Andrew R. Pepper

Islet cell transplantation for the treatment of type 1 diabetes: recent advances and future challenges

Islet transplantation is a well-established therapeutic treatment for a subset of patients with complicated type I diabetes mellitus. Prior to the Edmonton Protocol, only 9% of the 267 islet transplant recipients since 1999 were insulin independent for >1 year. In 2000, the Edmonton group reported the achievement of insulin independence in seven consecutive patients, which in a collaborative team effort propagated expansion of clinical islet transplantation centers worldwide in an effort to ameliorate the consequences of this disease. To date, clinical islet transplantation has established improved success with insulin independence rates up to 5 years post-transplant with minimal complications. In spite of marked clinical success, donor availability and selection, engraftment, and side effects of immunosuppression remain as existing obstacles to be addressed to further improve this therapy. Clinical trials to improve engraftment, the availability of insulin-producing cell sources, as well as alternative transplant sites are currently under investigation to expand treatment. With ongoing experimental and clinical studies, islet transplantation continues to be an exciting and attractive therapy to treat type I diabetes mellitus with the prospect of shifting from a treatment for some to a cure for all.

Revascularization of Transplanted Pancreatic Islets and Role of the Transplantation Site

Since the initial reporting of the successful reversal of hyperglycemia through the transplantation of pancreatic islets, significant research efforts have been conducted in elucidating the process of revascularization and the influence of engraftment site on graft function and survival. During the isolation process the intrinsic islet vascular networks are destroyed, leading to impaired revascularization after transplant. As a result, in some cases a significant quantity of the beta cell mass transplanted dies acutely following the infusion into the portal vein, the most clinically used site of engraftment. Subsequently, despite the majority of patients achieving insulin independence after transplant, a proportion of them recommence small, supplemental exogenous insulin over time. Herein, this review considers the process of islet revascularization after transplant, its limiting factors, and potential strategies to improve this critical step. Furthermore, we provide a characterization of alternative transplant sites, analyzing the historical evolution and their role towards advancing transplant outcomes in both the experimental and clinical settings.

Current status of clinical islet transplantation

Islet transplantation (IT) is today a well-established treatment modality for selected patients with type 1 diabetes mellitus (T1DM). After the success of the University of Alberta group with a modified approach to the immune protection of islets, the international experience grew along with the numbers of transplants in highly specialized centers. Yet, long-term analysis of those initial results from the Edmonton group indicated that insulin-independence was not durable and most patients return to modest amounts of insulin around the fifth year, without recurrent hypoglycemia events. Many phenomena have been identified as limiting factor for the islet engraftment and survival, and today all efforts are aimed to improve the quality of islets and their engrafting process, as well as more optimized immunosuppression to facilitate tolerance and ultimately, better long term survival. This brief overview presents recent progress in IT. A concise historical perspective is provided, along with the latest efforts to improve islet engraftment, immune protection and ultimately, prolonged graft survival. It is apparent that as the community continues to work together further optimizing IT, it is hopeful a cure for T1DM will soon be achievable.

Diabetes Is Reversed in a Murine Model by Marginal Mass Syngeneic Islet Transplantation Using a Subcutaneous Cell Pouch Device

Background Islet transplantation is a successful &bgr;-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. Methods Syngeneic mouse islets were transplanted subcutaneously within Sernova Corps 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. Results 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. Conclusions 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.

Biologic Agents in Islet Transplantation

Islet transplantation is today an accepted modality for treating selected patients with frequent hypoglycemic events or severe glycemic lability. Despite tremendous progress in islet isolation, culture, and preservation, clinical use is still restricted to a limited subset, and lifelong immunosuppression is required. Issues surrounding limited islet revascularization and immune destruction remain. One of the major challenges is to prevent alloreactivity and recurrence of autoimmunity against β-cells. These two hurdles can be effectively reduced by immunosuppressive therapy combining induction and maintenance treatments. The introduction of highly potent and selective biologic agents has significantly reduced the frequency of acute rejection and has prolonged graft survival, while minimizing the complications of this therapeutic scheme. This review will address the most important biological agents used in islet transplantation. We provide a historical perspective of their introduction into clinical practice and their role in current clinical protocols, aiming at improved engraftment efficiency, increased long-term survival, and better overall results of clinical islet transplantation.

Microbial Contamination of Clinical Islet Transplant Preparations Is Associated with Very Low Risk of Infection

BACKGROUND Several published studies have analyzed microbial contamination rates of islet products, ranging from 0% to 16%. However, few studies make reference to potential clinical consequences for transplant recipients and possible impact on islet survival. MATERIALS AND METHODS The current study defines rates of microbiological contamination of islet products under current good manufacturing practice conditions in 164 patients receiving 343 transplants at a single institution. RESULTS Nineteen (5.5%) islet preparations showed positive microbial growth with a majority (79.4%) due to Gram-positive organisms. The most frequently identified microorganism was coagulase-negative Staphylococcus (nine of 19 [47.3%]), followed by polymicrobial organisms (eight of 19 [42.1%]). No patient developed signs of clinical infection, and there were no hepatic abscesses evident on imaging by ultrasound or magnetic resonance imaging (none of 19 [0%]), despite the use of potent T-depletional induction. Finally, we could not demonstrate any negative impact of microbiological contamination on long-term islet graft survival. CONCLUSIONS Microbiological contamination of the final islet preparation appears to have little or no effect on patients or on islet survival when appropriate antibiotics are given. However, preparation sterility should be guaranteed at all cost in order maximize patient safety and avoid potential complications in immunosuppressed patients.

Glutathione ethyl ester supplementation during pancreatic islet isolation improves viability and transplant outcomes in a murine marginal islet mass model.

Background The success of pancreatic islet transplantation still faces many challenges, mainly related to cell damage during islet isolation and early post-transplant. The increased generation of reactive oxygen species (ROS) during islet isolation and the consumption of antioxidant defenses appear to be an important pathway related to islet damage. Methodology/Principal Findings In the present study we evaluated whether supplementation of glutathione-ethyl-ester (GEE) during islet isolation could improve islet viability and transplant outcomes in a murine marginal islet mass model. We also cultured human islets for 24 hours in standard CMRL media with or without GEE supplementation. Supplementation of GEE decreased the content of ROS in isolated islets, leading to a decrease in apoptosis and maintenance of islet viability. A higher percentage of mice transplanted with a marginal mass of GEE treated islets became euglycemic after transplant. The supplementation of 20 mM GEE in cultured human islets significantly reduced the apoptosis rate in comparison to untreated islets. Conclusions/Significance GEE supplementation was able to decrease the apoptosis rate and intracellular content of ROS in isolated islets and might be considered a potential intervention to improve islet viability during the isolation process and maintenance in culture before islet transplantation.

Harnessing the Foreign Body Reaction in Marginal Mass Device-less Subcutaneous Islet Transplantation in Mice.

Background Islet transplantation is a successful &bgr;-cell replacement therapy for selected patients with type 1 diabetes mellitus. However, despite early insulin independence, long-term graft attrition gradually reverts recipients to exogenous insulin dependency. Undoubtedly, as insulin producing stem cell therapies progress, a transplant site that is retrievable is desirable. This prerequisite is currently incompatible with intrahepatic islet transplantation. Herein, we evaluate the functional capacity of a prevascularized subcutaneous site to accommodate marginal islet mass transplantation in mice. Methods Syngeneic mouse islets (150) were transplanted either under the kidney capsule (KC), into a prevascularized subcutaneous device-less (DL) site, or into the unmodified subcutaneous (SC) tissue. The DL site was created 4 weeks before diabetes induction and islet transplantation through the transient placement of a 5-Fr vascular catheter. Recipient mice were monitored for glycemic control and intraperitoneal glucose tolerance. Results A marginal islet mass transplanted into the DL site routinely reversed diabetes (n = 13 of 18) whereas all SC islet recipients failed to restore glycemic control (n = 0 of 10, P < 0.01, log-rank). As anticipated, nearly all islet-KC mice (n = 15 of 16) became euglycemic posttransplant. The DL recipients’ glucose profiles were comparable to KC islet grafts, postintrapertioneal glucose tolerance testing, whereas SC recipients remained hyperglycemic postglucose challenge. All normoglycemic mice maintained graft function for 100 days until graft retrieval. DL and KC islet grafts stained positively for insulin, microvessels, and a collagen scaffold. Conclusions The device-less prevascularized approach supports marginal mass islet engraftment in mice.

Transplantation of Human Pancreatic Endoderm Cells Reverses Diabetes Post Transplantation in a Prevascularized Subcutaneous Site

Summary Beta-cell replacement therapy is an effective means to restore glucose homeostasis in select humans with autoimmune diabetes. The scarcity of “healthy” human donor pancreata restricts the broader application of this effective curative therapy. “β-Like” cells derived from human embryonic stem cells (hESC), with the capacity to secrete insulin in a glucose-regulated manner, have been developed in vitro, with limitless capacity for expansion. Here we report long-term diabetes correction in mice transplanted with hESC-derived pancreatic endoderm cells (PECs) in a prevascularized subcutaneous site. This advancement mitigates chronic foreign-body response, utilizes a device- and growth factor-free approach, facilitates in vivo differentiation of PECs into glucose-responsive insulin-producing cells, and reliably restores glycemic control. Basal and stimulated human C-peptide secretion was detected throughout the study, which was abolished upon graft removal. Recipient mice demonstrated physiological clearance of glucose in response to metabolic challenge and safely retrieved grafts contained viable glucose regulatory cells.

Lung-Derived Microscaffolds Facilitate Diabetes Reversal after Mouse and Human Intraperitoneal Islet Transplantation.

There is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. Endocrine micro-pancreata (EMPs) made up of acellular organ-derived micro-scaffolds seeded with human islets have been shown to express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than three months in vitro. The aim of this study was to investigate the capacity of EMPs to restore euglycemia in vivo after transplantation of mouse or human islets in chemically diabetic mice. We proposed that the organ-derived EMPs would restore the extracellular components of the islet microenvironment, generating favorable conditions for islet function and survival. EMPs seeded with 500 mouse islets were implanted intraperitoneally into streptozotocin-induced diabetic mice and reverted diabetes in 67% of mice compared to 13% of controls (p = 0.018, n = 9 per group). Histological analysis of the explanted grafts 60 days post-transplantation stained positive for insulin and exhibited increased vascular density in a collagen-rich background. EMPs were also seeded with human islets and transplanted into the peritoneal cavity of immune-deficient diabetic mice at 250 islet equivalents (IEQ), 500 IEQ and 1000 IEQ. Escalating islet dose increased rates of normoglycemia (50% of the 500 IEQ group and 75% of the 1000 IEQ group, n = 3 per group). Human c-peptide levels were detected 90 days post-transplantation in a dose-response relationship. Herein, we report reversal of diabetes in mice by intraperitoneal transplantation of human islet seeded on EMPs with a human islet dose as low as 500 IEQ.