[Show abstract][Hide abstract] ABSTRACT: Preserving β cell function during the development of obesity and insulin resistance would limit the worldwide epidemic of type 2 diabetes (T2DM). Endoplasmic reticulum (ER) calcium (Ca(2+)) depletion induced by saturated free fatty acids and cytokines causes β cell ER stress and apoptosis, but the molecular mechanisms behind these phenomena are still poorly understood. Here, we demonstrate that palmitate-induced sorcin (SRI) down-regulation, and subsequent increases in glucose-6-phosphatase catalytic subunit-2 (G6PC2) levels contribute to lipotoxicity. SRI is a calcium sensor protein involved in maintaining ER Ca(2+) by inhibiting ryanodine receptor activity and playing a role in terminating Ca(2+)-induced Ca(2+) release. G6PC2, a GWAS gene associated with fasting blood glucose, is a negative regulator of glucose-stimulated insulin secretion (GSIS). High fat feeding in mice and chronic exposure of human islets to palmitate decreases endogenous SRI expression while levels of G6PC2 mRNA increase. Sorcin null mice are glucose intolerant, with markedly impaired GSIS and increased expression of G6pc2. Under high fat diet, mice overexpressing SRI in the β cell display improved glucose tolerance, fasting blood glucose and GSIS, whereas G6PC2 levels are decreased and cytosolic and ER Ca(2+) are increased in transgenic islets. SRI may thus provide a target for intervention in T2DM.
[Show abstract][Hide abstract] ABSTRACT: The expansion of cells for regenerative therapy will require the genetic dissection of complex regulatory mechanisms governing
the proliferation of non-transformed human cells. Here we report the development of a high-throughput RNAi screening strategy
specifically for use in primary cells and demonstrate that silencing the cell cycle dependent kinase inhibitors CDKN2C/p18
or CDKN1A/p21 facilitates cell cycle entry of quiescent adult human pancreatic beta cells. This work identifies p18 and p21
as novel targets for promoting proliferation of human beta cells and demonstrates the promise of functional genetic screens
for dissecting therapeutically relevant state changes in primary human cells.
Preview · Article · Jan 2016 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Recent years have seen an increased focus on human islet biology, and exciting findings in the stem cell and genomic arenas highlight the need to define the key features of mature human islets and β-cells. Donor and organ procurement parameters impact human islet yield, although for research purposes islet yield may be secondary in importance to islet function. We examined the feasibility of a research-only human islet isolation, distribution, and biobanking program; and whether key criteria such as cold ischemia time (CIT) and metabolic status may be relaxed and still allow successful research-focused isolations, including from donors with type 1 diabetes (T1D) and type 2 diabetes (T2D). Through 142 isolations over ∾5 years we confirm that CIT and HbA1c each have weak negative impacts on isolation purity and yield, and extending CIT beyond the typical clinical isolation cutoff of 12 h (to ≥18 h) had only modest impact on islet function. Age and HbA1c/T2D status negatively impacted secretory function, however these and other biological (sex, body mass index) and procurement/isolation variables (CIT, time in culture) appear to make only a small contribution to the heterogeneity of human islet function. This work demonstrates the feasibility of extending acceptable CIT for research-focused human islet isolation and highlights the biological variation in function of human islets from donors with and without diabetes.
[Show abstract][Hide abstract] ABSTRACT: We report the long-term follow-up of the efficacy and safety of islet transplantation in seven type 1 diabetic subjects from the United States enrolled in the multicenter international Edmonton Protocol who had persistent islet function after completion of the Edmonton Protocol. Subjects were followed up to 12 years with serial testing for sustained islet allograft function as measured by C-peptide. All seven subjects demonstrated continued islet function longer than a decade from the time of first islet transplantation. One subject remained insulin independent without the need for diabetic medications or supplemental transplants. One subject who was insulin-independent for over 8 years experienced graft failure 10.9 years after the first islet transplant. The remaining six subjects demonstrated continued islet function upon trial completion, although three had received a supplemental islet transplant each. At trial completion, five subjects were receiving insulin and two remained insulin independent, although one was treated with liraglutide. The median hemoglobin A1c was 6.3% (45 mmol/mol). All subjects experienced progressive decline in the C-peptide/glucose ratio. No patients experienced severe hypoglycemia, opportunistic infection, or lymphoma. Thus, although the rate and duration of insulin independence was low, the Edmonton Protocol was safe in the long term. Alternative approaches to islet transplantation are under investigation.
No preview · Article · Oct 2015 · American Journal of Transplantation
[Show abstract][Hide abstract] ABSTRACT: Islet transplantation is a successful β-cell replacement therapy for selected patients with type 1 diabetes mellitus. Although high rates of early insulin independence are achieved routinely, long-term function wanes over time. Intraportal transplantation is associated with procedural risks, requires multiple donors, and does not afford routine biopsy. Stem cell technologies may require potential for retrievability, and graft removal by hepatectomy is impractical. There is a clear clinical need for an alternative, optimized transplantation site. The subcutaneous space is a potential substitute, but transplantation of islets into this site has routinely failed to reverse diabetes. However, an implanted device, which becomes prevascularized before transplantation, may alter this equation.
Syngeneic mouse islets were transplanted subcutaneously within Sernova Corp's Cell Pouch (CP). All recipients were preimplanted with CPs 4 weeks before diabetes induction and transplantation. After transplantation, recipients were monitored for glycemic control and glucose tolerance.
Mouse islets transplanted into the CP routinely restored glycemic control with modest delay and responded well to glucose challenge, comparable to renal subcapsular islet grafts, despite a marginal islet dose, and normoglycemia was maintained until graft explantation. In contrast, islets transplanted subcutaneously alone failed to engraft. Islets within CPs stained positively for insulin, glucagon, and microvessels.
The CP is biocompatible, forms an environment suitable for islet engraftment, and offers a potential alternative to the intraportal site for islet and future stem cell therapies.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.
[Show abstract][Hide abstract] ABSTRACT: In islet transplantation, deceased cardiac death (DCD) donation has been identified as a potential extended source. There are currently no studies comparing outcomes between these categories, and our goal was to compare islet isolation success rates and transplantation outcomes between DCD and neurological determination of death (NDD) donors.
Islet isolations from 15 DCD and 418 NDD were performed in our centre between September 2008 and September 2014. Donor variables, islet yields, metabolic function of isolated isled and insulin requirements at 1-month post transplant were compared.
Compared to NDD, pancreata from DCD were more often procured locally and donors required less vasopressive support (p<0.001 and p=0.023, respectively), but the other variables were similar between groups. Pre- and post-purification islet yields were similar between NDD and DCD (576 vs. 608 x10(3) islet equivalent, p=0.628 and 386 vs. 379, p=0.881, respectively). The metabolic function was similar between NDD and DCD, as well as the mean decrease in insulin-requirement at 1-month post-transplantation (NDD: 64.82%; DCD: 60.17% reduction, p=0.517).
These results support the broader use of DCD pancreata for islet isolation. A much larger DCD islet experience will be required to truly determine non-inferiority of both short and long-term outcomes. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
No preview · Article · Aug 2015 · Transplant International
[Show abstract][Hide abstract] ABSTRACT: There are potential advantages to the low-temperature (-196°C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years.
Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes.
The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation.
We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.
[Show abstract][Hide abstract] ABSTRACT: Transplantation of donor-derived islets into the liver is a successful cellular replacement therapy for individuals with diabetes. However, the hepatic vasculature is not an optimal transplant site for several reasons, including graft attrition and the inability to retrieve or image the islets. Here we describe islet transplantation into a prevascularized, subcutaneous site created by temporary placement of a medically approved vascular access catheter. In mice with streptozotocin (STZ)-induced diabetes, transplantation of ∼500 syngeneic islets into the resulting 'device-less' space reversed diabetes in 91% of mice and maintained normoglycemia for >100 days. The approach was also effective in mice with pre-existing diabetes, in another mouse strain that mounts a more vigorous inflammatory response, and across an allogeneic barrier. These results demonstrate that transient priming of a subcutaneous site supports diabetes-reversing islet transplantation in mouse models without the need for a permanent cell-encapsulation device.
No preview · Article · Apr 2015 · Nature Biotechnology
[Show abstract][Hide abstract] ABSTRACT: We report a unique first case of benign heterotopic pancreas arising within the proximal hepatic bile duct, containing a focus of intraductal papillary mucinous neoplasm (IPMN). The condition was diagnosed on pathological explant after left hepatic lobectomy with total extrahepatic bile duct excision.
[Show abstract][Hide abstract] ABSTRACT: Decades of research have brought islet transplantation from the dream of few to the reality of many. This procedure started as an experimental method to treat type 1 diabetes mellitus, which features β-cell dysfunction due to auto immunity. The burden of complication in these patients is cumulative and has become a major health problem, despite the availability of insulin therapy. The procedure relies on a sequence of finely orchestrated procedures starting in the donor and followed by several steps to isolate high quality islets. Extensive research is nowadays focussed in improving islet engraftment by providing a more beneficial environment to newly transplanted cells, coupled by more effective immunosuppressive drugs to avoid allo and auto immunity. Excellent results are now a reality in the most specialized centers. Yet, further steps are required to transform this low-risk treatment in a widely available and long-lasting therapy for diabetics worldwide.