Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a ␤ cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.immune-suppression ͉ rejection ͉ xeno-transplantation

BackgroundTransplantation 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 FindingsImplantation 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.ConclusionsThese 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.

OBJECTIVEDefining an optimal costimulatory blockade–based immune suppression protocol enabling engraftment and functional development of E42 pig embryonic pancreatic tissue in mice.RESEARCH DESIGN AND METHODSConsidering that anti-CD40L was found to be thrombotic in humans, we sought to test alternative costimulatory blockade agents already in clinical use, including CTLA4-Ig, anti-LFA1, and anti-CD48. These agents were tested in conjunction with T-cell debulking by anti-CD4 and anti-CD8 antibodies or with conventional immunosuppressive drugs. Engraftment and functional development of E42 pig pancreatic tissue was monitored by immunohistology and by measuring pig insulin blood levels.RESULTSFetal pig pancreatic tissue harvested at E42, or even as early as at E28, was fiercely rejected in C57BL/6 mice and in Lewis rats. A novel immune suppression comprising anti-LFA1, anti-CD48, and FTY720 afforded optimal growth and functional development. Cessation of treatment with anti-LFA1 and anti-CD48 at 3 months posttransplant did not lead to graft rejection, and graft maintenance could be achieved for >8 months with twice-weekly low-dose FTY720 treatment. These grafts exhibited normal morphology and were functional, as revealed by the high pig insulin blood levels in the transplanted mice and by the ability of the recipients to resist alloxan induced diabetes.CONCLUSIONSThis novel protocol, comprising agents that simulate those approved for clinical use, offer an attractive approach for embryonic xenogeneic transplantation. Further studies in nonhuman primates are warranted.

The use of germ-free (GF) isolators for microbiome-related research is exponentially increasing, yet limited by its cost, isolator size and potential for trans-contamination. As such, current isolator technology is highly limiting to researchers engaged in short period experiments involving multiple mouse strains and employing a variety of mono-inoculated microorganisms. In this study, we evaluate the use of positive pressure Isocages as a solution for short period studies (days to 2-3 weeks) of experimentation with GF mice at multiple simultaneous conditions. We demonstrate that this new Isocage technology is cost-effective and room-sparing, and enables maintenance of multiple simultaneous groups of GF mice. Using this technology, transferring GF mice from isolators to Isocage racks for experimentation, where they are kept under fully germ-free conditions, enables parallel inoculation with different bacterial strains and simultaneous experimentation with multiple research conditions. Altogether, the new GF Isocage technology enables the expansion of GF capabilities in a safe and cost-effective manner that can facilitate the growth, elaboration and flexibility of microbiome research.

Cell therapy as an alternative to orthotopic liver transplantation represents a major challenge, since negligible proliferation of isolated hepatocytes occurs after transplantation because of the stringent homeostatic control displayed by the host liver. Thus, different modalities of liver injury as part of the pretransplant conditioning are a prerequisite for this approach. The major objective of the present study was to test whether xenotransplantation of pig fetal liver fragments, in which potential cell-cell and cell-stroma interactions are spared, might afford more robust growth and proliferation compared with isolated pig fetal hepatoblasts. After transplantation into SCID mice, fetal liver tissue fragments exhibited marked growth and proliferation, in the setting of a quiescent host liver, compared with isolated fetal hepatoblasts harvested at the same gestational age (embryonic day 28). The proliferative advantage of fetal pig liver fragments was clearly demonstrated by immunohistochemical and morphometric assays and was observed not only after implantation into the liver but also into extrahepatic sites, such as the spleen and the subrenal capsule. The presence of all types of nonparenchymal liver cells that is crucial for normal liver development and regeneration was demonstrated in the implants. Preservation of the three-dimensional structure in pig fetal liver fragments enables autonomous proliferation of transplanted hepatic cells in the setting of a quiescent host liver, without any requirement for liver injury in the pretransplant conditioning. The marked proliferation and functional maturation exhibited by the pig fetal liver fragments suggests that it could afford a preferable source for transplantation. STEM CELLS 2008;