Cell therapy for diabetes using piscine islet tissue

The islet tissue, called Brockmann bodies, in certain teleost fish is anatomically distinct from the pancreatic exocrine tissue and can be easily identified macroscopically. Expensive islet isolation procedures, such as required when procuring islet tissue from mammalian pancreases, are unnecessary. Tilapia islets transplanted into diabetic athymic nude mice will produce long-term normoglycemia and mammalian-like glucose tolerance profiles. Encapsulated tilapia islets function well after transplantation into euthymic recipients. Additionally, tilapia are potentially ideal xenogeneic donors because of markedly lower donor production costs, minimal islet procurement costs, and possibly decreased xenozoonotic potential relative to mammalian donors. Tilapia islets appear to be appropriately glucose responsive with high insulin output, can be cryopreserved, and are much more resistant to hypoxia than mammalian islets. Because tilapia and human insulin differ by 17 amino acids, we have cloned, sequenced, and modified the tilapia insulin gene by site-directed mutagenesis resulting in a tilapia insulin gene that codes for "humanized" insulin while still maintaining all of the tilapia regulatory (noncoding) sequences. We have now produced small numbers of founder transgenic tilapia with incorporation of our humanized tilapia insulin gene construct, and we have shown transmission and expression of the transgene in the beta cells and serum of their Fl offspring. Our ultimate goal is to achieve homologous recombination and to breed for homozygosity for the hybrid insulin gene. Subsequent generations of transgenic tilapia will undergo a program of genomic optimization selecting for growth, survival, and fecundity as well as stability of the transgene. Islets from the resulting transgenic fish, after extensive characterization, could be harvested, encapsulated, and then transplanted into diabetic patients.     

Long-term survival and function of intraportal porcine and human islet xenografts in nondiabetic nude mice

Instant blood-mediated inflammatory reaction (IBMIR) is a serious obstacle to both clinical islet allotransplantation and future islet xenotransplantation via the portal vein. We have previously observed uniform long-term tilapia (fish) islet xenograft survival when islets were transplanted intraportally into nondiabetic nude mice (nDNM), but not in diabetic nude mice (DNM). In this study, we examined whether human islets (HI) and adult porcine islets (API) can tolerate intraportal transplantation into nDNM like tilapia islets. HI and API were transplanted intraportally into nDNM. Recipients were humanely killed either 14 or 28 days after transplantation and livers were processed for histology. Human insulin and human C-peptide were measured in the terminal serum samples of HI recipients. In six of seven HI and seven of seven API recipients, liver histology showed insulin-positive islet xenografts. In recipients with HI, the numbers of islets/ductal structures seen histologically correlated well with serum sample results. These results show that HI and API can survive and function long term after intraportal transplantation into nDNM recipients. Our previous and present data indicated that DNM and nDNM could be useful models to study "glucose toxicity" and the role of IBMIR in the fate of intraportal islet grafts.     

Evidence that tilapia islets do not express alpha-(1,3)gal: implications for islet xenotransplantation

BACKGROUND: Cell therapy for diabetes using teleost fish islet tissue has emerged as an intriguing alternative to the use of islet tissue from mammalian pancreases. The islet tissue, called Brockman bodies (BBs), is anatomically distinct from the pancreatic exocrine tissue and can be easily identified and isolated. Islets harvested from Nile tilapia (Oreochromis niloticus), when transplanted into streptozotocin-diabetic nude mice, produce long-term normoglycemia and achieve mammalian-like glucose tolerance profiles. We asked whether tilapia express the alpha-(1,3)gal epitope, the immunodominant target of human xenogeneic responses. METHODS AND RESULTS: Immunostaining with the alpha-(1,3)gal-specific IB4 lectin on tilapia BB, liver, heart, spleen, and head kidney was negative, as was staining with murine anti-alpha-gal-specific monoclonal antibodies. Absence of alpha-gal-specific binding of IB4 or murine anti-gal mAbs to dispersed BBs was confirmed by fluorescent-activated cell sorter analysis. Tilapia BB cell membranes failed to reduce binding of anti-alpha-(1,3)gal-specific mAb in an enzyme-linked immunosorbent assay (ELISA) inhibition assay, while porcine and murine tissue lysates did. Tilapia BB cell lysates were shown to be devoid of alpha-1,3 galactosyltransferase activity by ELISA. Transplantation of tilapia BBs into diabetic alpha-gal knockout (gal KO) mice was not associated with accelerated xenograft rejection when compared with wild type control recipients (mean survival time 6.5 days vs. 7.2 days). Tilapia BBs failed to induce a rise in anti-gal IgG and IgM titers in gal KO mice, while the transplant of wild type mouse islets into gal KO mice caused a significant rise in anti-gal IgG and IgM antibodies. CONCLUSIONS: We conclude that tilapia BBs are devoid of alpha-gal expression, and may offer an alternative to swine as a donor species for islet xenotransplantation.     

Transplantation of neonatal islets from LEA29Y‐transgenic pigs restores normoglycemia in streptozotocin‐diabetic NSG‐mice

Beta cell replacement therapy represents the only way towards complete restoration of the physiological glucose homeostasis in patients with Type 1 diabetes. Xenotransplantation of transgenic pig islets expressing immunomodulatory molecules might represent a promising approach to overcome the problems of lacking organ donors, adverse effects of the required systemic immunosuppression, and disappointing transplantation outcomes. LEA29Y, a second generation CTLA‐4‐Ig fusion protein exerts potent immunosuppressive effects by selective blockade of the B7 costimulatory pathway. To assess the impact of high local LEA29Y concentrations on xenogeneic islet graft rejection, a transgenic pig model with islet‐specific LEA29Y transgene expression was generated. Islet‐like clusters from neonatal pigs expressing LEA29Y under the control of the porcine insulin promoter and from wild‐type pigs were isolated by collagenase digestion and subsequent in vitro culture. After 6 days 2500 clusters/mouse were transplanted under the kidney capsule of streptozotocin‐diabetic NOD‐scidIL2Rgamma^null (NSG) mice. After an initial period of insulin dependency, mice developed stable normoglycemia within 8 weeks after transplantation. Transplanted mice of both groups (Tx, wt; Tx, LEA29Y) exhibited normal and as compared to non‐transplanted normoglycemic NSG mice even improved fasting glucose and glucose tolerance resulting from glucose‐responsive graft‐derived porcine insulin secretion. Beta cell‐specific expression of LEA29Y was detectable in neonatal porcine pancreas, in the grafts of transplanted mice, and was also measurable in the plasma of normoglycemic mice. Furthermore, mice with LEA29Y transgenic grafts exhibited a glucose‐dependent LEA29Y release during glucose tolerance testing. The present study demonstrates that LEA29Y transgenic islet‐like clusters display normal beta cell function and have the same potential as wild‐type islet clusters to restore normoglycemia in streptozotocin‐diabetic NSG mice after an in vivo maturation period towards a functional endocrine tissue. NSG mice with LEA29Y islet grafts may therefore represent a promising model to study the modulation of human‐anti‐pig xenograft rejection in detail. In ongoing experiments xenograft rejection is analyzed in “humanized” mice after the transfer of human PBMCs or isolated CD34⁺ stem cells.     

The potential of local immunomodulation and tolerance induction in porcine islet xenotransplantation

Transplantation of pancreas or isolated islet cells is currently the only option to cure type 1 diabetes. The success of islet transplantation is still limited by the requirement of large numbers of high quality islets and the shortage of organ donors. Porcine islets are a promising cell source, but the intensive immunosuppressive regimen required to suppress rejection prevents the translation into clinical practice. We aimed to develop a novel method to inhibit the human‐anti‐pig immune reaction by the expression of immunomodulatory molecules in porcine beta cells. Thus, a transgenic pig was generated expressing LEA29Y – a second generation human CTLA4‐Ig fusion protein, which inhibits activation of T cells by CD80/CD86‐CD28 costimulation – under the control of the porcine insulin promotor. Islet‐like clusters (ICC) from neonatal pigs were isolated and transplanted under the kidney capsule of diabetic NOD‐scid‐IL2γnull (NSG) mice. After an in vivo maturation period mice transplanted with wildtype (wt) as well as with LEA29Y transgenic (tg) ICCs developed normal glucose homeostasis. Within 30 days after the transfer of human PBMCs 80% of NSG mice transplanted with wt‐ICCs developed diabetes indicating xenograft rejection. By contrast, LEA‐tg ICCs were completely protected from rejection in all animals (1). Immunohistochemistry revealed a massive intra‐islet T cell infiltration, which was absent in the LEA‐tg ICCs. This proof of principle study suggests that specific expression of immunomodulatory molecules in beta cells does not disturb beta cell function and may have the potential to modulate immune response locally at the transplantation site without systemic immunosuppression. To overcome the strong xenogeneic barrier of the human and cellular immune system a combination of LEA29Y with additional immunomodulatory factors may be required. Recently, Yi and coworkers demonstrated that the treatment with in vitro expanded regulatory T cells (Treg) prevents porcine islet rejection in humanized NSG mice by the suppression of the T cell‐mediated graft destruction (2). Other potential candidates to induce a state of tolerance against porcine islets currently under investigation are molecules targeting innate immunity and factors that prevent the reoccurrence of autoimmunity. Recent advances in xenotransplantation suggest that it may be possible to start with clinical trials using porcine neonatal or adult islets within the near future. References: 1. KLYMIUK N, VAN BÜRCK L, BÄHR Aet al. Xenografted islet‐cell‐clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice. Diabetes 2012; 61:1527–1532. 2. YI S, JI M, WU J et al. Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin‐10 in humanized mice. Diabetes 2012; 61:1180–1191.     

The Introduction of Human Heme Oxygenase‐1 and Soluble Tumor Necrosis Factor‐α Receptor Type I With Human IgG1 Fc in Porcine Islets Prolongs Islet Xenograft Survival in Humanized Mice

Apoptosis during engraftment and inflammation induce poor islet xenograft survival. We aimed to determine whether overexpression of human heme oxygenase‐1 (HO‐1) or soluble tumor necrosis factor‐α receptor type I with human IgG₁ Fc (sTNF‐αR‐Fc) in porcine islets could improve islet xenograft survival. Adult porcine islets were transduced with adenovirus containing human HO‐1, sTNF‐αR‐Fc, sTNF‐αR‐Fc/HO‐1 or green fluorescent protein (control). Humanized mice were generated by injecting human cord blood–derived CD34⁺ stem cells into NOD‐scid‐IL‐2Rγ^null mice. Both HO‐1 and sTNF‐αR‐Fc reduced islet apoptosis under in vitro hypoxia or cytokine stimuli and suppressed RANTES induction without compromising insulin secretion. Introduction of either gene into islets prolonged islet xenograft survival in pig‐to‐humanized mice transplantation. The sTNF‐αR‐Fc/HO‐1 group showed the best glucose tolerance. Target genes were successfully expressed in islet xenografts. Perigraft infiltration of macrophages and T cells was suppressed with decreased expression of RANTES, tumor necrosis factor‐α and IL‐6 in treatment groups; however, frequency of pig‐specific interferon‐γ–producing T cells was not decreased, and humoral response was not significant in any group. Early apoptosis of islet cells was suppressed in the treatment groups. In conclusion, overexpression of HO‐1 or sTNF‐αR‐Fc in porcine islets improved islet xenograft survival by suppressing both apoptosis and inflammation. HO‐1 or sTNF‐αR‐Fc transgenic pigs have potential for islet xenotransplantation.     

Effect of glucose toxicity on intraportal tilapia islet xenotransplantation in nude mice

BACKGROUND: Discordant xenogeneic islets transplanted intraportally into athymic nude rats experience primary non-function and are rapidly destroyed. Recently, it has been reported that adult porcine islets transplanted intraportally into nude mice are also rapidly destroyed and that this constitutes a new model for instant blood-mediated inflammatory reaction (IBMIR). METHODS: Tilapia (fish) islets were harvested, mechanically broken into mammalian islet-sized fragments, cultured for 48 h, and transplanted via the portal vein into athymic or euthymic mice. RESULTS: There were several groups of recipient mice. Streptozotocin-diabetic nude mice received 400 islets via the portal vein (n = 12). Recipients were killed when hyperglycemic (>200 mg/dl); livers and native pancreases were examined histologically. Mean graft survival time, based on function, was 5.4 +/- 1.2 days; at autopsy, histology showed occasional viable islets. We also performed a group of transplants in non-diabetic nude mice (n = 6) and then killed the recipients 2 or 4 weeks later; all had abundant viable, well-granulated islet grafts based on histology. Therefore, the intraportal environs in nude mice are not incompatible with discordant fish islets; rather, it appears as if hyperglycemia adversely affects the intraportal islet grafts (i.e. 'glucose toxicity'). To test this hypothesis, transplants were performed into non-diabetic nude mice and allowed to engraft for either 3 days (n = 6) or 10 days (n = 8) prior to injection of streptozotocin (200 to 220 mg/kg i.v.) to destroy the beta-cells in the recipients' native islets (n.b. tilapia islets are exceedingly resistant to streptozotocin); these recipients were followed for 28 days post-transplantation (or until hyperglycemic) and then killed for histology. Mean graft function exceeded 25 days for both groups and viable well-granulated, tilapia islets grafts were readily identified in all recipients; in all but one, the native pancreases were markedly beta-cell depleted -- confirming that normoglycemia was due to functional fish islet xenografts. CONCLUSIONS: Our results suggest that 'glucose toxicity' plays a role in the immediate demise of intraportal tilapia islet xenografts.     

Amelioration of streptozotocin-induced diabetes in mice using human islet cells derived from long-term culture in vitro

BACKGROUND: Long-term maintenance of the phenotype of beta cells in vitro is difficult. The objective of this study was to examine an in vitro method for preserving the capacity of adult human beta cells to express insulin. We evaluated the use of long-term cultured islet cells for the treatment of diabetic SCID mice. METHODS: Human islets were isolated from cadaveric donors. The islets were cultured as monolayers and clusters in repeating cycles for 4 months. Thereafter, the cells were tested in vitro for their capacity to express insulin and to secrete insulin in response to glucose challenge. Finally, the cluster-cultured cells were transplanted under the kidney capsule and into the kidney tissue in streptozotocin (STZ)-induced diabetic SCID mice. RESULTS: Approximately 3.6% of cultured islet cells in cluster phase expressed insulin at 4 months and this was confirmed using immuno-gold-labeling electron microscopy. The cultured islet cells secreted insulin in response to glucose challenge in a dose-dependent manner. After transplantation, the islet cells redifferentiated and generated >20% insulin positive cells. The 4-month cultured cells rendered the blood glucose level near normal in mild diabetic mice (7.25 mM+/-1.595 vs. 15.225 mM+/-2.55, P<0.0025). CONCLUSION: It is possible to preserve the capacity of adult human islets to express insulin over a 4-month period in vitro, and this capacity was enhanced significantly by transplantation into SCID mice. The described system will be useful in studies of beta cell proliferation and differentiation.     

Enhanced effect of human mesenchymal stem cells expressing human TNF‐αR‐Fc and HO‐1 gene on porcine islet xenotransplantation in humanized mice

Background

Porcine islet xenotransplantation is considered an attractive alternative treatment for type 1 diabetes mellitus. However, it is largely limited because of initial rejection due to Instant Blood‐Mediated Inflammatory Reaction (IBMIR), oxidative stress, and inflammatory responses. Recently, soluble tumor necrosis factor‐ɑ receptor type I (sTNF‐αR) and heme oxygenase (HO)‐1 genes (HO‐1/sTNF‐αR) have been shown to improve the viability and functionality of porcine islets after transplantation.

Methods

In this study, genetically modified mesenchymal stem cells (MSCs) expressing the HO‐1/sTNF‐αR genes (HO‐1/sTNF‐αR‐MSC) were developed using an adenoviral system, and porcine islet viability and function were confirmed by in vitro tests such as GSIS, AO/PI, and the ADP/ATP ratio after coculturing with HO‐1/sTNF‐αR‐MSCs. Subsequently, isolated porcine islets were transplanted underneath the kidney capsule of diabetic humanized mice without MSCs, with MSCs or with HO‐1/sTNF‐αR‐MSCs.

Results

According to the results, the HO‐1/sTNF‐αR‐MSC‐treated group exhibited improved survival of porcine islets and could reverse hyperglycemia more than porcine islets not treated with MSCs or islets cotransplanted with MSCs. Moreover, the HO‐1/sTNF‐αR‐MSC group maintained its morphological characteristics and the insulin secretion pattern of transplanted porcine islets similar to endogenous islets in immunocompetent humanized mice.

Conclusions

Our results suggest that HO‐1/sTNF‐αR‐MSCs are efficient tools for porcine islet xenotransplantation, and this study may provide basic information for pre‐clinical animal models and future clinical trials of porcine islet xenotransplantation.     

Co-encapsulation of Sertoli enriched testicular cell fractions further prolongs fish-to-mouse islet xenograft survival

BACKGROUND: We previously demonstrated that alginate microencapsulation can prolong fish (tilapia) islet xenograft survival in diabetic animals. However, at present, microencapsulation does not provide complete immune protection to discordant islet xenografts, and long-term graft survival requires supplemental low-dose systemic immunosuppression. In the present study, fish islets were co-encapsulated with Sertoli enriched testicular cell fractions to find out whether this would further prolong fish islet graft survival in diabetic mice. METHODS: Sertoli enriched testicular cell fractions were enzymatically harvested from adult Balb/c or Wistar-Furth rats. They were cultured and co-encapsulated with fragmented tilapia islets in alginate microcapsules. Encapsulated islets alone or islets co-encapsulated with Sertoli cells were then intraperitoneally transplanted into streptozotocin-diabetic Balb/c mice, and graft survival times were compared. Encapsulated and co-encapsulated islet function was also confirmed in streptozotocin-diabetic athymic nude mice. RESULTS: Co-encapsulation with Sertoli enriched testicular cell fractions further prolonged mean fish islet graft survival time from 21+/-6.7 days (encapsulated islet cells alone) to >46+/-6.3 days (co-encapsulated with syngeneic murine Sertoli cells), without additional systemic immunosuppression. Testicular cells harvested from xenogeneic Wistar-Furth rats produced similar protective results (>46+/-10.9 days). CONCLUSIONS: Our results support the theory that Sertoli cells produce local immunosuppressive factors. These factors supplement the immune protective feature of alginate microcapsules in our model. Testicular cell fractions may be an important naturally occurring facilitator in the development of new microencapsulation systems for islet xenotransplantation.     

Functional assessment of microencapsulated porcine islets with agarose polystyrene sulfonic acid in vitro and in xenotransplantation

We evaluated the functional efficacy of microencapsulated porcine islet xenografts transplanted into nonobese diabetic (NOD) mice. Islets were isolated from the pancreata of CSK miniature swine by manual collagenase digestion and Ficoll purification. Purified porcine islets were immediately encapsulated into microbeads of agarose polystyrene sulfonic acid (Ag-PSSa). They remained morphologically intact by dithizone staining after 7 days in culture. Insulin secretion from encapsulated islets was determined in response to glucose challenge during perifusion. When encapsulated islets were exposed to 200 mg/dL glucose, within 5 minutes, insulin release became 5-fold greater than that at 80 mg/dL. However, a second phase insulin secretion appeared in response to 250 mg/dL glucose challenge. In xenotransplantation, microencapsulated porcine islets (1000 to 1800 MC islets) were transplanted into the peritoneal cavity of diabetic NOD mice (n = 4) without immunosuppression. The survival times after the onset of diabetes were observed after both MC islets transplanted NOD mice and nontransplanted NOD mice (n = 4). MC islets transplant recipients had significantly (P < .05) longer survival (47.5 +/- 18.6; mean +/- SD) than nontransplanted NOD mice (21.0 +/- 9.31), although random blood glucose levels were not normalized.     

Beneficial effects of the transgenic expression of human sTNF-αR-Fc and HO-1 on pig-to-mouse islet xenograft survival

Both human soluble tumor necrosis factor-α receptor-Fc (sTNF-αR-Fc) and heme oxygenase-1 (HO-1) transgenic pigs have been generated previously for xenotransplantation. Here, we investigated whether overexpression of sTNF-αR-Fc or HO-1 in pig islets prolongs islet xenograft survival. Adult porcine islets were isolated from human sTNF-αR-Fc or HO-1 transgenic and wild type pigs, and were transplanted into diabetic nude mice. Effects of the expression of both genes on islet apoptosis, chemokine expression, cellular infiltration, antibody production, and islet xenograft survival were analyzed. Human sTNF-αR-Fc transgenic pigs successfully expressed sTNF-αR-Fc in the islets; human HO-1 transgenic pigs expressed significant levels of HO-1 in the islets. Pig-to-mouse islet xenograft survival was significantly prolonged in both the sTNF-αR-Fc and HO-1 groups compared with that in the wild type group. Both the sTNF-αR-Fc and HO-1 groups exhibited suppressed intragraft expression of monocyte chemoattractant protein-1 (MCP-1) and decreased perigraft infiltration of immune cells. However, there was no difference in the anti-pig antibody levels between the groups. Apoptosis of islet cells during the early engraftment was suppressed only in the HO-1 group. Porcine islets from both sTNF-αR-Fc and HO-1 transgenic pigs prolonged xenograft survival by suppressing islet cell apoptosis or secondary inflammatory responses following islet death, indicating that these transgenic pigs might have applications in successful islet xenotransplantation.     

Native pancreatic α-cell adaptation in streptozotocin-induced diabetic primates: importance for pig islet xenotransplantation

Abstract: Background: Metabolic compatibility between donor and recipient species is an important matter for pig islet xenotransplantation. Glucagon is a key hormone for the function of pig islets as well as control of hypoglycemia in the recipients of the islets. Because a discrepancy exists in the composition of glucagon cells of pig and human/primate islets, the present study was designed to determine the role of native recipient glucagon cells in the treatment of diabetes by islet transplantation in a “pig‐to‐primate” model. Methods: Streptozotocin‐treated (50 mg/kg) monkeys (n = 12, follow‐up of 6 to 231 days) were compared with non‐diabetic animals (n = 5; follow‐up, 180 days). Metabolic [fasting and intravenous glucose tolerance tests (IVGTTs) for serum levels of glucose, insulin, glucagon] and morphologic (endocrine volume density and cell mass for insulin and glucagon) were compared between non‐diabetic and diabetic animals. Six additional diabetic primates were given transplants of 15 000 adult pig islet equivalents without immunosuppression to monitor glucose, glucagon, insulin, and porcine C‐peptide levels until 48 h after transplantation. Results: Elevated fasting blood glucose, pathologic IVGTT, destruction of 95% of β‐cell mass, and glycosylated hemoglobin (>13%) were assessed in diabetic monkeys. The serum glucagon levels and glucagon cell mass correlated significantly with diabetes time course of diabetes (R = 0.940, p = 0.005; R = 0.663, p = 0.019, respectively). A mean increase of 89% in glucagon cell mass was observed for primates suffering from diabetes >53 days. No response of glucagon secretion was observed for diabetic animals during IVGTT, because no increase of serum insulin levels followed glucose loading. Blood glucose levels dropped after pig islet xenografts in diabetic primates. This reduction was maintained by an insulin level >20 μU/ml over the period of time of xenograft function (porcine C‐peptide >0.1 ng/ml). A total restoration of native primate glucagon sensitivity to insulin was found after pig islets xenotransplantation as revealed by a reduction of 80% of the glucagon level. When graft dysfunction (>24 h post‐transplantation), the insulin level dropped and glucagon levels rose again (>50 pg/ml). Conclusions: Native glucagon cells provide morphologic and functional plasticity to diabetes. Adult pig islet xenotransplantation can restore the sensitivity of primate glucagon to insulin but cannot protect the diabetic recipient against hypoglycemia.     

Transplantation of porcine islets in nude mice: Implications for islet replacement therapy in humans

Abstract: Porcine islets obtained from adult donors were transplanted under the kidney capsule of streptozotocin diabetic nude mice. Over a period of 30 days or more, blood glucose levels fell to values lower than those of normal mice but comparable to those of normal pigs; this change in the mice was probably being driven by a lower set point for glucose-induced insulin secretion of porcine islets. Oral and intraperitoneal glucose tolerance tests had lower glucose profiles than those carried out in control mice. The mass of beta cell tissue in the porcine islet graft that cured the diabetes was about 1 mg, close to the normal beta cell mass of a mouse pancreas. When graft-bearing kidneys were perfused in situ, there was a marked increase of insulin secretion to challenges with glucose and arginine. These results suggest that porcine islets could be a good source of tissue for human islet replacement therapy. Some of the ramifications of this possibility are discussed.     

Long‐Term Controlled Normoglycemia in Diabetic Non‐Human Primates After Transplantation with hCD46 Transgenic Porcine Islets

Xenotransplantation of porcine islets into diabetic non‐human primates is characterized by (i) an initial massive graft loss possibly due to the instant blood‐mediated inflammatory reaction and (ii) the requirement of intensive, clinically unfriendly immunosuppressive therapy. We investigated whether the transgenic expression of a human complement‐regulatory protein (hCD46) on porcine islets would improve the outcome of islet xenotransplantation in streptozotocin‐induced diabetic Cynomolgus monkeys. Immunosuppression consisted of thymoglobulin, anti‐CD154 mAb for costimulation blockade, and mycophenolate mofetil. Following the transplantation of islets from wild‐type pigs (n = 2) or from 1,3‐galactosyltransferase gene‐knockout pigs (n = 2), islets survived for a maximum of only 46 days, as evidenced by return to hyperglycemia and the need for exogenous insulin therapy. The transplantation of islets from hCD46 pigs resulted in graft survival and insulin‐independent normoglycemia in four of five monkeys for the 3 months follow‐up of the experiment. One normalized recipient, selected at random, was followed for >12 months. Inhibition of complement activation by the expression of hCD46 on the pig islets did not substantially reduce the initial loss of islet mass, rather was effective in limiting antibody‐mediated rejection. This resulted in a reduced need for immunosuppression to preserve a sufficient islet mass to maintain normoglycemia long‐term.     

Engraftment of Adult Porcine Islet Xenografts in Diabetic Nonhuman Primates Through Targeting of Costimulation Pathways

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.     

Neonatal porcine islets exhibit natural resistance to hypoxia-induced apoptosis

BACKGROUND: Despite the success of the Edmonton protocol for human islet transplantation, an alternate source of islet tissue must be developed if beta-cell replacement therapy is to see widespread application. Neonatal porcine islets (NPI) represent one potential source of tissue. When human or rodent islets are transplanted, the majority of cells undergo hypoxia-induce apoptosis soon after the grafts are placed in the recipient. In the present study, we investigated whether NPI were similarly sensitive to hypoxia. METHODS: NPI were exposed to hypoxia and hypoxia/reoxygenation using an in vitro hypoxic chamber. Afterwards, viability, frequency of apoptosis, and beta-cell function were evaluated. NPI and adult porcine islets were transplanted into chemically diabetic, immunodeficient mice and graft apoptosis was assessed 24 hours and seven days posttransplant. RESULTS: NPI demonstrated a remarkable capacity to resist apoptosis and maintain insulin secretion despite severe stresses such as hypoxia/reoxygenation. One day after transplantation, NPI grafts showed limited apoptosis, confined to rare strongly insulin positive cells. In contrast, adult porcine islet grafts underwent widespread apoptosis. Western blotting revealed that NPI express high levels of at least one potent endogenous antiapoptotic protein (XIAP). CONCLUSIONS: The majority of cells within transplanted human islets undergo apoptosis soon after portal infusion. In contrast, NPI have the capacity to resist this early posttransplant apoptosis, with likely reduced antigen release and diminished immune stimulation. NPI appear to contain a population of insulin-low to insulin-negative pre-beta-cells, which are resistant to hypoxia-induced apoptosis and still capable of differentiating into mature beta-cells.     

Co‐Transplantation of Bone Marrow‐Derived Endothelial Progenitor Cells Improves Revascularization and Organization in Islet Grafts

Bone marrow‐derived early endothelial progenitor cells (BM‐EPCs) are a clinical tool for enhancing revascularization. However, the therapeutic efficacy of co‐transplantation of BM‐EPC with islets has not been investigated. In this study, marginal mass islets were co‐transplanted with or without BM‐EPCs under the kidney capsules of syngeneic streptozotocin‐induced diabetic mice. Using green fluorescent protein transgenic (GFP‐Tg) mice as BM‐EPC and islet donors or recipients, the role of EPCs in revascularization was assessed for graft morphology, vascular density and fate of EPCs by immunohistochemistry. Islet‐EPC co‐transplantation improved the outcome of islet transplantation as measured by glucose tolerance, serum insulin level and diabetes reversal rate, compared with transplantation of islets alone. Between groups, the morphology of islet grafts showed significant differences in size and composition of grafted endocrine tissues. Significantly more vessel density derived from donors and recipients was detected with islet‐EPC co‐transplantation. Abundant GFP‐Tg mice‐derived BM‐EPCs (GFP‐EPCs) were observed in or around islet grafts and incorporated into CD31‐positive capillaries. Remaining GFP‐EPCs expressed VEGF. In conclusion, co‐transplantation of islets with BM‐EPCs could improve the outcome of marginal mass islet transplantation by promoting revascularization and preserving islet morphology.     

Advantages of combined fetal and adult porcine islets as donor tissue for transplantation

Abstract: The molecular similarity between human and porcine insulin is the primary reason for selecting the pig as a donor for islet xenotransplantation in order to treat type I diabetes. Porcine islets can be prepared from adult, newborn, or fetal pigs. However, each of these islet types possesses advantages and disadvantages due to their anatomical, developmental, and physiological characteristics. We have evaluated both adult and fetal porcine islets for their ability to reverse diabetes using an allograft model. Based on our accumulated data, we recommend transplantation of combined adult and fetal porcine islets as a prospective approach for pig to man islet xenotransplantation. The advantages of combined adult and fetal islet transplantation are as follows: 1) Adult porcine islets can reverse hyperglycemia soon after transplantation; 2) well controlled blood glucose at the critical developmental stage of fetal islets will provide the time and environment necessary to achieve the optimal proliferation, growth and maturation of fetal β cells; 3) after 2 to 3 months, fetal islets will functionally mature and begin to share the insulin-producing load previously carried by adult islets, while new islets will continuously emerge and proliferate from stem cells; and 4) islets from fetal pigs will subsequently play a primary role in controlling hyperglycemia, and provide long-lasting insulin independence.     

CD40‐Specific Costimulation Blockade Enhances Neonatal Porcine Islet Survival in Nonhuman Primates

The widespread clinical implementation of alloislet transplantation as therapy for type 1 diabetes has been hindered by the lack of suitable islet donors. Pig‐to‐human islet xenotransplantation is one strategy with potential to alleviate this shortage. Long‐term survival of porcine islets has been achieved using CD154‐specific antibodies to interrupt the CD40/CD154 costimulation pathway; however, CD154‐specific antibodies seem unlikely candidates for clinical translation. An alternative strategy for CD40/CD154 pathway interruption is use of CD40‐specific antibodies. Herein, we evaluate the ability of a chimeric CD40‐specific monoclonal antibody (Chi220) to protect islet xenografts. Neonatal porcine islets (～50 000 IEQ/kg) were transplanted intraportally into pancreatectomized diabetic macaques. Immunosuppression consisted of induction therapy with Chi220 and the IL‐2 receptor‐specific antibody basiliximab, and maintenance therapy with sirolimus and the B7‐specific fusion protein belatacept. Chi220 effectively promoted xenoislet engraftment and survival, with five of six treated recipients achieving insulin‐independent normoglycemia (median rejection‐free survival 59 days; mean 90.8 days, maximum 203 days). No thromboembolic phenomena were observed. CD40 represents a promising alternative to CD154 as a therapeutic target, and the efficacy of CD40‐specific antibodies in islet xenotransplantation warrants further investigation.