Long-term survival of nonhuman primate islets implanted in an omental pouch on a biodegradable scaffold

Abstract

The aim of this study was to test whether an omental pouch can be used as an alternative site for islet implantation in diabetic monkeys. Here we report the successful engraftment of islets in diabetic cynomolgus monkeys when loaded on a synthetic biodegradable scaffold and placed in an omental pouch. One autologous and five allogeneic diabetic monkey transplants under the cover of steroid-free immune suppression (SFIS) were undertaken. Fasting blood glucose (FBG) and C-peptide (CP), exogenous insulin requirements (EIR), intravenous glucose tolerance test (IVGTT), A1C and histopathology were used to assess islet engraftment and survival. All animals achieved CP levels > 1.0 ng/mL following transplant, a 66-92% posttransplant decrease in EIR and reduced A1C. Following graft removal, CP became negative and histopathological analysis of the explanted grafts demonstrated well-granulated and well-vascularized, insulin-positive islets, surrounded by T-cell subsets and macrophages. Compared to intrahepatic allogeneic islet transplants (n = 20), there was a delayed engraftment for omental pouch recipients but similar levels of CP production were ultimately achieved, with a broad range of IEQ/kg transplanted in both sites. Our results suggest this extrahepatic transplantation site has potential as an alternative site for clinical islet cell transplantation.

Figure 1. Exogenous insulin requirements (EIR), fasting blood glucose (FBG) (upper panel) and c-peptide and rapamycin levels (lower panel)

A. Animal 1 was pancreatectomized on POD −2 and received 5,093 autologous IEQ/kg on POD 0 onto 2 scaffolds, each one wrapped in an omental pouch. The bilateral omental pouches were explanted on POD 124. For recipients of allogeneic islets (animals 2–5), diabetes was induced with streptozotocin. With the exception of animal 4 (2 scaffolds), the other monkeys were implanted with one scaffold; each scaffold was wrapped in an omental pouch, which was explanted on the indicated day. B. Animal 2 received 4,200 IEQ/kg onto one scaffold, which was explanted on POD 376. C. Animal 3 received 9,441 IEQ/kg onto one scaffold; the animal died during a technical procedure on POD 139. D. Animal 4 received 8,540 IEQ/kg onto 2 scaffolds, which were explanted on POD 223. E. Animal 5 received 10,291 IEQ/kg onto one scaffold, which was explanted on POD 145. All animals gradually became c-peptide positive, with reduction of EIR and varying degrees of improvement in glycemic control (upper panels). With the exception of animal 3 (died with functioning islets), all recipients became c-peptide negative after explant (lower panels).

Figure 1. Exogenous insulin requirements (EIR), fasting blood glucose (FBG) (upper panel) and c-peptide and rapamycin levels (lower panel)

A. Animal 1 was pancreatectomized on POD −2 and received 5,093 autologous IEQ/kg on POD 0 onto 2 scaffolds, each one wrapped in an omental pouch. The bilateral omental pouches were explanted on POD 124. For recipients of allogeneic islets (animals 2–5), diabetes was induced with streptozotocin. With the exception of animal 4 (2 scaffolds), the other monkeys were implanted with one scaffold; each scaffold was wrapped in an omental pouch, which was explanted on the indicated day. B. Animal 2 received 4,200 IEQ/kg onto one scaffold, which was explanted on POD 376. C. Animal 3 received 9,441 IEQ/kg onto one scaffold; the animal died during a technical procedure on POD 139. D. Animal 4 received 8,540 IEQ/kg onto 2 scaffolds, which were explanted on POD 223. E. Animal 5 received 10,291 IEQ/kg onto one scaffold, which was explanted on POD 145. All animals gradually became c-peptide positive, with reduction of EIR and varying degrees of improvement in glycemic control (upper panels). With the exception of animal 3 (died with functioning islets), all recipients became c-peptide negative after explant (lower panels).

Figure 1. Exogenous insulin requirements (EIR), fasting blood glucose (FBG) (upper panel) and c-peptide and rapamycin levels (lower panel)

A. Animal 1 was pancreatectomized on POD −2 and received 5,093 autologous IEQ/kg on POD 0 onto 2 scaffolds, each one wrapped in an omental pouch. The bilateral omental pouches were explanted on POD 124. For recipients of allogeneic islets (animals 2–5), diabetes was induced with streptozotocin. With the exception of animal 4 (2 scaffolds), the other monkeys were implanted with one scaffold; each scaffold was wrapped in an omental pouch, which was explanted on the indicated day. B. Animal 2 received 4,200 IEQ/kg onto one scaffold, which was explanted on POD 376. C. Animal 3 received 9,441 IEQ/kg onto one scaffold; the animal died during a technical procedure on POD 139. D. Animal 4 received 8,540 IEQ/kg onto 2 scaffolds, which were explanted on POD 223. E. Animal 5 received 10,291 IEQ/kg onto one scaffold, which was explanted on POD 145. All animals gradually became c-peptide positive, with reduction of EIR and varying degrees of improvement in glycemic control (upper panels). With the exception of animal 3 (died with functioning islets), all recipients became c-peptide negative after explant (lower panels).

Figure 1. Exogenous insulin requirements (EIR), fasting blood glucose (FBG) (upper panel) and c-peptide and rapamycin levels (lower panel)

A. Animal 1 was pancreatectomized on POD −2 and received 5,093 autologous IEQ/kg on POD 0 onto 2 scaffolds, each one wrapped in an omental pouch. The bilateral omental pouches were explanted on POD 124. For recipients of allogeneic islets (animals 2–5), diabetes was induced with streptozotocin. With the exception of animal 4 (2 scaffolds), the other monkeys were implanted with one scaffold; each scaffold was wrapped in an omental pouch, which was explanted on the indicated day. B. Animal 2 received 4,200 IEQ/kg onto one scaffold, which was explanted on POD 376. C. Animal 3 received 9,441 IEQ/kg onto one scaffold; the animal died during a technical procedure on POD 139. D. Animal 4 received 8,540 IEQ/kg onto 2 scaffolds, which were explanted on POD 223. E. Animal 5 received 10,291 IEQ/kg onto one scaffold, which was explanted on POD 145. All animals gradually became c-peptide positive, with reduction of EIR and varying degrees of improvement in glycemic control (upper panels). With the exception of animal 3 (died with functioning islets), all recipients became c-peptide negative after explant (lower panels).

Figure 1. Exogenous insulin requirements (EIR), fasting blood glucose (FBG) (upper panel) and c-peptide and rapamycin levels (lower panel)

A. Animal 1 was pancreatectomized on POD −2 and received 5,093 autologous IEQ/kg on POD 0 onto 2 scaffolds, each one wrapped in an omental pouch. The bilateral omental pouches were explanted on POD 124. For recipients of allogeneic islets (animals 2–5), diabetes was induced with streptozotocin. With the exception of animal 4 (2 scaffolds), the other monkeys were implanted with one scaffold; each scaffold was wrapped in an omental pouch, which was explanted on the indicated day. B. Animal 2 received 4,200 IEQ/kg onto one scaffold, which was explanted on POD 376. C. Animal 3 received 9,441 IEQ/kg onto one scaffold; the animal died during a technical procedure on POD 139. D. Animal 4 received 8,540 IEQ/kg onto 2 scaffolds, which were explanted on POD 223. E. Animal 5 received 10,291 IEQ/kg onto one scaffold, which was explanted on POD 145. All animals gradually became c-peptide positive, with reduction of EIR and varying degrees of improvement in glycemic control (upper panels). With the exception of animal 3 (died with functioning islets), all recipients became c-peptide negative after explant (lower panels).

Figure 2. Plasma glucose (left panel) and c-peptide (right panel) during an intravenous glucose tolerance test (IVGTT) at different times post-transplant

A: animal 1; B: animal 2; C: animal 3; D: animal 4 and E: animal 5.

Figure 2. Plasma glucose (left panel) and c-peptide (right panel) during an intravenous glucose tolerance test (IVGTT) at different times post-transplant

A: animal 1; B: animal 2; C: animal 3; D: animal 4 and E: animal 5.

Figure 3. Body weight in an autologous (inset, animal 1) and in four allogeneic transplanted animals

Closed circle: animal 2; open circle: animal 3; closed triangle: animal 4 and open triangle: animal 5.

Figure 4. A1C levels in an autologous (inset, animal 1) and in four allogeneic recipients

Closed circle: animal 2; open circle: animal 3; closed triangle: animal 4 and open triangle: animal 5.

Figure 5. Histopathological assessment of omental pouch explants from an autologous (A, animal 1, explanted on POD 124) and from an allogeneic islet recipient (B, animal 5, explanted on POD 145)

Confocal immunofluorescence showing expression of insulin (red) and CD4+, CD8+ or CD68+ T cells (green) at a magnification of 40X (bar indicates 200 μm), H&E staining at 50X and immunohistochemistry staining for insulin (brown, 50X).

Figure 6. Vascularization of transplanted islets

Confocal fluorescent images of transplanted islets. Insulin (red), blood vessels (combination of anti-CD31 and anti-CD34, green), nuclei (blue). White bar indicates 100 μm, insets are control staining with primary antibodies omitted. A: animal 1; B: animal 2; C: animal 3; D: animal 4; and E: animal 5.