In the year of 2000, the Edmotion's protocol1 of islet allotransplantation achieved a remarkable success in treatment of type 1 diabetes mellitus (T1DM). However, the shortage of donors greatly restricts the development of clinical islet transplantation.2 Xenotranspalntation may provide an effective resolution for this restriction. Among the possible candidate tissues for xenotransplantation, pig islets are ideal for future clinical applications.3-8 Although encouraging results have been achieved in the pig-to-primate islet xenotransplantation models,9-14 the potential clinical application of pig islets still faces major challenges including inadequate supply of high-quality functional islets and xenogeneic immune rejection. This review focuses on the optimal pig donor for islet xenotranspalntation, pig islet grafts preparation, immune xenorejection and immune tolerance induction, and the results of recent preliminary clinical trials.
Original of pig islets
Islets obtained from embryonic, fetal, neonatal, or adult pig have been selected as the graft for xenotranspalntation. Fetal pig islet-like cell clusters (ICCs) had a poor insulin response to glucose.15-17 Typically, ICCs take 8-12 weeks to mature before achieving blood glucose normalization in vivo.18 Therefore, the immaturity may be the major disadvantage for ICCs as xenografts. Another disadvantage is the higher expression (approximately 19%)19 of alpha-1, 3-galactose (Gal) on the surface of the ICCs. Gal antigen makes ICCs more susceptible to humoral immune rejection. In addition, only a small amount of pig ICCs can be obtained because of the small size of fetal pancreas, therefore, many fetuses should be sacrificed in order to provide sufficient islets to normalize hyperglycemia in diabetic primates. This not only evokes ethical problems but also limits further large-scale clinical application.
Neonatal pig islets (NPIs) comprise pancreatic endocrine cells (approximately 35%) and primarily epithelial cells (approximately 57%), which is also regarded as the islet precursor cells.20,21 NPIs are able to functionally correct hyperglycemia in diabetic animal models, which is mainly due to striking differentiation of endocrine precursor cells into β cells and the β cell expansion.22,23 Though the insulin secretion of NPIs is also delayed, the maturation time is usually earlier than ICCs. Moreover, NPIs not only exhibit stronger resistance to inflammatory and hypoxia-induced injury, but also induce a lower T-cell reactivity than adult pig islets (APIs).24,25 Thus, NPIs are considered as a potential valid alternative source of islet grafts. The major disadvantage of using NPIs as islet source for clinical application is its lower yield: about only 50 000 cell aggregates could be obtained per pancreas after enzymatic digestion and culturing.20
Adult pigs are considered as the major donor source of islets for xenotransplantation, which provide a sufficient number of functional islet grafts.26-28 Usually, insulin independence in diabetic primates can be achieved when a sufficient islet cell mass (≥10 000 islet equivalents (IEQs)/kg body weight) is transplanted, which requires pooling of islets from 2-4 adult pig donors or 7-10 neonatal donors.28 In contrast to ICCs and NPIs, APIs respond to hyperglycemia immediately after the transplantation,29 mainly due to their mature status. The antigenicity of APIs mainly derives from N-linked sugars,30 but not the Gal antigen,19,31 APIs are more difficult to be isolated and more fragile than the immature islets. Over 2-year-old adult pigs (including retired breeders) are considered to be the optimal donors,32,33 which can not only provide higher islet yields, but also preserve intact morphology during isolation and culturing process.
Pig breeds should also be considered before islets isolation. It was suggested that Landrace pigs were the most suitable islet donors because of the greatest number of large-size islets (>250 μm) and islet volume density.34 However, a published study showed that the pancreases of the adult Chicago Medical School (CMS) miniature pigs contained larger islet sizes and higher islet yields (up to (9 589±2 838) IEQs/g) than that of market pigs or other miniature pigs.35 The CMS miniature pigs could be bred under specific pathogen-free (SPF) conditions. All these make this pig breed one of the best donors for clinical islet xenotransplantation.36 Recently, the Chinese Wuzhishan (WZS) miniature pigs provide another feasible source of islet grafts for xenotransplantation.37 The anatomical structure of the inbred WZS pigs' pancreas is similar to human pancreas. After isolation, the WZS breed shows higher yields of functional islets than that of the market pig ((6 078±1 105) IEQs/g vs. (2 500±625) IEQs/g).
Gender is another determining factor for successful islets isolation. A few studies suggested that compared with female pigs, as donors, male ones providing a more favorable islet yield in miniature pig strains.33,38
Preparation of pig islets
Both ICCs and NPIs can be easily isolated by enzymatic digestion (not lot- or type-dependent) after extensive flush of pancreas during and after surgical procedure.20,39
The isolation procedure of APIs from pigs is similar to islets acquisition from non-heart-beating humans. During the process of pancreas harvesting, several factors including quality of donor pancreas, blood exsanguinations, warm ischemia time (WIT), cold ischemia time (CIT), and perfusate would eventually affect the islet yield and viability.27,33,40-42 Simply, complete exsanguination and extensive flushing of the pancreas should be performed, and the WIT during the procedures should be reduced as much as possible to prevent autolysis of donor pancreas by proteolytic and lipolytic enzymes. It was suggested that WIT shortened within 10 minutes was essential for successful pig-to-primates islets xenotransplantation.27 The CIT within 60-90 minutes is usually considered as the appropriate storage time for successful islet yield and activity in adult pigs.11 However, studies demonstrated that viable APIs could be also successfully isolated after prolonged ischemia (up to 7-hour preservation) utilizing perfluorocarbon (PFC) or perfluorohexyloctan (F6H8) for pancreas oxygenation during cold storage.43,44 Moreover, compared with static cold storage, the new technique of hypothemic machine perfusion (HMP) significantly improves the quality of pig pancreas preservation, facilitates processing of enzymatic digestion, and consequently results in a greater islet yield and purity.45,46 Before storage of the pancreas in cold preservation solution, the morphological screening and rapid assessment of pancreas must be conducted. The accurate islet yield could be significantly predicted only by IEQs per mm2 (IEQs/mm2) of the splenic lobe of pancreas.47 Donor pig pancreas containing more than 82 islets/cm2 and 42% composition of large islets (>100 μm) should be selected for following digestion/isolation processes.27
Since the introduction of an efficient enzymatic/mechanical method for pig pancreas digestion by Ricordi et al,48 several similar, semiautomatic methods for pancreatic tissue digestion have been developed. With modifications of the digestion/filtration devices, the improved semiautomatic Ricordi's chambers have been widely used for pig islets isolation with the advantages of labor-saving, controlled digestion temperature, high islet yields, less over-digestion and mechanical injuries, and no contaminations.49,50
The efficiency of pig islet isolation is strongly based upon the digestion enzymes' activity. Liberase PI batches containing an endotoxin content <30 EU/mg is recommended for successful pancreas digestion.27 Recently, a novel enzyme product, Liberase MTF C/T, characterized by less degradation of Class I collagenase and low endotoxin content (<10 EU/mg),51 has been shown resulted in better islet quality than that of Liberase PI from adult pigs.38 Although remarkable progress has been gained for successful islet isolation by collagenase digestion, there are still several significant disadvantages such as collagenase-induced damage, lot-to-lot variability, and high cost.27,52,53 Thus, the selective osmotic shock (SOS) method is introduced for pig islet isolation.54 This method is based on the “selection” of glucose-responsive islets by a high-glucose solution treatment, then followed by a zero-glucose solution treatment. The cell yield, purity, and insulin secretion from islets isolated by SOS were proved superior to islets isolated by Liberase in the traditional fashion. The SOS may be the promising method for large-scale pig islet isolation with other advantages including no endotoxins, less cost, easy manipulation, and potentiality of full automatic processing.
Purification is the next necessary step to separate the islet from the exocrine tissue, especially for the APIs preparation. Nowadays, the process using cell processor (COBE) with density gradient solution is the standard protocol for pig islet purification. Compared with the traditionally widely used Ficoll density gradient solution, the midly hypertonic iodixanol-based solution could significantly improve the number, viability, and insulin secretory response of purified pig islets.55 Moreover, the iodixanol-based solution could also significantly reduce cytokine/chemokine production during the islet isolation, as well as the loss of β cell mass during pre-transplantation culture.56
Immunological rejection of pig islets xenotransplantation
Immediate blood-mediated inflammatory reaction
Immunological rejection is still the major obstacle for successful pig islet xenotransplantation and its' clinical applications. When the pig islets were infused into the portal vein of diabetic primates, the elevated expression of tissue factor from the islets initiated the immediate blood-mediated inflammatory reaction (IBMIR), which was characterized by platelet aggregation on the islet surface, infiltration of leukocytes, activation of coagulation and complement systems.57-59 IBMIR occurs in the early phase of post-transplantation, and is closely associated with the considerable early islet loss (approximately 60%-80%).60 IBMIR is also likely to amplify the subsequent specific immune response to pig islet xenografts.
Unlike vascularized organ transplants, pig islets do not undergo hyperacute rejection (HAR) and acute vascular rejection (AVR) after implantation. The possible explanations are: first, the pig islets are not vascularized at the time of transplantation and the xenografts are revascularized by endothelial cells of host origin;61 second, the Gal molecules expressed on pig islets (5% of APIs)19 are much lower than solid organs. The Gal is usually considered as the blood group antigen foreign to primates, and the humoral rejection is largely characterized by abundant anti-Gal antibody production. The study of pig-to-nonhuman primates (NHPs) islet xenotranplantation showed no significant increasing levels of circulating Gal-specific IgG/IgM, as well as Gal-specific staining on islets.62 Therefore, the Gal antigens are not the major targets for humoral xenorejection to pig islets. Perhaps the non-Gal specific antibodies play an important role in the humoral xenorejection of pig islets.63-65
If the transplanted pig islet escapes the acute injury caused by IBMIR and additional humoral rejection, it will undergo acute cellular xenograft rejection (ACXR). The xenogeneic T-cell response appears to play a major role in the cellular rejection.60,66,67 In the diabetic rheus macaques without immunosuppressive therapy, the pig islet xenografts were mainly subject to ACXR mediated by CD4+ T cells, CD8+ T cells, and macrophages.68 Similar phenomenon was also observed in the immunosuppressed macaques with diabetes.62 Gene analysis of the intragrafts indicated that CXCR3, interferon-inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig) were involved in T cell recruitment during acute islet xenogrft rejection.69 Additionally, both of direct and the indirect pathways of donor antigen presentation are involved in the initiation of primary T-cell response to islet xenografts.70 Thus, the treatments targeting different pathways affecting T-cell activation/function can effectively induce a long-term pig islet survival and produce a sustained host hyporeactivity.
Methods to relieve xenogeneic immune rejection
Encapsulation, which hides the islet grafts from recipients' immune system and allows free exchange of metabolic substance with external environments, is becoming an effective strategy to protect pig islets from rejection.71,72 Currently, there are two forms of immunoisolation devices: microencapsulation and macroencapsulation. The microcapsules are superior to macrocapsules because of the advantages including easier implantation, better diffusion of oxygen and nutrients, and more sensitive response to hyperglycemia.
The biocompatibility of encapsulation membrane is the most crucial factor for islet survival with regard to escaping the nonspecific rejection which leads to fibrotic overgrowth and consequent islet loss.73 Various artificial materials including protamine-heparin complex, cellulose, and agarose were used to produce encapsulated pig islet grafts, as a result, a long-term euglycemia was maintained after implantation.74-76 However, the most promising materials for pig islet immunoisolation is the alginate based capsules, which reverse diabetes in NHPs up to 6 months without immunosuppressants.77,78 In addition, the suitable sites are needed for successful encapsulated islet implantation. Kidney subcapsular and subcutaneous spaces seems appropriate for clinical application in the future.79,80
T-cell activation requires double signaling namely the T-cell receptor (TCR) signaling and signaling mediated by co-stimulatory molecules. The lack of co-stimulation drives T-cell into a nonresponsive state, known as anergy. Therefore, co-stimulatory signaling blocking is supposed to be an attractive therapeutic strategy to avoid cellular rejection. In rodent xenotransplantion models, blockage of the CD80/86-CD28 (by CTLA4Ig fusion protein) or/and CD40 L(CD154)-CD40 (by anti-CD40 L monoclonal antibody) signaling pathways of co-stimulation significantly promoted prolonged survival and function of pig islet without maintaining immunosuppression.70,81,82 The CD40Lspecific antibody seems to be an inappropriate candidate for clinical translation due to high risk of thromboembolic complications,83 therefore, the CD40-specific antibody presents an alternative target for enhancing pig islet survival in diabetic NHPs.14 However, in pig-to-NHPs models, immune tolerance would not be achieved by utilizing these co-stimulatory blocking agents alone, unless they are applied with other immunosuppressants in long-term treatments (Table 1). Because of the synergistic effect among the co-stimulatory pathways, only a single pathway blocking may not achieve a stable immune tolerance. Multi-channel joint blocking combined with other specific immunosuppressive drugs may be promising.
Genetic modification of pigs
Genetically modified pigs/islets have shown considerable advantages in protecting donor pig islets from the xenogenic immune response and reversing molecular incompatibilities between pig and primate (Table 1).8,84-86 Up to now, there are several strategies to acquire genetic modification in pigs, including knocking out/knocking down the genes responsible for the expression of major antigens targeted by natural anti-pig antibodies, transferring human complement- and coagulation-regulatory genes, and inserting T-cell co-stimulation blocking genes. The islet grafts from N-acetylglucosaminyltransferase-III (GnTIII) transgenic pigs showed significantly higher survival rate and less xenoantigenicity than wild-type pigs in diabetic monkeys.65 Tissue factor (TF) knockdown in pig islets resulted in an effective suppression of IBMIR.87 The xenografted islets from INSLEA29Y (a high-affinity variant of CTLA4Ig) transgenic pigs reversed hyperglycemia and prevented immune rejection in humanized rodents;88 this finding might provide a novel method for pig-to-NHPs islet xenotransplantation without adverse effects of systemic immunosuppression.
Recent studies show the capacity of mesenchymal stem cells (MSCs) to suppress alloreactive T-cell responses responsible for graft damage after allogeneic islet transplantation.89,90 Other published studies also demonstrated that MSCs obtained from genetically modified pig (GTKO/CD46 pig) could significantly downregulate human T cell responses to pig antigens.91,92 Thus, islets from genetically modified pig, if cotransplanted with syngeneic MSCs, would potentially improve a long-term engraftment of pig islets and induce T-cell tolerance in human recipients. Altogether, as a heterogeneous islet donor, transgenic pigs will exert great prospects for mechanic study and clinical application.
Islet graft revascularization
In order to pursue prolonged survival and sustained function, the transplanted islet grafts must be revascularized. Usually, the complete revascularization is generated at 10-14 days after transplantation.93 During this period, islet grafts rely on the diffusion of nutrients and oxygen from the surrounding tissues. The loss of vascularity may lead to early loss of islets. Thus, accelerating the vascularization procedure and protecting newly formed microvasculature from rejection-mediated injuries would contribute a lot to islet survival and function, especially for islet xenografts.
Previously, MSCs and endothelial cells (ECs) coated islets showed beneficial effects on revascularization; the vascular sprouts on the islet were enhanced by MSCs.94 Similar results were confirmed in the studies of syngeneic islet transplantation.95,96 Considering the effective proangiogenic and immunomodulatory properties of MSCs, for pig islet xenotranslantation, pretreatment of donor pig islets with recipient-derived MSCs and ECs may be useful to promote islet revascularization and improve xenograft engraftment.
Currently, several encouraging studies suggest the tremendous potential of pig islet xenotransplantation in curing diabetes in NHPs.12-14,62,86 Based on the data showing diabetes reversal and long-term survival of pig islets in diabetic NHPs, the International Xenotranplantation Association (IXA) released a consensus statement on conditions for undertaking clinical trials of pig islets in T1DM patients.97 Although there are potential limitations and restrictions in the pig-to-NHP islet transplantation,98,99 the pre-clinical trials still powerfully rationalize the clinical applications of pig islets.
The long-term viability, function and safety of pig islets in diabetic patient were assessed by Elliott et al.71 The insulin requirement of the T1DM patient was significantly reduced after intraperitoneal alginate-encapsulated NPIs implantation. In the 10-year follow-up study, there were still a large number of functional islets throughout the peritoneal tissue, and no signs of porcine endogenous retrovirus (PERV) infection were observed.
Living Cell Technology Co., Ltd. (LCT) developed the commercial encapsulated pig islets (called Diabecell), which was tested in the phase I/IIa clinical study in Moscow since 2007.100,101 All diabetic patients showed improved blood glucose control as reflected by decreased glycated haemoglobin (%HbA1c) levels after implantation. Two patients were independent with insulin administration for 8 months, moreover, no contagious pathogens from pigs were found in the recipients. Currently, the additional phase I/IIa trials are conducting in New Zealand and Argentina.
Building on the remarkable progresses in the experimental studies, it appears that pig islets xenotransplantation have the grate potentiality in treatment of diabetes in NHPs and furthermore in humans. In the light of development of suitable sources of genetically modified pigs and the modification of isolation/purification technology, as well as the improvement of specific immunosuppressive methods, together with the clinically applicable regimens, a proper and tangible therapy would benefit the patients with diabetes in the near future.
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