Dynamic Proteomic Analysis of Pancreatic Mesenchyme Reveals Novel Factors That Enhance Human Embryonic Stem Cell to Pancreatic Cell Differentiation

Current approaches in human embryonic stem cell (hESC) to pancreatic beta cell differentiation have largely been based on knowledge gained from developmental studies of the epithelial pancreas, while the potential roles of other supporting tissue compartments have not been fully explored. One such tissue is the pancreatic mesenchyme that supports epithelial organogenesis throughout embryogenesis. We hypothesized that detailed characterization of the pancreatic mesenchyme might result in the identification of novel factors not used in current differentiation protocols. Supplementing existing hESC differentiation conditions with such factors might create a more comprehensive simulation of normal development in cell culture. To validate our hypothesis, we took advantage of a novel transgenic mouse model to isolate the pancreatic mesenchyme at distinct embryonic and postnatal stages for subsequent proteomic analysis. Refined sample preparation and analysis conditions across four embryonic and prenatal time points resulted in the identification of 21,498 peptides with high-confidence mapping to 1,502 proteins. Expression analysis of pancreata confirmed the presence of three potentially important factors in cell differentiation: Galectin-1 (LGALS1), Neuroplastin (NPTN), and the Laminin α-2 subunit (LAMA2). Two of the three factors (LGALS1 and LAMA2) increased expression of pancreatic progenitor transcript levels in a published hESC to beta cell differentiation protocol. In addition, LAMA2 partially blocks cell culture induced beta cell dedifferentiation. Summarily, we provide evidence that proteomic analysis of supporting tissues such as the pancreatic mesenchyme allows for the identification of potentially important factors guiding hESC to pancreas differentiation

Synergy of a Hepatitis C Virus (HCV) NS4A Antagonist in Combination with HCV Protease and Polymerase Inhibitors▿

Rapid emergence of resistance to monotherapy with virus-specific inhibitors necessitates combination therapy. ACH-806 is a hepatitis C virus NS4A inhibitor with a novel mechanism of action and resistance pathway. This compound was synergistic with NS3 protease inhibitors and NS5B nucleoside and nonnucleoside polymerase inhibitors

Synergy of Small Molecular Inhibitors of Hepatitis C Virus Replication Directed at Multiple Viral Targets

Chronic hepatitis C virus (HCV) infection is a significant worldwide health problem with limited therapeutic options. A number of novel, small molecular inhibitors of HCV replication are now entering early clinical trials in humans. Resistance to small molecular inhibitors is likely to be a significant hurdle to their use in patients. A systematic assessment of combinations of interferon and/or novel anti-hepatitis C virus agents from several different mechanistic classes was performed in vitro. Combinations of inhibitors with different mechanisms of action consistently demonstrated more synergy than did compounds with similar mechanisms of action. These results suggest that combinations of inhibitors with different mechanisms of action should be prioritized for assessment in clinical trials for chronic hepatitis C virus infection

β-Cell Insulin Secretion Requires the Ubiquitin Ligase COP1

SummaryA variety of signals finely tune insulin secretion by pancreatic β cells to prevent both hyper-and hypoglycemic states. Here, we show that post-translational regulation of the transcription factors ETV1, ETV4, and ETV5 by the ubiquitin ligase COP1 (also called RFWD2) in β cells is critical for insulin secretion. Mice lacking COP1 in β cells developed diabetes due to insulin granule docking defects that were fully rescued by genetic deletion of Etv1, Etv4, and Etv5. Genes regulated by ETV1, ETV4, or ETV5 in the absence of mouse COP1 were enriched in human diabetes-associated genes, suggesting that they also influence human β-cell pathophysiology. In normal β cells, ETV4 was stabilized upon membrane depolarization and limited insulin secretion under hyperglycemic conditions. Collectively, our data reveal that ETVs negatively regulate insulin secretion for the maintenance of normoglycemia