Engraftment of insulin-producing cells from porcine islets in non-immune-suppressed rats or nonhuman primates transplanted previously with embryonic pig pancreas

Abstract

Transplantation therapy for diabetes is limited by unavailability of donor organs and outcomes complicated by immunosuppressive drug toxicity. Xenotransplantation is a strategy to overcome supply problems. Implantation of tissue obtained early during embryogenesis is a way to reduce transplant immunogenicity. Insulin-producing cells originating from embryonic pig pancreas obtained very early following pancreatic primordium formation (embryonic day 28 (E28)) engraft long-term in non-immune, suppressed diabetic rats or rhesus macaques. Morphologically, similar cells originating from adult porcine islets of Langerhans (islets) engraft in non-immune-suppressed rats or rhesus macaques previously transplanted with E28 pig pancreatic primordia. Our data are consistent with induction of tolerance to an endocrine cell component of porcine islets induced by previous transplantation of embryonic pig pancreas, a novel finding we designate organogenetic tolerance. The potential exists for its use to enable the use of pigs as islet cell donors for humans with no immune suppression requirement.

Figure 1

Sections of the islet-implanted kidney from a rat (a, b) or rhesus macaque (c–g) 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); or hybridized to an antisense (f) or sense (g) probe for porcine proinsulin mRNA. PT: proximal tubule. Arrows: positively staining cells (a, c, and e). Scale bars 10 um (a, b); 15 um (c, d), 7.5 um (e–g). Reproduced with permission from the American Society for Investigative Pathology [7] and from Organogenesis [8].

Figure 2

Fluorescence microscopy of tissue sections originating from (a) a normal porcine pancreas or (b) 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 (b). Scale bar 10 um. Reproduced with permission from Organogenesis [8].

Figure 3

RT-PCR: (a) shown left to right are DNA molecular weights (Mr); amplification of bands using primers specific for porcine proinsulin from 1 ug 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: kidney, heart, spleen, lung, a negative control for porcine-specific primers (no RNA); amplification of bands using primers specific for monkey proinsulin from 1 ug of pig pancreas RNA; monkey pancreas; 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 ug 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: kidneys, mesenteric lymph node (MLN) spleen, liver, a negative control for porcine-specific primers (no RNA); amplification of bands using primers specific for monkey proinsulin from 2 ug 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. Reproduced with permission from Organogenesis [8].

Figure 4

Levels of insulin (uU/mL) measured over 60 min in vitro after addition of glucose to tissue following time 0: (a) from a macaque kidney implanted with porcine islets following transplantation of E28 pig pancreatic primordia in mesentery; (b) the contralateral nonimplanted kidney. Reproduced with permission from Organogenesis [8].

Figure 5

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 um. Reproduced with permission from Organogenesis [8].