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Perfluorodecalin-enriched fibrin matrix for human islet culture.
Disruption of microenvironment and decrease in oxygen supply during isolation and culture lead to pancreatic islet injury and their poor survival after transplantation. This study aimed to create a matrix for culturing islets, using fibrin as scaffold and perfluorodecalin as oxygen diffusion enhancing medium. Human pancreatic islets were divided in four groups: control, islets cultured in fibrin, islets in fibrin containing non-emulsified perfluorodecalin, and finally islets in fibrin supplemented with emulsified perfluorodecalin. After an overnight culture, cell damage (viability, proinsulin and insulin unregulated release, apoptosis (caspase-3 activation), secretory function, and presence of hypoxia markers (HIF-1a and VEGF expression) were assessed. Islets cultured in a matrix, had similar islet viability to controls (no matrix) but decreased levels of active caspase-3 and unregulated hormone release, but high level of hypoxia markers expression. Although the supplementation of fibrin with non-emulsified perfluorodecalin improves secretory response, there was no decrease in hypoxia markers expression. In contrast, emulsified perfluorodecalin added to the matrix improved islet function, islet viability and maintained level of hypoxia markers similar to control. Fibrin matrix supplemented with emulsified perfluorodecalin can provide a beneficial physical and chemical environment for improved pancreatic human islet function and viability in vitro.
The importance of pancreatic embryonic epithelium for mesenchyme-to-epithelial transition during islet development.
Stem or progenitor cells are a promising potential alternative source of pancreatic islets for transplantation in the treatment of juvenile-onset diabetes. However, to derive islets from such cells, it is important to elucidate the mechanisms of normal pancreatic development. Previous work in our laboratory has shown that, contrary to previous thinking, pancreatic mesenchyme when combined with pancreatic epithelium can contribute cells to islets. However, the signals and role of individual tissues involved in this mesenchyme-to-epithelial transition (MET) have yet to be elucidated. The aim of this study was to investigate whether MET can occur in the absence of pancreatic epithelium. Chick and quail eggs were incubated for 4 days and the dorsal pancreatic buds and stomach rudiments were microdissected. Mesenchyme and epithelium of the organ rudiments were separated after collagenase treatment. Separated pancreatic mesenchyme were cultured alone and in combination with stomach (nonpancreatic). After 7 days of culture, the specimens were analysed using immunohistochemistry for quail-specific nucleolar antigen (QCPN), insulin, and islet precursor cell marker (ISL-1). Pancreatic mesenchyme when cultured in the absence of epithelium did not differentiate into islets, but differentiated into fibroblast-like cells. When pancreatic mesenchyme were cultured in combination with stomach epithelium, there was no evidence of mesenchymally derived islets. We have demonstrated that pancreatic mesenchyme require pancreatic epithelium to differentiate into islet cells. These findings further increase our understanding of normal pancreatic islet development and may help to elucidate the molecular mechanisms of MET in islet development.
Comparison of the collagen VI content within the islet-exocrine interface of the head, body, and tail regions of the human pancreas.
Efficient islet isolation depends on the use of collagenase to digest the extracellular matrix within the islet-exocrine interface, the molecular structure of which is poorly understood. Recently it has been reported that transplantable yields of islets can be isolated from the tail segment of the pancreas alone. This study aimed to quantify and compare the amount of collagenase-resistant collagen VI within the islet-exocrine interface of the head, body, and tail of the human pancreas. Human adult pancreata (n = 5) were retrieved from heart-beating donors (age range, 40-62 years; cold ischemia times <10 hours). Tissue blocks from the head, body, and tail region of each pancreas were fixed in formalin and processed for immuno-labelling of collagen VI, which was quantified in the islet-exocrine interface using a Zeiss KS-400 image analysis system. Data were expressed as area of collagen at the interface relative to the islet area. Statistical analysis was done using paired t test. The mean islet areas in the head, body, and tail regions were not significantly different. Collagen VI was uniformly present within the islet-exocrine interface of all regions of the pancreas and was 0.326 +/- 0.064, 0.324 +/- 0.060, and 0.334 +/- 0.052 microm(2)/islet area (P = .441) in the head, body, and tail, respectively. The content of collagen VI within the islet-exocrine interface was uniform throughout all parts of the adult pancreas. Targeting this collagen subtype with novel collagenase blends may result in consistently improved islet yields and enable a wider number of available donor pancreata to be used.
Foregut Mesenchyme Contributes Cells to Islets during Pancreatic Development in a 3-Dimensional Avian Model.
Current interest in the potential use of pancreatic stem-cells in the treatment of insulin dependent diabetes mellitus has led to increased research into normal pancreatic development. Pancreatic organogenesis involves branching morphogenesis of undifferentiated epithelium within surrounding mesenchyme. Current understanding is that the pancreatic islets develop exclusively from the epithelium of the embryonic buds. However, a cellular contribution to islets by mesenchyme has not been conclusively excluded. We present evidence that the mesenchyme of both the dorsal pancreatic bud and stomach rudiment make a substantial contribution of cells to islets during development in a three-dimensional avian model. These data suggest that mesenchyme can be a source not only of signals but also of cells for the definitive epithelia, making pancreatic organogenesis more akin to that of the kidney than to other endodermal organs. This raises the possibility for the use of mesenchymal cells as stem-or progenitor-cells for islet transplantation.
Gastroschisis: a national cohort study to describe contemporary surgical strategies and outcomes.
BACKGROUND: Information on adoption of newer surgical strategies for gastroschisis and their outcomes is largely limited to hospital-based studies. The aim of this study was to use a new UK national surveillance system to identify cases and thus to describe the contemporary surgical management and outcomes of gastroschisis. METHODS: We conducted a national cohort study using the British Association of Paediatric Surgeons Congenital Anomalies Surveillance System to identify cases between October 2006 and March 2008. RESULTS: All 28 surgical units in the United Kingdom and Ireland participated (100%). Data were received for 95% of notified cases of gastroschisis (n = 393). Three hundred thirty-six infants (85.5%) had simple gastroschisis; 45 infants (11.5%) had complex gastroschisis. For 12 infants (3.0%), the type of gastroschisis could not be categorized. Operative primary closure (n = 170, or 51%) and staged closure after a preformed silo (n = 120, or 36%) were the most commonly used intended techniques for simple gastroschisis. Outcomes for infants with complex gastroschisis were significantly poorer than for simple cases, although all deaths occurred in the simple group. CONCLUSIONS: This study provides a comprehensive picture of current UK practice in the surgical management of gastroschisis. Further follow-up data will help to elucidate additional prognostic factors and guide future research.
Insulin storage and glucose homeostasis in mice null for the granule zinc transporter ZnT8 and studies of the type 2 diabetes-associated variants.
OBJECTIVE: Zinc ions are essential for the formation of hexameric insulin and hormone crystallization. A nonsynonymous single nucleotide polymorphism rs13266634 in the SLC30A8 gene, encoding the secretory granule zinc transporter ZnT8, is associated with type 2 diabetes. We describe the effects of deleting the ZnT8 gene in mice and explore the action of the at-risk allele. RESEARCH DESIGN AND METHODS: Slc30a8 null mice were generated and backcrossed at least twice onto a C57BL/6J background. Glucose and insulin tolerance were measured by intraperitoneal injection or euglycemic clamp, respectively. Insulin secretion, electrophysiology, imaging, and the generation of adenoviruses encoding the low- (W325) or elevated- (R325) risk ZnT8 alleles were undertaken using standard protocols. RESULTS: ZnT8(-/-) mice displayed age-, sex-, and diet-dependent abnormalities in glucose tolerance, insulin secretion, and body weight. Islets isolated from null mice had reduced granule zinc content and showed age-dependent changes in granule morphology, with markedly fewer dense cores but more rod-like crystals. Glucose-stimulated insulin secretion, granule fusion, and insulin crystal dissolution, assessed by total internal reflection fluorescence microscopy, were unchanged or enhanced in ZnT8(-/-) islets. Insulin processing was normal. Molecular modeling revealed that residue-325 was located at the interface between ZnT8 monomers. Correspondingly, the R325 variant displayed lower apparent Zn(2+) transport activity than W325 ZnT8 by fluorescence-based assay. CONCLUSIONS: ZnT8 is required for normal insulin crystallization and insulin release in vivo but not, remarkably, in vitro. Defects in the former processes in carriers of the R allele may increase type 2 diabetes risks.
Pancreatic islet transplantation.
Pancreatic islet transplantation is a minimally invasive treatment that has the potential to prevent diabetes after total pancreatectomy for benign disease (islet autotransplantation) and to reverse diabetes in those with type 1 diabetes (islet allotransplantation). In both scenarios, the treatment involves extracting sufficient numbers of pancreatic islets from the resected or donor pancreas and infusing these through the portal vein into the patient's liver. The results for both forms of this treatment have been encouraging over recent years, with up to 85% of adult patients remaining insulin independent for at least 1 year after transplantation. However, there are a number of challenges that need to be overcome before these treatments can be used routinely in children. The aim of this review is to outline the rationale, methodology, and current results of islet allotransplantation, and to discuss the potential new developments that could enable this important treatment to be widely applicable to children. Although islet autotransplantation is not discussed in detail, the overall rationale and methods are similar. However, as the procedure involves the transplantation of the patient's own islets, and the indication for the procedure is not for autoimmune disease, there are none of the difficult immune challenges of allotransplantation.
Vascular endothelial growth factor as a survival factor for human islets: effect of immunosuppressive drugs.
AIMS/HYPOTHESIS: Rapamycin, part of the immunosuppressive regimen of the Edmonton protocol, has been shown to inhibit vascular endothelial growth factor (VEGF) production and VEGF-mediated survival signalling in tumour cell lines. This study investigates the survival-promoting activities of VEGF in human islets and the effects of rapamycin on islet viability. MATERIALS AND METHODS: Levels of VEGF and its receptors in isolated human islets and whole pancreas was determined by western blotting and immunostaining. Islet viability following VEGF or immunosuppressive drug treatment was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Islet VEGF release was measured by ELISA. Mouse islets infected with an adenovirus expressing the gene for VEGF were transplanted syngeneically into streptozotocin-induced diabetic mice, with blood glucose levels measured three times per week. RESULTS: Isolated human islets produced multiple isoforms of VEGF and VEGF receptors 1, 2 and 3 and the coreceptor neuropilin 1. Exogenous VEGF (10 ng/ml) prevented human islet death induced by serum starvation, which suggests that VEGF can act as a survival factor for human islets. Transplantation of mouse islets infected with a VEGF-expressing adenovirus in a syngeneic model, improved glycaemic control at day 1 post-transplantation (p < 0.05). Rapamycin at 10 and 100 ng/ml significantly reduced islet VEGF release (by 37 +/- 4% and 43 +/- 6%, respectively; p < 0.05) and at 100 ng/ml reduced islet viability (by 36 +/- 9%) and insulin release (by 47 +/- 7%, all vs vehicle-treated controls; p < 0.05). Tacrolimus had no effect on islet VEGF release or viability. CONCLUSIONS/INTERPRETATION: Our data suggest that rapamycin may have deleterious effects on islet survival post-transplantation, both through a direct effect on islet viability and indirectly through blockade of VEGF-mediated revascularisation.