The Science of Stem Cell Biobanking: Investing in the Future

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Systematic Chromosomal Analysis of Cultured Mouse Neural Stem Cell Lines

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Cx36 makes channels coupling human pancreatic β-cells, and correlates with insulin expression

Previous studies have documented that the insulin-producing β-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes β-cells, leading to secretory defects reminiscent of those observed in type 2 diabetes. Since human islets differ in several respects from those of laboratory rodents, we have now screened human pancreas, and islets isolated thereof, for expression of a variety of connexin genes, tested whether the cognate proteins form functional channels for islet cell exchanges, and assessed whether this expression changes with β-cell function in islets of control and type 2 diabetics. Here, we show that (i) different connexin isoforms are differentially distributed in the exocrine and endocrine parts of the human pancreas; (ii) human islets express at the transcript level different connexin isoforms; (iii) the membrane of β-cells harbors detectable levels of gap junctions made of Cx36; (iv) this protein is concentrated in lipid raft domains of the β-cell membrane where it forms gap junctions; (v) Cx36 channels allow for the preferential exchange of cationic molecules between human β-cells; (vi) the levels of Cx36 mRNA correlated with the expression of the insulin gene in the islets of both control and type 2 diabetics. The data show that Cx36 is a native protein of human pancreatic islets, which mediates the coupling of the insulin-producing β-cells, and contributes to control β-cell function by modulating gene expressio

Cx36 makes channels coupling human pancreatic β-cells, and correlates with insulin expression

Previous studies have documented that the insulin-producing beta-cells of laboratory rodents are coupled by gap junction channels made solely of the connexin36 (Cx36) protein, and have shown that loss of this protein desynchronizes beta-cells, leading to secretory defects reminiscent of those observed in type 2 diabetes. Since human islets differ in several respects from those of laboratory rodents, we have now screened human pancreas, and islets isolated thereof, for expression of a variety of connexin genes, tested whether the cognate proteins form functional channels for islet cell exchanges, and assessed whether this expression changes with beta-cell function in islets of control and type 2 diabetics. Here, we show that (i) different connexin isoforms are differentially distributed in the exocrine and endocrine parts of the human pancreas; (ii) human islets express at the transcript level different connexin isoforms; (iii) the membrane of beta-cells harbors detectable levels of gap junctions made of Cx36; (iv) this protein is concentrated in lipid raft domains of the beta-cell membrane where it forms gap junctions; (v) Cx36 channels allow for the preferential exchange of cationic molecules between human beta-cells; (vi) the levels of Cx36 mRNA correlated with the expression of the insulin gene in the islets of both control and type 2 diabetics. The data show that Cx36 is a native protein of human pancreatic islets, which mediates the coupling of the insulin-producing beta-cells, and contributes to control beta-cell function by modulating gene expression.The Swiss National Science Foundation (310000-122430 to P.Me), the Juvenile Diabetes Research Foundation (1-2005-1084 to V.C., 1-2007-158 to P.Me), the National Institute of Health (DK55183 to V.C.), the European Union (FP6-Integrated Project EuroDia LSHM-CT-2006-518153 to P.Ma; FP-7 BETAIMAGE 222980 to P.Me), Novo Nordisk (to P.Me) and The Larry L. Hillblom Foundation (to V.C.). Image analysis was performed at The National Center for Microscopy and Imaging Research (NIH grant RR4050 to M. Ellisman). Fresh human islets were provided by the Cell Isolation and Transplantation Cente

Endothelium-Derived Netrin-4 Supports Pancreatic Epithelial Cell Adhesion and Differentiation through Integrins α2β1 and α3β1

BACKGROUND: Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1-3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified. METHODOLOGY/PRINCIPAL FINDINGS: Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins α2β1 and α3β1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins α2β1 and α3β1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and up-regulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated. CONCLUSIONS/SIGNIFICANCE: Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors α2β1 and α3β1 integrins

SEL1L Regulates Adhesion, Proliferation and Secretion of Insulin by Affecting Integrin Signaling

<div><p>SEL1L, a component of the endoplasmic reticulum associated degradation (ERAD) pathway, has been reported to regulate the (<i>i</i>) differentiation of the pancreatic endocrine and exocrine tissue during the second transition of mouse embryonic development, (<i>ii</i>) neural stem cell self-renewal and lineage commitment and (<i>iii</i>) cell cycle progression through regulation of genes related to cell-matrix interaction. Here we show that in the pancreas the expression of SEL1L is developmentally regulated, such that it is readily detected in developing islet cells and in nascent acinar clusters adjacent to basement membranes, and becomes progressively restricted to the islets of Langherans in post-natal life. This peculiar expression pattern and the presence of two inverse RGD motifs in the fibronectin type II domain of SEL1L protein indicate a possible interaction with cell adhesion molecules to regulate islets architecture. Co-immunoprecipitation studies revealed SEL1L and ß1-integrin interaction and, down-modulation of SEL1L in pancreatic ß-cells, negatively influences both cell adhesion on selected matrix components and cell proliferation likely due to altered ERK signaling. Furthermore, the absence of SEL1L protein strongly inhibits glucose-stimulated insulin secretion in isolated mouse pancreatic islets unveiling an important role of SEL1L in insulin trafficking. This phenotype can be rescued by the ectopic expression of the ß1-integrin subunit confirming the close interaction of these two proteins in regulating the cross-talk between extracellular matrix and insulin signalling to create a favourable micro-environment for ß-cell development and function.</p></div

SEL1L expression in fetal and adult mouse pancreas.

<p>Representative images of pancreatic sections from E16.5 mouse embryos (<b>A</b>–<b>F</b>) and 8-weeks-old mice (<b>G</b>–<b>L</b>) immunostained for SEL1L (green; <b>A</b>, <b>D</b>, <b>G</b> and <b>J</b>), glucagon (red; <b>B</b> and <b>H</b>) and insulin (red; <b>E</b> and <b>K</b>). Dual-color immunoflurescence showed SEL1L specific immunoreactivity (<i>green, </i><b>C </b><i>and </i><b>F</b>) in the nascent acinar tissue and in the developing islets (<i>asterisks</i>) stained for glucagon (red, <b>C</b>) and insulin (<i>red, </i><b>F</b>). While exocrine tissue, in the adult mouse, didn’t show any SEL1L immunoreactivity (<i>green, </i><b>I </b><i>and </i><b>J</b>), endocrine cells revealed a marked expression of SEL1L protein with a strong cytoplasmic immunoreactivity in α-cells (stained for glucagon in red, <b>I</b>) and a moderate expression in β-cells (stained for insulin in red, <b>L</b>). Scale bar = 50 µm.</p

SEL1L interacts with integrins.

<p>(<b>A</b>) Co-immunoprecipitation for integrin subunits followed by western blotting for SEL1L (<i>left panel</i>) and viceversa (<i>right panel</i>); arrows indicate 130-kDa mature β1 integrin and 110-kDa precursor. (<b>B</b>) MIN6 cells immunostained for SEL1L (<i>green</i>) and β1 integrin (<i>red</i>); a higher magnification of SEL1L/ß1 integrin co-localization to plasma membranes is shown in the inset of the right panel. Nuclei (<i>blue</i>) are counterstained with Hoechst 33258.</p

Impact of SEL1L down-modulation on insulin secretion and cell proliferation.

<p>Insulin release of MIN6 transfected (<b>A</b>) and mouse islet nucleofected (<b>B</b>) cells was quantified after 1-hour stimulation with 22.8 mM glucose. Values are normalized by DNA content and are expressed as a mean ± SD of fold increase in insulin release over basal glucose concentration from three separate experiments. MIN6 transfected cells (<b>C</b>) and mouse islet nucleofected cells (<b>D</b>) were assayed by qPCR for effective <i>Sel1l</i> down-modulation by siRNA. Values are expressed as fold expression ± SD relative to un-treated sample (value = 1) and normalized to <i>Hprt</i>. (<b>E</b>) To assess SEL1L-dependent cell proliferation, MIN6 transfected and untransfected cells were pulsed for 1 hour with BrdU, fixed and immunostained. Frequency of BrdU-positive cells were represented as percentage of total number of nuclei counted and expressed as means ± SD from three independent experiments. (<b>F</b>) Changes in the expression of the key regulators of the cell cycle progression Cyclin D1 (<i>Ccdn1</i>) and p21 (<i>Cdkn1a</i>) were validated by qPCR; values are expressed as fold expression relative to un-treated sample (value = 1) and normalized to <i>Hprt</i>.</p