Engraftment of cells from porcine islets of Langerhans following transplantation of pig pancreatic primordia in non-immunosuppressed diabetic rhesus macaques

Abstract

Transplantation therapy for human diabetes is limited by the toxicity of immunosuppressive drugs. If toxicity can be minimized, there will still be a shortage of human donor organs. Xenotransplantation of porcine islets is a strategy to overcome supply problems. Xenotransplantation in mesentery of pig pancreatic primordia obtained very early during organogenesis [embryonic day 28 (E28)] is a way to obviate the need for immunosuppression in rats or rhesus macaques and to enable engraftment of a cell component originating from porcine islets implanted beneath the renal capsule of rats. Here, we show engraftment in the kidney of insulin and porcine proinsulin mRNA-expressing cells following implantation of porcine islets beneath the renal capsule of diabetic rhesus macaques transplanted previously with E28 pig pancreatic primordia in mesentery. Donor cell engraftment is confirmed using fluorescent in situ hybridization (FISH) for the porcine X chromosome and is supported by glucose-stimulated insulin release in vitro. Cells from islets do not engraft in the kidney without prior transplantation of E28 pig pancreatic primordia in mesentery. This is the first report of engraftment following transplantation of porcine islets in non-immunosuppressed, immune-competent non-human primates. The data are consistent with tolerance to a cell component of porcine islets induced by previous transplantation of E28 pig pancreatic primordia.

Figure 1

Photographs of mesentery from rhesus macaques. (A) Rhesus macaque transplanted with E28 pig pancreatic primordia in mesentery and implanted with porcine islets in one kidney. Arrow parallels lymphatic vessel (LN, lymph node). (B) Rhesus macaque implanted with porcine islets in one kidney without prior transplantation of E28 pig pancreatic primordia. Scale bar, 2 cm.

Figure 2

Sections of a mesenteric lymph node from a macaque transplanted with E28 pig pancreatic primordia in mesentery, followed by porcine islets in the renal subcapsular space stained using anti-insulin antibodies (A, C and E) or control antiserum (B, D and F) or hybridized to an antisense (G) or sense (H) probe for porcine proinsulin mRNA. Arrows, positively staining cells (A, C, E and G). Scale bars, 80 µm (A and B); 25 µm (C and D); 15 µm (E–H).

Figure 3

Photographs of kidneys from rhesus macaques. (A) From a macaque transplanted with E28 pig pancreatic primordia in mesentery and implanted with porcine islets in one kidney; A whitish well-demarcated triangle-shaped graft (arrows) is delineated. (B) A non-implanted kidney from a macaque transplanted with E28 pig pancreatic primordia in mesentery and implanted with porcine islets in the contralateral kidney; and (C) From a macaque implanted with porcine islets in the kidney with no prior transplantation of E28 pig pancreatic primordia. Scale bar, 0.8 cm.

Figure 4

Sections of the islet-implanted kidney from a macaque transplanted with E28 pig pancreatic primordia in mesentery followed by porcine islets in the renal subcapsular space stained using anti-insulin antibodies (A and C) or control antiserum (B) or hybridized to an anti-sense (D) or sense (E) probe for porcine proinsulin mRNA (PT, proximal tubule). Arrows, positively staining cells (A, C and D). Scale bars 15 µm (A and B); 7.5 µm (C–E).

Figure 5

Sections of the non-implanted kidney (A and B) from a macaque transplanted with E28 pig pancreatic primordia in mesentery, followed by porcine islets in the renal subcapsular space of the contralateral kidney, or the implanted kidney from a macaque implanted with porcine islets without prior transplantation of E28 pig pancreatic primordia (C) stained using anti-insulin antibodies (A and C) or control antiserum (B) Arrowheads, subcapsular space (A–C). Scale bar 15 µm.

Figure 6

Fluorescence microscopy of tissue sections originating from (A and C) a normal porcine pancreas or (B) a mesenteric lymph node from a rhesus macaque that had been transplanted with embryonic pig pancreas in mesentery and subsequently with porcine islets in kidney or (D) a subcapsular section from a kidney of a rhesus macaque transplanted with embryonic pig pancreas in mesentery and subsequently with porcine islets in that kidney (PT, proximal tubule). Arrows delineate pig X chromosomes. Arrowheads renal capsule (D). Scale bar 10 µm.

Figure 7

RT-PCR: (A) Shown left to right are DNA molecular weights (Mr); amplification of bands using primers specific for porcine proinsulin from 1 µg RNA extracted from pig pancreas; or from a rhesus macaque (monkey) transplanted with E28 pig pancreatic primordia in mesentery, followed by implantation of porcine islets in the renal subcapsular space (Diab-E28-Islets), kidney, heart, spleen, lung, a negative control for porcine-specific primers (no RNA); and amplification of bands using primers specific for monkey proinsulin from 1 µg of pig pancreas RNA, monkey pancreas and a second negative control for macaque-specific primers. (B) Shown left to right are DNA molecular weights (Mr); amplification of bands using primers specific for porcine proinsulin from 2 µg RNA extracted from pig pancreas; or from a rhesus macaque (monkey) implanted with porcine islets in the renal subcapsular space with no previous transplantation of E28 pig pancreatic primordia (Diab-Islets), kidneys, mesenteric lymph node (MLN) spleen, liver, a negative control for porcine-specific primers (no RNA); and amplification of bands using primers specific for monkey proinsulin from 2 µg of pig pancreas RNA, monkey pancreas and a second negative control for macaque-specific primers. Pig primers amplify a 193 bps band. Monkey primers amplify a 199 bps band.

Figure 8

Electron micrograph of rhesus macaque kidney following sequential transplantation of E28 pig pancreatic primordia in mesentery and implantation of porcine islets in the kidney. Arrows, endocrine granules. Scale bar a 2 µm.

Figure 9

Intravenous glucose tolerance in rhesus macaques. Glucose in peripheral venous blood was measured in (A) three macaques in the Diab-E28-Islets group and (B) three macaques in the Diab-Islets group before any transplantations prior to administration STZ (pre-STZ) and 5 days following administration of STZ (post-STZ). Glucose tolerance was measured in Diab-E28 Islets macaques 4 weeks post-transplantation of E28 pig pancreatic primordia (post-E28) and again 7 weeks after subsequent implantation of porcine islets (post-E28-I) and in Diab-Islets group macaques 7 weeks following implantation of porcine islets (post-islets). Data are shown as mean ± SE (three macaques).

Figure 10

Levels of insulin (µU/ml) measured over 60 min in DMEM-containing weight-matched tissue originating from (A) a kidney implanted with porcine islets (159 mg) and (B) the contralateral non-implanted kidney (158 mg) of the same macaque in the Diab-E28-Islets group. Immediately after measurements were made at time 0, glucose was added to DMEM so as to render the glucose concentration 20 mM. Data are representative of three determinations.