A major obstacle to successful islet transplantation for both type 1 and 2 diabetes is an inadequate supply of insulin-producing tissue. This need for transplantable human islets has stimulated efforts to expand existing pancreatic islets and͞or grow new ones. To test the hypothesis that human adult duct tissue could be expanded and differentiated in vitro to form islet cells, digested pancreatic tissue that is normally discarded from eight human islet isolations

Diabetes is a chronic debilitating disease that results from insufficient production of insulin from pancreatic β-cells. Islet cell replacement can effectively treat diabetes but is currently severely limited by the reliance upon cadaveric donor tissue. We have developed a protocol to efficiently differentiate commercially available human embryonic stem cells (hESCs) in vitro into a highly enriched PDX1+ pancreatic progenitor cell population that further develops in vivo to mature pancreatic endocrine cells. Immature pancreatic precursor cells were transplanted into immunodeficient mice with streptozotocin-induced diabetes, and glycemia was initially controlled with exogenous insulin. As graft-derived insulin levels increased over time, diabetic mice were weaned from exogenous insulin and human C-peptide secretion was eventually regulated by meal and glucose challenges. Similar differentiation of pancreatic precursor cells was observed after transplant in immunodeficient rats. Throughout the in vivo maturation period hESC-derived endocrine cells exhibited gene and protein expression profiles that were remarkably similar to the developing human fetal pancreas. Our findings support the feasibility of using differentiated hESCs as an alternative to cadaveric islets for treating patients with diabetes.

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes.

We describe a new technique for microencapsulation with high-mannuronic acid (high-M) alginate crosslinked with BaCl 2 without a traditional permselective component, which allows the production of biocompatible capsules that allow prolonged survival of syngeneic and allogeneic transplanted islets in diabetic BALB/c and NOD mice for >350 days. The normalization of the glycemia in the transplanted mice was associated with normal glucose profiles in response to intravenous glucose tolerance tests. After explantation of the capsules, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated islets. The retrieved capsules were free of cellular overgrowth and islets responded to glucose stimulation with a 5-to 10-fold increase of insulin secretion. Transfer of splenocytes isolated from transplanted NOD mice to NOD/SCID mice adoptively transferred diabetes, indicating that NOD recipients maintained islet-specific autoimmunity. In conclusion, we have developed a simple technique for microencapsulation that prolongs islet survival without immunosuppression, providing complete protection against allorejection and the recurrence of autoimmune diabetes. Diabetes 50: 1698 -1705, 2001 I slet transplantation represents an important alternative for the treatment of type 1 diabetes but still requires immunosuppressive agents with their serious side effects (1). One approach to avoid such treatment is to protect islets from the host's immune system with a semipermeable, biocompatible membrane (2,3). Transplantation of islets contained in alginate-poly-L-lysine (PLL) capsules was first described by Lim and Sun (4). Numerous studies have shown successful reversal of diabetes by transplantation of islets enclosed in alginate-PLL capsules in streptozotocin-induced animals (5-9). However, limited success has been reported in spontaneously diabetic NOD mice, a model of autoimmune diabetes (10 -13). Various factors have been implicated in the failure of encapsulated islets. A cellular reaction surrounding the capsules has often been observed, which could lead to depletion of oxygen and nutrients (14) or production of toxic cytokines (15). This accumulation of cells could be due to an immune response to the contained islets or to bioincompatibility of the capsular materials (16,17). Failure might also be attributed to problems with ␤-cell viability in the capsules (18).Protection of porcine islets remains a goal of encapsulation, but xenografts might be more difficult to protect than allografts, as suggested by studies performed with permeable polymer membranes (19,20). This concept is important because of the improved prospects for obtaining an abundant supply of human ␤-cells from precursor cells (21,22). The goal of this study was to determine whether stable biocompatible alginate microcapsules without a permselective component, such as PLL or polyethylene-glycol, would be able to protect mouse islets against allorejection and autoimmunity. RESEARCH DESIGN AND METHODSIslet isolation. Islets were isolated from ma...

Efficient differentiation of hESC-derived pancreatic endocrine cells can occur in a macroencapsulation device, yielding glucose-responsive insulin-producing cells capable of reversing diabetes.

Despite improvements in outcomes for human islet transplantation, characterization of islet preparations remains poorly defined. This study used both light (LM) and electron microscopy (EM) to characterize 33 islet preparations used for clinical transplants. EM allowed accurate identification and quantification of cell types with measured cell number fractions (mean ± SEM) 35.6 ± 2.1% β-cells, 12.6 ± 1.0% non-β-islet cells, (48.3 ± 2.6% total islet cells), 22.7 ± 1.5% duct cells, and 25.3 ± 1.8% acinar cells. Of the islet cells, 73.6 ± 1.7% were β cells. For comparison to the literature, estimates of cell number fraction, cell volume, and extracellular volume were combined to convert number fraction data to volume fractions applicable to cells, islets, and the entire preparation. The mathematical framework for this conversion was developed. By volume, β cells were 86.5 ± 1.1% of the total islet cell volume and 61.2 ± 0.8% of intact islets (including the extracellular volume), which is similar to that of islets in the pancreas. Our estimates gave 1560 ± 20 cells in an islet equivalent (volume of 150-μm diameter sphere), of which 1140 ± 15 were β cells. To test if LM analysis of the same tissue samples could provide reasonable estimates of purity of the islet preparations, volume fraction islet tissue was measured on thin sections available from 27 of the clinical preparations by point counting morphometrics. Islet purity (islet volume fraction) of individual preparations determined by LM and EM analysis correlated linearly with excellent agreement (R2 = 0.95). However, islet purity by conventional dithizone staining was substantially higher with a 20-30% overestimation. Thus, both EM and LM provide accurate methods to determine the cell composition of human islets preparations and can help us understand many of the discrepancies of islet composition in the literature.

The aim of this study was to test whether an omental pouch can be used as an alternative site for islet implantation in diabetic monkeys. Here we report the successful engraftment of islets in diabetic cynomolgus monkeys when loaded on a synthetic biodegradable scaffold and placed in an omental pouch. One autologous and 5 allogeneic diabetic monkey transplants under the cover of steroid-free immune suppression were undertaken. Fasting blood glucose (FBG) and c-peptide (CP), exogenous insulin requirements (EIR), intravenous glucose tolerance test (IVGTT), A1C and histopathology were used to assess islet engraftment and survival. All animals achieved CP levels > 1.0 ng/ml following transplant, a 66 – 92% post-transplant decrease in EIR and reduced A1C. Following graft removal, CP became negative and histopathological analysis of the explanted grafts demonstrated well granulated and well vascularized, insulin positive islets, surrounded by T cell subsets and macrophages. Compared to intrahepatic allogeneic islet transplants (n=20), there was a delayed engraftment for omental pouch recipients but similar levels of CP production were ultimately achieved, with a broad range of IEQ/kg transplanted in both sites. Our results suggest this extrahepatic transplantation site has potential as an alternative site for clinical islet cell transplantation.

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