Embryonic Pig Pancreatic Tissue for the Treatment of Diabetes in a Nonhuman Primate Model

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

Gil Hechta,1, Smadar Eventov-Friedmana,1, Chava Rosena, Elias Shezena, Dalit Tchorsha, Anna Aronovicha, Enrique Freudb, Hana Golana, Ronit El-Hasidc, Helena Katchmana, Bernhard J. Heringd, Amnon Zunge, Zipi Kra-Ozf, Pninit Shaked-Mishanf, Alex Yusimg, Alex Shtabskyh, Pavel Idelevitcha, Ana Tobari, Alon Harmelinj, Esther Bachar-Lustiga, and Yair Reisnera,2

aDepartment of Immunology, Weizmann Institute of Science, Rehovot 76100, Israel; bDepartment of Pediatric Surgery, Schneider Children’s Medical Center of Israel, Petach Tikvah 49202, Israel; cDepartment of Pediatric Hemato-Oncology, Meyer Children Hospital, Haifa 31096, Israel; dDiabetes Institute for Immunology Transplantation, University of Minnesota, Minneapolis, MN, 55455; eDepartment of Pediatrics, Kaplan Medical Center, Rehovot 76100, Israel; fDepartment of Virology, Rambam Medical Center, Haifa 31096, Israel; gDepartment of Transplantation, Rabin Medical Center, Petah Tikva, Israel; hDepartment of Pathology, Sourasky Medical Center, Tel-Aviv 64239, Israel; iDepartment of Pathology, Rabin Medical Center, Petah Tikva, Israel; and jDepartment of Veterinary Resources, Weizmann Institute of Science, Rehovot 76100, Israel

Edited by George Klein, Karolinska Institutet, Stockholm, Sweden, and approved April 3, 2009 (received for review December 10, 2008) Xenotransplantation of pig tissues has great potential to overcome (unpublished), spleen (8), pancreas, liver, and lung (7) into SCID the shortage of organ donors. One approach to address the mice. Once this threshold was established, we were able to vigorous immune rejection associated with xenotransplants is the further characterize an optimal gestational window for trans- use of embryonic precursor tissue, which induces and utilizes host plantation based on growth potential and immunogenicity. Thus, vasculature upon its growth and development. Recently, we while the optimal window for kidney transplantation was defined showed in mice that embryonic pig pancreatic tissue from embry- around E28, our data suggested E42 as the optimal gestational onic day 42 (E42) exhibits optimal properties as a ␤ cell replacement time point for pig donor tissue in pancreatic transplantation (9). therapy. We now demonstrate the proof of concept in 2 diabetic Importantly, upon growth and development of the embryonic Cynomolgus monkeys, followed for 393 and 280 days, respectively. pancreatic implant, acini gradually disappear and the tissue is A marked reduction of exogenous insulin requirement was noted predominantly comprised of islets and stroma identified by

by the fourth month after transplantation, reaching complete vimentin straining. Thus, the risk of potential ongoing destruc- MEDICAL SCIENCES independence from exogenous insulin during the fifth month after tion of the growing organ by local proteolytic enzymes, which transplantation, with full physiological control of blood glucose might be released by the exocrine components of the implant, is levels. The porcine origin of insulin was documented by a radio- miniml (9). These mouse studies also demonstrated the curative immunoassay specific for porcine C-peptide. Furthermore, the potential of such implants in diabetic mice under relatively growing tissue was found to be predominantly vascularized with tolerable immune suppression. However, considering that xeno- host blood vessels, thereby evading hyperacute or acute rejection, geneic transplantation into primates is far more complex, due to which could potentially be mediated by preexisting anti-pig anti- preexisting anti-porcine antibodies, studies in a nonhuman pri- bodies. Durable graft protection was achieved, and most of the late mate (NHP) model for diabetes were required. Our working complications could be attributed to the immunosuppressive pro- hypothesis, suggesting that embryonic tissue transplantation tocol. While fine tuning of immune suppression, tissue dose, and would not be adversely affected by such a humoral response was implantation techniques are still required, our results demonstrate based on 2 major observations: (i) hyperacute and acute rejec- that porcine E-42 embryonic pancreatic tissue can normalize blood tion are mainly mediated by complement activation in blood glucose levels in primates. Its long-term proliferative capacity, its vessels, mediated by preformed anti-pig antibodies recognizing revascularization by host endothelium, and its reduced immuno- ␣-gal and other carbohydrates expressed on endothelial cells genicity, strongly suggest that this approach could offer an attrac- (10), and (ii) data from our mouse studies showing that the tive replacement therapy for diabetes. embryonic implants predominantly induce host-type vasculature to support their growth and development in the recipient, immune-suppression ͉ rejection ͉ xeno-transplantation resulting in an organ comprised mainly of porcine epithelial cells and host endothelium. llogeneic islet cell replacement can restore normoglycemia To further assess the curative potential of E42 pig pancreatic Ain patients with type 1 diabetes; yet, the shortage of tissue, we have now evaluated its capacity to correct hypergly- available organs for transplantation continues to severely limit cemia under tolerable immune suppression in an NHP model for this therapeutic option. A potential solution for this shortage is diabetes. the use of pig organs and tissues as a source for transplantation. Results However, the immunological barriers to xenotransplantation are formidable (1). Thus, new approaches that could further reduce Defining a Tolerable Immune Suppression Protocol. Diabetes was the intensity of the required immune suppression are critical for induced in 4 cynomologous monkeys by the administration of successful application of pig pancreatic tissue as a novel source of islet cells. One means to address this challenge could be Author contributions: G.H., S.E.-F., C.R., E.F., B.J.H., and Y.R. designed research; G.H., S.E.-F., provided by the use of embryonic porcine tissue. This strategy is C.R., E.S., D.T., A.A., E.F., H.G., R.E.-H., H.K., A.Z., Z.K.-O., P.S.-M., P.I., A.T., and A.H. based on the growing evidence, over the past 5 decades, dem- performed research; G.H., S.E.-F., C.R., E.S., D.T., A.A., A.Z., Z.K.-O., P.S.-M., A.S., A.T., and onstrating maternal immune tolerance to the fetus (2, 3), and on Y.R. analyzed data; and G.H., S.E.-F., A.Y., E.B.-L., and Y.R. wrote the paper. recent observations that embryonic tissues exhibit reduced im- Conflict of interest: Y.R. is a scientific consultant and holds equity with Tissera, Inc., which munogenicity in various transplantation settings (4–7). Consid- supported this work. ering that very early embryonic tissues are associated with a This article is a PNAS Direct Submission. substantial risk of teratoma formation, we have attempted, 1G.H. and S.E.-F. contributed equally to this work. during the past several years, to define the earliest gestational 2To whom correspondence should be addressed. E-mail: [email protected]. time point that does not pose a teratoma risk for transplantation This article contains supporting information online at www.pnas.org/cgi/content/full/ of different embryonic pig tissues, including kidney (6), heart 0812253106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812253106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 25, 2021 Implantation of Conditioning AA pancreatic precursors Protocol

Day -33-33 -10 -7-7 -5-4 -3-3 0 44 55 1414 2828 …………….. …………………… Abatacept STZ ATG Simulect Rituxan FTY720 daily B C Everolimus daily

Fig. 1. Immune suppression protocol in STZ-treated NHP

150 mg/kg of streptozotocin (STZ). About 1 month after STZ administration, E42 porcine tissue was implanted in the omen- B C tum as described in Methods. Immune suppression was induced D 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 D previously shown to be effective and well-tolerated in allogeneic E islet transplantation in the NHP model (11–14). Fig. 2. Developmental and functional characteristics of E42 pig pancreatic The study consisted of 2 cohorts, with 2 animals in each. In the implant, 3 months after intra-omental implantation in animal #2. (A) Macro- first cohort, central line-associated infections eventually led to scopic appearance of the graft removed postmortem (pancreatic nodes are the death of both NHP #1 and #2, 2 and 3 months following marked by arrows). Each scale mark represents 1 mm. (B) Demonstration of transplantation, respectively. Apart from the adverse effects insulin-positive cells in the graft islets by immunoperoxidase staining (brown). associated with the central line catheters, the ATG dose (total (C) Minimal intra-graft presence of CD3 positive T cells (brown, arrows). of 70 mg/kg) used in monkey #1 was associated with severe (D) Proliferating Ki67-positive cells (red ﬂuorescence) within a pancreatic islet, outlined by cytokeratin positivity (green ﬂuorescence). (Scale bars in anemia and coagulation disorders, requiring extensive blood B–D,50␮m.) 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 regenerative capability of the pancreatic tissue, in agreement NHP are 6 times higher than in humans (12). However, sub- with our previous mouse studies (9). stantial reduction of ATG dose in monkey #2 to 40 mg/kg was The E42 pancreatic tissue used for transplantation was not well-tolerated and did not affect engraftment or acceptance of manipulated ex vivo before transplantation. Marked expression the graft, as evidenced by histology (Fig. 2). In contrast to NHP of ␣-gal on endothelial but not on epithelial cells was found by #1 and #2, which were followed for a relatively short time, which double staining of the graft tissue before transplantation. Thus, was not sufficient to allow a significant progression toward as can be seen in Fig. 3A, ␣-gal positive cells stained with insulin independence, the changes made in the treatment of the Banderia Simplicifolia isolectin 4 are distinguished from fetal animals in cohort 2 (i.e., avoiding use of central line and using epithelial structures stained with anti-cytokeratin. In contrast, a lower ATG dose) enabled these animals to survive for 393 staining of the graft at 3 months posttransplant with anti-human (NHP #3) and 280 days (NHP #4) posttransplantation. CD31 antibodies (cross-reactive with monkey but not porcine Similarly to NHP #2, NHP #3 was transplanted using a total endothelial cells), revealed that the islet-like structures, as well dose of 40 mg/kg ATG. This animal exhibited CMV reactivation, as other cells within the stroma, were primarily vascularized by which required continuous treatment (see Materials and Meth- host-type (primate) endothelium and only slight staining of pig endothelial cells was observed with Banderia Simplicifolia lectin ods), and therefore in animal #4, the immune suppression (Fig. 3B). protocol was further reduced to comprise a total dose of 20 The predominance of host vascularization can be clearly seen mg/kg ATG. Postmortem immunohistological analysis of mes- upon examination of islets under high magnification (Fig. 3 C enteric lymph nodes and spleen of this animal revealed pre- and D). served T and B cell follicles (SI), while as can be expected from Thus, the ability of the growing implant to use host vasculature the use of FTY720 in the protocol, low lymphocyte blood levels might be critical for graft survival and growth in the recipient NHP. (average 0.68 k/␮L Ϯ 0.37) were recorded throughout the follow-up period (SI). Implantation of E42 Pig Embryonic Pancreatic Tissue Restores an Enduring Euglycemic State. Long-term follow-up of cohort 2 Islets and Stroma Within the Growing Pig Embryonic Pancreas Use demonstrated the ability of the grafts to cure diabetes. As can be Host Vasculature and Evade Humoral Hyperacute and Acute Rejection. seen in Fig. 4, both animals of the second cohort exhibited a very NHP #1 and #2 exhibited impressive growth of the implants similar pattern of recovery of glucose control, in parallel to the (Fig. 2A) as seen upon postmortem examination. Clusters of growth and development of the implant. Thus, similarly to our islets, with insulin-containing cells were evident (Fig. 2B), while results in the mouse model, a marked reduction of exogenous only a few incidental lymphocytes were observed (Fig. 2C), insulin requirement (i.e., less than 10% of the daily insulin dose indicating the absence of rejection. Furthermore, an active used before transplantation) was noted by the fourth month after proliferating pool of cells, stained for Ki67 was found within the transplantation, reaching complete exogenous insulin indepen- islets (Fig. 2D). This observation emphasizes the long-term dence during the fifth posttransplantation month (Fig. 4 A and

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812253106 Hecht et al. Downloaded by guest on September 25, 2021 Fig. 3. Vascularization pattern of E42 pancreatic tissue. (A) Marked expression of ␣-gal on endothelial but not on epithelial cells in the graft tissue before transplantation, indicated by double staining. Thus, ␣-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 sup- MEDICAL SCIENCES porting 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 ␮m.)

B), with marked physiological control of blood glucose levels Fig. 4. Assessment of E42 fetal porcine xenograft function in streptozotocin- (Fig. 4 A and B). A radioimmunoassay (RIA)-specific for induced diabetic Cynomolgus monkeys of cohort 2. Insulin requirement (blue) porcine C-peptide (i.e., non-cross-reactive with primate C- and fasting glucose levels (yellow) in monkeys #3 and #4 are shown in panels peptide) confirmed the presence of basal porcine C-peptide A and B, respectively. Speciﬁc pig C-peptide (red) and insulin (green) levels levels as early as 3 months following transplantation (0.24 and after IVGTT in monkey #3 (C) and #4 (D) were determined by ELISA speciﬁc for 0.35 ng/mL for monkeys #3 and #4, respectively), and marked pig C-peptide (non-cross-reactive with monkey C-peptide) and for total insulin elevation of porcine C-peptide blood levels was demonstrated in (using an antibody that does not distinguish between the 2 species), at 393 both animals after i.v. glucose challenge test (IVGTT), in days and 280 days posttransplant, respectively. K values for the clearance of accordance with the enhanced insulin levels (Fig. 4 C and D). 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 the immune suppression, necessitated euthanizing of the animal significant elevation of primate C-peptide could be detected by at 393 days posttransplant (SI). this assay following glucose challenge, in contrast to the eleva- A postmortem immunohistochemical analysis of the graft re- tion of porcine C-peptide (SI). This constant and unresponsive vealed intact insulin-positive islet fields (Fig. 5B and C), and only base line level could be due to some cross-reactivity with rare CD3ϩ lymphocytes could be recorded in the proximity of undefined blood components or to some level of regeneration of transplanted islets (Fig. 5B) ruling out potential immune rejection. undifferentiated or nonfunctional insulin-positive beta cells in In NHP #4 insulin independence was sustained for more than the recipient. However, the absence of insulin-positive beta cells 17 weeks (average fasting glucose during this period was 113 Ϯ within the host pancreas upon postmortem (Fig. 5A) strongly 20) without any complications. At the beginning of the eighth suggests that insulin independence, attained in both animals month posttransplant, technical failure to sustain Everolimus upon growth and development of the implant, is most likely not levels above the threshold of 3 ng/mL (SI) seemed initially to be associated with the potential recovery of host pancreatic activity. associated with partial loss of graft function (Fig. 4B). A glucose The animals exhibited an Hb A1C of 6.1% and 5.2% at 333 and challenge test performed 2 weeks earlier revealed robust pig 195 days following transplant, respectively. pancreatic activity (maximal C-peptide of 3.79 ng/mL), suggest- Correction of hyperglycemia was adversely affected twice in ing that the acute reduction in function might indeed be attrib- monkey #3. Two weeks after reaching insulin independence, low uted to the parallel drop of Everolimus blood levels. This Everolimus levels were detected and partial rejection was sus- decrease in functionality was accompanied by clinical deterio- pected as insulin requirement relapsed to an average of 15% of ration, which was manifested by anorexia, and led to euthanizing maximal levels during week 33 (Fig. 4A and SI). However, a of the animal at 280 days posttransplantation (SI). Postmortem gradual restoration of graft function, to a minimal requirement examination revealed essentially similar findings to those found of insulin supplementation, less than 3.7% of the insulin require- in animal #3 (see SI). Thus, the presence of intact islets fields ment before transplantation, was observed (Fig. 4A). Eventually, with no evidence of rejection indicates that an infectious process clinical complications likely associated with the continuous of unknown etiology could be responsible for the partial loss of CMV infection and the combined toxicity of anti-viral drugs and function in both animals.

Hecht et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 25, 2021 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. How- ever, 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 Fig. 5. Immunohistochemical characterization of islets in the E42 pig pancreatic implant versus the host pancreas, at 13 months after intra-omental adverse effects of Rapamycin on islet function (19–21). implantation in animal #3. (A) Immunoperoxidase labeling of insulin (brown Beta cell replacement can be achieved by either whole organ- staining) reveals minimal residual presence of insulin-positive cells within the pancreas allogeneic transplantation or transplantation of iso- host pancreas (each bar represents 50 ␮m). Insulin-containing islets within the lated islet cells. The transplantation of a whole pancreas achieves graft (B and C) were labeled by immunofluorescence labeling of insulin (blue a longer graft survival and functionality than isolated islets (67% fluorescence). Few incidental CD3-positive T cells (green fluorescence) can be after 10 years and 10% after 5 years, respectively) (22, 23). This detected outside the islets (blue) (B). discrepancy could be attributed to the deleterious effects en- countered during islet preparation by enzyme digestion, pro- Discussion longed ischemia time, and the loss of surrounding mesenchyme. The active involvement of the surrounding mesenchyme in Initial clinical attempts of fetal porcine pancreas transplantation pancreatic development and its role in endocrine cell differen- were carried out early in the 1990s by Groth et al. (15). In these tiation and proliferation has been documented (24, 25). Thus, studies, patients were transplanted with E66-E81 porcine fetal the embryonic pancreatic tissue growing in the context of its own islet-like cell clusters. Blood glucose levels increased shortly stroma as documented here, might survive longer than infused after transplantation and insulin administration had to be in- neonate or adult islets growing heterotopically in the liver. creased, so that eventually it did not differ from the pretrans- Indeed, following graft injury and partial loss of function in plant requirements. We have shown recently in a mouse model monkey #3, the growing pig implant exhibited a marked capacity that this outcome could be attributed, in part, to the relatively to withstand stress and regenerate. This characteristic of the late stage of gestation at which the embryonic pancreatic tissue was harvested. Thus, based on growth potential and immuno- graft, which could be related to mesenchymal support, is of genicity, we demonstrated that the optimal ‘‘window’’ for these particular relevance considering that although 1-year success transplants could be provided at around E42 (7, 9). rates for islet transplantation are high, the long-term success rate In our present study, the almost identical pattern of insulin is unacceptably low, even in the pioneering Edmonton Center reduction exhibited in both animals of cohort 2 (Fig. 4 A and B) (23). The poor long-term clinical outcome, taken together with demonstrates the reproducibility of the time frame of growth and the recent study of Koulmanda et al. (26), indicates that one of differentiation required to achieve normoglycemia, and provides the reasons for long-term failure may be due to the low islet cell proof of concept for the curative potential of the E42 pig mass that actually survives the harvesting and transplant proce- pancreatic implants in NHP. dures, as well as islet attrition by exhaustion. The potential Furthermore, the growing tissue makes use of host vascula- importance of islet replacement with E42 graft that will expand ture, and thereby evades hyperacute or acute rejection, which with time could afford a major advantage. could potentially be mediated by preexisting anti-pig antibodies Recent rodent studies on islet transplantation suggest that it (10). This finding supports our working hypothesis that growing might be possible to use s.c. sites for implantation (27). Such a the embryonic pig tissue de novo within the NHP recipient, noninvasive mode of transplantation could enable continuous provides an advantageous source for organ transplantation monitoring of the implant growth and will avoid the potential compared to adult pig organ transplantation, which requires risk of developing sclerosing encapsulating peritonitis that might anastomosis of donor blood vessels. Further NHP studies with arise (as suspected in animal #4) upon infection or graft other precursor tissues such as embryonic kidney (6), liver (7), rejection at the omental site. and spleen (8), for which appropriate embryonic sources have Finally, it should be noted that the embryonic precursor tissue been recently characterized, are warranted. used in our study differs from islet transplantation in its dose/ Very recently, the capacity of neonatal and adult porcine islets response relationship. While islet transplantation exhibits linear to restore normoglycemia in diabetic NHP was demonstrated dose dependency, and the islet dose must be adjusted to the body (16, 17). However, the immune suppression required to maintain weight of the recipient, the embryonic precursor tissue, which the implants was either too toxic or included anti-CD40L, which exhibits remarkable growth potential, can adopt itself to the size cannot be used in human patients due to its thrombotic prop- of the recipient through a poorly defined complex mechanism of erties. Other studies suggested that E28 pancreatic tissue might organ size control (28, 29). Thus, the same dose of embryonic be accepted in nonimmunosuppressed rats (33) and monkeys tissue could potentially give rise to final organs of different size, (34). In contrast, our mouse (9) and rat (35) data has revealed depending on the species or weight of the recipient and its fierce rejection of pig embryonic tissue of any gestational time physiological demands. In the present study, we chose to test our point in the absence of immune suppression, while demonstrat- implants under conditions in which tissue dose was not limiting. ing that the immunogenicity of E42 tissue was relatively lower Having demonstrated the proof of concept, further studies will

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812253106 Hecht et al. Downloaded by guest on September 25, 2021 be performed to define the minimal tissue dose for attainment Anesthesia. After the i.m. administration of a combination of midzolam and of insulin independence. ketamine as a premedication, an i.v. line was inserted, and the monkey was In conclusion, although further fine tuning of immune sup- intubated using an orotracheal tube. Thereafter, ventilation with a gas mix- pression, tissue dose, and implantation techniques are war- ture containing Isofluran was carried out for anesthesia maintenance. Tem- perature was maintained within the normal range using a heating mattress. ranted, our results demonstrate that porcine E-42 embryonic Heart rate, temperature, blood oxygenation, and blood pressure were mon- pancreatic tissue can correct hyperglycemia under a tolerable itored during the surgical procedure. immune suppression protocol. The long-term proliferative ca- Surgical procedures. The surgical procedure included transplantation of E42 pacity of these grafts, their ability to induce revascularization by fetal porcine pancreas, isolated shortly before the surgery, in the omental host endothelium, and their reduced immunogenicity, strongly folds. Pig embryos were routinely obtained from the Lahav Institute of Animal suggest that porcine embryonic xenotransplantation could offer Research (Kibbutz Lahav, Israel). Pregnant sows were operated on at precise an attractive replacement therapy for diabetes. stages of their pregnancy under general anesthesia, and embryos were extracted. Warm ischemia time was less than 10 min, and the embryos were Methods transferred in cold PBS. Pig pancreas precursors for transplantation were extracted under a light microscope and kept under sterile conditions at 4 °C in Animals. Juvenile Macaca fascicularis (cynomolgus) male monkeys (2–3 years RPMI 1640 (Biological Industries) before transplantation without any attempt of age, 2–3.5 kg) bred at a local Israeli farm (Moshav Mazor) from a colony to remove exocrine elements and without any other ex vivo manipulation. derived from Mauritius were used as transplant recipients. The animals were Cold ischemia time until transplantation was routinely less than 2 h. tested annually for all known pathogenic viruses and for tuberculosis and In all operations, an anterior abdominal approach was used. Following were treated with anti-helmintics. All of the procedures were monitored by exposure of the omentum, an omental pocket was created by circular suture the veterinarian of the Veterinary Resources Unit of the Weizmann Institute legation followed by embryonic tissue implantation into the pocket. In each and approved by the Institutional Animal Care and Use Committee (IACUC). transplantation, approximately 6 omental pockets were created, and in each Monkeys were maintained in pairs. The cages (Lab. Products) were designed pocket, 10 E42 pig pancreatic fragments were implanted. The abdominal wall with free moving rooms attached to them. All cages and rooms were equipped was closed in layers using conventional technique. with enrichment accessories. Cages were cleaned daily and sterilized weekly. Postoperative management. For the first 12 h following operation, no oral Fresh fruits and vegetables were washed with soap and water before feeding. feeding was permitted. Afterward, the monkey was allowed to drink and eat freely, if clinically feasible. Antibiotic treatment was continued for 3 days, Immunosuppressive Protocol. The immune suppression protocol consisted of according to the surgeon’s orders. All post-operative wound treatments and induction therapy with Rituxan (Rituximab, 20 mg/kg; La Roche LTD ) on day blood tests were performed after the administration of ketamine and mida- Ϫ10 before transplant, and ATG (Thymoglobulin, rabbit anti-human thymo- zolam. Pain relief in the immediate surgical period and afterward was pro- cyte globulin; 10–20 mg/kg; Sangstat) on days Ϫ4 and Ϫ3, before transplan- vided by Buprenex i.m. injections. MEDICAL SCIENCES tation, as described for animals #2, #3, and #4. Animal #1 received a total of During the first week after the transplantation surgery, blood tests for 70 mg over days Ϫ5, Ϫ4, and Ϫ3. Induction was completed with 2 doses of complete blood cell count, kidney and liver functions, electrolytes, cholesterol Simulect (10 mg; Novartis Pharma Stein AG) on at the day of the transplant and and triglycerides, porcine insulin, Ig and complement levels, and immunosup- on day 4 posttransplant [postoperative day (POD) 4]. Maintenance was con- pressive drug levels were taken at a frequently based on the clinical condition, tinued with human CTLA4-Ig (Abetacept; Bristol-Myers, 20 mg/kg on POD 0 followed by 1–2 blood tests weekly, unless clinical signs of infection, fever, and 4, and repeated every 14 days), Everolimus [0.075–0.15 mg/kg daily acute rejection, or other complications appeared. The maximal blood volume administered s.c.; the dose was adjusted according to trough blood levels (4–8 drawn over any 2-week period was not permitted to exceed 1% of the ng/mL)], and FTY720 (0.1 mg/kg daily PO, starting 5 days after transplanta- monkey’s body weight. tion). All intravenous (i.v.) drug administrations were performed under anesthesia. Everolimus and FTY720 were supplied by Novartis Pharma Stein AG ELISA Measurements of Insulin. The porcine/human insulin kit (K6219; Dako) under MTA. was used to follow insulin levels according to the manufacturer’s instructions. Each ATG administration was preceded by prophylactic hydrocortisone NHP serum samples were loaded in a blinded fashion into the wells. (5–10 mg/kg i.v.), Promethazine [0.5–1 mg/kg intramuscular (i.m.)], and parac- etamol (15–20 mg/kg rectally). On the first 2 days, ATG (10–20 mg/kg), diluted Determination of Hemoglobin A1C. Total Hb and HbA1c concentrations were in 50 mL Ringer’s lactate or saline 0.9%, was injected i.v. at a rate of 10 mL/kg/h determined using COBAS INTEGRA system, according to the manufacturer’s twice daily. instructions. Briefly, total Hb was measured colorimetrically. HbA1c was de- Animals were monitored closely for possible side effects, including rash, termined by an immunoturbidimetric assay. The ratio of both concentrations respiratory symptoms, and body temperature elevation during and following yielded the final percent HbA1c result (HbA1c %). treatment with immunosuppressive agents. If clinical signs suggesting pul- monary edema or allergic reaction developed, then treatment with diuretics Everolimus (RAD) Blood Levels. Blood samples were analyzed routinely once (furosemide 1 mg/kg i.v. or i.m.) and antihistamines was added. weekly at the Sheba Medical Center in Tel-Aviv, by Innofluor Certican fluorescence polarization immunoassay (Seradyn). Results were obtained within Prophylactic and Preemptive Treatment. CMV infection has a significant neg- 2–3 days. Targeted trough RAD levels were between 4 and 8 ng/mL. ative impact on graft function and recipient survival (30). Prophylactic Gan- ciclovir (Cytovene 2.5 mg/kg i.m. in a divided dose, 1.0 mg/kg AM, 1.5 mg/kg Porcine C-Peptide Levels. RIA for pig C-peptide was performed using Porcine PM) treatment was therefore initiated 2 days before ATG administration. C-Peptide RIA kit (catalog no. PCP-22K; Linco Research) according to the RT-PCR monitoring of rhCMV replication was performed twice a week at the manufacturer’s instructions. virology laboratory of the Rambam Hospital, Haifa, Israel. When rhCMV replication was observed (Ͼ3,500 copies/mL), the Ganciclovir dose was in- Primate C-Peptide Levels. RIA for human C-peptide was performed using creased to 10 mg/kg i.m. and, in cases where the development of viral Human C-Peptide RIA Kit (catalog no. HCP-20K; Linco Research) according to tolerance was suspected, Cidofovir treatment (5 mg/kg once per week) was the manufacturer’s instructions. added until rhCMV levels became undetectable. Bacterial infection was defined as positive blood and/or wound culture in combination with leukocytosis Diabetes Induction and Management. STZ protocol for the induction of diabetes in or leukopenia, abnormal body temperature, and/or general clinical deterio- NHP. 1. STZ (Zanosar, Amersham Pharmacia and Upjohn) was given after ration. For suspected infection, blood cultures (and, if infected, wound cul- overnight fasting and antiemetic treatment (Zofran). The animal was sedated ture) were obtained, and then empiric antibiotic therapy was initiated using with 10 mg/kg Ketamine i.m. and 0.04 mg/kg atropine i.m. Diabetes was enrofloxacin (5 mg/kg i.m. 2dd2 daily doses) and stopped when cultures induced using a single high dose i.v. STZ injection (150 mg/kg) (31). High remained negative after 48 h of incubation and the clinical condition of the fasting blood glucose levels were observed in all monkeys within 24 h follow- animal improved. For each confirmed infection, appropriate antimicrobial ing STZ injection. Implantation of the embryonic tissues was conducted at therapy was chosen based on the in vitro susceptibilities. least 30 days after diabetes induction. Diabetes management. Daily diabetes management included the administration Surgery. The animals were fasted 8 h before the surgery. Prophylactic antibi- of Lantus (Sanofi-Aventis U.S. LLC), once or twice daily, and Lispro (Humalog; Eli otic and anti-inflammatory treatments were administered. Lilly) as required. The monkeys were fed 3 low carbohydrate meals a day. Uneaten

Hecht et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 25, 2021 food was removed after 45 min. Blood for glucose measurement was taken from CD31 (clone JC/70A) for the demonstration of NHP’s endothelium. For immu- the finger tip just before meals, and then Lispro was administered according to noperoxidase labeling, the following second antibodies and reagents were the following scale: Glucose levels of 300–400 mg/dL, 1 unit; 400–500, 2 units; and used: Dako peroxidase envision system for detection of mouse and rabbit 500–600, 3 units. Insulin doses were reevaluated and adjusted for each monkey antibodies, and Sigma biotinylated anti-goat antibody (followed by extra- according to blood glucose measurements 2 h after Lispro administration. Lantus avidin peroxidase reagent) for detection of goat antibodies. Diaminobenzi- dose was adjusted according to fasting morning blood glucose levels, 12 or 24 h dine was used as the chromogen for peroxidase labeling. Tissue sections were post-Lantus administration. Close monitoring of blood glucose was performed at counterstained with hematoxylin and embedded in Entellan. night to avoid unexpected hyperglycemia and to minimize the risk of hypogly- For single and multiple immunofluorescence labeling, the following sec- cemia or ketoacidosis. ondary antibodies were used: Donkey anti-mouse and donkey anti-rabbit Glucose Tolerance Test. The test was taken after an 18-h fast and insulin antibodies, conjugated with CY2 or TxR (Jackson); goat anti-rabbit antibody, treatment withdrawal for 26 h (Lantus) and 6 h (Lispro). Each animal was conjugated with Alexa Fluor 350 (Invitrogen); and donkey anti-guinea pig sedated with ketamine hydrochloride (10 mg/kg i.m.) before placement of antibody, conjugated with biotin (Jackson). Griffonia Simplicifolia, isolectin 4, bilateral saphenous vein catheters for the infusion of glucose and blood conjugated with FITC (L-2895Sigma), was used for demonstration of ␣- sampling, respectively. The glucose tolerance test was performed by injecting galactose expressing blood vessels of pig origin. Rabbit anti-human IgG, a solution of 12.5% dextrose (0.5 g/kg) over a 30-s period at t ϭ 0 and conjugated with FITC (Dako), was used to evaluate immunoglobin deposition. determining the subsequent blood glucose concentrations with an Elite XL Nuclei were counterstained with the fluorescent nuclear dyes, Hoechst 33342 glucometer (Bayer) at 0, 1, 3, 5, 10, 15, 20, 30, 60, and 90 min post-injection. and Hoechst Yellow (Molecular Probes). The histological sections were em- Blood for insulin and porcine C-peptide determination was collected at the bedded in Cytomation Fluorescent Mounting Medium (Dako). same time. The glucose disappearance rate (KG) was calculated from the portion of the curve occurring between the 5 and 30 min time points. The K ACKNOWLEDGMENTS. Professor Yair Reisner holds the Henry H. Drake Pro- value represents the rate of reduction in plasma blood glucose as a percentage fessorial Chair in Immunology. The authors wish to thank Prof. N. Kenyon, per minute, and was calculated using the following formula (32): KG ϭ (Miami University), Dr. W. Schuler (Novartis, Switzerland), and Melanie Gra- [LN(glucose level at 5 min) Ϫ LN(glucose level at 30 min)/25] ϫ 100. ham (University of Minnesota) for valuable advice in establishing the NHP model. We also thank Prof. David Sutherland (University of Minnesota) and the Juvenile Diabetes Research Foundation for valuable discussions. This work Histology and Immunohistochemistry. Sections were routinely stained by he- was supported in part by Tissera, Inc., Mrs. Erica Drake, D Cure-Diabetes Care matoxylin and eosin. Histochemical and immunohistochemical labeling was in Israel (R.A.), the Seymour Spira Fund, the Russell Berrie Foundation, and the performed as previously described (9) with the addition of mouse anti-human Gabriella Rich Center for Transplantation Biology Research.

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