Embryonic pig pancreatic tissue for the treatment of diabetes in a nonhuman primate model

Abstract

Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a β cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.

Allogeneic islet cell replacement can restore normoglycemia in patients with type 1 diabetes; yet, the shortage of available organs for transplantation continues to severely limit this therapeutic option. A potential solution for this shortage is the use of pig organs and tissues as a source for transplantation. However, the immunological barriers to xenotransplantation are formidable (1). Thus, new approaches that could further reduce the intensity of the required immune suppression are critical for successful application of pig pancreatic tissue as a novel source of islet cells. One means to address this challenge could be provided by the use of embryonic porcine tissue. This strategy is based on the growing evidence, over the past 5 decades, demonstrating maternal immune tolerance to the fetus (2, 3), and on recent observations that embryonic tissues exhibit reduced immunogenicity in various transplantation settings (4–7). Considering that very early embryonic tissues are associated with a substantial risk of teratoma formation, we have attempted, during the past several years, to define the earliest gestational time point that does not pose a teratoma risk for transplantation of different embryonic pig tissues, including kidney (6), heart (unpublished), spleen (8), pancreas, liver, and lung (7) into SCID mice. Once this threshold was established, we were able to further characterize an optimal gestational window for transplantation based on growth potential and immunogenicity. Thus, while the optimal window for kidney transplantation was defined around E28, our data suggested E42 as the optimal gestational time point for pig donor tissue in pancreatic transplantation (9). Importantly, upon growth and development of the embryonic pancreatic implant, acini gradually disappear and the tissue is predominantly comprised of islets and stroma identified by vimentin straining. Thus, the risk of potential ongoing destruction of the growing organ by local proteolytic enzymes, which might be released by the exocrine components of the implant, is miniml (9). These mouse studies also demonstrated the curative potential of such implants in diabetic mice under relatively tolerable immune suppression. However, considering that xenogeneic transplantation into primates is far more complex, due to preexisting anti-porcine antibodies, studies in a nonhuman primate (NHP) model for diabetes were required. Our working hypothesis, suggesting that embryonic tissue transplantation would not be adversely affected by such a humoral response was based on 2 major observations: (i) hyperacute and acute rejection are mainly mediated by complement activation in blood vessels, mediated by preformed anti-pig antibodies recognizing α-gal and other carbohydrates expressed on endothelial cells (10), and (ii) data from our mouse studies showing that the embryonic implants predominantly induce host-type vasculature to support their growth and development in the recipient, resulting in an organ comprised mainly of porcine epithelial cells and host endothelium.

To further assess the curative potential of E42 pig pancreatic tissue, we have now evaluated its capacity to correct hyperglycemia under tolerable immune suppression in an NHP model for diabetes.

Results

Defining a Tolerable Immune Suppression Protocol.

Diabetes was induced in 4 cynomologous monkeys by the administration of 150 mg/kg of streptozotocin (STZ). About 1 month after STZ administration, E42 porcine tissue was implanted in the omentum as described in Methods. Immune suppression was induced and maintained in all 4 animals using a protocol based on induction with a single dose of anti-CD20 antibody (Rituximab) followed by a short course of ATG and 2 doses of anti-CD25 antibody (Basiliximab). Immune suppression was maintained by Everolimus, FTY720, and biweekly treatment with CTLA4-Ig (Abatacept) (Fig. 1). All these immunosuppressive agents were previously shown to be effective and well-tolerated in allogeneic islet transplantation in the NHP model (11–14).

Fig. 1.

Immune suppression protocol in STZ-treated NHP

The study consisted of 2 cohorts, with 2 animals in each. In the first cohort, central line-associated infections eventually led to the death of both NHP #1 and #2, 2 and 3 months following transplantation, respectively. Apart from the adverse effects associated with the central line catheters, the ATG dose (total of 70 mg/kg) used in monkey #1 was associated with severe anemia and coagulation disorders, requiring extensive blood transfusions. The choice of this relatively high ATG dose was based on previous suggestions that rabbit anti-human ATG is less active in NHP than in humans and that equivalent doses in NHP are 6 times higher than in humans (12). However, substantial reduction of ATG dose in monkey #2 to 40 mg/kg was well-tolerated and did not affect engraftment or acceptance of the graft, as evidenced by histology (Fig. 2). In contrast to NHP #1 and #2, which were followed for a relatively short time, which was not sufficient to allow a significant progression toward insulin independence, the changes made in the treatment of the animals in cohort 2 (i.e., avoiding use of central line and using a lower ATG dose) enabled these animals to survive for 393 (NHP #3) and 280 days (NHP #4) posttransplantation.

Fig. 2.

Developmental and functional characteristics of E42 pig pancreatic implant, 3 months after intra-omental implantation in animal #2. (A) Macroscopic appearance of the graft removed postmortem (pancreatic nodes are marked by arrows). Each scale mark represents 1 mm. (B) Demonstration of insulin-positive cells in the graft islets by immunoperoxidase staining (brown). (C) Minimal intra-graft presence of CD3 positive T cells (brown, arrows). (D) Proliferating Ki67-positive cells (red fluorescence) within a pancreatic islet, outlined by cytokeratin positivity (green fluorescence). (Scale bars in B–D, 50 μm.)

Similarly to NHP #2, NHP #3 was transplanted using a total dose of 40 mg/kg ATG. This animal exhibited CMV reactivation, which required continuous treatment (see Materials and Methods), and therefore in animal #4, the immune suppression protocol was further reduced to comprise a total dose of 20 mg/kg ATG. Postmortem immunohistological analysis of mesenteric lymph nodes and spleen of this animal revealed preserved T and B cell follicles (SI), while as can be expected from the use of FTY720 in the protocol, low lymphocyte blood levels (average 0.68 k/μL ± 0.37) were recorded throughout the follow-up period (SI).

Islets and Stroma Within the Growing Pig Embryonic Pancreas Use Host Vasculature and Evade Humoral Hyperacute and Acute Rejection.

NHP #1 and #2 exhibited impressive growth of the implants (Fig. 2A) as seen upon postmortem examination. Clusters of islets, with insulin-containing cells were evident (Fig. 2B), while only a few incidental lymphocytes were observed (Fig. 2C), indicating the absence of rejection. Furthermore, an active proliferating pool of cells, stained for Ki67 was found within the islets (Fig. 2D). This observation emphasizes the long-term regenerative capability of the pancreatic tissue, in agreement with our previous mouse studies (9).

The E42 pancreatic tissue used for transplantation was not manipulated ex vivo before transplantation. Marked expression of α-gal on endothelial but not on epithelial cells was found by double staining of the graft tissue before transplantation. Thus, as can be seen in Fig. 3A, α-gal positive cells stained with Banderia Simplicifolia isolectin 4 are distinguished from fetal epithelial structures stained with anti-cytokeratin. In contrast, staining of the graft at 3 months posttransplant with anti-human CD31 antibodies (cross-reactive with monkey but not porcine endothelial cells), revealed that the islet-like structures, as well as other cells within the stroma, were primarily vascularized by host-type (primate) endothelium and only slight staining of pig endothelial cells was observed with Banderia Simplicifolia lectin (Fig. 3B).

Fig. 3.

Vascularization pattern of E42 pancreatic tissue. (A) Marked expression of α-gal on endothelial but not on epithelial cells in the graft tissue before transplantation, indicated by double staining. Thus, α-gal-positive cells are stained with Banderia Simplicifolia isolectin 4 (green fluorescence), and fetal epithelial structures are stained with anti-cytokeratin (blue). (B) Predominant vascularization of the porcine graft by host blood vessels is demonstrated by staining with anti-human CD31 antibody that cross-reacts with monkey but not with pig endothelial cells (red fluorescence). Only slight staining of pig endothelial cells was observed with Banderia Simplicifolia lectin (green). Islet epithelium is outlined by cytokeratin expression (blue). (C) Higher magnification of anti-cytokeratin-positive islets (blue) observed within the marked frame in inset B. (D) Higher magnification of the vasculature network supporting the islets observed within the marked frame in inset B. Double staining with anti-human CD31 (red) and Banderia Simplicifolia lectin (green) reveals predominance of endothelial cells of monkey origin. Nuclei were counterstained by Hoechst Yellow (yellowish). (Scale bars, 50 μm.)

The predominance of host vascularization can be clearly seen upon examination of islets under high magnification (Fig. 3 C and D).

Thus, the ability of the growing implant to use host vasculature might be critical for graft survival and growth in the recipient NHP.

Implantation of E42 Pig Embryonic Pancreatic Tissue Restores an Enduring Euglycemic State.

Long-term follow-up of cohort 2 demonstrated the ability of the grafts to cure diabetes. As can be seen in Fig. 4, both animals of the second cohort exhibited a very similar pattern of recovery of glucose control, in parallel to the growth and development of the implant. Thus, similarly to our results in the mouse model, a marked reduction of exogenous insulin requirement (i.e., less than 10% of the daily insulin dose used before transplantation) was noted by the fourth month after transplantation, reaching complete exogenous insulin independence during the fifth posttransplantation month (Fig. 4 A and B), with marked physiological control of blood glucose levels (Fig. 4 A and B). A radioimmunoassay (RIA)-specific for porcine C-peptide (i.e., non-cross-reactive with primate C-peptide) confirmed the presence of basal porcine C-peptide levels as early as 3 months following transplantation (0.24 and 0.35 ng/mL for monkeys #3 and #4, respectively), and marked elevation of porcine C-peptide blood levels was demonstrated in both animals after i.v. glucose challenge test (IVGTT), in accordance with the enhanced insulin levels (Fig. 4 C and D).

Fig. 4.

Assessment of E42 fetal porcine xenograft function in streptozotocin-induced diabetic Cynomolgus monkeys of cohort 2. Insulin requirement (blue) and fasting glucose levels (yellow) in monkeys #3 and #4 are shown in panels A and B, respectively. Specific pig C-peptide (red) and insulin (green) levels after IVGTT in monkey #3 (C) and #4 (D) were determined by ELISA specific for pig C-peptide (non-cross-reactive with monkey C-peptide) and for total insulin (using an antibody that does not distinguish between the 2 species), at 393 days and 280 days posttransplant, respectively. K values for the clearance of glucose were 1.84 and 2.0, respectively.

A RIA for human C-peptide (cross-reactive with primate) revealed some baseline level of primate C-peptide. However, no significant elevation of primate C-peptide could be detected by this assay following glucose challenge, in contrast to the elevation of porcine C-peptide (SI). This constant and unresponsive base line level could be due to some cross-reactivity with undefined blood components or to some level of regeneration of undifferentiated or nonfunctional insulin-positive beta cells in the recipient. However, the absence of insulin-positive beta cells within the host pancreas upon postmortem (Fig. 5A) strongly suggests that insulin independence, attained in both animals upon growth and development of the implant, is most likely not associated with the potential recovery of host pancreatic activity. The animals exhibited an Hb A1C of 6.1% and 5.2% at 333 and 195 days following transplant, respectively.

Fig. 5.

Immunohistochemical characterization of islets in the E42 pig pancreatic implant versus the host pancreas, at 13 months after intra-omental implantation in animal #3. (A) Immunoperoxidase labeling of insulin (brown staining) reveals minimal residual presence of insulin-positive cells within the host pancreas (each bar represents 50 μm). Insulin-containing islets within the graft (B and C) were labeled by immunofluorescence labeling of insulin (blue fluorescence). Few incidental CD3-positive T cells (green fluorescence) can be detected outside the islets (blue) (B).

Correction of hyperglycemia was adversely affected twice in monkey #3. Two weeks after reaching insulin independence, low Everolimus levels were detected and partial rejection was suspected as insulin requirement relapsed to an average of 15% of maximal levels during week 33 (Fig. 4A and SI). However, a gradual restoration of graft function, to a minimal requirement of insulin supplementation, less than 3.7% of the insulin requirement before transplantation, was observed (Fig. 4A). Eventually, clinical complications likely associated with the continuous CMV infection and the combined toxicity of anti-viral drugs and the immune suppression, necessitated euthanizing of the animal at 393 days posttransplant (SI).

A postmortem immunohistochemical analysis of the graft revealed intact insulin-positive islet fields (Fig. 5 B and C), and only rare CD3+ lymphocytes could be recorded in the proximity of transplanted islets (Fig. 5B) ruling out potential immune rejection.

In NHP #4 insulin independence was sustained for more than 17 weeks (average fasting glucose during this period was 113 ± 20) without any complications. At the beginning of the eighth month posttransplant, technical failure to sustain Everolimus levels above the threshold of 3 ng/mL (SI) seemed initially to be associated with partial loss of graft function (Fig. 4B). A glucose challenge test performed 2 weeks earlier revealed robust pig pancreatic activity (maximal C-peptide of 3.79 ng/mL), suggesting that the acute reduction in function might indeed be attributed to the parallel drop of Everolimus blood levels. This decrease in functionality was accompanied by clinical deterioration, which was manifested by anorexia, and led to euthanizing of the animal at 280 days posttransplantation (SI). Postmortem examination revealed essentially similar findings to those found in animal #3 (see SI). Thus, the presence of intact islets fields with no evidence of rejection indicates that an infectious process of unknown etiology could be responsible for the partial loss of function in both animals.

Discussion

Initial clinical attempts of fetal porcine pancreas transplantation were carried out early in the 1990s by Groth et al. (15). In these studies, patients were transplanted with E66-E81 porcine fetal islet-like cell clusters. Blood glucose levels increased shortly after transplantation and insulin administration had to be increased, so that eventually it did not differ from the pretransplant requirements. We have shown recently in a mouse model that this outcome could be attributed, in part, to the relatively late stage of gestation at which the embryonic pancreatic tissue was harvested. Thus, based on growth potential and immunogenicity, we demonstrated that the optimal “window” for these transplants could be provided at around E42 (7, 9).

In our present study, the almost identical pattern of insulin reduction exhibited in both animals of cohort 2 (Fig. 4 A and B) demonstrates the reproducibility of the time frame of growth and differentiation required to achieve normoglycemia, and provides proof of concept for the curative potential of the E42 pig pancreatic implants in NHP.

Furthermore, the growing tissue makes use of host vasculature, and thereby evades hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies (10). This finding supports our working hypothesis that growing the embryonic pig tissue de novo within the NHP recipient, provides an advantageous source for organ transplantation compared to adult pig organ transplantation, which requires anastomosis of donor blood vessels. Further NHP studies with other precursor tissues such as embryonic kidney (6), liver (7), and spleen (8), for which appropriate embryonic sources have been recently characterized, are warranted.

Very recently, the capacity of neonatal and adult porcine islets to restore normoglycemia in diabetic NHP was demonstrated (16, 17). However, the immune suppression required to maintain the implants was either too toxic or included anti-CD40L, which cannot be used in human patients due to its thrombotic properties. Other studies suggested that E28 pancreatic tissue might be accepted in nonimmunosuppressed rats (33) and monkeys (34). In contrast, our mouse (9) and rat (35) data has revealed fierce rejection of pig embryonic tissue of any gestational time point in the absence of immune suppression, while demonstrating that the immunogenicity of E42 tissue was relatively lower compared to tissues harvested at E56 or beyond (9). Therefore, we proposed that mild immune suppression might suffice to permit engraftment and growth of the E42 embryonic tissue.

In the present study, we chose a relatively strong immune suppression, so as to avoid hurdles associated with tissue rejection. This protocol is likely less nephrotoxic than current allogeneic islet transplantation protocols, such as that recently described by Bellin et al. (18) comprising induction with ATG and maintenance with Everolimus and Cyclosporine A. However, although the present immune suppression protocol used in animal #4 was well tolerated, further assessment is required to define the minimal immune suppression that can support graft survival while avoiding toxicity and infections. Preliminary results in the mouse model, evaluating potential substitutes for Evorolimus with co-stimulatory blockade agents other than anti-CD40L have shown encouraging results (35). Such substitutes could be of particular importance considering the skin lesions found in animal #3 (see SI for details) and other reported adverse effects of Rapamycin on islet function (19–21).

Beta cell replacement can be achieved by either whole organ-pancreas allogeneic transplantation or transplantation of isolated islet cells. The transplantation of a whole pancreas achieves a longer graft survival and functionality than isolated islets (67% after 10 years and 10% after 5 years, respectively) (22, 23). This discrepancy could be attributed to the deleterious effects encountered during islet preparation by enzyme digestion, prolonged ischemia time, and the loss of surrounding mesenchyme. The active involvement of the surrounding mesenchyme in pancreatic development and its role in endocrine cell differentiation and proliferation has been documented (24, 25). Thus, the embryonic pancreatic tissue growing in the context of its own stroma as documented here, might survive longer than infused neonate or adult islets growing heterotopically in the liver. Indeed, following graft injury and partial loss of function in monkey #3, the growing pig implant exhibited a marked capacity to withstand stress and regenerate. This characteristic of the graft, which could be related to mesenchymal support, is of particular relevance considering that although 1-year success rates for islet transplantation are high, the long-term success rate is unacceptably low, even in the pioneering Edmonton Center (23). The poor long-term clinical outcome, taken together with the recent study of Koulmanda et al. (26), indicates that one of the reasons for long-term failure may be due to the low islet cell mass that actually survives the harvesting and transplant procedures, as well as islet attrition by exhaustion. The potential importance of islet replacement with E42 graft that will expand with time could afford a major advantage.

Recent rodent studies on islet transplantation suggest that it might be possible to use s.c. sites for implantation (27). Such a noninvasive mode of transplantation could enable continuous monitoring of the implant growth and will avoid the potential risk of developing sclerosing encapsulating peritonitis that might arise (as suspected in animal #4) upon infection or graft rejection at the omental site.

Finally, it should be noted that the embryonic precursor tissue used in our study differs from islet transplantation in its dose/response relationship. While islet transplantation exhibits linear dose dependency, and the islet dose must be adjusted to the body weight of the recipient, the embryonic precursor tissue, which exhibits remarkable growth potential, can adopt itself to the size of the recipient through a poorly defined complex mechanism of organ size control (28, 29). Thus, the same dose of embryonic tissue could potentially give rise to final organs of different size, depending on the species or weight of the recipient and its physiological demands. In the present study, we chose to test our implants under conditions in which tissue dose was not limiting. Having demonstrated the proof of concept, further studies will be performed to define the minimal tissue dose for attainment of insulin independence.

In conclusion, although further fine tuning of immune suppression, tissue dose, and implantation techniques are warranted, our results demonstrate that porcine E-42 embryonic pancreatic tissue can correct hyperglycemia under a tolerable immune suppression protocol. The long-term proliferative capacity of these grafts, their ability to induce revascularization by host endothelium, and their reduced immunogenicity, strongly suggest that porcine embryonic xenotransplantation could offer an attractive replacement therapy for diabetes.

Methods

Animals.

Juvenile Macaca fascicularis (cynomolgus) male monkeys (2–3 years of age, 2–3.5 kg) bred at a local Israeli farm (Moshav Mazor) from a colony derived from Mauritius were used as transplant recipients. The animals were tested annually for all known pathogenic viruses and for tuberculosis and were treated with anti-helmintics. All of the procedures were monitored by the veterinarian of the Veterinary Resources Unit of the Weizmann Institute and approved by the Institutional Animal Care and Use Committee (IACUC). Monkeys were maintained in pairs. The cages (Lab. Products) were designed with free moving rooms attached to them. All cages and rooms were equipped with enrichment accessories. Cages were cleaned daily and sterilized weekly. Fresh fruits and vegetables were washed with soap and water before feeding.

Immunosuppressive Protocol.

The immune suppression protocol consisted of induction therapy with Rituxan (Rituximab, 20 mg/kg; La Roche LTD ) on day −10 before transplant, and ATG (Thymoglobulin, rabbit anti-human thymocyte globulin; 10–20 mg/kg; Sangstat) on days −4 and −3, before transplantation, as described for animals #2, #3, and #4. Animal #1 received a total of 70 mg over days −5, −4, and −3. Induction was completed with 2 doses of Simulect (10 mg; Novartis Pharma Stein AG) on at the day of the transplant and on day 4 posttransplant [postoperative day (POD) 4]. Maintenance was continued with human CTLA4-Ig (Abetacept; Bristol-Myers, 20 mg/kg on POD 0 and 4, and repeated every 14 days), Everolimus [0.075–0.15 mg/kg daily administered s.c.; the dose was adjusted according to trough blood levels (4–8 ng/mL)], and FTY720 (0.1 mg/kg daily PO, starting 5 days after transplantation). All intravenous (i.v.) drug administrations were performed under anesthesia. Everolimus and FTY720 were supplied by Novartis Pharma Stein AG under MTA.

Each ATG administration was preceded by prophylactic hydrocortisone (5–10 mg/kg i.v.), Promethazine [0.5–1 mg/kg intramuscular (i.m.)], and paracetamol (15–20 mg/kg rectally). On the first 2 days, ATG (10–20 mg/kg), diluted in 50 mL Ringer's lactate or saline 0.9%, was injected i.v. at a rate of 10 mL/kg/h twice daily.

Animals were monitored closely for possible side effects, including rash, respiratory symptoms, and body temperature elevation during and following treatment with immunosuppressive agents. If clinical signs suggesting pulmonary edema or allergic reaction developed, then treatment with diuretics (furosemide 1 mg/kg i.v. or i.m.) and antihistamines was added.

Prophylactic and Preemptive Treatment.

CMV infection has a significant negative impact on graft function and recipient survival (30). Prophylactic Ganciclovir (Cytovene 2.5 mg/kg i.m. in a divided dose, 1.0 mg/kg AM, 1.5 mg/kg PM) treatment was therefore initiated 2 days before ATG administration. RT-PCR monitoring of rhCMV replication was performed twice a week at the virology laboratory of the Rambam Hospital, Haifa, Israel. When rhCMV replication was observed (>3,500 copies/mL), the Ganciclovir dose was increased to 10 mg/kg i.m. and, in cases where the development of viral tolerance was suspected, Cidofovir treatment (5 mg/kg once per week) was added until rhCMV levels became undetectable. Bacterial infection was defined as positive blood and/or wound culture in combination with leukocytosis or leukopenia, abnormal body temperature, and/or general clinical deterioration. For suspected infection, blood cultures (and, if infected, wound culture) were obtained, and then empiric antibiotic therapy was initiated using enrofloxacin (5 mg/kg i.m. 2dd2 daily doses) and stopped when cultures remained negative after 48 h of incubation and the clinical condition of the animal improved. For each confirmed infection, appropriate antimicrobial therapy was chosen based on the in vitro susceptibilities.

Surgery.

The animals were fasted 8 h before the surgery. Prophylactic antibiotic and anti-inflammatory treatments were administered.

Anesthesia.

After the i.m. administration of a combination of midzolam and ketamine as a premedication, an i.v. line was inserted, and the monkey was intubated using an orotracheal tube. Thereafter, ventilation with a gas mixture containing Isofluran was carried out for anesthesia maintenance. Temperature was maintained within the normal range using a heating mattress. Heart rate, temperature, blood oxygenation, and blood pressure were monitored during the surgical procedure.

Surgical procedures.

The surgical procedure included transplantation of E42 fetal porcine pancreas, isolated shortly before the surgery, in the omental folds. Pig embryos were routinely obtained from the Lahav Institute of Animal Research (Kibbutz Lahav, Israel). Pregnant sows were operated on at precise stages of their pregnancy under general anesthesia, and embryos were extracted. Warm ischemia time was less than 10 min, and the embryos were transferred in cold PBS. Pig pancreas precursors for transplantation were extracted under a light microscope and kept under sterile conditions at 4 °C in RPMI 1640 (Biological Industries) before transplantation without any attempt to remove exocrine elements and without any other ex vivo manipulation. Cold ischemia time until transplantation was routinely less than 2 h.

In all operations, an anterior abdominal approach was used. Following exposure of the omentum, an omental pocket was created by circular suture legation followed by embryonic tissue implantation into the pocket. In each transplantation, approximately 6 omental pockets were created, and in each pocket, 10 E42 pig pancreatic fragments were implanted. The abdominal wall was closed in layers using conventional technique.

Postoperative management.

For the first 12 h following operation, no oral feeding was permitted. Afterward, the monkey was allowed to drink and eat freely, if clinically feasible. Antibiotic treatment was continued for 3 days, according to the surgeon's orders. All post-operative wound treatments and blood tests were performed after the administration of ketamine and midazolam. Pain relief in the immediate surgical period and afterward was provided by Buprenex i.m. injections.

During the first week after the transplantation surgery, blood tests for complete blood cell count, kidney and liver functions, electrolytes, cholesterol and triglycerides, porcine insulin, Ig and complement levels, and immunosuppressive drug levels were taken at a frequently based on the clinical condition, followed by 1–2 blood tests weekly, unless clinical signs of infection, fever, acute rejection, or other complications appeared. The maximal blood volume drawn over any 2-week period was not permitted to exceed 1% of the monkey's body weight.

ELISA Measurements of Insulin.

The porcine/human insulin kit (K6219; Dako) was used to follow insulin levels according to the manufacturer's instructions. NHP serum samples were loaded in a blinded fashion into the wells.

Determination of Hemoglobin A1C.

Total Hb and HbA1c concentrations were determined using COBAS INTEGRA system, according to the manufacturer's instructions. Briefly, total Hb was measured colorimetrically. HbA1c was determined by an immunoturbidimetric assay. The ratio of both concentrations yielded the final percent HbA1c result (HbA1c %).

Everolimus (RAD) Blood Levels.

Blood samples were analyzed routinely once weekly at the Sheba Medical Center in Tel-Aviv, by Innofluor Certican fluorescence polarization immunoassay (Seradyn). Results were obtained within 2–3 days. Targeted trough RAD levels were between 4 and 8 ng/mL.

Porcine C-Peptide Levels.

RIA for pig C-peptide was performed using Porcine C-Peptide RIA kit (catalog no. PCP-22K; Linco Research) according to the manufacturer's instructions.

Primate C-Peptide Levels.

RIA for human C-peptide was performed using Human C-Peptide RIA Kit (catalog no. HCP-20K; Linco Research) according to the manufacturer's instructions.

Diabetes Induction and Management.

STZ protocol for the induction of diabetes in NHP.

1. STZ (Zanosar, Amersham Pharmacia and Upjohn) was given after overnight fasting and antiemetic treatment (Zofran). The animal was sedated with 10 mg/kg Ketamine i.m. and 0.04 mg/kg atropine i.m. Diabetes was induced using a single high dose i.v. STZ injection (150 mg/kg) (31). High fasting blood glucose levels were observed in all monkeys within 24 h following STZ injection. Implantation of the embryonic tissues was conducted at least 30 days after diabetes induction.

Diabetes management.

Daily diabetes management included the administration of Lantus (Sanofi-Aventis U.S. LLC), once or twice daily, and Lispro (Humalog; Eli Lilly) as required. The monkeys were fed 3 low carbohydrate meals a day. Uneaten food was removed after 45 min. Blood for glucose measurement was taken from the finger tip just before meals, and then Lispro was administered according to the following scale: Glucose levels of 300–400 mg/dL, 1 unit; 400–500, 2 units; and 500–600, 3 units. Insulin doses were reevaluated and adjusted for each monkey according to blood glucose measurements 2 h after Lispro administration. Lantus dose was adjusted according to fasting morning blood glucose levels, 12 or 24 h post-Lantus administration. Close monitoring of blood glucose was performed at night to avoid unexpected hyperglycemia and to minimize the risk of hypoglycemia or ketoacidosis.

Glucose Tolerance Test.

The test was taken after an 18-h fast and insulin treatment withdrawal for 26 h (Lantus) and 6 h (Lispro). Each animal was sedated with ketamine hydrochloride (10 mg/kg i.m.) before placement of bilateral saphenous vein catheters for the infusion of glucose and blood sampling, respectively. The glucose tolerance test was performed by injecting a solution of 12.5% dextrose (0.5 g/kg) over a 30-s period at t = 0 and determining the subsequent blood glucose concentrations with an Elite XL glucometer (Bayer) at 0, 1, 3, 5, 10, 15, 20, 30, 60, and 90 min post-injection. Blood for insulin and porcine C-peptide determination was collected at the same time. The glucose disappearance rate (K_G) was calculated from the portion of the curve occurring between the 5 and 30 min time points. The K value represents the rate of reduction in plasma blood glucose as a percentage per minute, and was calculated using the following formula (32): K_G = [LN(glucose level at 5 min) − LN(glucose level at 30 min)/25] × 100.

Histology and Immunohistochemistry.

Sections were routinely stained by hematoxylin and eosin. Histochemical and immunohistochemical labeling was performed as previously described (9) with the addition of mouse anti-human CD31 (clone JC/70A) for the demonstration of NHP's endothelium. For immunoperoxidase labeling, the following second antibodies and reagents were used: Dako peroxidase envision system for detection of mouse and rabbit antibodies, and Sigma biotinylated anti-goat antibody (followed by extra-avidin peroxidase reagent) for detection of goat antibodies. Diaminobenzidine was used as the chromogen for peroxidase labeling. Tissue sections were counterstained with hematoxylin and embedded in Entellan.

For single and multiple immunofluorescence labeling, the following secondary antibodies were used: Donkey anti-mouse and donkey anti-rabbit antibodies, conjugated with CY2 or TxR (Jackson); goat anti-rabbit antibody, conjugated with Alexa Fluor 350 (Invitrogen); and donkey anti-guinea pig antibody, conjugated with biotin (Jackson). Griffonia Simplicifolia, isolectin 4, conjugated with FITC (L-2895Sigma), was used for demonstration of α-galactose expressing blood vessels of pig origin. Rabbit anti-human IgG, conjugated with FITC (Dako), was used to evaluate immunoglobin deposition. Nuclei were counterstained with the fluorescent nuclear dyes, Hoechst 33342 and Hoechst Yellow (Molecular Probes). The histological sections were embedded in Cytomation Fluorescent Mounting Medium (Dako).