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Find video protocols related to scientific articles indexed in Pubmed.
Clinical Immunosuppressants Inhibit Inflammatory, Proliferative And Reprogramming Potential, But Not Angiogenesis Of Human Pancreatic Duct Cells.
Cell Transplant
PUBLISHED: 09-09-2014
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The presence of pancreatic duct cells in clinical islet grafts may affect long-term metabolic success. Human pancreatic duct cells express factors that may exert both protective and damaging effects on islet cells in the graft. Here, we studied the potential of commonly used immunosuppressive drugs in islet transplantation - sirolimus, tacrolimus and mycophenolate mofetil (MMF) - to influence the inflammatory and angiogenic capacity of human pancreatic duct cells in addition to their proliferation and reprogramming abilities. Our data show that the expression of specific pro-inflammatory cytokines by the human pancreatic duct cells was either unaltered or inhibited by the immunosuppressants studied, especially tacrolimus and MMF, whereas expression of chemotactic and angiogenic factors was unaffected. Although none of the immunosuppressants directly led to duct cell death, MMF prevented duct cell proliferation and sirolimus inhibited Ngn-3-mediated duct-to-(neuro)endocrine cell reprogramming. Our data indicate that the immunosuppressant tacrolimus was the least aggressive on the angiogenic, proliferative, and reprogramming potential of human pancreatic duct cells, while it was most powerful in inhibiting inflammatory cytokines, which may influence the outcome of islet transplantation.
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In vitro reprogramming of pancreatic alpha cells towards a beta cell phenotype following ectopic HNF4? expression.
Mol. Cell. Endocrinol.
PUBLISHED: 08-21-2014
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There is currently a shortage of organ donors available for pancreatic beta cell transplantation into diabetic patients. An alternative source of beta cells is pre-existing pancreatic cells. While we know that beta cells can arise directly from alpha cells during pancreatic regeneration we do not understand the molecular basis for the switch in phenotype. The aim of the present study was to investigate if hepatocyte nuclear factor 4 alpha (HNF4?), a transcription factor essential for a normal beta cell phenotype, could induce the reprogramming of alpha cells towards potential beta cells. We utilised an in vitro model of pancreatic alpha cells, the murine ?TC1-9 cell line. We initially characterised the ?TC1-9 cell line before and following adenovirus-mediated ectopic expression of HNF4?. We analysed the phenotype at transcript and protein level and assessed its glucose-responsiveness. Ectopic HNF4? expression in the ?TC1-9 cell line induced a change in morphology (1.7-fold increase in size), suppressed glucagon expression, induced key beta cell-specific markers (insulin, C-peptide, glucokinase, GLUT2 and Pax4) and pancreatic polypeptide (PP) and enabled the cells to secrete insulin in a glucose-regulated manner. In conclusion, HNF4? reprograms alpha cells to beta-like cells.
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Downregulation of Sox9 expression associates with hepatogenic differentiation of human liver mesenchymal stem/progenitor cells.
Stem Cells Dev.
PUBLISHED: 05-05-2014
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Understanding the mechanisms triggering hepatogenic differentiation of stem/progenitor cells would be useful for studying postnatal liver regeneration and development of liver cell therapies. Many evidences support the involvement of Sox9 transcription factor in liver development. Here, we investigate the possibility of liver mesenchymal stem/progenitor cells to constitutively express Sox9 by using reverse transcription-quantitative polymerase chain reaction, immunocytochemistry, and western blotting. The involvement of Sox9 in hepatogenic differentiation was assessed by following its expression at different steps of the process, evaluating the impact of its altered expression, and analyzing its expression in human liver disease specimen. Liver mesenchymal stem/progenitor cells constitutively express Sox9 at both the mRNA and protein levels. Upon hepatogenic differentiation, Sox9 expression is downregulated mainly in the maturation step after oncostatin M treatment. Induction of Sox9 expression using transforming growth factor beta is accompanied with a decrease of the quality of hepatogenic differentiation. Blunting Sox9 expression using specific ShRNA clearly alters the levels of several hepatic markers, an effect confirmed in HepG2 cells. In human liver disease specimen, Sox9 expression is enhanced at both the mRNA and protein levels compared with healthy donors. The current data demonstrate that Sox9 may play a pivotal role in hepatocyte lineage development, including adult liver mesenchymal stem/progenitor cells. Further studies on the identification of pathways regulated by or regulating Sox9 will certainly gain insight into the molecular networks controlling hepatogenic differentiation.
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IL-6-dependent proliferation of alpha cells in mice with partial pancreatic-duct ligation.
Diabetologia
PUBLISHED: 03-27-2014
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IL-6 was recently shown to control alpha cell expansion. As beta cells expand following partial pancreatic-duct ligation (PDL) in adult mice, we investigated whether PDL also causes alpha cells to expand and whether IL-6 signalling is involved. As alpha cells can reprogramme to beta cells in a number of beta cell (re)generation models, we examined whether this phenomenon also exists in PDL pancreas.
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Prospectively isolated NGN3-expressing progenitors from human embryonic stem cells give rise to pancreatic endocrine cells.
Stem Cells Transl Med
PUBLISHED: 02-03-2014
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Pancreatic endocrine progenitors obtained from human embryonic stem cells (hESCs) represent a promising source to develop cell-based therapies for diabetes. Although endocrine pancreas progenitor cells have been isolated from mouse pancreata on the basis of Ngn3 expression, human endocrine progenitors have not been isolated yet. As substantial differences exist between human and murine pancreas biology, we investigated whether it is possible to isolate pancreatic endocrine progenitors from differentiating hESC cultures by lineage tracing of NGN3. We targeted the 3' end of NGN3 using zinc finger nuclease-mediated homologous recombination to allow selection of NGN3eGFP(+) cells without disrupting the coding sequence of the gene. Isolated NGN3eGFP(+) cells express PDX1, NKX6.1, and chromogranin A and differentiate in vivo toward insulin, glucagon, and somatostatin single hormone-expressing cells but not to ductal or exocrine pancreatic cells or other endodermal, mesodermal, or ectodermal lineages. This confirms that NGN3(+) cells represent pancreatic endocrine progenitors in humans. In addition, this hESC reporter line constitutes a unique tool that may aid in gaining insight into the developmental mechanisms underlying fate choices in human pancreas and in developing cell-based therapies.
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Combining MK626, a novel DPP-4 inhibitor, and low-dose monoclonal CD3 antibody for stable remission of new-onset diabetes in mice.
PLoS ONE
PUBLISHED: 01-01-2014
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Combining immune intervention with therapies that directly influence the functional state of the ?-cells is an interesting strategy in type 1 diabetes cure. Dipeptidyl peptidase-4 (DPP-4) inhibitors elevate circulating levels of active incretins, which have been reported to enhance insulin secretion and synthesis, can support ?-cell survival and possibly stimulate ?-cell proliferation and neogenesis. In the current study, we demonstrate that the DPP-4 inhibitor MK626, which has appropriate pharmacokinetics in mice, preceded by a short-course of low-dose anti-CD3 generated durable diabetes remission in new-onset diabetic non-obese diabetic (NOD) mice. Induction of remission involved recovery of ?-cell secretory function with resolution of destructive insulitis and preservation of ?-cell volume/mass, along with repair of the islet angioarchitecture via SDF-1- and VEGF-dependent actions. Combination therapy temporarily reduced the CD4-to-CD8 distribution in spleen although not in pancreatic draining lymph nodes (PLN) and increased the proportion of effector/memory T cells as did anti-CD3 alone. In contrast, only combination therapy amplified Foxp3+ regulatory T cells in PLN and locally in pancreas. These findings open new opportunities for the treatment of new-onset type 1 diabetes by introducing DPP-4 inhibitors in human CD3-directed clinical trials.
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Reversal of hyperglycemia in diabetic mice by a marginal islet mass together with human blood outgrowth endothelial cells is independent of the delivery technique and blood clot-induced processes.
Islets
PUBLISHED: 11-08-2013
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We recently reported that human blood outgrowth endothelial cells (BOEC) are supportive to reverse hyperglycemia in marginal islet mass-transplanted diabetic mice. In this report, we investigated whether the observed effect was evoked by islet packing in a blood clot prior to transplantation or could be mimicked by another method of islet/cell delivery. A marginal islet mass with or without BOEC was grafted underneath the kidney capsule of diabetic recipient mice via a (blood clot-independent) tubing system and compared with previous islet packing in a blood clot. The effect on metabolic outcome of both delivery techniques as well as the additive effect of BOEC was subsequently evaluated. Marginal islet mass transplantation via a tubing system required more islets per recipient than via a blood clot. Using the tubing method, transplantation of a marginal islet mass combined with 5 x 10 (5) BOEC resulted in reversal of hyperglycemia, improved glucose tolerance and increased kidney insulin content. The present study provides evidence that (1) previous packing in a blood clot results in more effective islet delivery compared with tubing; (2) BOEC exert a beneficial effect on marginal islet transplantation, independent of grafting technique and potential blood clot-induced processes. These data further support the use of BOEC in (pre-) clinical studies that aim to improve current islet transplantation protocols.
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The inactivation of Arx in pancreatic ?-cells triggers their neogenesis and conversion into functional ?-like cells.
PLoS Genet.
PUBLISHED: 10-01-2013
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Recently, it was demonstrated that pancreatic new-born glucagon-producing cells can regenerate and convert into insulin-producing ?-like cells through the ectopic expression of a single gene, Pax4. Here, combining conditional loss-of-function and lineage tracing approaches, we show that the selective inhibition of the Arx gene in ?-cells is sufficient to promote the conversion of adult ?-cells into ?-like cells at any age. Interestingly, this conversion induces the continuous mobilization of duct-lining precursor cells to adopt an endocrine cell fate, the glucagon(+) cells thereby generated being subsequently converted into ?-like cells upon Arx inhibition. Of interest, through the generation and analysis of Arx and Pax4 conditional double-mutants, we provide evidence that Pax4 is dispensable for these regeneration processes, indicating that Arx represents the main trigger of ?-cell-mediated ?-like cell neogenesis. Importantly, the loss of Arx in ?-cells is sufficient to regenerate a functional ?-cell mass and thereby reverse diabetes following toxin-induced ?-cell depletion. Our data therefore suggest that strategies aiming at inhibiting the expression of Arx, or its molecular targets/co-factors, may pave new avenues for the treatment of diabetes.
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Conditional Hypovascularization and Hypoxia in Islets Do Not Overtly Influence Adult ?-Cell Mass or Function.
Diabetes
PUBLISHED: 08-23-2013
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It is generally accepted that vascularization and oxygenation of pancreatic islets are essential for the maintenance of an optimal ?-cell mass and function and that signaling by vascular endothelial growth factor (VEGF) is crucial for pancreas development, insulin gene expression/secretion, and (compensatory) ?-cell proliferation. A novel mouse model was designed to allow conditional production of human sFlt1 by ?-cells in order to trap VEGF and study the effect of time-dependent inhibition of VEGF signaling on adult ?-cell fate and metabolism. Secretion of sFlt1 by adult ?-cells resulted in a rapid regression of blood vessels and hypoxia within the islets. Besides blunted insulin release, ?-cells displayed a remarkable capacity for coping with these presumed unfavorable conditions: even after prolonged periods of blood vessel ablation, basal and stimulated blood glucose levels were only slightly increased, while ?-cell proliferation and mass remained unaffected. Moreover, ablation of blood vessels did not prevent ?-cell generation after severe pancreas injury by partial pancreatic duct ligation or partial pancreatectomy. Our data thus argue against a major role of blood vessels to preserve adult ?-cell generation and function, restricting their importance to facilitating rapid and adequate insulin delivery.
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Transient cytokine treatment induces acinar cell reprogramming and regenerates functional beta cell mass in diabetic mice.
Nat. Biotechnol.
PUBLISHED: 07-12-2013
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Reprogramming of pancreatic exocrine cells into cells resembling beta cells may provide a strategy for treating diabetes. Here we show that transient administration of epidermal growth factor and ciliary neurotrophic factor to adult mice with chronic hyperglycemia efficiently stimulates the conversion of terminally differentiated acinar cells to beta-like cells. Newly generated beta-like cells are epigenetically reprogrammed, functional and glucose responsive, and they reinstate normal glycemic control for up to 248 d. The regenerative process depends on Stat3 signaling and requires a threshold number of Neurogenin 3 (Ngn3)-expressing acinar cells. In contrast to previous work demonstrating in vivo conversion of acinar cells to beta-like cells by viral delivery of exogenous transcription factors, our approach achieves acinar-to-beta-cell reprogramming through transient cytokine exposure rather than genetic modification.
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Mouse beta cell proliferation is inhibited by thymidine analogue labelling.
Diabetologia
PUBLISHED: 06-06-2013
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Long-term labelling of mice with halogenated thymidine analogues is an established method for quantifying the contribution of beta cell proliferation to in vivo beta cell mass expansion in (re)generation models. The method is believed to give accurate information on the accrued number of cycling beta cells over a period of time. Multiple thymidine analogue labelling is applied for evaluating the duration of postmitotic quiescence in beta cells. We hypothesise, however, that long-term labelling by thymidine analogues hampers beta cell proliferation.
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Suppression of epithelial-to-mesenchymal transitioning enhances ex vivo reprogramming of human exocrine pancreatic tissue toward functional insulin-producing ?-like cells.
Diabetes
PUBLISHED: 04-22-2013
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Because of the lack of tissue available for islet transplantation, new sources of ?-cells have been sought for the treatment of type 1 diabetes. The aim of this study was to determine whether the human exocrine-enriched fraction from the islet isolation procedure could be reprogrammed to provide additional islet tissue for transplantation. The exocrine-enriched cells rapidly dedifferentiated in culture and grew as a mesenchymal monolayer. Genetic lineage tracing confirmed that these mesenchymal cells arose, in part, through a process of epithelial-to-mesenchymal transitioning (EMT). A protocol was developed whereby transduction of these mesenchymal cells with adenoviruses containing Pdx1, Ngn3, MafA, and Pax4 generated a population of cells that were enriched in glucagon-secreting ?-like cells. Transdifferentiation or reprogramming toward insulin-secreting ?-cells was enhanced, however, when using unpassaged cells in combination with inhibition of EMT by inclusion of Rho-associated kinase (ROCK) and transforming growth factor-?1 inhibitors. Resultant cells were able to secrete insulin in response to glucose and on transplantation were able to normalize blood glucose levels in streptozotocin diabetic NOD/SCID mice. In conclusion, reprogramming of human exocrine-enriched tissue can be best achieved using fresh material under conditions whereby EMT is inhibited, rather than allowing the culture to expand as a mesenchymal monolayer.
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Adult duct-lining cells can reprogram into ?-like cells able to counter repeated cycles of toxin-induced diabetes.
Dev. Cell
PUBLISHED: 04-16-2013
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It was recently demonstrated that embryonic glucagon-producing cells in the pancreas can regenerate and convert into insulin-producing ?-like cells through the constitutive/ectopic expression of the Pax4 gene. However, whether ? cells in adult mice display the same plasticity is unknown. Similarly, the mechanisms underlying such reprogramming remain unclear. We now demonstrate that the misexpression of Pax4 in glucagon(+) cells age-independently induces their conversion into ?-like cells and their glucagon shortage-mediated replacement, resulting in islet hypertrophy and in an unexpected islet neogenesis. Combining several lineage-tracing approaches, we show that, upon Pax4-mediated ?-to-?-like cell conversion, pancreatic duct-lining precursor cells are continuously mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon(+) and a ?-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition. Importantly, these processes can repeatedly regenerate the whole ? cell mass and thereby reverse several rounds of toxin-induced diabetes, providing perspectives to design therapeutic regenerative strategies.
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Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis.
EMBO J.
PUBLISHED: 02-03-2013
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Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5(+) stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.
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Spatiotemporal patterns of multipotentiality in Ptf1a-expressing cells during pancreas organogenesis and injury-induced facultative restoration.
Development
PUBLISHED: 01-16-2013
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Pancreatic multipotent progenitor cells (MPCs) produce acinar, endocrine and duct cells during organogenesis, but their existence and location in the mature organ remain contentious. We used inducible lineage-tracing from the MPC-instructive gene Ptf1a to define systematically in mice the switch of Ptf1a(+) MPCs to unipotent proacinar competence during the secondary transition, their rapid decline during organogenesis, and absence from the mature organ. Between E11.5 and E15.5, we describe tip epithelium heterogeneity, suggesting that putative Ptf1a(+)Sox9(+)Hnf1?(+) MPCs are intermingled with Ptf1a(HI)Sox9(LO) proacinar progenitors. In the adult, pancreatic duct ligation (PDL) caused facultative reactivation of multipotency factors (Sox9 and Hnf1?) in Ptf1a(+) acini, which undergo rapid reprogramming to duct cells and longer-term reprogramming to endocrine cells, including insulin(+) ?-cells that are mature by the criteria of producing Pdx1(HI), Nkx6.1(+) and MafA(+). These Ptf1a lineage-derived endocrine/?-cells are likely formed via Ck19(+)/Hnf1?(+)/Sox9(+) ductal and Ngn3(+) endocrine progenitor intermediates. Acinar to endocrine/?-cell transdifferentiation was enhanced by combining PDL with pharmacological elimination of pre-existing ?-cells. Thus, we show that acinar cells, without exogenously introduced factors, can regain aspects of embryonic multipotentiality under injury, and convert into mature ?-cells.
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Reversal of hyperglycemia by insulin-secreting rat bone marrow- and blastocyst-derived hypoblast stem cell-like cells.
PLoS ONE
PUBLISHED: 01-01-2013
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?-cell replacement may efficiently cure type 1 diabetic (T1D) patients whose insulin-secreting ?-cells have been selectively destroyed by autoantigen-reactive T cells. To generate insulin-secreting cells we used two cell sources: rat multipotent adult progenitor cells (rMAPC) and the highly similar rat extra-embryonic endoderm precursor (rXEN-P) cells isolated under rMAPC conditions from blastocysts (rHypoSC). rMAPC/rHypoSC were sequentially committed to definitive endoderm, pancreatic endoderm, and ?-cell like cells. On day 21, 20% of rMAPC/rHypoSC progeny expressed Pdx1 and C-peptide. rMAPCr/HypoSC progeny secreted C-peptide under the stimulus of insulin agonist carbachol, and was inhibited by the L-type voltage-dependent calcium channel blocker nifedipine. When rMAPC or rHypoSC differentiated d21 progeny were grafted under the kidney capsule of streptozotocin-induced diabetic nude mice, hyperglycemia reversed after 4 weeks in 6/10 rMAPC- and 5/10 rHypoSC-transplanted mice. Hyperglycemia recurred within 24 hours of graft removal and the histological analysis of the retrieved grafts revealed presence of Pdx1-, Nkx6.1- and C-peptide-positive cells. The ability of both rMAPC and HypoSC to differentiate to functional ?-cell like cells may serve to gain insight into signals that govern ?-cell differentiation and aid in developing culture systems to commit other (pluripotent) stem cells to clinically useful ?-cells for cell therapy of T1D.
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Lineage tracing evidence for transdifferentiation of acinar to duct cells and plasticity of human pancreas.
Gastroenterology
PUBLISHED: 04-06-2011
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Animal studies have indicated that pancreatic exocrine acinar cells have phenotypic plasticity. In rodents, acinar cells can differentiate into ductal precursors that can be converted to pancreatic ductal adenocarcinoma or insulin-producing endocrine cells. However, little is known about human acinar cell plasticity. We developed nongenetic and genetic lineage tracing methods to study the fate of human acinar cells in culture.
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Clusters of conserved beta cell marker genes for assessment of beta cell phenotype.
PLoS ONE
PUBLISHED: 03-21-2011
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The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators.
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Pdx1- and Ngn3-Cre-mediated PLAG1 expression in the pancreas leads to endocrine hormone imbalances that affect glucose metabolism.
Cell Transplant
PUBLISHED: 02-03-2011
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Pleomorphic adenoma gene-like 1 (PLAGL1) has been linked to transient neonatal diabetes mellitus. Here, we investigated the role of the related pleomorphic adenoma gene 1 (PLAG1) in glucose homeostasis. PLAG1 transgenic mice in which expression of the PLAG1 transgene can be targeted to different organs by Cre-mediated modulation were crossed with Pdx1-Cre or Ngn3-Cre mice, resulting in double transgenic P1-Pdx1Cre or P1-Ngn3Cre mice, respectively. P1-Pdx1Cre and P1-Ngn3Cre mice developed hyperplasia of pancreatic islets due to increased ?- and ?- but not ?-cell proliferation. In young P1-Pdx1Cre mice (less than 15 weeks) there was a balanced increase in the pancreatic content of insulin and somatostatin, which was associated with normoglycemia. In older P1-Pdx1Cre mice the pancreatic somatostatin content far exceeded that of insulin, leading to the progressive development of severe hypoglycemia beyond 30 weeks. In contrast, in older P1-Ngn3Cre mice the relative increase of the pancreatic insulin content exceeded that of somatostatin and these mice remained normoglycemic. In conclusion, forced expression of PLAG1 under the control of the Pdx1 or Ngn3 promoter in murine pancreas induces different degrees of endocrine hormone imbalances within the pancreas, which is associated with hypoglycemia in P1-Pdx1Cre mice but not P1-Ngn3Cre mice. These results suggest that once stem cell-derived islet transplantations become possible, the appropriate balance between different hormone-producing cells will need to be preserved to prevent deregulated glucose metabolism.
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Pancreatic beta-cells: from generation to regeneration.
Semin. Cell Dev. Biol.
PUBLISHED: 06-03-2010
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The pancreas is composed of two main compartments consisting of endocrine and exocrine tissues. The majority of the organ is exocrine and responsible for the synthesis of digestive enzymes and for their transport via an intricate ductal system into the duodenum. The endocrine tissue represents less than 2% of the organ and is organized into functional units called islets of Langerhans, comprising alpha-, beta-, delta-, epsilon- and PP-cells, producing the hormones glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide (PP), respectively. Insulin-producing beta-cells play a central role in the control of the glucose homeostasis. Accordingly, absolute or relative deficiency in beta-cells may ultimately lead to type 1 and/or type 2 diabetes, respectively. One major goal of diabetes research is therefore to understand the molecular mechanisms controlling the development of beta-cells during pancreas morphogenesis, but also those underlying the regeneration of adult injured pancreas, and assess their significance for future cell-based therapy. In this review, we will therefore present new insights into beta-cell development with focus on beta-cell regeneration.
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Derepression of Polycomb targets during pancreatic organogenesis allows insulin-producing beta-cells to adopt a neural gene activity program.
Genome Res.
PUBLISHED: 04-15-2010
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The epigenome changes that underlie cellular differentiation in developing organisms are poorly understood. To gain insights into how pancreatic beta-cells are programmed, we profiled key histone methylations and transcripts in embryonic stem cells, multipotent progenitors of the nascent embryonic pancreas, purified beta-cells, and 10 differentiated tissues. We report that despite their endodermal origin, beta-cells show a transcriptional and active chromatin signature that is most similar to ectoderm-derived neural tissues. In contrast, the beta-cell signature of trimethylated H3K27, a mark of Polycomb-mediated repression, clusters with pancreatic progenitors, acinar cells and liver, consistent with the epigenetic transmission of this mark from endoderm progenitors to their differentiated cellular progeny. We also identified two H3K27 methylation events that arise in the beta-cell lineage after the pancreatic progenitor stage. One is a wave of cell-selective de novo H3K27 trimethylation in non-CpG island genes. Another is the loss of bivalent and H3K27me3-repressed chromatin in a core program of neural developmental regulators that enables a convergence of the gene activity state of beta-cells with that of neural cells. These findings reveal a dynamic regulation of Polycomb repression programs that shape the identity of differentiated beta-cells.
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Ectopic expression of E2F1 stimulates beta-cell proliferation and function.
Diabetes
PUBLISHED: 03-18-2010
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Generating functional beta-cells by inducing their proliferation may provide new perspectives for cell therapy in diabetes. Transcription factor E2F1 controls G(1)- to S-phase transition during the cycling of many cell types and is required for pancreatic beta-cell growth and function. However, the consequences of overexpression of E2F1 in beta-cells are unknown.
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Pancreatic exocrine duct cells give rise to insulin-producing beta cells during embryogenesis but not after birth.
Dev. Cell
PUBLISHED: 07-02-2009
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A longstanding unsettled question is whether pancreatic beta cells originate from exocrine duct cells. We have now used genetic labeling to fate map embryonic and adult pancreatic duct cells. We show that Hnf1beta+ cells of the trunk compartment of the early branching pancreas are precursors of acinar, duct, and endocrine lineages. Hnf1beta+ cells subsequent form the embryonic duct epithelium, which gives rise to both ductal and endocrine lineages, but not to acinar cells. By the end of gestation, the fate of Hnf1beta+ duct cells is further restrained. We provide compelling evidence that the ductal epithelium does not make a significant contribution to acinar or endocrine cells during neonatal growth, during a 6 month observation period, or during beta cell growth triggered by ligation of the pancreatic duct or by cell-specific ablation with alloxan followed by EGF/gastrin treatment. Thus, once the ductal epithelium differentiates it has a restricted plasticity, even under regenerative settings.
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The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.
Cell
PUBLISHED: 05-13-2009
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We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.
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Investigation and characterization of the duct cell-enriching process during serum-free suspension and monolayer culture using the human exocrine pancreas fraction.
Pancreas
PUBLISHED: 02-10-2009
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We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells.
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Notch signaling as gatekeeper of rat acinar-to-beta-cell conversion in vitro.
Gastroenterology
PUBLISHED: 01-16-2009
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Exocrine acinar cells in the pancreas are highly differentiated cells that retain a remarkable degree of plasticity. After isolation and an initial phase of dedifferentiation in vitro, rodent acinar cells can convert to endocrine beta-cells when cultured in the presence of appropriate factors. The mechanisms regulating this phenotypic conversion are largely unknown.
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Gene delivery to pancreatic exocrine cells in vivo and in vitro.
BMC Biotechnol.
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Effective gene transfer to the pancreas or to pancreatic cells has remained elusive although it is essential for studies of genetic lineage tracing and modulation of gene expression. Different transduction methods and viral vectors were tested in vitro and in vivo, in rat and mouse pancreas.
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Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming.
PLoS ONE
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Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it.
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Role of the ductal transcription factors HNF6 and Sox9 in pancreatic acinar-to-ductal metaplasia.
Gut
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Growing evidence suggests that a phenotypic switch converting pancreatic acinar cells to duct-like cells can lead to pancreatic intraepithelial neoplasia and eventually to invasive pancreatic ductal adenocarcinoma. Histologically, the onset of this switch is characterised by the co-expression of acinar and ductal markers in acini, a lesion called acinar-to-ductal metaplasia (ADM). The transcriptional regulators required to initiate ADM are unknown, but need to be identified to characterise the regulatory networks that drive ADM. In this study, the role of the ductal transcription factors hepatocyte nuclear factor 6 (HNF6, also known as Onecut1) and SRY-related HMG box factor 9 (Sox9) in ADM was investigated.
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Short-term overexpression of VEGF-A in mouse beta cells indirectly stimulates their proliferation and protects against diabetes.
Diabetologia
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Vascular endothelial growth factor (VEGF) has been recognised by loss-of-function experiments as a pleiotropic factor with importance in embryonic pancreas development and postnatal beta cell function. Chronic, non-conditional overexpression of VEGF-A has a deleterious effect on beta cell development and function. We report, for the first time, a conditional gain-of-function study to evaluate the effect of transient VEGF-A overexpression by adult pancreatic beta cells on islet vasculature and beta cell proliferation and survival, under both normal physiological and injury conditions.
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