The International Diabetes Federation (IDF) estimates that diabetes currently affects more than 223 million people worldwide, and the figure is expected to reach 333 million by 2025, approximately 6.3% of the global population. The number of people in China with diabetes or pre-diabetic conditions, such as impaired glucose tolerance, has increased dramatically in recent years. Islet transplantation was considered an experimental procedure until 2000, when substantial progress was made by Shapiro's team in Edmonton, Canada, who were the first to report successful clinical trials of islet transplantation in humans.1 This approach, now known as the “Edmonton protocol”, involves islet transplantation in combination with a novel glucocorticoid-free immunosuppressive regimen and resulted in 100% (n=7) of the patients with type 1 diabetes being insulin-independent for at least 1 year. Compared with pancreas transplantation, islet transplantation/infusion is a less demanding method of β-cell replacement because it does not involve major surgery, involves less immunosuppression, and is potentially less expensive for the recipient. Based on data from the 2008 Collaborative Islet Transplant Registry (CITR), the desired outcomes of 325 islet recipients with type 1 diabetes include elimination of severe hypoglycemic events, restoration of hypoglycemia awareness, improved control of blood glucose, remarkable reductions in HbA1C levels and, ideally, independence from injected insulin. Although significant progress in clinical islet transplantation has occurred in recent years, many challenges remain. Unclear long-term results, high costs, and the relatively high incidence of major and minor adverse events make it difficult to convincingly argue for the expansion of islet transplantation to the general population.2 Based on our previous studies,3,4 we describe the current limitations of islet transplantation and discuss possible new strategies that are necessary to encourage widespread use of islet transplantation in patients with diabetes.
OVERCOMING THE SHORTAGE OF AVAILABLE DONOR ORGANS
A minimum of 10 000 islet equivalents (IEs)/kg is required to restore insulin dependence by the current accepted standard. Therefore, islets are typically collected from multiple donors for the engraftment of a sufficient number of insulin-producing cells to achieve insulin independence. Meanwhile, even in centers with highest rates of transplant success, insulin independence is achieved in only a few cases with a single transplant; most patients require more than one infusion of islets. In addition, the yield, quality and purity of islet cells isolated from pancreases are limited by many factors including age, body weight, plasma glucose, cause of death, and ischemic time of the pancreas. Consequently, there is an enormous short-fall between supply and demand.
Many researchers agree that the recent improvements in purity, yield, and viability of islet preparations render single donor islet transplants are sufficient for insulin independence in recipients. For example, Hering et al5 reported that an average of 7271 IEs/kg, isolated from a single cadaveric donor, was sufficient for all eight recipients to achieve insulin independence, and that five of these patients remained insulin independent for longer than 1 year.
Living donor islet transplantation may also offer an alternative approach to overcome the current obstacles of islet transplantation. The first successful living donor islet transplantation was reported in 2005 by Matsumoto et al in Japan.6 In that report, 408 144 IEs were isolated from the freshly harvested distal pancreas of a mother with brittle diabetes and were transplanted in an unpurified state. The recipient became insulin independent 22 days after transplant and had a normal oral glucose tolerance test 37 days after transplant.
Meanwhile, numerous alternative forms of insulin-secreting tissues have been proposed to replace human islets for β-cell-replacement therapy, including xenogeneic islets (predominantly from pigs), embryonic stem cells7 or adult stem cells, induced pluripotent stem (iPS) cells8 and other transdifferentiated somatic cells. Many studies are underway, in an attempt to improve the efficiency, potency, and safety of such cell sources. Recently, Zhou et al9 identified a specific combination of three transcription factors, Ngn3, Pdx1, and Mafa, which reprogram differentiated pancreatic exocrine cells in adult mice into cells that closely resemble β-cells. Similarly, recent studies using iPS cells10 have shown that skin fibroblast-derived iPS cells have the potential for differentiation into islet-like clusters through definitive and pancreatic endoderm, raising the possibility that patient-specific iPS cells could provide a treatment for diabetes in the future.
TECHNICAL ASPECTS OF ISLET PREPARATION AND TRANSPLANTATION
Many studies have focused on improving islet isolation techniques. The technologies currently used in human islet-cell processing are principally based on an automated method by Camillo Ricordi. After injection of collagenase into the pancreatic duct, the pancreas undergoes gradual digestion into fragments of decreasing size, until cell clusters within the volume range of islets are collected. A final purification step is carried out by density-gradient centrifugation using Ficoll or other non-ionic radiologic contrast gradients, and is now performed using a COBE2991 cell processor. The level of purification of a successful islet preparation is controversial. Injection of unpurified islets into the portal vein can cause portal hypertension and even portal venous thrombus. Meanwhile, the main cause for rejection and immunogenicity has been found to be the exocrine tissue surrounding the islets. However, the establishment of an islet-isolation/purification laboratory that meets current good manufacturing practice (cGMP) guidelines is an expensive undertaking for many clinical institutions.
The prevention of early islet loss, which is estimated to occur for up to 50% of the transplanted mass, is crucial to improve the outcomes of transplantation.11 Several strategies to improve islet function are based on maximizing islet survival and preventing of apoptosis via gene therapy approaches, such as the induction of heme-oxygenase-1.12 Another novel strategy includes the use of protein transduction domains (PTDs). Embury et al13 described a highly efficient system of transiently transferring anti-apoptotic proteins into pancreatic islets, which protected the islets from the destructive apoptotic signals generated during islet isolation. The study showed that the PTD-fusion proteins can transduce pancreatic islets with great efficiency ex vivo without affecting the insulin secretion capability of the islets.
The implantation technique itself is rather simple and, compared with open surgery for whole-pancreas transplantation, islet cell transplantation is much less invasive. However, the native pancreatic bed is relatively inaccessible and attempts to deliver islet grafts into the splenic vasculature resulted in significant morbidity, including infarction, rupture and gastric perforation. Currently, islets are delivered into the portal vasculature and the liver has been shown to offer the greatest clinical success to date. However, complications associated with islet infusion into the liver have included bleeding, portal venous thrombosis, and portal hypertension. Because of these problems, it is reasonable to consider the use of non-hepatic sites. Indeed, Cantarelli et al14 reported that infusion of islets into the bone marrow were more likely to achieve euglycemia compared with islets infused into the liver. Thus, bone marrow as a target infusion site is an attractive and safe alternative to the liver for pancreatic islet transplantation. In addition, the spleen, renal capsule, testes, brain, peritoneal cavity and omentum have all been proposed as potential sites for islet infusion.15 To aid monitoring of the implanted grafts, some researchers have focused on the in vivo detection of transplanted islets grafts in real time using noninvasive imaging methods, such as magnetic resonance imaging (MRI).16
IMMUNOLOGICAL STRATEGIES FOR ISLET TRANSPLANTATION
Immunosuppression is critical for islet transplantation because islet grafts are prone to immune destruction not only by allo-rejection, but also by the recurrence of autoimmunity. According to the 2008 report from the CITR, at the time of the first infusion, the majority (59%) of islet transplant recipients were placed on a daclizumab, sirolimus and tacrolimus immunosuppression regimen, now known as the Edmonton Protocol. More recently, however, over two-thirds of all islet cell transplants have received alternative immunosuppressive regimens, including alemtuzumab, anti-thymocyte globulin, etanercept, efalizumab and methylprednisolone. Mycophenolate mofetil (MMF) has been often used instead of sirolimus. However, irrespective of which immunosuppressive agents are used, these drugs are expensive and may increase the risk for specific malignancies and opportunistic infections. Furthermore, these agents are often associated with specific toxicities; calcineurin inhibitors, including tacrolimus and cyclosporin, are very nephrotoxic, for example. Ojo et al17 have reported that, of patients receiving other-than-kidney allografts, 7%-21% develop renal failure as a result of the transplant and/or subsequent immunosuppression. Thus, many clinicians believe that long-term use of maintenance immunosuppressive drugs will lead to worse outcomes than long-term insulin therapy. In addition, and somewhat ironically, the most commonly used agents are also associated with insulin resistance and/or decreased β-cell function and may promote the development of diabetes.18
Studies focusing on newer immunotherapeutic strategies are expected to provide means to protect islet allografts and prolong their survival. Prolonged culture of islet cells in high (95%) oxygen and low temperature, or exposure to ultraviolet radiation can deplete the islets of passenger leucocytes, which are thought to be the main cause for rejection. However, the feasibility of these methods in clinical use has yet to be confirmed. Meanwhile, cell encapsulation with an artificial, semi-permeable membrane may protect transplanted islet cells from immune rejection and limit the need for immunosuppression.19 The pore size of this membrane is such that only essential nutrients, gases and insulin can pass though it, whereas immune cells and antibodies are unable to reach the islets. Therefore, no rejection process is initiated. Calafiore et al20 reported the engraftment of 400 000 and 600 000 microencapsulated islets (islet equivalents normalized to 150 μ) in two patients, and both patients showed a rise in their serum C-peptide levels several weeks after transplantation, decreases in their mean daily glucose levels, and a progressive decline in exogenous insulin requirements. Another approach to overcome immunosuppression is by the induction of tolerance, including the blockade of critical co-stimulatory molecules on the surface of T cells and the use of lymphocyte-depleting agents. CD3-specific monoclonal antibodies may induce stable disease remission by restoring tolerance to pancreatic β-cells.21 Herold et al22 reported that treatment with a CD3-specific monoclonal antibody maintained or improved insulin production after 1 year in nine of 12 patients and speculated that the mechanism of action of the anti-CD3 monoclonal antibody may involve direct effects on pathogenic T cells, the induction of populations of regulatory cells, or both.
LIMITED LONG-TERM OUTCOMES OF ISLET TRANSPLANTATION
Although short-term results published by various islet transplant programs indicate that 63% of participants who underwent islet transplantation once were free of exogenous insulin 6 months later, the published data for the long-term clinical outcomes of islet transplantation are limited by small numbers of patients, the small number of transplant centers, and the short duration of follow-up. In one study, Ryan et al23 reported that the 5-year post-islet transplantation graft survival was approximately 80%, as measured by C-peptide positivity, but insulin independence was more difficult to maintain, with a rate close to 10% at 5 years. In 2006, a study24 conducted by the National Institutes of Health revealed the feasibility and reproducibility of islet transplantation with the Edmonton protocol. In that study, 36 patients with type 1 diabetes underwent islet transplantation at nine international sites. Of the 36 subjects, 21 were insulin independent and had good glycemic control at any point throughout the trial. However, few of the patients achieved long-term (>2 years) insulin independence. Nevertheless, a few uncontrolled preliminary studies have shown that the restoration of islet function is probably protective against long-term diabetic complications, particularly cardiomyopathy and angiopathy,25 nephropathy,26 retinopathy,27 and neuropathy.28 Large-scale studies are needed to confirm these preliminary findings.
Although significant advances in basic research and clinical islet transplantation have been achieved in recent years, many obstacles must still be overcome before islet transplantation can take its place as a conventional therapeutic option. Two of the most important limitations are the limited supply of islets for transplantation and the inadequate methods of preventing islet rejection. Innovative strategies, including single donor or living donor islet transplantation, stem cell application and gene therapy, may overcome the current limited supply of islet cells. The Edmonton protocol and other attempts to improve immunological tolerance, such as the use of monoclonal antibodies or blockade of critical co-stimulatory molecules on the surface of T cells, should help promote islet survival and protect islets in the long term from immunological injury. The current data suggest that the best candidates for islet transplantation are those with type 1 diabetes, severe hypoglycemia and marked glycemic fluctuations. On the other hand, islet after kidney transplantation or simultaneous kidney and pancreas transplantation are favorable for those patients with end-stage renal disease as a result of type 1 diabetes.29
The discovery of insulin in 1922 has changed the course of diabetes treatments. However, insulin treatment is still unable to fully prevent chronic complications or eliminate the risk of severe hypoglycemia. Islets not only make their own insulin, but they are capable of strictly regulating the quantity and timing of insulin release.30 Accordingly, islet transplantation offers a cure for diabetes, not just a treatment. We know that there are several obstacles ahead but, through the synergy of important innovative strategies, including stem cell applications, immunomodulation and gene therapy, islet cell transplantation will offer hope to millions of patients with diabetes who envision a life free of glucose checks and insulin injections.
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