Engraftment of cells from porcine islets of Langerhans and normalization of glucose tolerance following transplantation of pig pancreatic primordia in nonimmune-suppressed diabetic rats

Abstract

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. However, even if toxicity can be minimalized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets may be a strategy to overcome these supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] can obviate the need for immune suppression in rats or rhesus macaques. Here, in rats transplanted previously with E28 pig pancreatic primordia in the mesentery, we show normalization of glucose tolerance in nonimmune-suppressed streptozotocin-diabetic LEW rats and insulin and porcine proinsulin mRNA-expressing cell engraftment in the kidney following implantation of porcine islets beneath the renal capsule. Donor cell engraftment was confirmed using fluorescent in situ hybridization for the porcine X chromosome and electron microscopy. In contrast, cells from islets did not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in the mesentery. This is the first report of prolonged engraftment and sustained normalization of glucose tolerance following transplantation of porcine islets in nonimmune-suppressed, immune-competent rodents. The data are consistent with tolerance induction to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.

Figure 1

Levels of blood glucose (A, C, and E) and rat insulin (B) or porcine insulin (D and F) before (time 0) and following i.v. glucose administration to nondiabetic rats (A and B) or STZ-diabetic rats implanted with porcine islets (C and D) or STZ-diabetic rats transplanted with E28 pig pancreatic primordia and subsequently with porcine islets (E and F). Data are mean ± SE of n = 4 rats in each group.

Figure 2

Photomicrographs of mesenteric lymph node (A–F) from a STZ-diabetic rat into which E28 pig pancreatic primordia had been transplanted in the mesentery four weeks previously (A–D) or a nontransplanted nondiabetic rat (E and F) or a pancreas from a nondiabetic nontransplanted rat (G and H) stained using anti-insulin antibody (A, C, E, and G) or control serum (B, D, F, and H). Arrows delineate tissue that stains positive for insulin (red-brown) (A and C) or negative staining tissue (B). Arrowheads delineate islet of Langerhans (G and H). Scale bars: 80 μm (A and B); 30 μm (C–F); 100 μm (G and H).

Figure 3

Photomicrographs of mesenteric lymph node from a formerly diabetic rat into which E28 pig pancreatic primordia and pig islets had been transplanted in the mesentery and kidney respectively stained using anti-insulin antibody (A and C) or control antibody (B and D) and sections hybridized to antisense (E) or sense (F) porcine proinsulin mRNA probes. Arrows delineate tissue that stains positive for insulin (red-brown) (A and C) or negative staining tissue (B) or positive staining for porcine proinsulin mRNA (E). Scale bars: 80 μm (A and B) and 25 μm (C–F).

Figure 4

Photograph of kidney from a STZ-treated rat transplanted previously with embryonic pig pancreas in mesentery. The photo was taken four weeks after implantation of pig islets in kidney. The whitish well-demarcated graft (white arrow) and the origin of a venous blood vessel (black arrow) are shown.

Figure 5

Photomicrographs of kidney from a diabetic rat into which embryonic pig pancreas had been transplanted in mesentery and pig islets had been transplanted subsequently in kidney stained using anti-insulin antibody (A and C) or control antibody (B and D) and sections hybridized to antisense (E) or sense (F) porcine proinsulin mRNA probes. Arrowheads delineate an expanded subcapsular space (A and B). Arrows delineate tissue in the subcapsular space that stains positive for insulin (red-brown) (C) or positive staining for porcine proinsulin mRNA (E). PT, A and B. Scale bars 80 μm (A and B) and 10 μm (C–F).

Figure 6

Photomicrographs of the contralateral kidney from a diabetic rat into which embryonic pig pancreas had been transplanted in the mesentery and pig islets had been implanted subsequently in the other kidney (A–D) or of a kidney from a diabetic rat in which pig islets had been implanted without prior transplantation of E28 pig pancreatic primordia (E and F) stained using anti-insulin antibody (A, C, and E) or control antiserum (B, D, and F). Arrowheads delineate a normal sized subcapsular space (A, C) or expanded subcapsular space (E). PT, A, B, E, and F. M, medulla (A and B). Scale bars: 100 μm (A and B), 10 μm (C and D), or 40 μm (E and F).

Figure 7

Fluorescence microscopy of tissue sections originating from a normal porcine pancreas (A) or a diabetic rat that had been transplanted with embryonic pig pancreas (B–D) in mesentery and subsequently with porcine islets in kidney: B, a subcapsular section from kidney; T, tubule, RC, renal capsule; C, a section of mesenteric lymph node, GC, germinal center, inset enlargement; and D, renal cortex, T, tubule. Arrows (A–C) delineate pig X chromosomes. Scale bar: 10 μm (D).

Figure 8

Electron micrographs of rat kidney following sequential transplantation of E28 pig pancreatic primordia in mesentery and implantation of porcine islets in kidney. A, Subcapsular space. T, renal tubule. Cell containing granules with a crystalline core surrounded by a clear space is delineated by an arrow; macrophages are delineated by arrowheads. B, Enlargement of macrophages. C, Enlargement of granules with a crystalline core surrounded by a clear space. Scale bar: 5 μm (A).

Figure 9

Levels of fasting glucose measured over time in STZ diabetic rats transplanted with E28 pig pancreatic primordia (E28) after measurements on week 1 and implanted with porcine islets (islets) after measurements on week 8. Data are mean ± SE of n = 4 rats (weeks 1–12) or n = 2 rats (weeks 16–28).

Figure 10

Photomicrographs of kidney from a diabetic rat into which embryonic pig pancreas had been transplanted in mesentery and pig islets had been transplanted subsequently in kidney stained using anti-insulin antibody (A) or control antibody (B). Tissue was obtained 20 weeks after islet transplantation. Arrows delineate cells in the subcapsular space with β cell morphology. RC, renal capsule; PT, A and B. Scale bar: 10 μm (A).