Embryonic pig pancreatic tissue transplantation for the treatment of diabetes

Abstract

Background: Transplantation of embryonic pig pancreatic tissue as a source of insulin has been suggested for the cure of diabetes. However, previous limited clinical trials failed in their attempts to treat diabetic patients by transplantation of advanced gestational age porcine embryonic pancreas. In the present study we examined growth potential, functionality, and immunogenicity of pig embryonic pancreatic tissue harvested at different gestational ages.

Methods and findings: Implantation of embryonic pig pancreatic tissues of different gestational ages in SCID mice reveals that embryonic day 42 (E42) pig pancreas can enable a massive growth of pig islets for prolonged periods and restore normoglycemia in diabetic mice. Furthermore, both direct and indirect T cell rejection responses to the xenogeneic tissue demonstrated that E42 tissue, in comparison to E56 or later embryonic tissues, exhibits markedly reduced immunogenicity. Finally, fully immunocompetent diabetic mice grafted with the E42 pig pancreatic tissue and treated with an immunosuppression protocol comprising CTLA4-Ig and anti-CD40 ligand (anti-CD40L) attained normal blood glucose levels, eliminating the need for insulin.

Conclusions: These results emphasize the importance of selecting embryonic tissue of the correct gestational age for optimal growth and function and for reduced immunogenicity, and provide a proof of principle for the therapeutic potential of E42 embryonic pig pancreatic tissue transplantation in diabetes.

Figure 1. Pig Insulin Secretion by Embryonic Pancreatic Precursors of Different Gestational Ages Transplanted under the Kidney Capsule of NOD-SCID Mice in Response to Glucose Stimulation

Mice were tested 6 wk after transplantation, and pig insulin levels were measured before ( T ₀) and 30 min after ( T ₃₀) glucose challenge. Each dot represents one mouse. Groups of mice grafted with E24, E28, E42, E56, E80, and E100 donor pancreatic tissues included seven, 11, 16, 14, 13, and seven recipients, respectively.

Figure 2. Pig Insulin Secretion and Histological Appearance of Long-Standing Embryonic Pancreatic Grafts

(A) Pig insulin levels following transplantation of E28, E42, E56, and E80 pig pancreas tissues under the kidney capsule of NOD-SCID mice. The data are based on average ± standard deviation pig insulin level measured in seven independent experiments, each of which includes comparison among pig pancreatic precursors of two to three different gestational ages (*, p < 0.05; ***, p < 0.005; comparing E42 or E56 with E28 pig insulin levels). (B) E42 pig pancreatic grafts 5 mo after transplantation under the kidney capsule of NOD-SCID mice. Macroscopic appearance reveals a large viable graft that covers the kidney and contains abundant blood vessels (panel A); the graft is marked by an arrow. Histological analysis of the grafts demonstrates mainly dense islets of different sizes (panel B) (Hematoxylin and Eosin staining; islets marked by arrows). The ability of these islets to produce hormones is evident by positive staining for insulin (panel C), glucagon (panel D), and pancreatic polypeptide (panel E). Close proximity between islets and ducts is occasionally seen (panel C, magnified inset). The epithelial cells are widely stained for cytokeratin 20 (panel F).

Figure 3. Insulin Secretion following Transplantation of E42 and E56 Pig Embryonic Pancreatic Tissues in the “Humanized” SCID Mouse Model

Pig insulin levels were measured 4 wk after transplantation of E42 and E56 pig pancreas into NOD-SCID mice in the absence (black bars) and presence (grey bars) of 80 × 10 ⁶ adoptively transferred hu-PBMCs infused into the grafted mice 1–3 d after transplant. Four experiments were carried out, each comparing pig insulin levels secreted by the grafts from two gestational time points. The bars represent average ± standard deviation pig insulin levels measured in all experiments. The number of grafted NOD-SCID mice without and with hu-PBMCs, respectively, was as follows: 19 and 17 for E42, and 15 and 14 for E56.

Figure 4. Expression of Endocrine, Exocrine, and Proliferative Markers in Long-Standing E42 Pig Pancreatic Grafts

E42 pig pancreatic tissue was transplanted into NOD-SCID mice. Grafts were histologically analyzed at 3 (A), 6 (B), 8 (C), and 10 mo (D) after transplant. Endocrine expression is tracked by anti-insulin staining (blue), exocrine expression by anti-trypsin and anti-amylase staining (green), and proliferation status by anti-Ki67 staining (red). Cells coexpressing insulin and Ki67 are marked by arrows (A and B).

Figure 5. E42 Pig Pancreatic Tissue Normalizes Blood Glucose Levels in Diabetic Mice

(A) Glucose levels in durably grafted (E42 embryonic pancreas) and non-grafted alloxan-treated NOD-SCID mice. Grafted (blue lines) and non-grafted (red lines) NOD-SCID mice were injected with alloxan 4 mo after transplantation. All non-grafted mice died within 2–18 d. Grafted mice exhibiting pig insulin levels below 120 pmol/l prior to the alloxan treatment failed to control hyperglycemia (broken blue lines); however, grafted mice demonstrating pig insulin levels above 120 pmol/l before alloxan injection maintained their glucose levels within the normal range (unbroken blue lines). Removal of the left kidney bearing the pig pancreatic graft at 41 or 61 d after alloxan treatment caused irreversible hyperglycemia. (B) Functionality of E42 pig pancreatic grafts in alloxan-treated NOD-SCID mice. After a 10-h fast period, 3 g/kg glucose was administered intraperitoneally. Glucose (black line) and pig insulin (broken black line) were followed at different time points spanning 150 min. The data represent three experiments and include 15 alloxan-treated NOD-SCID mice grafted with E42 pig pancreas evaluated 4 mo after transplant. (C) Long-term follow-up of average glucose (black line) and pig insulin (broken black line) in streptozotocin-treated NOD-SCID mice grafted with E42 pig pancreas. Of 19 animals treated with streptozotocin, ten survived up to 14 wk following transplantation and eventually became independent of exogenous insulin. (D and E) Pig insulin is highly expressed in the alloxan- (D) and the streptozotocin-treated (E) NOD-SCID mice 4 mo after transplantation, as detected by specific staining of nephrectomized kidneys bearing the E42 pancreatic grafts.

Figure 6. Rejection of the E42 Pig Pancreas in Different Immunologically Mutant Mice

Grafts of E42 pig pancreas were transplanted under the kidney capsule of NOD-SCID (A), C57BL/6 (B), XID (C), and nude (D) mice and harvested 17 d after transplant. Note the extensive fibrosis and infiltration indicating rejection in the C57BL/6 and XID mice, while pancreatic components are seen in the NOD-SCID and nude mice. Each group included five mice.

Figure 7. Pig Insulin Secretion and Histological Findings of E42 Pig Embryonic Pancreas Transplanted in NOD-SCID and Immunocompetent Mice Treated with Costimulatory Blockade

(A) Pig insulin secretion by E42 pig pancreatic grafts implanted under the kidney capsule of NOD-SCID mice (black bars) or C57BL/6 mice treated every 2 wk with CTLA4-Ig and anti-CD40L (grey bars). No pig insulin could be detected in negative control transplanted C57BL/6 mice in the absence of immunosuppression, therefore these results are not shown. Each group included 13 mice. (B) Histological findings 3 mo following E42 pig pancreas transplantation under the kidney capsule of NOD-SCID mice (panel A), immunocompetent C57BL/6 mice (panel B), and C57BL/6 mice treated biweekly with CTAL4-Ig and anti-CD40L (panel C). Note the fierce rejection in C57BL/6 mice evident by implant destruction and fibrosis in (panel B), while intact graft development is demonstrated in C57BL/6 mice treated with CTLA4-Ig and anti-CD40L (panel C) revealing positive staining for insulin (panel D). A small number of mouse CD3 cells (panel E) and macrophages (panel F, stained by F4/80) infiltrated the graft parenchyma (marked by arrowheads) without causing apparent damage to the pancreatic structures.