Adult Porcine Islets Research Articles

Xenotransplantation of Embryonic Pig Kidney or Pancreas to Replace the Function of Mature Organs

Lack of donor availability limits the number of human donor organs. The need for host immunosuppression complicates transplantation procedures. Ultrastructurally precise kidneys differentiate in situ following xenotransplantation in mesentery of embryonic pig renal primordia. The developing organ attracts its blood supply from the host, obviating humoral rejection. Engraftment of pig renal primordia transplanted directly into rats requires host immune suppression. However, insulin-producing cells originating from embryonic pig pancreas obtained very early following initiation of organogenesis [embryonic day 28 (E28)] engraft long term in nonimmune-suppressed diabetic rats or rhesus macaques. Engraftment of morphologically similar cells originating from adult porcine islets of Langerhans (islets) occurs in rats previously transplanted with E28 pig pancreatic primordia. Here, we review recent findings germane to xenotransplantation of pig renal or pancreatic primordia as a novel organ replacement strategy.

Superiority of Visipaque (Iodixanol)-Controlled Density Gradient Over Ficoll-400 in Adult Porcine Islet Purification

Objective Sufficient and favorable biological functions of islets are major problems hindering xenotransplantation. The aim of the present study was to evaluate the effects on harvesting, purity, viability, and function of using improved Visipaque (iodixanol) and Ficoll-400 for adult porcine islet purification. Methods Twelve adult porcine pancreata were randomly divided into an Iodixanol-University of Wisconsin (UW) group and a Ficoll-400-UW group according to the purification method. Porcine pancreata were isolated by collagenase digestion. After isolation and purification, the islet yield and purity were evaluated by dithizone staining, and islet function assessed by in vitro insulin release assays and in vivo islet xenotransplantation. Results There were no marked differences in the islet yield before purification (5254.67 ± 189.44 IEQ/g vs 5092.67 ± 178.94 IEQ/g, P > .05). After purification, there were significantly more islets harvested in Iodixanol-UW group than in the Ficoll-400-UW group: 4222.00 ± 228.84 IEQ/g vs 3036.83 ± 79.60 IEQ/g ( P < .05). Islets from the two groups showed satisfactory insulin secretory ability. There were no significant differences in islet survival times between the two groups in diabetic rats: 8.2 ± 1.619 days vs 6.9 ± 1.197 days ( P > .05). Conclusion The improved iodixanol-UW density gradient method was superior to Ficoll-400 method to improve the number, viability, and insulin secret of purified adult porcine islets although the benefits did not improve in vivo survival.

Enhanced Prediction of Porcine Islet Yield and Posttransplant Outcome Using a Combination of Quantitative Histomorphometric Parameters and Flow Cytometry

Prediction of islet yield and posttransplant outcome is essential for clinical porcine islet xenotransplantation. Although several histomorphometric parameters of biopsied porcine pancreases are predictive of islet yield, their role in the prediction of in vivo islet potency is unknown. We investigated which histomorphometrical parameter best predicts islet yield and function, and determined whether it enhanced the predictive value of in vitro islet function tests for the prediction of posttransplant outcome. We analyzed the histomorphometry of pancreases from which 60 adult pig islet isolations were obtained. Islet function was assessed using the beta-cell viability index based on flow cytometry analysis, oxygen consumption rate, ADP/ATP ratio, and/or concurrent transplantation into NOD/SCID mice. Receiver operating characteristic (ROC) analysis revealed that only islet equivalent (IEQ)/cm(2) and the number of islets >200 microm in diameter significantly predicted an islet yield of >2000 IEQ/g (p < 0.001 for both) and in vivo islet potency (p = 0.024 and p = 0.019, respectively). Although not predictive of islet yield, a high proportion of large islets (>100 microm in diameter) best predicted diabetes reversal (p = 0.001). Multiple regression analysis revealed that the beta-cell viability index (p = 0.003) and the proportion of islets >100 microm in diameter (p = 0.048) independently predicted mean posttransplant blood glucose level (BGL). When BGL was estimated using both these parameters [area under the ROC curve (AUC), 0.868; 95% confidence interval (CI), 0.730-1.006], it predicted posttransplant outcome more accurately than the beta-cell viability index alone (AUC, 0.742; 95% CI, 0.544-0.939). In conclusion, we identified the best histomorphometric predictors of islet yield and posttransplant outcome. This further enhanced the predictive value of the flow cytometry analysis. These parameters should be useful for predicting islet yield and in vivo potency before clinical adult porcine islet xenotransplantation.

Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts

Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.

Long-Term Metabolic Control of Autoimmune Diabetes in Spontaneously Diabetic Nonobese Diabetic Mice by Nonvascularized Microencapsulated Adult Porcine Islets

The long-term metabolic function of microencapsulated xenogeneic adult porcine islets (API) was assessed in a murine model of type 1 diabetes mellitus. API were encapsulated in barium-gelled alginate and transplanted intraperitoneally in diabetic nonobese diabetic (NOD) mice given no immunosuppression or given costimulatory blockade (CoB; CTLA4-Ig+anti-CD154 mAb). Control mice received nonencapsulated API under the kidney capsule. Graft function was monitored by measurement of random blood glucose levels, serum glycosylated hemoglobin (HbA1c), serum porcine C peptide, in vivo glucose tolerance tests, and histologic analyses of host pancreas and graft biopsies. Host immune responses to the islet xenografts were characterized by phenotyping peritoneal cellular infiltrates and by measuring serum antiporcine antibody levels. Without immunosuppression, nonencapsulated API functioned for less than 1 week, and microencapsulated API functioned for 35+/-14 days before rejection, associated with both a cellular and a humoral immune response. With continuous CoB, nonencapsulated API functioned for 27+/-4 days, whereas microencapsulated API functioned for >450 days with measurable levels of serum porcine C peptide, near normal in vivo glucose tolerance tests and HbA1c levels, and intact microcapsules containing viable, insulin-positive porcine islets. Microencapsulated API restored normoglycemia for more than 1 year in spontaneously diabetic NODs given dual CoB. To our knowledge, this is the first study to document long-term normalized HbA1c, porcine C peptide, and near normal glucose tolerance in immunosuppressed diabetic NOD mice transplanted intraperitoneally with microencapsulated API. Our study suggests that transplantation of microencapsulated porcine islet xenografts may be a future treatment for patients with type 1 diabetes mellitus.

Chapter 3: Pig islet product manufacturing and release testing

This chapter provides recommendations on pig islet product manufacturing and release testing to scientific and corporate programs interested in future clinical studies using xenogeneic porcine pancreatic islet cell products for the treatment of type 1 diabetes.To facilitate control of manufacturing as well as reproducibility and consistency of product lots, the manufacturing process, and the manufacturing facility must be in compliance with current Good Manufacturing Practices regulations. Data must be provided to demonstrate that islet products can be consistently prepared that would meet basic lot release requirements. To facilitate product safety: (i) materials used in the manufacturing process, including the pig pancreas, must be free of adventitious agents; (ii) islets must be manufactured using aseptic processing; and (iii) final product must undergo tests for sterility, mycoplasma (if cultured) and endotoxin. Safety specifications for pig islet product release include a negative Gram stain and an endotoxin content of <5.0 EU/kg recipient body weight. Product post-release assessments must include sterility cultures on the final product. Because results for sterility are available only retrospectively, a plan of action must be in place for patient notification and treatment in case the sterility culture results are positive for contamination. Product characterization information must address important aspects of lot release testing such as identity/purity (cell composition), quantity [islet equivalents (IE), cell number] and potency (insulin secretory capacity, oxygen consumption rate corrected for DNA or transplant bioassay in immunoincompetent diabetic mice). This information is also critical to demonstrate manufacturing control and product consistency across multiple islet preparations (lots). Providing islet products containing an islet mass sufficient to restore euglycemia in trial participants (>or=10 000 IE/kg) requires pooling of islets from multiple donor pancreata (two to four from adult donors and seven to 10 from neonatal donors). Demonstration of product consistency across products from individual pancreata would warrant release testing to be performed on a sample of the pooled product. As product development and clinical trials advance, the increasingly more detailed specifications of potency assays on adult porcine islet products are expected to be predictive of post-transplant glycemic control. The immaturity of fetal and neonatal porcine islet tissue precludes the use of in vitro insulin secretion as a potency test as part of lot release testing; another measure of potency appropriate to fetal and neonatal cells will need to be developed for product release testing and evaluation of aliquots of these products in mouse transplant bioassays should be performed to provide meaningful post-release information.

Pathogenesis of coxsackievirus‐B5 acquired from intra‐renal porcine islet cell xenografts in diabetic mice

We previously demonstrated the ability of a human isolate of coxsackievirus-B5 (CVB5) to infect productively adult porcine islet cells (PICs) in vitro. PICs infected with CVB5 remain viable, and upon transplantation reversed diabetes in C56BL/6 mice for up to 5 days. In the present work, we expanded this graft-to-host xenozoonosis model by examining the long-term functionality of CVB5-infected PIC xenografts in immunosuppressed mice. And, we characterized the pathogenesis of CVB5 infection in mice resulting from directional transmission of the virus from PIC xenografts to surrounding tissues in a mouse model for immunosuppressed human PIC xenograft recipients. Both acutely (12 h) and chronically (72 h) infected PIC xenografts functioned in vivo to reverse diabetes in mice. The efficacy of both infected and un-infected PICs was transient beyond 5 days post-inoculation and the long-term functionality of the grafts was compromised by host-to-graft rejection. CVB5-infected PIC xenografts transmitted infectious virus to immunosuppressed recipient mice resulting in extensive histopathologic changes. The virus replicated in the heart, liver, spleen, kidney, pancreas, brain and skeletal muscle in higher levels in severe-combined immunodeficient (SCID) mice that were directly inoculated with virus when compared to controls. In addition, infectious virus was recovered for up to 22 days after inoculation in SCID mice whereas it was only detected up to Day 4 PI in non-SCID mice. Immunosuppressed PIC xenograft recipients may be more susceptible to infection with CVB5 which could target the xenograft leading to disseminated infection in the host.

Anticarbohydrate Antibody Repertoires in Patients Transplanted with Fetal Pig Islets Revealed by Glycan Arrays

Ten patients with type I diabetes were transplanted with porcine fetal islet-like cell clusters (ICC) between 1990 and 1993. A significant rise in the anti-α-Gal antibody titers was seen posttransplant, but also non-α-Gal-specific antibodies were detected in some patients. We have reanalyzed the carbohydrate specificity of antibodies in the sera from seven of these patients taken before transplantation, 1, 6 and 12 months posttransplantation using a glycan array with 200 structurally defined glycans. The main findings were: (i) prepig ICC transplantation patients had antibodies reactive with terminal α-GalNAc (e.g. the Forssman antigen, but not the blood group A determinant in blood group A patients), α-Gal (except blood group B determinants in B individuals), β3-linked Gal especially Galβ1,3GlcNAc even if terminally sulfated or sialylated, β-GlcNAc except if β1,3-linked and oligomannosyl compounds; (ii) the titers of all carbohydrate-specific antibodies detected before transplantation rose after transplantation; (iii) the kinetics of the antibody responses differed between patients; (iv) in some patients antibodies reacting with Galα1,3Lex and several structures terminated with Neu5Gc appeared after transplantation. In conclusion, anti-α-Gal antibodies are the predominant anticarbohydrate antibodies detected after porcine ICC transplantation, with some patients also developing Neu5Gc-specific antibodies. Their clinical significance needs to be established.

Pig Pancreatic Islet Transplantation Into Spontaneously Diabetic Dogs

Introduction Pig islet xenotransplantation represents an attractive way to solve our human organ shortage. In this preclinical protocol, we implanted adult porcine islets microencapsulated in alginate-polylysin into insulin-dependent diabetic dogs. Methods Pancreata were obtained from animals weighing 100 to 150 kg in a slaughterhouse. The islets were isolated by collagenase digestion. The encapsulation technique was a modification of Sun's method. Isolated islets (5000 islet equivalents per kilogram of dog weight) were mixed with 1.6% low-viscocity alginate. Microcapsules were cultured for 36 hours before implantation. The five dogs were in healthy prior to induction of diabetes mellitus at least 1 year prior. Under sedation, we implanted microcapsules. We performed determinations of peripheral blood insulin at baseline and every 3 months as well as glycosylated hemoglobin at baseline and every 4 months. During follow-up, glycemia was estimated twice a day at 3 hours after morning and night meals using a blood glucose monitoring system. Results We observed significant decrease (20%–80%) in insulin needs ( P < .01). Of note, before the procedure no hormone was detected in the blood at 6 to 12 months after transplantation, plasma insulin had improved significantly ( P < .05) and glycosylated hemoglobin also showed a significant decrease ( P < .01). All owners subjectively claimed that their animals were enjoying a better quality of life. Discussion Our preliminary data suggested that pig islet microencapsulation achieved metabolic control in type I diabetic dogs without the risk of immunosuppression using one or two procedures per year.

Improved Islet Yields After Purification Following the Novel Endogenous Trypsin Inhibitor and Histidine-Tryptophan-Ketoglutarate Treatment in Pigs

Background Adult porcine islet xenotransplantation into humans is greatly diminished by the difficulty to isolate islets because of their fragility. The goal of this study was to improve the efficacy of islet yields using endogenous trypsin inhibitor and histidine-tryptophan-ketoglutarate (HTK) perfusate. Method We compared two porcine islet isolation protocols: Eurocollins solution for in situ pancreas perfusion without use of an endogenous trypsin inhibitor versus HTK solution including endogenous trypsin inhibitor for pancreas perfusion. Results Endogenous trypsin inhibitor and HTK strategies significantly improved total islet yield, recovery, and islet index after purification ( P < .05), whereas unpurified islet yield did not increase. An average of 228,000 ± 95,000 islet equivalents (IEQ) ( n = 20) purified islets were obtained in the first group compared with 115,000 ± 56,000 IEQ ( n = 18) in the second group. The average islet index was significantly increased in the first group compared with the second group before and after purification: before: 0.28 versus 0.49 versus after: 0.25 versus 0.4 ( P < .05). At this time, islet purity, viability, and stimulation index did not show a significant difference between groups. Conclusion Our study showed that endogenous trypsin inhibitor and HTK strategies significantly improved purified islet isolation efficacy because of reduction of islet fragility.

Biocompatibility and Immune Acceptance of Adult Porcine Islets Transplanted Intraperitoneally in Diabetic NOD Mice in Calcium Alginate Poly-L-lysine Microcapsules versus Barium Alginate Microcapsules without Poly-L-lysine

If alginate microcapsules are to be used clinically for therapeutic cell transplants, capsule formulations must be designed to enhance optimal biocompatibility and immune acceptance. Microcapsules were generated using highly purified, endotoxin-free, ultra-low viscosity, high mannuronic acid alginate. The capsules differed with respect to gelling cation (50 mM barium or 100 mM calcium), alginate concentration (2.0% or 3.3%), alginate density (homogeneous or inhomogeneous), and the presence or absence poly-L-lysine (PLL) coating. Four types of empty capsules were implanted intraperitoneally (i.p.) in normal NOD mice, and their biocompatibility was evaluated after various time periods in vivo. Encapsulated adult porcine islets (APIs) were transplanted i.p. in diabetic NOD mice, and immune acceptance was evaluated by graft survival times, host cell adherence to capsule surfaces, and flow cytometric analysis of peritoneal host cells. All empty alginate capsules were biocompatible in vivo, but barium-gelled alginate capsules without PLL were clearly the most biocompatible, since 99% of these empty capsules had no host cell adherence up to 9 months in vivo. In diabetic NOD mice, APIs functioned significantly longer in barium-alginate capsules without PLL than in calcium-alginate capsules with PLL and had strikingly less host cell adherence, although large numbers of host cells (predominantly macrophages and eosinophils) infiltrated the peritoneal cavities of recipients with APIs in both types of capsules. Addition of PLL coatings to barium-alginate capsules dramatically decreased graft survival. Inhomogeneous barium-gelled alginate capsules without PLL are the optimal candidates for clinical trials, based on their enhanced biocompatibility and immune acceptance in vivo.

Dissecting the instant blood‐mediated inflammatory reaction in islet xenotransplantation

A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non-human primates. The detrimental instant blood-mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen. Low molecular weight dextran sulfate (LMW-DS) and the complement inhibitor Compstatin were used in a combination of in vitro and in vivo studies designed to dissect the xenogeneic IBMIR in a non-human primate model of pancreatic islet transplantation. Adult porcine islets (10,000 IEQs/kg) were transplanted intraportally into three pairs of cynomolgus monkeys that had been treated with LMW-DS or heparin (control), and the effects on the IBMIR were characterized. Porcine islets were also incubated in human blood plasma in vitro to assess complement inhibition by LMW-DS and Compstatin. Morphological scoring and immunohistochemical staining revealed that the severe islet destruction and macrophage, neutrophilic granulocyte, and T-cell infiltration observed in the control (heparin-treated) animals were abrogated in the LMW-DS-treated monkeys. Both coagulation and complement activation were significantly reduced in monkeys treated with LMW-DS, but IgM and complement fragments were still found on the islet surface. This residual complement activation could be inhibited by Compstatin in vitro. The xenogeneic IBMIR in this non-human primate model is characterized by an immediate binding of antibodies that triggers deleterious complement activation and a subsequent clotting reaction that leads to further complement activation. The effectiveness of LMW-DS (in vivo and in vitro) and Compstatin (in vitro) in inhibiting this IBMIR provides the basis for a protocol that can be used to abrogate the IBMIR in pig-human clinical islet transplantation.

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.

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.

Transcriptional profiling of stress response in cultured porcine islets

Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7mM), inflammatory cytokine addition (IL-1β, TNF-α, and IFN-γ), or both, for 48h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and β cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.

Neonatal Porcine Islets Exhibit Natural Resistance to Hypoxia-Induced Apoptosis

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. 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. 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). 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.

Pretransplant Culture Selects for High-quality Porcine Islets

The pig is generally considered a suitable alternative donor for clinical islet transplantation. However, adult pig islets are difficult to isolate and culture, often behave variably in in vitro assays, and do not consistently cure diabetic nude mice. In this study, we compared the in vivo function of freshly isolated and cultured adult porcine islets by transplantation in diabetic nude mice. Freshly isolated and cultured islets were transplanted in different doses to diabetic nude mice (N = 48). Average islet yield was 1924 islet-equivalents per gram of pancreas, purity 96%, and the viability that was measured by acridine orange and propidium iodide was greater than 80% in all freshly isolated islet preparations. Grafts of freshly isolated islets failed to reduce hyperglycemia in 17 of 18 recipients. Although after 1 day of culture islet recovery was only 21%, grafts of these islets cured 12 of 17 mice. After 7 to 14 days of culture, the recovery had decreased to 11%; however, these islets reversed hyperglycemia in all mice (13/13) and showed shorter time-to-normoglycemia and more tightly regulated blood glucose. Although freshly isolated adult porcine islets survive culture and transplantation poorly, islets selected by prolonged culture are of high potential.

Parameters favouring successful adult pig islet isolations for xenotransplantation in pig‐to‐primate models

In the near future, adult porcine islets of Langerhans appear as an unlimited source of insulin-producing cells which could play a major role for treating diabetes mellitus. There is, however, an obvious lack of pre-clinical results and data in the pig-to-primate model. One of the main hurdles of this model is certainly related to the difficulty of reproducing regularly successful porcine islet isolation. This experimental work was designed to provide guidelines applicable in pig pancreas procurement and islet isolation for successful islet xenotransplantation into primates. Pancreases were harvested from adult Belgium Landrace pigs (n = 79) in a single centre. The impact on islet yield of (1) pancreas procurement (blood exsanguination and warm ischaemia time (WIT)), (2) cold storage solutions (classic UW and modified UW (without hydroxyethyl starch and inverse K+/Na+ concentration)), (3) a dynamic or static method of pancreas digestion, and (4) the endotoxin content and enzymatic activity from five different batches of Liberase PI was studied. In addition, pancreatic biopsies (n = 18), performed before isolation, were retrospectively analyzed to study the impact of histomorphometry on porcine islet yield. Finally, two diabetic cynomolgus monkeys were transplanted without immunosuppression with 15,000 pig islet equivalents/kg body weight of recipient to assess in vivo the function of freshly isolated islets. Univariate and multivariate analyses were performed. By multiple linear regression, the most significant variables that significantly improved islet yield were, firstly, the presence of <30 EU (endotoxin units) of endotoxin in Liberase batches, followed by a WIT under 10 min and the use of blood exsanguination before pancreas harvesting (P < 0.005). In contrast, isolation method (dynamic vs. static) and the solution used for storage (short-term) (UW vs. modified UW) did not significantly influence islet yield. The correlation of retrospective histomorphometry analysis of native pancreas and extemporaneous biopsy before isolation clearly determined a positive relationship between isolated islet number and the number of islets/cm2 (r = 0.708, P < 0.01) or with the percentage of large islets (r = 0.680, P < 0.01) found in pancreas biopsies. Pig pancreases containing more than 82 islets/cm2 and more than 42% of large islets (>100 microm) thus enabled more than 120,000 islet equivalents to be obtained in 90% of the cases, which is an ideal amount of islets to transplant into a primate of 4 to 5 kg. In vivo, a reduction of blood glucose (<200 mg/dl), associated with porcine C-peptide production, was observed in two primates after transplantation with adult pig islets. At day 7 post-transplantation, however, loss of islet function was associated with graft destruction and immune reaction. Morphological screening of the pig pancreas before isolation, optimal blood exsanguination, WIT <10 min, and an endotoxin content <30 EU/mg in Liberase PI batches determine successful pig islet isolation for xenotransplantation in primates.

Pig islet xenotransplantation: activation of porcine endogenous retrovirus in the immediate post‐transplantation period

Porcine endogenous retroviruses (PERV) are considered as the main infectious barrier in islet xenotransplantation. PERV has been shown to infect, but not to cause symptomatic disease in mice after islet transplantation. In vivo activation of PERV have so far not been examined. Expression of PERV was examined in adult and fetal porcine islets with or without the presence of known retroviral inducers or after transplantation to rats. Isolated adult and fetal porcine islets were cultured under normal conditions or in the presence of dexamethasone or 5-azacytidine and 5-iodo-2-deoxyuridine. PERV mRNA content was analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and culture supernatants were analyzed for the presence of retroviral RT. Also, fetal islets were transplanted under the kidney capsule of immunocompetent or nude athymic rats. Expression of PERV mRNA in the grafts was evaluated by real-time quantitative RT-PCR. Infiltration of immunocompetent cells were evaluated by immunohistochemistry. Both fetal and adult islets in culture produced small or even undetectable amounts of PERV mRNA and retroviral RT. PERV expression was not enhanced by retroviral inducers. In contrast, activation of PERV expression was observed the first day after transplantation of fetal islet-like cell clusters in both athymic and normal rats. PERV expression peaked after 1 to 3 days and was then rapidly returned to background levels. PERV expression neither correlated with the innate immune response seen in athymic rats nor with the specific process of rejection in normal rats. Both fetal and adult islets produce low amounts of PERV mRNA in culture. After transplantation PERV expression is induced, seemingly independent of both the unspecific inflammatory response and the specific T-cell-mediated rejection process. It is speculated that PERV expression is correlated with the level of hypoxia in the islet xenograft.

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

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). 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. 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. Our results suggest that ''glucose toxicity'' plays a role in the immediate demise of intraportal tilapia islet xenografts.