Project description:We have developped a novel human pancreatic beta cell line: EndoC-βH5. EndoC-βH5 cells are ready-to-use and storable cells with physiological insulin secretion. EndoC-βH5 cells were generated by integrative gene transfer of immortalizing transgenes hTERT and SV40 large T along with Herpes Simplex Virus-1 thymidine kinase into human fetal pancreas. Immortalizing transgenes were removed after amplification using CRE activation and remaining non-excized cells eliminated using ganciclovir. Resulting cells were distributed as ready to use EndoC-βH5 cells. We performed comparative transcriptome analysis with EndoC-βH1 cells , extensive functional and immunological assays. RNA-seq confirmed abundant expression of beta cell transcription factors and functional markers, including incretin receptors.
Project description:We report the actively transcribed chromatin regions in EndoC-βH1 Cells that are associated with histone H3 lysine 27 acetylation mark.
Project description:EndoC-βH1 is emerging as a critical human β cell model to study the genetic and environmental etiologies of β cell (dys)function and diabetes. Comprehensive knowledge of its molecular landscape is lacking, yet required, for effective use of this model. Here, we report chromosomal (spectral karyotyping), genetic (genotyping), epigenomic (ChIP-seq and ATAC-seq), chromatin interaction (Hi-C and Pol2 ChIA-PET), and transcriptomic (RNA-seq and miRNA-seq) maps of EndoC-βH1. Analyses of these maps define known (e.g., PDX1 and ISL1) and putative (e.g., PCSK1 and mir-375) β cell-specific transcriptional cis-regulatory networks and identify allelic effects on cis-regulatory element use. Importantly, comparison with maps generated in primary human islets and/or β cells indicates preservation of chromatin looping but also highlights chromosomal aberrations and fetal genomic signatures in EndoC-βH1. Together, these maps, and a web application we created for their exploration, provide important tools for the design of experiments to probe and manipulate the genetic programs governing β cell identity and (dys)function in diabetes.
Project description:The aim of the study was to characterize the role of PCSK9 in human beta cells. We performed siRNA-mediated knockdown of PCSK9 in human beta cell line EndoC-bH1 and compared the expression profiles against control siRNA-treated cells.
Project description:To investigate the glucocorticoid-mediated transcriptomic changes in human pancreatic islets and the human insulin-secreting EndoC-βH1 cells in order to uncover genes and molecular pathways involved in β-cell steroid stress-response processes.
Project description:EndoC-BH1 cells were plated in normal media and forward transfected with lipofectamine 3000 (2ug cDNA) of empty, OAS1, OAS2, or OAS3 plasmids from Vector builder
Project description:Most obese and insulin resistant individuals do not develop diabetes. This is the result of the capacity of β-cells to adapt and produce enough insulin to cover the needs of the organism. The underlying mechanism of β-cell adaptation in obesity, however, remains unclear. Previous studies have suggested a role for STAT3 in mediating β-cell development and human glucose homeostasis, but little is known about its role in β-cells in obesity. We observed enhanced cytoplasmatic expression of STAT3 in severe obese and diabetic subjects. To address the functional role of STAT3 in adult β-cells, we generated mice with tamoxifen-inducible partial or full deletion of STAT3 in β-cells and fed them a high fat diet before analysis. Interestingly, β-cell homozygous and heterozygous STAT3 deficient obese mice showed glucose intolerance when compared to controls. Gene expression analysis by RNA-seq showed reduced expression of mitochondrial genes in STAT3 knocked-down human EndoC-βH1 cells and was confirmed in FACS-purified β-cells from STAT3 deficient mice. Moreover, knockdown of STAT3 impaired mitochondria activity in EndoC-βH1 and human islets, suggesting a mechanism for STAT3-regulated β-cell function. We propose non-canonical STAT3 activity as a marker of β-cell identity, improving glucose induced insulin secretion in obesity.
Project description:EndoC-bH1 cells were incubated with control siRNA or PCSK9 siRNA for 72 hours. The experiment was powered based PCSK9 knockdown in a pilot experiment. Sample size: 12x12.
