Neonatal Porcine Islets Research Articles

Dual Islet Transplantation Modeling of the Instant Blood-Mediated Inflammatory Reaction

Islet xenotransplantation is a potential treatment for diabetes without the limitations of tissue availability. Although successful experimentally, early islet loss remains substantial and attributed to an instant blood-mediated inflammatory reaction (IBMIR). This syndrome of islet destruction has been incompletely defined and characterization in pig-to-primate models has been hampered by logistical and statistical limitations of large animal studies. To further investigate IBMIR, we developed a novel in vivo dual islet transplant model to precisely characterize IBMIR as proof-of-concept that this model can serve to properly control experiments comparing modified xenoislet preparations. WT and α1,3-galactosyltransferase knockout (GTKO) neonatal porcine islets were studied in nonimmunosuppressed rhesus macaques. Inert polyethylene microspheres served as a control for the effects of portal embolization. Digital analysis of immunohistochemistry targeting IBMIR mediators was performed at 1 and 24 h after intraportal islet infusion. Early findings observed in transplanted islets include complement and antibody deposition, and infiltration by neutrophils, macrophages and platelets. Insulin, complement, antibody, neutrophils, macrophages and platelets were similar between GTKO and WT islets, with increasing macrophage infiltration at 24 h in both phenotypes. This model provides an objective and internally controlled study of distinct islet preparations and documents the temporal histology of IBMIR.

Neonatal Porcine Islet-Mesenchymal Stem Cells Co-Transplantation in Diabetes Rhesus Monkeys.

Objective In this study, we investigated the effects of monkeyes menchamal Stem Cell (MSC) on neonatal porcine islets (NPI) survival and insulin secretion in vitro and in vivo. Methods NPI were cultured with or without MSC, and exposed to hypoxia (O2 ≤ 1%, 12h) /reoxygenation (48h) respectively. Apoptotic cells were quantified using Annexin V. The effect of MSC on the viability of NPI was detected by CCK8. Islet function was evaluated by GSIS assay. mRNA expression of HIF-1α, HO-1, COX-2 and iNOS were detected by qRT-PCR. 30,000 IEQ/kg NPI were infusedinto the mesenteric vein of diabetic monkeys with or without MSCs (2×106/kg, 5 times). ATG, tacrolimus and sirolimus administration were used. Results When islets were cocultured with MSC in ratio of 10:1, MSC can decrease H/R-induced apoptotic cells (46.2 % vs. 64.3%), increase the islet metabolism activity at 38 %, maintained the stimulation index of GSIS at higher level (1.35 vs. 1.15), increase HIF-1a, HO-1, COX-2, and iNOS mRNA expression.Figure: No Caption available.In islet-MSC co-transplantion group, 4 of the 5 monkeys attained insulin independence for 1-2 months, and then with reduced insulin injection for another 3 months. In the NPI transplantation alone group (n=6), 2 recipients had reduced insulin injection for 5 months. Conclusion Monkey MSC can protect islet vitality and function from H/R-induced injury. NPI and MSCs co-transplantation can improve the porcine islets functional differentiation and the success rate of pig-monkey islet transplantation.

Therapeutic Effects of Islets Isolated From Genetically Engineered Pigs Transgenic for hCD46 and hCD39 in Immune-Deficient Mice.

Background: Xenotransplantation using neonatal porcine islets is being considered as an alternative to allotransplantation as treatment for type 1 diabetes mellitus due to a more stable supply of islets. To overcome severe immunological responses, genetically engineered (GE) pigs have been developed with islets that may be quiescent to the human immune system. In this study, we investigated therapeutic effects of islets isolated from two different types of neonatal GE pigs. Group 1 GE pigs (GT KO+hCD46), the epitope alpha1, 3-galactose (αGal) was absent (GT KO), and transgenic for a human complement regulatory protein (hCD46). Group 2 GE pigs (GT KO+hCD46+hCD39) had the same genetic modification as Group 1 GE pigs but also were transgenic for a human anticoagulant gene (hCD39). Methods: Pancreases were harvested from Group 1 GE pigs aged 5 days, and from Group 2 GE pigs aged 2, 6 and 7 days. Islets were isolated with collagenase processing and followed by 7-day culture purification. Morphological and cellular composition of the islets were determined by immunohistochemical analysis and 2000 islet equivalents were transplanted under the kidney capsule of Sterptozotocin-induced diabetic immune-deficient mice to assess their function in vivo. Results: Morphological evaluation of the islets from Group 1 and Group 2 GE pigs showed no difference in the insulin and glucagon-positive cells. In addition, neonatal islets from both groups had no detectable αGal-positive cells. Immunoflorescence staining of pancreas sections taken from pigs cloned from the same exact cells showed strong CD46 and CD39 expression. The in vivo study showed that six of the 10 mice that received islets from Group 1 GE pigs and two of the four mouse recipients of islets from 6 day-old Group 2 GE pigs reached normal glycemia after 54.0 and 55.5 days of transplantation respectively. The mouse recipients of islets from 2 and 7 day-old Group 2 GE pigs remained diabetic for 113 days post-transplantation. Conclusions: In this study, islets from both types of GE pigs showed therapeutic effect on chemically induced diabetic immune-deficient mice. Furthermore, it remains to be determined whether islets from GT KO+hCD46 ± hCD39 pigs will be less susceptible to rejection mediated by human immune cells. DISCLOSURES:Bianchi, J.: Employee, Revivicor/United Therapeutics. Ayares, D.: Employee, Revivicor/United Therapeutics.

Clinical Porcine Islet Xenotransplantation Under Comprehensive Regulation

BackgroundXenotransplantation with porcine islets is a promising approach to overcome the shortage of human donors. This is the first report of phase 1/2a xenotransplantation study of encapsulated neonatal porcine islets under the current framework of regulations for xenotransplantation in New Zealand. MethodsNewborn piglets were anesthetized and bled, and the pancreata were removed with the use of sterile technique and processed. Encapsulated neonatal porcine islets were implanted with the use of laparoscopy into the peritoneal cavity of 14 patients with unstable type 1 diabetes without any immunosuppressive drugs. The patients received encapsulated islets of 5,000 (n = 4; group 1), 10,000 (n = 4; group 2), 15,000 (n = 4; group 3), or 20,000 (n = 2; group 4) islet equivalents per kg body weight. Outcome was determined from adverse event reports, HbA1c, total daily insulin dose, and frequency of unaware hypoglycemic events. To assess graft function, transplant estimated function (TEF) scores were calculated. Sufficient or marginal numbers of encapsulated neonatal porcine islets were transplanted into streptozotocin-induced diabetic B6 mice as an in vivo functional assay. ResultsThere were 4 serious adverse events, of which 3 were considered to be possibly related to the procedure. Tests for porcine endogenous retrovirus DNA and RNA were all negative. The numbers of unaware hypoglycemia events were reduced after transplantation in all groups. Four of 14 patients attained HbA1c <7% compared with 1 at baseline. The average TEF scores were 0.17, 0.02, −0.01, and 0.08 in groups 1, 2, 3, and 4 respectively. The in vivo study demonstrated that a sufficient number of the transplanted group reversed diabetes with positive porcine C-peptide. ConclusionsTransplantation of encapsulated neonatal porcine islets was safe and was followed by a reduction in unaware hypoglycemia events in unstable type 1 diabetic patients. The mouse in vivo assessment data demonstrated certain graft function.

Control of IBMIR in Neonatal Porcine Islet Xenotransplantation in Baboons

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.

Encapsulated piscine (tilapia) islets for diabetes therapy: studies in diabetic NOD and NOD‐SCID mice

Our goal was to improve islet transplantation as a therapy for patients with type I diabetes mellitus. Because human donor islets are scarce, we are studying islet xenografts in the diabetic NOD mouse model. We hypothesize that optimal xenoislet survival will be achieved by the combination of donor islet immunoisolation with recipient immunosuppression. We and others have studied adult and neonatal porcine islets as sources of tissue for microencapsulated islet xenografts, but we believe it is also advantageous to consider using islets from fish, which can be raised in large numbers relatively quickly and economically. Therefore, in this study, we have evaluated the function of microencapsulated xenogeneic piscine (tilapia) islets transplanted intraperitoneally (IP) in NOD mice in the presence of CD4(+) T-cell depletion and/or costimulatory blockade. Spontaneously diabetic NOD mice or streptozotocin (STZ)-diabetic NOD-SCID mice were transplanted IP with microencapsulated tilapia islets. Recipient immunosuppression included anti-CD4 mAb, CTLA4-Ig, anti-CD80 mAb, anti-CD86 mAb, or anti-CD154 mAb, alone or in combination. Graft function was evaluated by blood glucose (BG) levels, intravenous (IV) and oral glucose tolerance tests (GTTs), histologic and immunohistochemical analyses of grafts, and flow cytometric analysis of peritoneal cells. Encapsulated tilapia islets normalized random BG levels for up to 210 days in NOD-SCID mice. In diabetic NOD mice, encapsulated tilapia islets were rejected on day 11 ± 4 with a peritoneal infiltrate of macrophages, eosinophils, B cells, occasional neutrophils, but few T cells. Immunohistochemical staining demonstrated the presence of murine IgG on tilapia islets within capsules of rejecting, non-immunosuppressed mice, as well as murine IgG-positive lymphocytes in the layer of host cells surrounding those capsules. These findings suggested that our barium (Ba)-gelled alginate capsules are permeable to IgG and that anti-piscine antibodies may be involved in the rejection of encapsulated tilapia islets in untreated mice. No single immunosuppressive agent prolonged encapsulated tilapia islet survival in NOD mice, but the combination of CTLA4-Ig plus anti-CD154 mAb extended tilapia islet graft survival until rejection at 119 ± 20 days and inhibited host cell recruitment to the peritoneal cavity. Triple treatment with CTLA4-Ig, anti-CD154 mAb, and anti-CD4 mAb allowed graft survival for 157 ± 35 days with little evidence of a host cellular reaction. IV and oral glucose tolerance tests (GTTs) of recipients with functioning xenografts demonstrated remarkably normal metabolic function. We conclude that microencapsulated tilapia islets can survive long term with excellent metabolic control in diabetic mice given targeted immunosuppression, suggesting that cross-species physiological incompatibility may not compromise the applicability of this novel approach for future clinical applications. We predict that an improved microcapsule that prevents the entrance of IgG will enhance tilapia islet survival in this model, possibly allowing the application of this technique with limited or no immunosuppression.

Bioengineering a highly vascularized matrix for the ectopic transplantation of islets

Islet transplantation is a promising treatment for Type 1 diabetes; however limitations of the intra-portal site and poor revascularization of islets must be overcome. We hypothesize that engineering a highly vascularized collagen-based construct will allow islet graft survival and function in alternative sites. In this study, we developed such a collagen-based biomaterial. Neonatal porcine islets (NPIs) were embedded in collagen matrices crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide containing combinations of chondroitin-6-sulfate, chitosan, and laminin, and compared with controls cultured in standard media. Islets were examined for insulin secretory activity after 24 h and 4 d and for apoptotic cell death and matrix integrity after 7 d in vitro. These same NPI/collagen constructs were transplanted subcutaneously in immunoincompetent B6.Rag−/− mice and then assessed for islet survival and vascularization. At all time points assessed during in vitro culture there were no significant differences in insulin secretory activity between control islets and those embedded in the collagen constructs, indicating that the collagen matrix had no adverse effect on islet function. Less cell death was observed in the matrix with all co-polymers compared with the other matrices tested. Immunohistochemical analysis of the grafts post-transplant confirmed the presence of intact insulin-positive islets; grafts were also shown to be vascularized by von Willebrand factor staining. This study demonstrates that a collagen, chondroitin-6-sulfate, chitosan, and laminin matrix supports islet function in vitro and moreover allows islet survival and vascularization post-transplantation; therefore, this bio-engineered vascularized construct is capable of supporting islet survival.

Long-Term Survival of Neonatal Porcine Islets Without Sertoli Cells in Rabbits

Cell-based therapy is a promising treatment for metabolic disorders such as type-1 diabetes. Transplantation protocols have investigated several anatomical sites for cell implantation; however, some of these procedures, such as intraportal infusion, can cause organ failure or thrombosis secondarily. Bio-artificial organs could be the choice, although concerns still remain. Using a subcutaneous device, we are able to preserve neonatal porcine islets without sertoli cells in healthy New Zealand rabbits. Devices were implanted in the back of the animals underneath the skin, and after 3 months the islets were transplanted. Histology showed the presence of inflammatory cells, predominantly eosinophils; however, insulin- and glucagon-positive cell clusters were identified inside the device at different time points for at least 90 days, and porcine C-peptide was also detected during the follow-up, indicating graft functionality. We have found that our device induces the deposition of a fibrous matrix enriched in blood vessels, which forms a good place for cell grafting, and this model is probably able to induce an immunoprivileged site. Under these conditions, transplanted porcine islet cells have the capability of producing insulin and glucagon for at least three months.

Evidence of safety and efficacy of two forms of neonatal cell transplants

On behalf of DOL and LCT A clinical trial in 14 adult type 1 diabetics intra‐peritoneal alginate microencapsulated neonatal porcine islets transplants commenced in NZ over 2 years ago and the last patient has just completed the required 12 months of follow up.To meet regulatory requirements for xenotransplantation, an elaborate program on xenovirology has been developed on one unique herd of pigs that was free from all conventional pathogens and was qualified as “null” pigs, i.e. they do not have a transmittable pig endogenous retrovirus (PERV). These pigs are the animal founders for the DPF donor herd. A comprehensive program for patients’ microbiology follow‐up was also developed. This program includes assays developed for monitoring potential infection with PERV and other potentially xenotic pathogens.All of the patients selected had unaware hypoglycaemia. The dose of islets used varied from 5000/kg (n = 4) 10 000/kg (n = 4) 15 000/kg (n = 4), and 20 000/kg (n = 2). The patients experience little if any side effects and there was no evidence of xenosis. Unaware hypoglycaemia was lessened in all groups but more so in the 10 000/kg group. Insulin dose was lowered and HbA1c improved in some but not all subjects. The patients with maximum insulin dose drop received 5000/kg dose. There was no clear cut relationship of return of hypoglycaemia awareness and glucagon or adrenergic responses to induced hypoglycaemia. There was direct laboratory evidence of islet beta cell function. Clinical follow up at 2 years of some patients indicates persistence of the benefits. A further clinical trial involving a different dosing schedule and some technical improvements is in progress.Choroid plexus (CP) ependymal epithelium secretes a wide variety of neurotrophic and neural support molecules. Micro‐encapsulated CP delivered intra‐cerebrally into rat models of stroke, Huntington's disease and Parkinson's disease show remarkable functional and histological recovery. In the quinolinic acid primate model of Huntington's and the MTPP model of Parkinson's functional recovery and relevant histological evidence of neuro‐regeneration were seen. The treatment was safe. A clinical trial of this form of treatment in humans with Parkinson's is being undertaken.Conclusions: Xenotransplantation of microencapsulated islets and other cell types into human and non‐human primates from a biocertified herd of pigs prepared under GMP conditions can be conducted with safety and efficacy. Further improvements in clinical outcomes are expected with technical advances.

Real‐time assessment of encapsulated neonatal porcine islets prior to clinical xenotransplantation

Porcine islet transplantation is emerging as an attractive option for the treatment of patients with type 1 diabetes, with the possibility of providing islets of higher and more consistent quality and in larger volumes than available from human pancreata. The use of encapsulated neonatal porcine islets (ENPI) is appealing because it can address islet supply limitations while reducing the need for anti-rejection therapy. Pre-transplant characterization of ENPI viability and potency is an essential component of the production process. We applied the validated assay for oxygen consumption rate normalized for DNA content (OCR/DNA) to characterize ENPI viability. ENPI of low viscosity and high m alginate were prepared according to standard methods and characterized at various culture time points up to 5 weeks. The OCR/DNA (nmol/min·mgDNA ± SEM) of ENPI (235 ± 10, n = 9) was comparable to that of free NPI (255 ± 14, n = 13). After encapsulation, NPI OCR/DNA was sustained over a culture period of up to 5 weeks. The average OCR/DNA of ENPI cultured longer than 9 days was higher than that of freshly encapsulated NPI. This is the first characterization of ENPI by a validated and more sensitive method for product viability. The NPI encapsulation process does not compromise viability as measured by OCR/DNA, and ENPI can be cultured for up to 5 weeks with maintenance of viability. ENPI meet or exceed current adult porcine islet product release criteria (established at the University of Minnesota) for preclinical xenotransplantation in terms of OCR/DNA.

A humanized mouse model to study human immune response in xenotransplantation

A major barrier to the clinical application of xenotransplantation as a treatment option for patients is T cell-mediated rejection. Studies based on experimental rodent models of xenograft tolerance or rejection in vivo have provided useful information about the role of T cell immune response in xenotransplantation. However not all observations seen in rodents faithfully recapitulate the human situation. This study aimed to establish a humanized mouse model of xenotransplantation, which mimics xenograft rejection in the context of the human immune system. NOD-SCID IL2rgamma-/- mice were transplanted with neonatal porcine islet cell clusters (NICC) followed by reconstitution of human peripheral blood mononuclear cells (PBMC). Human leukocyte engraftment and islet xenograft rejection were confirmed by flow cytometric and histological analyses. In the absence of human PBMC, porcine NICC transplanted into NOD-SCID IL2rgamma-/- mice revealed excellent graft integrity and endocrine function. Human PBMC demonstrated a high level of engraftment in NOD-SCID IL2rgamma-/- mice. Reconstitution of NICC recipient NOD-SCID IL2rgamma-/- mice with human PBMC led to the rapid destruction of NICC xenografts in a PBMC number-dependent manner. Human PBMC-reconstituted NOD-SCID IL2rgamma-/- mice provide an ideal model to study human immune responses in xenotransplantation. Studies based on this humanized mouse model will provide insight for improving the outcomes of clinical xenotransplantation.

Alternative Immunomodulatory Strategies for Xenotransplantation: CD40/154 Pathway-Sparing Regimens Promote Xenograft Survival

Immunosuppressive therapies that block the CD40/CD154 costimulatory pathway have proven to be uniquely effective in preclinical xenotransplant models. Given the challenges facing clinical translation of CD40/CD154 pathway blockade, we examined the efficacy and tolerability of CD40/CD154 pathway-sparing immunomodulatory strategies in a pig-to-nonhuman primate islet xenotransplant model. Rhesus macaques were rendered diabetic with streptozocin and given an intraportal infusion of ≈ 50 000 islet equivalents/kg wild-type neonatal porcine islets. Base immunosuppression for all recipients included maintenance therapy with belatacept and mycophenolate mofetil plus induction with basiliximab and LFA-1 blockade. Cohort 1 recipients (n = 3) were treated with the base regimen alone; cohort 2 recipients (n = 5) were additionally treated with tacrolimus induction and cohort 3 recipients (n = 5) were treated with alefacept in place of basiliximab, and more intense LFA-1 blockade. Three of five recipients in both cohorts 2 and 3 achieved sustained insulin-independent normoglycemia (median rejection-free survivals 60 and 111 days, respectively), compared to zero of three recipients in cohort 1. These data show that CD40/CD154 pathway-sparing regimens can promote xenoislet survival. Further optimization of these strategies is warranted to aid the clinical translation of islet xenotransplantation.

Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice.

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ−/− mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.

Tissue Factor Knockdown in Porcine Islets: An Effective Approach to Suppressing the Instant Blood-Mediated Inflammatory Reaction

Tissue factor (TF) expression on islets has been shown to trigger instant blood-mediated inflammatory reaction (IBMIR), leading to rapid islet loss in portal vein islet transplantation. This study investigated whether antisense RNA-mediated TF gene knockdown in islets could suppress IBMIR as a strategy to overcome IBMIR. Neonatal porcine islet cell clusters (NICCs) were transfected with or without TF-specific antisense RNA or a nonspecific RNA by a lipid-based method. Expression of both TF gene and protein in NICCs was analyzed after transfection by real-time PCR, Western blot, and FACS, respectively. The impact of antisense RNA transfection on NICC viability and in vitro function was examined by FACS and insulin release test, respectively. The effect of TF knockdown in NICCs on IBMIR was assessed with an in vitro tubing loop assay using human blood. A significant reduction in TF gene and protein expression was achieved in TF antisense RNA but not control RNA transfected NICCs, which did not affect NICCs' viability or their insulin secreting capacity. Incubation of TF antisense RNA transfected with human blood resulted in a considerable reduction in blood clot formation, platelet consumption, and complement and coagulation activation compared to that observed in the loops containing human blood and untreated or control RNA transfected NICCs. Consistent with these findings, infiltrating neutrophils in the blood clots with entrapped TF antisense RNA transfected NICCs was also reduced substantially compared to that seen in the clots containing untreated or control RNA transfected NICCs. This study presents a nontoxic TF antisense RNA-mediated TF knockdown in porcine islets that leads to an effective suppression of IBMIR, suggesting a potentially new strategy to improve islet transplantation outcomes.

Protection of Porcine Islet Xenografts in Mice Using Sertoli Cells and Monoclonal Antibodies

To remedy the shortage of human donor islets, xenotransplantation of neonatal porcine islets (NPI) provides an attractive alternative source of donor tissue so long as graft rejection can be circumvented. Thus, in this study, we sought to determine whether cotransplantation of NPI with Sertoli cells (SC) combined with a short-course treatment of monoclonal antibody (mAb) could provide long-term islet xenograft survival. NPI alone or NPI cotransplanted with neonatal porcine SC were transplanted into diabetic C57BL/6 mice. These mice were left untreated or were treated with a short course of antileukocyte function associated antigen-1 (LFA-1), anti-CD154, or anti-CD45RB mAb. Blood glucose levels were monitored twice a week to assess graft function. At more than 100 days posttransplantation or on the day of rejection, graft-bearing kidneys were collected for characterization using immunohistochemistry. None of the untreated control mice transplanted with NPI alone (0/5) or NPI cotransplanted with SC (0/8) achieved normoglycemia. However, of the mice receiving NPI alone, 3 of 7 treated with anti-LFA-1 mAb, 2 of 7 treated with anti-CD154 mAb, and 1 of 7 treated with anti-CD45RB mAb achieved long-term graft survival (>100 days). These proportions improved considerably when NPI were cotransplanted with SC, as 15 of 15 mice treated with anti-LFA-1 mAb, 7 of 8 mice treated with anti-CD154 mAb, and 4 of 9 mice treated with anti-CD45RB mAb achieved long-term graft survival. These results show that transient administration of anti-LFA-1 mAb or anti-CD154 mAb is efficacious in prolonging NPI xenograft survival when islets are cotransplanted with SC. Interleukin-4 and Serpina3n may be important mediators of protection observed in this model.

Islet Xenotransplantation Using Gal-Deficient Neonatal Donors Improves Engraftment and Function

Significant deficiencies in understanding of xenospecific immunity have impeded the success of preclinical trials in xenoislet transplantation. Although galactose-α1,3-galactose, the gal epitope, has emerged as the principal target of rejection in pig-to-primate models of solid organ transplant, the importance of gal-specific immunity in islet xenotransplant models has yet to be clearly demonstrated. Here, we directly compare the immunogenicity, survival and function of neonatal porcine islets (NPIs) from gal-expressing wild-type (WT) or gal-deficient galactosyl transferase knockout (GTKO) donors. Paired diabetic rhesus macaques were transplanted with either WT (n = 5) or GTKO (n = 5) NPIs. Recipient blood glucose, transaminase and serum xenoantibody levels were used to monitor response to transplant. Four of five GTKO versus one of five WT recipients achieved insulin-independent normoglycemia; transplantation of WT islets resulted in significantly greater transaminitis. The WT NPIs were more susceptible to antibody and complement binding and destruction in vitro. Our results confirm that gal is an important variable in xenoislet transplantation. The GTKO NPI recipients have improved rates of normoglycemia, likely due to decreased susceptibility of xenografts to innate immunity mediated by complement and preformed xenoantibody. Therefore, the use of GTKO donors is an important step toward improved consistency and interpretability of results in future xenoislet studies.

SiRNA-mediated tissue factor knockdown in porcine neonatal islet cell clusters in vitro

To determine the genetic modification on neonatal porcine islet cell clusters (NICC) by small interfering RNA (siRNA)-mediated tissue factor (TF) knockdown in vitro. Porcine NICC were transfected with 5 pairs of designed siRNA respectively or in different combinations with lipofectamine 2000. Transfected NICC were analyzed for TF gene by real-time PCR to select the siRNA which worked best. Meanwhile, the viability of NICC after the TF siRNA transfection was examined by FACS. The efficiency of TF gene and protein suppression was measured by real-time PCR and and FACS respectively. Real-time PCR and FACS showed that a 60% reduction in the TF gene expression and a 50% reduction in the protien level of TF on NICC were achieved by transfecting 3 pairs of selected siRNA. The siRNA transfection had no significant effect on the viability of NICC which was analyzed by FACS. The expression of TF on porcine NICC is efficiently suppressed by 3 pairs of designed siRNA in vitro.

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.

Islets transplantation: New Zealand experience

Xenotransplantation might alleviate the problem of donor organ shortage for the treatment of life threatening conditions. In 2009 the New Zealand Government approved a phase I/IIa clinical trial using porcine islet cells. That was the conclusion of 14 years of regulatory work on xenotransplantation in New Zealand. In 1995 Diatranz received an approval for a pilot study on islet xenotransplantation. Four subjects with and two without microencapsulation of neonatal porcine islets were transplanted. In 1997 the pilot study was voluntary stopped by the company due to the theoretical threat of PERV transmission to a human recipient. The company initiated a project on Xenovirology including intensive PERV study. The company also initiated a dialogue with the Ministry of Health regarding future clinical trials using porcine islets. The recommendation from New Zealand Regulatory Authority, Medsafe, was to follow the FDA guidelines on xenotransplantation. The Ministry also pointed out the need to have a nationwide consultation on xenotransplantation. In 2005 the Bioethics Council of New Zealand held a nationwide consultation where cultural, ethical and spiritual aspects of animal-to-human transplantation were discussed. The recommendation from the Council included that xenotransplantation should be allowed to be developed in the country, and that the government should develop a regulatory and decision-making framework, set a monitoring body and a national register for xenotransplant recipients.The Ministry of Health also pointed out that there should be certain prerequisites for any xenotrial application, namely a donor herd free from all detectable viruses and harmful bacteria, that the Sponsor should have a GMP facility for product manufacturing, and an IANZ accredited diagnostic laboratory with the exhaustive virology program for patients’ follow-up. LCT received a GMP license in 2001 and IANZ accreditation in 2007. An elaborate program on Xenovirology has been under development and implementation since 1997 (1–6). This program allowed LCT to allocate one specific herd of pigs that was free from all conventional pathogens and was also qualified as “null” pigs or pigs that do not have a transmittable PERV. These pigs are the animal-founders for a DPF donor herd. Compliance with the requirements from NZ government allowed LCT to successfully complete its application for a clinical trial using porcine islets. In October 2009 the first New Zealand patient was transplanted with DIABECELL®. The primary objectives for the phase I/IIa clinical trial is safety and improvement in blood glucose level reflected in a decrease of HbA1c (%) level. The secondary objectives include glucose lability, reduction of insulin dose and decrease in hypoglycaemia frequency. To date LCT reports that the trial is meeting objectives for safety. A reduction in the number of unaware hypoglycaemic episodes is also reported in transplanted patients. Prolonged insulin dose reduction for up to 30% is also reported. Safety data shows the absence of evidence of pig viruses’ transmission to the recipients. Data relating to all other endpoints will be reported once the trial has completed and is unblinded.

Effect of erythropoietin on the proliferation and apoptosis of neonatal porcine islet cells.

To investigate the effect of erythropoietin on the proliferation,differentiation, and apoptosis of the cultured neonatal porcine islet cells in vitro. Neonatal porcine islet cells were separated and pured from neonatal pigs with collagenase digestion and tissue culture, and their viability and purity were tested. The neonatal porcine islet cells were divided into a control group and an experimental group.The experimental group was treated with erythropoietin but not the control group, and the insulin secretion responsiveness induced by low and high glucose stimulation in the islet was tested after 5 days. Cells were counted and the activation of amplification was determined by MTT chromatometry. The rates of cell apoptosis were observed by ethidium bromide/acridine orange (EB/AO) of fluorescent light staining and flow cytometry, and the cell cycle was analyzed by flow cytometry. The expression of bcl-2, bax, caspase-3, glucose transporter 2 (GlUT-2), and pancreatic duodenal homeobox-1 (PDX-1) mRNA was tested by RT-PCR. After erythropoietin was treated in the cell culture, the neonatal porcine islet cells had normal morphology, function, and reaction of insulin secretion to the glucose stimulation. Cell count showed more cells in the experimental group than in the control group (P<0.05). MTT chromatometry showed the optical absorbance tended to increase with time, and compared with the control group, the optical absorbance was higher in the experimental group (P<0.05), the expression of PDX-1 mRNA was slightly up-regulated (P<0.05). The expression of GLUT-2 mRNA had no difference in the 2 groups (P=0.34). In the experimental group, the apoptisis rate was lower than that in the control group by flow cytometry and EB/AO fluoscence staining (P<0.01), and the expression of bcl-2 mRNA was higher. Howerer bax mRNA and caspase-3 mRNA were obviously lower than those in the control group (P<0.01). Erythropoietin can promote the proliferation but has no effect on the function of neonatal porcine islet cells in vitro. Erythropoietin can protect neonatal porcine islet cells from apoptosis through up-regulating bcl-2 mRNA and downreguling bax and caspase-3 mRNA.