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Joglekar M.V.,National Center for Cell Science | Joglekar M.V.,St. Vincents Institute | Hardikar A.A.,National Center for Cell Science | Hardikar A.A.,OBrien Institute | Hardikar A.A.,University of Melbourne
Cell Cycle

In vitro generation of insulin-producing cells from stem/progenitor cells presents a promising approach to overcome the scarcity of donor pancreas for cell replacement therapy in diabetes. In this regard, pancreatic islet-derived progenitors are proposed to be a better alternative as they are obtained from cells that can efficiently produce insulin under physiological conditions and are supposed to retain the epigenetic memory for producing 'insulin' even after transition to a mesenchymal-like cell type. However, in last few years there has been significant debate in understanding the origin of such islet-derived mesenchymal-like progenitor cells in vitro. The initial idea proposed that human insulin-producing β-cells contribute to generation of a population of islet-derived endocrine progenitor cells by a process of epithelial-to- mesenchymal transition (EMT) in vitro. This idea was challenged by a series of lineage-tracing studies in mice demonstrating the non-beta origin of mesenchymal cells in culture. However, recent observations made by two independent groups confirm that human islet insulin-producing cells can proliferate and contribute to mesenchymal-like cell populations in vitro. Here, we provide a fact sheet about the observations that are till now reported by several groups regarding origin of mesenchymal-like cells in the cultures of pancreatic islets. © 2010 Landes Bioscience. Source

Phillips B.W.,Singapore Institute of Medical Biology | Crook J.M.,OBrien Institute

The need for new and improved pharmacotherapies in medicine, high late-stage compound attrition in drug discovery, and upcoming patent expirations is driving interest by the pharmaceutical industry in pluripotent stem cells for in vitro modeling and early-stage testing of toxicity and target engagement. In particular, human embryonic and induced pluripotent stem cells represent potentially cost-effective and accessible sources of organ-specific cells that foretell in vivo human tissue response to new chemical entities. Here we consider the potential of these cells as novel tools for drug development, including toxicity screening and metabolic profiling. We hold that despite various challenges to translating proof-of-concept screening platforms to industrial use, the promise of research is considerable, and close to being realized. © 2010 Adis Data Information BV. Source

Chan E.C.,University of Melbourne | Peshavariya H.M.,University of Melbourne | Liu G.-S.,University of Melbourne | Jiang F.,Shandong University | And 2 more authors.
Biochemical and Biophysical Research Communications

The synthesis of extracellular matrix including collagen during wound healing responses involves signaling via reactive oxygen species (ROS). We hypothesized that NADPH oxidase isoform Nox4 facilitates the stimulatory effects of the profibrotic cytokine transforming growth factor (TGF) β1 on collagen production in vitro and in vivo. TGFβ1 stimulated collagen synthesis and hydrogen peroxide generation in mouse cardiac fibroblasts, and both responses were attenuated by a scavenger of superoxide and hydrogen peroxide (EUK-134). Furthermore, by expressing a dominant negative form of Nox4 (Adv-Nox4ΔNADPH) in fibroblasts, TGFβ1-induced hydrogen peroxide production and collagen production were abrogated, suggesting that Nox4-dependent ROS are important for TGFβ1 signaling in collagen production. This was confirmed by the inhibitory effect of an adenovirus carrying siRNA targeting Nox4 (Adv-Nox4i) on TGFβ1-induced collagen synthesis and expression of activated myofibroblasts marker smooth muscle alpha actin. Finally we used a mouse model of subcutaneous sponge implant to examine the role of Nox4 in the local stimulatory effects of TGFβ1 on collagen accumulation in vivo. TGFβ1-induced collagen accumulation was significantly reduced when the sponges were instilled with Adv-Nox4ΔNADPH. In conclusion, Nox4 acts as an intermediary in the signaling of TGFβ1 to facilitate collagen synthesis. © 2012 Elsevier Inc. Source

Denham M.,University of Melbourne | Denham M.,Lund University | Thompson L.H.,Lund University | Thompson L.H.,Florey Neuroscience Institute | And 5 more authors.
Stem Cells

Generation of mesencephalic dopamine (mesDA) neurons from human embryonic stem cells (hESCs) requires several stages of signaling from various extrinsic and intrinsic factors. To date, most methods incorporate exogenous treatment of Sonic hedgehog (SHH) to derive mesDA neurons. However, we and others have shown that this approach is inefficient for generating FOXA2+ cells, the precursors of mesDA neurons. As mesDA neurons are derived from the ventral floor plate (FP) regions of the embryonic neural tube, we sought to develop a system to derive FP cells from hESC. We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to FP lineage. The GLI1+ cells coexpressed FP markers, FOXA2 and Corin, and displayed exocrine SHH activity by ventrally patterning the surrounding neural progenitors. This system results in 63% FOXA2+ cells at the neural progenitor stage of hESC differentiation. The GLI1-transduced cells were also able to differentiate to neurons expressing tyrosine hydroxylase. This study demonstrates that GLI1 is a determinant of FP specification in hESC and describes a highly robust and efficient in vitro model system that mimics the ventral neural tube organizer. © AlphaMed Press. Source

Wu Y.,University of Western Ontario | Li J.,University of Western Ontario | Saleem S.,University of Western Ontario | Yee S.-P.,University of Western Ontario | And 3 more authors.
Laboratory Investigation

Recent evidence has shown that stem cell factor (SCF) and its receptor, c-Kit, have an important role in pancreatic islet development by promoting islet cell differentiation and proliferation. In this study, we examined the role of c-Kit and SCF in the differentiation and proliferation of insulin- and glucagon-producing cells using a human pancreatic duct cell line (PANC-1). Our study showed that increased expression of endocrine cell markers (such as insulin and glucagon) and transcription factors (such as PDX-1 and PAX-6) coincided with a decrease in CK19 and c-Kit cells (P0.001) during PANC-1 cell differentiation, determined by immunofluorescence and qRT-PCR. Cells cultured with exogenous SCF showed an increase in insulin (26%) and glucagon (35%) cell differentiation (P0.01), an increase in cell proliferation (P0.05) and a decrease in cell apoptosis (P0.01). siRNA knockdown of c-Kit resulted in a decrease in endocrine cell differentiation with a reduction in PDX-1 and insulin mRNA, as well as the number of cells immunostaining for PDX-1 and insulin. Taken together, these results show that c-Kit/SCF interactions are involved in mediating islet-like cluster formation and islet-like cell differentiation in a human pancreatic duct cell line. © 2010 USCAP, Inc All rights reserved. Source

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