Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.     

Organogenetic tolerance

Transplantation therapy for humans is limited by insufficient availability of donor organs and outcomes are complicated by the toxicity of immunosuppressive drugs. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce immunogenicity of transplants. Insulin-producing cells originating from embryonic pig pancreas obtained very early following initiation of organogenesis [embryonic day 28 (E28)] engraft long-term in non-immune suppressed diabetic rats or rhesus macaques. Recently, we demonstrated engraftment of morphologically similar cells originating from adult porcine islets of Langerhans (islets) in rats previously transplanted with E28 pig pancreatic primordia. Our findings are consistent with induction of tolerance to a cell component of porcine islets induced by previous transplantation of embryonic pig pancreas, a phenomenon we designate organogenetic tolerance. Induction of organogenetic tolerance to porcine islets in humans with diabetes mellitus would enable the use of pigs as islet donors with no host immune suppression requirement. Adaptation of methodology for transplanting embryonic organs other than pancreas so as to induce organogenetic tolerance would revolutionize transplantation therapy.     

Organogenetic tolerance

Transplantation therapy for humans is limited by insufficient availability of donor organs and outcomes are complicated by the toxicity of immunosuppressive drugs. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce immunogenicity of transplants. Insulin-producing cells originating from embryonic pig pancreas obtained very early following initiation of organogenesis [embryonic day 28 (E28)] engraft long-term in non-immune suppressed diabetic rats or rhesus macaques. Recently, we demonstrated engraftment of morphologically similar cells originating from adult porcine islets of Langerhans (islets) in rats previously transplanted with E28 pig pancreatic primordia. Our findings are consistent with induction of tolerance to a cell component of porcine islets induced by previous transplantation of embryonic pig pancreas, a phenomenon we designate organogenetic tolerance. Induction of organogenetic tolerance to porcine islets in humans with diabetes mellitus would enable the use of pigs as islet donors with no host immune suppression requirement. Adaptation of methodology for transplanting embryonic organs other than pancreas so as to induce organogenetic tolerance would revolutionize transplantation therapy.     

Development of a novel xenotransplantation strategy for treatment of diabetes mellitus in rat hosts and translation to non-human primates

Transplantation therapy for diabetes is limited by unavailability of donor organs and outcomes complicated by immunosuppressive drug toxicity. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce transplant immunogenicity. Insulin-producing cells originating from embryonic pig pancreas obtained very early following pancreatic primordium formation [embryonic day 28 (E28)] engraft long-term in inbred diabetic Lewis or Zucker Diabetic Fatty (ZDF) rats or rhesus macaques. Endocrine cells originating from embryonic pig pancreas transplanted in host mesentery migrate to mesenteric lymph nodes, engraft, normalize glucose tolerance in rats and improve glucose tolerance in rhesus macaques without the need for immune suppression. Engraftment of primordia is permissive for engraftment of an insulin-expressing cell component from porcine islets implanted subsequently without immune suppression. Similarities between findings in inbred rat and non-human primate hosts bode well for successful translation to humans of what could be a novel xenotransplantation strategy for the treatment of diabetes.     

Development of a novel xenotransplantation strategy for treatment of diabetes mellitus in rat hosts and translation to non-human primates

Transplantation therapy for diabetes is limited by unavailability of donor organs and outcomes complicated by immunosuppressive drug toxicity. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce transplant immunogenicity. Insulin-producing cells originating from embryonic pig pancreas obtained very early following pancreatic primordium formation [embryonic day 28 (E28)] engraft long-term in inbred diabetic Lewis or Zucker Diabetic Fatty (ZDF) rats or rhesus macaques. Endocrine cells originating from embryonic pig pancreas transplanted in host mesentery migrate to mesenteric lymph nodes, engraft, normalize glucose tolerance in rats and improve glucose tolerance in rhesus macaques without the need for immune suppression. Engraftment of primordia is permissive for engraftment of an insulin-expressing cell component from porcine islets implanted subsequently without immune suppression. Similarities between findings in inbred rat and non-human primate hosts bode well for successful translation to humans of what could be a novel xenotransplantation strategy for the treatment of diabetes.     

Normalization of glucose post-transplantation into diabetic rats of pig pancreatic primordia preserved in vitro

Embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of non-immunosuppresed steptozotocin (STZ)-diabetic Lewis rats normalize levels of circulating glucose within 2–4 weeks. Exocrine tissue does not differentiate after transplantation of pancreatic primordia. Rather individual endocrine (beta) cells engraft within the mesentery.To determine whether transplanted pig pancreatic primordia engraft, differentiate and function in rat hosts after preservation in vitro, we implanted pig pancreatic primordia into STZ-diabetic rats either directly or after 24 hours of suspension in ice-cold University of Wisconsin (UW) preservation solution with added growth factors. Here we show engraftment in mesentery and mesenteric lymph nodes and normalization of glucose levels in STZ-diabetic rat hosts following transplantation of preserved E28 pig pancreatic primordia comparable to glucose normalization after transplantation of non-preserved E28 pancreatic primordia.Key words: beta cell, diabetes mellitus, transplantation, xenotransplantation     

Normalization of glucose post-transplantation into diabetic rats of pig pancreatic primordia preserved in vitro

Embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of non-immunosuppresed steptozotocin (STZ)-diabetic Lewis rats normalize levels of circulating glucose within 2-4 weeks. Exocrine tissue does not differentiate after transplantation of pancreatic primordia. Rather individual endocrine (beta) cells engraft within the mesentery. To determine whether transplanted pig pancreatic primordia engraft, differentiate, and function in rat hosts after preservation in vitro, we implanted pig pancreatic primordia into STZ-diabetic rats either directly or after 24 hours of suspension in ice-cold University of Wisconsin (UW) preservation solution with added growth factors. Here we show engraftment in mesentery and mesenteric lymph nodes and normalization of glucose levels in STZ-diabetic rat hosts following transplantation of preserved E28 pig pancreatic primordia comparable to glucose normalization after transplantation of non-preserved E28 pancreatic primordia.     

Organogenesis of Kidney and Endocrine Pancreas

Growing new organs in situ by implanting developing animal organ primordia (organogenesis) represents a novel solution to the problem of limited supply for human donor organs that offers advantages relative to transplanting embryonic stem (ES) cells or xenotransplantation of developed organs. Successful transplantation of organ primordia depends on obtaining them at defined windows during embryonic development within which the risk of teratogenicity is eliminated, growth potential is maximized, and immunogenicity is reduced. We and others have shown that renal primordia transplanted into the mesentery undergo differentiation and growth, become vascularized by blood vessels of host origin, exhibit excretory function and support life in otherwise anephric hosts. Renal primordia can be transplanted across isogeneic, allogeneic or xenogeneic barriers. Pancreatic primordia can be transplanted across the same barriers undergo growth, and differentiation of endocrine components only and secrete insulin in a physiological manner following mesenteric placement. Insulin-secreting cells originating from embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of streptozotocin-diabetic (type 1) Lewis rats or ZDF diabetic (type 2) rats or STZ-diabetic rhesus macaques engraft without the need for host immune-suppression. Our findings in diabetic macaques represent the first steps in the opening of a window for a novel treatment of diabetes in humans.     

Long-term survival of neonatal porcine islets in nonhuman primates by targeting costimulation pathways

We evaluated the ability of neonatal porcine islets to engraft and restore glucose control in pancreatectomized rhesus macaques. Although porcine islets transplanted into nonimmunosuppressed macaques were rapidly rejected by a process consistent with cellular rejection, recipients treated with a CD28-CD154 costimulation blockade regimen achieved sustained insulin independence (median survival, >140 days) without evidence of porcine endogenous retrovirus dissemination. Thus, neonatal porcine islets represent a promising solution to the crucial supply problem in clinical islet transplantation.     

Depletion of donor Ia+ cells before transplantation does not prolong islet allograft survival

Culture of pancreatic islets reduces their immunogenicity and results in prolonged graft survival after allotransplantation. The mechanism by which immunogenicity is reduced by culture is not known, but it has been suggested that prolonged graft survival is the result of the depletion of Ia+ cells from the graft. We studied the effect of eliminating Ia+ cells from islets before allotransplantation. Freshly isolated islets were incubated with anti-Ia serum plus complement or with monoclonal antibody to IAk plus complement or were left untreated before transplantation beneath the renal capsule of diabetic recipients. Incubation with anti-Ia serum plus complement eliminated intra-islet IA+ cells as demonstrated by indirect immunofluorescence staining. Incubation with monoclonal antibody to IAk plus complement significantly reduced but did not eliminate IA+ cells. Neither pretreatment regimen influenced survival of islet allografts placed beneath the renal capsule. However, untreated islets injected into the portal circulation were rejected in a low percentage of cases. We conclude that decreased immunogenicity observed after culture is not due solely to the depletion of Ia+ cells and that the site of engraftment has an important impact on graft survival.     

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)‐silenced islet cell clusters (ICC). Single‐cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2‐microglobulin or class II transactivator, respectively. SLA‐silenced ICCs‐derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs‐derived cells. Xenogeneic T cell immune responses, NK cell and antibody‐mediated cellular‐dependent immune responses were significantly decreased in SLA‐silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single‐cell engineering and post‐transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.     

Magnetic resonance-guided, real-time targeted delivery and imaging of magnetocapsules immunoprotecting pancreatic islet cells

In type I diabetes mellitus, islet transplantation provides a moment-to-moment fine regulation of insulin. Success rates vary widely, however, necessitating suitable methods to monitor islet delivery, engraftment and survival. Here magnetic resonance-trackable magnetocapsules have been used simultaneously to immunoprotect pancreatic beta-cells and to monitor, non-invasively in real-time, hepatic delivery and engraftment by magnetic resonance imaging (MRI). Magnetocapsules were detected as single capsules with an altered magnetic resonance appearance on capsule rupture. Magnetocapsules were functional in vivo because mouse beta-cells restored normal glycemia in streptozotocin-induced diabetic mice and human islets induced sustained C-peptide levels in swine. In this large-animal model, magnetocapsules could be precisely targeted for infusion by using magnetic resonance fluoroscopy, whereas MRI facilitated monitoring of liver engraftment over time. These findings are directly applicable to ongoing improvements in islet cell transplantation for human diabetes, particularly because our magnetocapsules comprise clinically applicable materials.     

Mast cells accumulate in the renal capsule adjacent to transplanted pancreatic islets in rats

Mast cells are important mediators of normal angiogenesis, and participate in normal would healing, i.e. processes involved in pancreatic islet engraftment. The aim of the study was to evaluate if mast cells are present in islet grafts. For this purpose, male normoglycaemic Wistar-Furth rats were either untreated or syngeneically implanted with 250 islets under the renal capsule. The animals were killed 1 month later, and the kidneys and endogenous pancreas were removed, fixed and embedded in paraffin. The distribution of mast cells was studied in Alcian Blue stained sections. Mast cells were rarely encountered in endogenous islets, but were frequent in the renal capsule adjacent to islet grafts. Mast cells interspersed between graft endocrine cells were as rare as in the endogenous pancreas. We conclude that mast cells may contribute to the engraftment after islet transplantation.     

Vascularized composite islet-kidney transplantation in a miniature swine model

Previous work from this laboratory has demonstrated that transplantation of allogeneic thymic tissue as part of a composite vascularized graft is far more successful in terms of both engraftment and long-term survival than transplantation of thymic tissue or cells alone. We have subsequently extended this concept to transplantation of allogeneic islets, comparing survival of islet cell suspensions to that of vascularized composite islet-kidneys (IK), prepared by injection of autologous islets underneath the renal capsule 2-3 months prior to allogeneic transplantation of the composite organ. We have utilized partially inbred miniature swine with defined MHC loci as the experimental large animals for this study, permitting reproducible transplantation across specific MHC barriers. Composite IK have been transplanted successfully across minor and full MHC mismatch barriers, using treatment regimens previously demonstrated to induce long-term tolerance of kidney allografts across these barriers. IK allografts containing ≥5000 islet equivalents (IE)/kg recipient body weight were found capable of reversing surgically induced diabetes, while injection of comparable numbers of purified islets via the portal vein or under the renal capsule did not. Studies are also being directed toward preparation of autologous “thymo-islet-kidneys” (TIK), for potential use as xenografts, in which the thymic component is intended to induce tolerance and the islets to reverse diabetic hyperglycemia. The use of both types of composite organ transplants may eventually be applicable to the treatment of type I diabetic patients suffering from end-stage diabetic nephropathy.     

Integration of nano‐ and biotechnology for beta‐cell and islet transplantation in type‐1 diabetes treatment

Regenerative medicine using human or porcine β‐cells or islets has an excellent potential to become a clinically relevant method for the treatment of type‐1 diabetes. High‐resolution imaging of the function and faith of transplanted porcine pancreatic islets and human stem cell–derived beta cells in large animals and patients for testing advanced therapy medicinal products (ATMPs) is a currently unmet need for pre‐clinical/clinical trials. The iNanoBIT EU H2020 project is developing novel highly sensitive nanotechnology‐based imaging approaches allowing for monitoring of survival, engraftment, proliferation, function and whole‐body distribution of the cellular transplants in a porcine diabetes model with excellent translational potential to humans. We develop and validate the application of single‐photon emission computed tomography (SPECT) and optoacoustic imaging technologies in a transgenic insulin‐deficient pig model to observe transplanted porcine xeno‐islets and in vitro differentiated human beta cells. We are progressing in generating new transgenic reporter pigs and human‐induced pluripotent cell (iPSC) lines for optoacoustic imaging and testing them in transplantable bioartificial islet devices. Novel multifunctional nanoparticles have been generated and are being tested for nuclear imaging of islets and beta cells using a new, high‐resolution SPECT imaging device. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological toolboxes for the xenotransplantation and ATMP field, and thus reinforces the European healthcare supply chain for regenerative medicinal products.     

Pancreas and Kidney Transplantation Using Embryonic Donor Organs

One novel solution to the shortage of human organs available for transplantation envisions ‘growing’ new organs in situ. This can be accomplished by transplantation of developing organ anlagen/primordia. We and others have shown that renal anlagen (metanephroi) transplanted into animal hosts undergo differentiation and growth, become vascularized by blood vessels of host origin and exhibit excretory function. Metanephroi can be stored for up to 3 days in vitro prior to transplantation with no impairment in growth or function post-implantation. Metanephroi can be transplanted across both concordant (rat to mouse) and highly disparate (pig to rodent) xenogeneic barriers. Similarly, pancreatic anlagen can be transplanted across concordant and highly disparate barriers, and undergo growth, differentiation and secrete insulin in a physiological manner following intra-peritoneal placement. Implantation of the embryonic pancreas, is followed by selective differentiation of islet components. Here we review studies exploring the potential therapeutic applicability for organogenesis of the kidney or endocrine pancreas.Key Words: cell therapy, diabetes mellitus, end-stage renal disease, metanephros, organogenesis, xenotransplantation     

Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species

Islet transplantation is a promising therapy for Type 1 diabetes, but many attempts have failed due to early graft hypoxia or immune rejection, which generate reactive oxygen species (ROS). In the current study, we determined that transgenic overexpression of the antioxidant metallothionein (MT) in pancreatic beta cells provided broad resistance to oxidative stress by scavenging most kinds of ROS including H2O2, peroxynitrite radical released from streptozotocin, 3-morpholinosydnonimine (SIN-1), and superoxide radical produced by xanthine/xanthine oxidase. MT also reduced nitric oxide-induced beta cell death. A direct test of hypoxia/reperfusion sensitivity was made by exposing FVB and MT islets to hypoxia (1% O2). MT markedly reduced ROS production and improved islet cell survival. Because MT protected beta cells from a broad spectrum of ROS and from hypoxia, we considered it to be an ideal candidate for improving islet transplantation. We first tested syngeneic transplantation by implanting islets under the kidney capsule of the same strain, FVB mice, thereby eliminating the immune rejection component. Under these conditions, MT islets maintained much greater insulin content than control islets. Allotransplantation was then tested. MT transgenic and normal FVB islets were implanted under the kidney capsule of BALB/c mice that were previously treated with streptozotocin to induce diabetes. We found that MT islets extended the duration of euglycemia 2-fold longer than nontransgenic islets. The benefit of MT was due to protection from ROS since nitrotyrosine staining, an indicator of free radical damage, was much lower in MT grafts than in FVB grafts. The time course of protection suggested that the major mode of MT action may have been protection from hypoxia or hypoxia/reperfusion. These data demonstrate that treatment with a broad spectrum antioxidant protects islets from ROS damage such as that produced during the early phase of islet transplantation.     

The role of endothelial cells on islet function and revascularization after islet transplantation

Islet transplantation has become a widely accepted therapeutic option for selected patients with type 1 diabetes mellitus. However, in order to achieve insulin independence a great number of islets are often pooled from 2 to 4 pancreata donors. Mostly, it is due to the massive loss of islets immediately after transplant. The endothelium plays a key role in the function of native islets and during the revascularization process after islet transplantation. However, if a delayed revascularization occurs, even the remaining islets will also undergo to cell death and late graft dysfunction. Therefore, it is essential to understand how the signals are released from endothelial cells, which might regulate both differentiation of pancreatic progenitors and thereby maintenance of the graft function. New strategies to facilitate islet engraftment and a prompt revascularization could be designed to intervene and might lead to improve future results of islet transplantation.     

From Langerhans Islets to Pancreas Transplantation over the past 30 years: A Review with Personal Contributions

Organ, tissue and cell banking is currently an important method used to prevent death of cells in replacement transplantation therapy. From 1975 to 1985 the author performed experimental transplantations of islets isolated by collagenase digestion through injection into the liver through the portal vein, or by transplantation of minced neonatal pancreases under the renal capsule of alloxan or spreptozotocin into severely chronic diabetic rats and mice. Seventy of the 256 transplanted rats and mice were cured for 1 year, i.e. one-third of their life span, at the 3rd to 15th inbreeding and only 2 weeks of immunosuppression by azathioprine or cyclosporin A. The author compares his results with those achieved later with diabetic patients by whole pancreas transplantation, including prevention of diabetic renal and ocular complications, infertility, health of progeny of cured rats, and slow rejection and possibility of cure by repeated transplantation. He welcomes the return to islet transplantation, and possibly also of immunologically tolerant pancreatic stem cell transplantation, or transplantation of subcutaneous fibroblasts, transfected with a complex insulin gene, which will produce adequate insulin to prevent hyperglycemia, as does hepatic pyruvate kinase from an insulin analog, a therapy that will not need permanent immunosuppression.     

Small rat islets are superior to large islets in in vitro function and in transplantation outcomes

Barriers to the use of islet transplantation as a practical treatment for diabetes include the limited number of available donor pancreata. This project was designed to determine whether the size of the islet could influence the success rate of islet transplantations in rats. Islets from adult rats were divided into two groups containing small (diameter <125 microm) or large (diameter >150 microm) islets. An average pancreas yielded three times more small islets than large. Smaller islets were approximately 20% more viable, with large islets containing a scattered pattern of necrotic and apoptotic cells or central core cell death. Small islets in culture consumed twice as much oxygen as large islets when normalized for the same islet equivalents. In static incubation, small islets released three times more insulin under basal conditions than did large islets. During exposure to high glucose conditions, the small islets released four times more insulin than the same islet equivalencies of large islets, and five times more insulin was released by the small islets in response to glucose and depolarization with K+. Most importantly, the small islets were far superior to large islets when transplanted into diabetic animals. When marginal islet equivalencies were used for renal subcapsular transplantation, large islets failed to produce euglycemia in any recipient rats, whereas small islets were successful 80% of the time. The results indicate that small islets are superior to large islets in in vitro testing and for transplantation into the kidney capsule of diabetic rats.