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.     

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.     

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.     

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.     

Stem cell-based approaches to solving the problem of tissue supply for islet transplantation in type 1 diabetes

Type 1 diabetes is a debilitating condition, affecting millions worldwide, that is characterized by the autoimmune destruction of insulin-producing pancreatic islets of Langerhans. Although exogenous insulin administration has traditionally been the mode of treatment for this disease, recent advancements in the transplantation of donor-derived insulin-producing cells have provided new hope for a cure. However, in order for islet transplantation to become a widely used technique, an alternative source of cells must be identified to supplement the limited supply currently available from cadaveric donor organs. Stem cells represent a promising solution to this problem, and current research is being aimed at the creation of islet-endocrine tissue from these undifferentiated cells. This review presents a summary of the research to date involving stem cells and cell replacement therapy for type 1 diabetes. The potential for the differentiation of embryonic stem (ES) cells to islet phenotype is discussed, as well as the possibility of identifying and exploiting a pancreatic progenitor/stem cell from the adult pancreas. The possibility of creating new islets from adult stem cells derived from other tissues, or directly form other terminally differentiated cell types is also addressed. Finally, a model for the isolation and maturation of islets from the neonatal porcine pancreas is discussed as evidence for the existence of an islet precursor cell in the pancreas.     

Peripheral blood progenitor cell mobilization and leukapheresis in pigs

BACKGROUND AND PURPOSE: The pig is being investigated as an organ donor for humans. Induction of immunologic tolerance to pig tissues in primates would overcome the major immunologic barriers to xenotransplantation. A proven method of inducing tolerance to allografts is by the induction of mixed hematopoietic chimerism by bone marrow transplantation. We are therefore investigating induction of mixed hematopoietic chimerism in the pig-to-baboon model. METHODS: To obtain large numbers of pig hematopoietic cells, leukapheresis was used to collect blood cell products in miniature swine (n = 5) after progenitor cell mobilization by use of a course of hematopoietic growth factors (cytokines), consisting of porcine interleukin 3, porcine stem cell factor, and human granulocyte colony-stimulating factor. RESULTS: Cytokine therapy and leukapheresis were well tolerated. Cytokine therapy increased the total white blood cell count and allowed large numbers of leukocytes (60 x 10(10)) to be obtained by apheresis, of which approximately 0.1% were granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming units (CFU-GEMMs), which are considered to be representative of hematopoietic progenitors with multi-lineage potential. CONCLUSIONS: The combination of cytokine therapy and leukapheresis enables hematopoietic progenitor cells to be obtained safely from miniature swine.     

Islet cell engraftment and control of diabetes in rats after transplantation of pig pancreatic anlagen

The insufficient supply of tissue, loss posttransplantation, and limited potential for expansion of beta-cells restrict the use of islet allotransplantation for diabetes. A way to overcome the supply and expansion problems is to xenotransplant embryonic tissue. We have shown that whole rat pancreatic anlagen isotransplanted into the omentum of rats, or xenotransplanted into costimulatory blocked mice, undergo growth and differentiate into islets surrounded by stoma without exocrine tissue. Isotransplants normalize glucose tolerance in diabetic hosts. Here, we show that embryonic day 29 porcine pancreas transplanted into the omentum of adult diabetic rats undergoes endocrine tissue differentiation over 20 wk and normalizes body weights and glucose tolerance. Unlike rat-to-rodent transplants, individual alpha- and beta-cells engraft without a stromal component, and no immunosuppression is required for pig-to-rat transplants. Herein is described a novel means to effect the xenotransplantation of individual islet cells across a highly disparate barrier.     

Normal development in porcine thymus grafts and specific tolerance of human T cells to porcine donor MHC

The induction of T cell tolerance is likely to play an essential role in successful xenotransplantation in humans. In this study, we show that porcine thymus grafts in immunodeficient mice support normal development of polyclonal, functional human T cells. These T cells were specifically tolerant to MHC Ags of the porcine thymus donor and responded to nondonor porcine xenoantigens and alloantigens. Exogenous IL-2 did not abolish tolerance, suggesting central clonal deletion rather than anergy as the likely tolerance mechanism. Our study suggests that the thymic transplantation approach to achieving tolerance with restoration of immunocompetence may be applicable to xenotransplantation of pig tissues to humans.     

The reversal of hyperglycemia after transplantation of mouse embryonic stem cells induced into early hepatocyte-like cells in streptozotocin-induced diabetic mice

Cellular replacement therapy is a potential therapeutic strategy for diabetes. In this study, we investigated the effect of transplantation of induced mouse embryonic stem cells (mESCs) into endoderm and early hepatocyte-like cells in streptozotocin (STZ)-diabetic mice. After embryoid body (EB) formation from mESC, the EBs were cultured in the presence of dexamethasone (DEX) and insulin for 4 days then was added acidic fibroblast growth factor (aFGF), hepatocyte growth factor (HGF) and oncostatin M (OSM) for 10 days, respectively. Blood glucose levels, intraperitoneal glucose tolerance (IGT) test and islet histology were assessed. The result revealed that transplantation of induced mESCs into early hepatocyte-like cells could repair pancreatic islets of control group. Blood glucose levels and intraperitoneal glucose tolerance test were significantly improved in test group compared to control group. Furthermore, there was significant increase in the number of islets in test group compared to control group. The findings declare that induced mESCs into endoderm and early hepatocyte-like cells, are appropriate candidate for regenerative therapy of pancreatic islets in type I diabetes.     

Changes of DNA methylation level and spatial arrangement of primordial germ cells in embryonic day 15 to embryonic day 28 pig embryos

The mammalian germline is generally assumed to undergo extensive epigenetic reprogramming during embryonic development, including a nearly complete erasure of DNA methylation. This assumption does, however, to large degree rely on data from mouse, and despite a well-grounded picture the general nature of these data needs to be validated by investigations of other mammalian species. This study represents such a contribution in the examination of the germline in the domestic pig (Sus scrofa). Semiquantitative immunohistochemistry was used to investigate the level of DNA methylation in the POU5F1-positive primordial germ cells (PGCs) compared with neighboring somatic cells in porcine embryos at Embryonic Day 15 (E15), E17, E20, E21, and E28. We show that, in agreement with the mouse model, a significantly lower level of DNA methylation was observed in the early migrating PGCs. This level was decreasing until a stage coinciding with the entrance of the PGCs to the genital ridge. After this, the methylation level increased. Using whole-mount immunostaining, we determined the spatial arrangement of the porcine PGCs in the period between E15 and E28, allowing some comparison with the migration of the murine germline. The overall conclusion from the obtained data is that the DNA methylation changes in porcine PGCs, as well as the migration of these cells, parallels the picture reported for the mouse.     

Islet cell transplantation

Islet cell transplantation is an attractive alternative therapy to conventional insulin treatment or vascularized whole pancreas transplantation for type 1 diabetic patients. It represents a successful example of somatic cell therapy in humans based on complex procedures for islet isolation from whole pancreas. The islets, that are only 1% of the total pancreas tissue, are isolated by two steps method starting with collagenase digestion that operates a rapid dissociation of the stromal component of the gland, while preserving islet anatomical integrity. After digestion, islets are then separated from exocrine tissue by centrifugation in density gradients. Transplantation consists of a simple injection of few milliliter-purified tissue in the portal vein through a percutaneous trans-hepatic approach performed in local anesthesia. Several studies have now demonstrated that islet transplant can replace pancreatic endocrine function without major side effects and with liver viability preservation in selected patients affected by long-term type 1 diabetes. It can restore endogenous insulin secretion, achieve insulin independence in more than 80% of patients, and recover the metabolism of glucose, protein and lipids. Improved control of glycated HbA1c, reduced risk of recurrent hypoglycemia and of diabetic complications are also seen as important benefits of islet cell transplantation, irrespective of the status of insulin independence. Many protocols are now on going for reduction of immunosuppression therapy in recipients, induction of tolerance, and prolongation of graft function.     

Generation of insulin-producing beta cells from stem cells--perspectives for cell therapy in type 1 diabetes

Cell based therapy for the treatment of type 1 diabetes is limited by the overall shortage of donor organs for transplantation. This is the rationale for the research on the generation of insulin-producing beta cells from an inexhaustible source of cells such as the stem cells. Stem cells are progenitor cells which possess the capacity of self-renewing and differentiation in fully mature cells depending on the culture conditions. The fundamental question is how to make terminally matured pancreatic beta cells. During the last years different approaches for the neogenesis of beta cells have been described using embryonic stem cells, adult stem cells residing in the pancreas, or other nonpancreatic cell types. Although fully functional islets have not yet been derived from any stem cells, the use of stem cells is still the most promising approach on the way to establish a treatment protocol for the cure of type 1 diabetes in the future.     

Porcine islet isolation, cellular composition and secretory response

Porcine islets were isolated by infusion of a warm collagenase solution into whole pancreata followed by static incubation at 37 degrees C for 15 minutes. The pancreata were then chopped into small pieces and the free islets purified by filtration and centrifugation over a ficoll gradient. The insulin:amylase ratio of the islets compared to that in the intact pancreas was determined in 19 pancreata and indicates that the isolated islets were of a high degree of purity. The distribution of insulin, glucagon, somatostatin and pancreatic polypeptide containing cells in pig pancreas sections was compared with that in rat. Porcine islets were much smaller and less well defined than rat islets with infiltration of acinar material even into the islet core. The levels of insulin, glucagon and somatostatin in porcine pancreas and isolated porcine islets were measured using conventional radioimmunoassay techniques. The ratio of these hormones in the pancreas was 105.1:5.8:1 respectively, and in the islets 105.1:0.68:0.087 respectively. Fragmentation of the islets during the isolation may have led to the loss of glucagon and somatostatin-containing cells. Islets cultured overnight and tested with a range of glucose concentrations for one hour did not show a significant stimulation of insulin secretion in the presence of 8.3 mM or 16.7 mM glucose compared to that in 2.8 mM glucose. However freshly isolated islets challenged with 8.3 mM, 13.9 mM and 22.2 mM glucose showed a 1.8 fold, 2.0 fold and 2.3 fold response respectively, over that in 2.8 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stem-cell-based approaches for regenerative medicine

Recent success in transplantation of islets raises the hopes of diabetic patients that replacement therapies may be a feasible treatment of their disease. Although several lines of evidence suggest that stem cells exist in the pancreas, it is still technically hard for us to isolate or maintain the stem cells in vitro. The establishment of human embryonic stem (ES) cells has excited scientists regarding their potential medical use in tissue replacement therapy. When applied with appropriate signals, ES cells can be directed to differentiate into a specific cell lineage. Therefore, ES cells are no doubt an excellent source not only for regenerative medicine but also for studies of early events of pancreatic development, and to portray the pancreatic progenitor cells. Despite many attempts that have been tried, the efficiency of differentiation of ES cells into islets is still very low. This low efficiency reflects our lack of understanding of the intrinsic and extrinsic signals which regulate the developmental processes of the pancreas. In this review, I present a summary of recent works on ES cells, the identification of pancreatic progenitor cells from the adult pancreas, and refer to the possibilities of transdifferentiation from adult stem cells derived from other tissues.     

Nonhuman primate models for islet transplantation in type 1 diabetes research

Insulin-dependent diabetes mellitus is an autoimmune disease that causes a progressive destruction of the pancreatic beta cells. As a result, the patient requires exogenous insulin to maintain normal blood glucose levels. Both the pancreas and the islets of Langerhans have been transplanted successfully in humans and in animal models, resulting in full normalization of glucose homeostasis. However, insulin independence, transient or persistent, was documented in only a small fraction of cases until recently. The chronic immunosuppression required to avoid immunological rejection appears to be toxic to the islets and adds the risk of lymphoproliferative disease reported earlier. For islet transplantation to become the method of choice, it is essential first to identify islet-friendly immunosuppressive regimens and/or to develop methods that induce donor-specific tolerance and improve islet isolation and transplantation protocols. Indeed, researchers have already successfully allografted islets in the presence of nonsteroidal immunosuppression in a process known as the Edmonton protocol. An alternative method, gene therapy, could replace these other methods and better meet the insulin requirement of an individual without requiring pancreatic or islet transplantation. This alternative, however, requires animal models to develop and test clinical protocols and to demonstrate the feasibility of preclinical trials. Nonhuman primates are ideally suited to achieve these goals. The efforts toward developing a nonhuman primate diabetic model with demonstrable insulin dependence are discussed and include pancreatic and islet transplant trials to reverse the diabetic state and achieve insulin independence. Also described are the various protocols that have been tested in primates to circumvent immunosuppression by using tolerance induction strategies in lieu of immunosuppression, thus exploring the field of donor-specific tolerance that extends beyond islet transplantation.     

Proteomic analysis of pancreas derived from adult cloned pig

The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreas were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis.     

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.