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.     

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.     

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.     

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.     

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.     

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.     

A new diabetic strain of rat (WBN/Kob)

A new, spontaneously occurring diabetic syndrome has been observed in the aged males of an inbred strain of Wistar rats, WBN/Kob. The main clinical sign, glycosuria, was first detected at about 60 weeks of age, and thereafter some animals developed hyperlipidaemia and gradual emaciation. Prior to the onset of glucosuria, male rats showed impaired glucose tolerance after a glucose load at 21 weeks of age. The histopathologic lesions of the pancreas in the diabetic males consisted of multifocal fibrosis, decreased in number and size of islets and atrophy of exocrine tissue. Multifocal inflammatory foci of varying stages were the main pancreatic lesion in prediabetic male rats. This inflammatory change was detected even in 12-week-old rats and tended to occur around the islets. Therefore focal fibrosis and the decrease in the number and size of islets were considered to result from post-inflammatory scarring. The maturity-onset of this syndrome and the impaired glucose tolerance in younger animals suggested that diabetes mellitus of this rat strain is insulin-independent type II. However, the histological lesions of the pancreas were somewhat different from previous reports of both type I and II diabetes mellitus in man and animals.     

Evidence for a lentiviral etiology in an epizootic of immune deficiency and lymphoma in stump-tailed macaques (Macaca arctoides).

A retrospective study determined that an epizootic of immune suppression and lymphoma in stump-tailed macaques (Macaca arctoides) that began in 1976 was associated with a horizontally spread lentivirus infection. This conclusion was based on serology, epidemiology, pathology, and virus isolation. The lesions found in the stump-tailed macaques were more compatible with lesions seen in SIV-infected rhesus than those seen in rhesus macaques infected with type D retroviruses. A lentivirus, isolated from a rhesus inoculated with lymph node homogenate from a stump-tailed macaque, was designed SIVstm and was pathogenic for rhesus macaques. The isolate was antigenically related to other SIVs as well as to HIV-1 and HIV-2. Two surviving stump-tailed macaques sent to another colony carried SIVstm latently for at least 7 years and disseminated it throughout that colony.     

Functional value of the pancreatic microtransplant in rats after intraductal injection of chloroprene

Microsurgery permits the carrying out of pancreatic isografts in inbred rats. By this experimental pattern, it is possible to study the technical problems of pancreatic transplantation without interference from the immune reaction. The research of suppressing exocrine function in the graft by intraductal chloroprene injection and the study of the short-term effects of this procedure on endocrine function of the pancreas are the aim of this work. These experiments show that injection of chloroprene into the main pancreatic duct is an effective method of selective suppression of pancreatic exocrine function without interference with endocrine function. This procedure prevents the high mortality and morbidity which characterizes the other procedures with pancreatic juice derivation.     

The locus encoding an oligomorphic family of <Emphasis Type="Italic">MHC-A</Emphasis> alleles (<Emphasis Type="Italic">Mane-A</Emphasis>*06/<Emphasis Type="Italic">Mamu-A</Emphasis>*05) is present at high frequency in several macaque species

Several macaques species are used for HIV pathogenesis and vaccine studies, and the characterization of their major histocompatibility complex (MHC) class I genes is required to rigorously evaluate the cellular immune responses induced after immunization and/or infection. In this study, we demonstrate that the gene expressing the Mane-A*06 allele of pig-tailed macaques is an orthologue of the locus encoding the Mamu-A*05 allele family in rhesus macaques. Analysis of the distribution of this locus in a cohort of 63 pig-tailed macaques revealed that it encodes an oligomorphic family of alleles, highly prevalent (90%) in the pig-tailed macaque population. Similarly, this locus was very frequently found (62%) in a cohort of 80 Indian rhesus macaques. An orthologous gene was also detected in cynomolgus monkeys originating from four different geographical locations, but was absent in two African monkey species. Expression analysis in pig-tailed macaques revealed that the Mane-A*06 alleles encoded by this locus are transcribed at 10- to 20-fold lower levels than other MHC-A alleles (Mane-A*03 or Mane-A*10). Despite their conservation and high prevalence among Asian macaque species, the alleles of the Mane-A*06 family and, by extension their orthologues in rhesus and cynomolgus monkeys, may only modestly contribute to cellular immune responses in macaques because of their low level of expression.     

Dominant-negative mutant hepatocyte nuclear factor 1α induces diabetes in transgenic-cloned pigs

Pigs have been recognized as an excellent biomedical model for investigating a variety of human health issues. We developed genetically modified pigs that exhibit the apparent symptoms of diabetes. Transgenic cloned pigs carrying a mutant human hepatocyte nuclear factor 1α gene, which is known to cause the type 3 form of maturity-onset diabetes of the young, were produced using a combined technology of intracytoplasmic sperm injection-mediated gene transfer and somatic cell nuclear transfer. Although most of the 22 cloned offspring obtained died before weaning, four pigs that lived for 20–196 days were diagnosed as diabetes mellitus with nonfasting blood glucose levels greater than 200 mg/dl. Oral glucose tolerance test on a cloned pig also revealed a significant increase of blood glucose level after glucose loading. Histochemical analysis of pancreas tissue from the cloned pigs showed small and irregularly formed Langerhans Islets, in which poor insulin secretion was detected.     

Improvement of glucose tolerance with immunomodulators in type 2 diabetic animals

Cytokine-inducers prevent insulin-dependent diabetes mellitus (IDDM) in animal models. We extended this therapy to non-insulin-dependent diabetes mellitus (NIDDM), because it was reported that diabetes of KK-Ay mice, a model for NIDDM, was recovered by allogenic bone-marrow transplantation that also prevented IDDM in animal models. An i.p. or i.v. injection of streptococcal preparation (OK 432) lowered fasting blood glucose (FBG) levels and markedly improved glucose tolerance test (GTT) in KK-Ay mice for more than 32 h regardless of the glucose loading routes (oral, i.v. or i.p.), while an i.v. injection of BCG improved FBG and GTT for more than 4 wks without body weight loss. The improvement of FBG and GTT with OK-432 was brought about in other NIDDM animals, GK rats and Wistar fatty rats. Among various cytokines possibly induced by OK-432 and BCG, IL-1α, TNFα and lymphotoxin significantly improved FBG and GTT in KK-Ay mice, whereas IL-2 and IFN_γ did not. There were no differences between the OK-432-treated KK-Ay mice and control in histology of the pancreas, degree of insulin-induced decrease in blood glucose levels, and muscle glycogen synthase activities. As to insulin secretion, there is a tendency that the OK-432-treatment less than 1 week did not affect insulin levels during GTT, whereas the treatment more than 2 weeks increased the insulin levels. Thus, cytokine-inducers improved FBG and glucose tolerance of NIDDM animals probably via cytokines. The results imply a role of the cytokines in glucose tolerance of NIDDM, although precise immune and metabolic mechanisms remain to be elucidated.     

Prevalence of glucose intolerance in free-ranging Macaca fascicularis of Mauritius

Oral glucose tolerance tests (OGTTs) were carried out on 30 free-ranging long-tailed macaques (Macaca fascicularis) on the island of Mauritius, following the suggestion that severe glucose intolerance and diabetes mellitus might be prevalent in this macaque population. OGTTs revealed no evidence of frank diabetes mellitus in the sample. However, 13% of individuals showed impaired glucose tolerance, with preserved insulin secretion, suggesting the presence of the target tissue resistance to insulin characteristic of human noninsulin-dependent diabetes mellitus (NIDDM). The macaques with impaired glucose tolerance were neither obese nor aged. Glucose levels at all time points of the OGTT in normal macaques in our free-ranging sample were lower than reported in captive populations, perhaps due to greater physical activity. Our observations demonstrate that a genetic predisposition to glucose intolerance does exist in M. fascicularis, and that this condition, well documented in laboratory macaques, is not simply an artifact of captivity.     

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.     

A Large‐Scale SNP‐Based Genomic Admixture Analysis of the Captive Rhesus Macaque Colony at the California National Primate Research Center

Some breeding facilities in the United States have crossbred Chinese and Indian rhesus macaque (Macaca mulatta) founders either purposefully or inadvertently. Genetic variation that reflects geographic origins among research subjects has the potential to influence experimental outcomes. The use of animals from different geographic regions, their hybrids, and animals of varying degrees of kinship in an experiment can obscure treatment effects under study because high interanimal genetic variance can increase phenotypic variance among the research subjects. The intent of this study, based on a broad genomic analysis of 2,808 single nucleotide polymorphisms (SNPs), is to ensure that only animals estimated to be of pure Indian or Chinese ancestry, based on both demographic and genetic information, are used as sources of infants for derivation and expansion of the California National Primate Research Center's (CNPRC) super‐Specific Pathogen Free (SSPF) rhesus macaque colony. Studies of short tandem repeats (STRs) in Indian and Chinese rhesus macaques have reported that heterozygosity of STRs is higher in Chinese rhesus macaques than in Indian rhesus macaques. The present study shows that heterozygosity of SNPs is actually higher in Indian than in Chinese rhesus macaques and that the Chinese SSPF rhesus macaque colony is far less differentiated from their founders compared to the Indian‐origin animals. The results also reveal no evidence of recent gene flow from long‐tailed and pig‐tailed macaques into the source populations of the SSPF rhesus macaques. This study indicates that many of the long‐tailed macaques held in the CNPRC are closely related individuals. Most polymorphisms shared among the captive rhesus, long‐tailed, and pig‐tailed macaques likely predate the divergence among these groups. Am. J. Primatol. 74:747‐757, 2012. © 2012 Wiley Periodicals, Inc.