donor pancreata were obtained from the Beta Cell Bank of the JDRF Centre for Beta Cell Therapy in Diabetes (Brussels, Belgium), from Pancreatic Islet Processing (ECIT center) of Diabetes Research Institute at the IRCCS San Raffaele Scientific Institute (Milan, Italy) and from the DRWF Human Islet Isolation Facility (Oxford, England). Full written consent for use of donor material for research was obtained according to Belgian, Italian and English laws. This project was approved by the Medical Ethical Committee of all institutions. Cells were cultured in 3D suspension culture for four days in Advanced RPMI supplemented with 5% FBS.

2020-07-18 | GSE154629 | GEO

Expression data from day of isolation and day 4 suspension cultured human pancreatic exocrine cells

2020-07-18 | GSE154628 | GEO

Endocrine cell type sorting and mature architecture in the islets of Langerhans require expression of Roundabout receptors in β cells

Project description:The spatial architecture of the islets of Langerhans is hypothesized to facilitate synchronized insulin secretion among β cells, yet testing this in vivo in the intact pancreas is challenging. Robo βKO mice, in which the genes Robo1 and Robo2 are deleted selectively in β cells, provide a unique model of altered islet spatial architecture without loss of β cell differentiation or islet damage from diabetes. Combining Robo βKO mice with intravital microscopy, we show here that Robo βKO islets have reduced synchronized intra-islet Ca2+ oscillations among β cells in vivo. We provide evidence that this loss is not due to a β cell-intrinsic function of Robo, mis-expression or mis-localization of Cx36 gap junctions, or changes in islet vascularization or innervation, suggesting that the islet architecture itself is required for synchronized Ca2+ oscillations. These results have implications for understanding structure-function relationships in the islets during progression to diabetes as well as engineering islets from stem cells.

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Transcription profiling of mouse pancreatic islets from wild type and DcR3 transgenic mice to investigate primary non-function (PNF) in islet transplantation

Project description:The islet primary non-function (PNF) is a serious problem in islet transplantation. In this study, we investigated whether DcR3-secreting transgenic (Tg) islets could reduce PNF. We generated transgenic mice expressing human DcR3. The transgenically expressed DcR3 protected islets from IFN-gama plus IL-1beta, or TNF-alpha plus IL-1beta-induced dysfunction and apoptosis in vitro. The Tg islets presented significantly reduced PNF after transplantation. <br><br> Three independent batches of islet isolation were carried out. For each batch, islets were obtained from 4 Tg and 4 WT mice, and pooled respectively. The pooled islets (Tg or WT) were then divided into 4 groups: 2 were cultured in the presence of IFN-gamma (0.5 ug/ml) plus IL-1beta (0.5 ng/ml) with 1 harvested at 24 h and 1 harvested at 48 h; 2 were cultured in the presence of TNF-alpha (100 ng/ml) plus IL-1beta (0.5 ng/ml) with 1 harvested at 24 h and 1 harvested at 48 h. Each treatment employed 3 chips using RNA from 3 different batches of islet isolation. For each treatment, genes with a mean signal strength difference above 2-fold between Tg and WT islets were selected for reverse PCR confirmation.

2009-08-15 | E-MEXP-2306 | biostudies-arrayexpress

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