Detection of donor cells without transmission of porcine C-type endogenous retrovirus (PERV) in host tissues following pig proislet xenotransplantation in fetal lambs and NOD-scid mice
Introduction In view of in vitro transmission of PERV from pig cell lines to cultured human cells, we set out to ascertain whether PERV transmission can occur in vivo following xenotransplantation of fetal pig proislets to recipient fetal lambs and NOD-scid mice. These models provide optimal conditions for retroviral infection, i.e. rapidly dividing host cells (fetal lamb) and no immunosuppression (both).
Materials and methods Fetal lambs in utero (56 days) were thymectomized and pig proislets were grafted to the thymic space. At 5-84 days post-transplant, host liver, spleen, lung, pancreas, heart, brain and kidney were harvested and frozen in liquid nitrogen for DNA and RNA extraction. Controls included untransplanted lamb tissues, pig proislets and PK15 cell line. For transplantation to NOD-scid mice, pig proislets were placed beneath the kidney capsule; xenografts, host liver and spleen were harvested at 96 days. PCR and RT-PCR using PERV A-, B- and C-specific or PERV ABC-common primers were used to identify PERV env sequences in extracted DNA and RNA (cDNA). Nested PCR using primers for pig cytochrome oxidase (CO II) was used to identify pig cells that had migrated to host tissues (microchimerism).
Results PERV sequences were clearly detected in DNA and cDNA of pig proislets but not of fetal lamb liver, spleen, pancreas, heart, lung, kidney and brain; COII sequences were detected in spleen DNA samples of transplanted fetal lambs at 7 and 23 days. In NOD-scid recipient mice, PERV env DNA and cDNA were identified in 3/3 long-term pig proislet xenografts but not in host tissues. COII sequences were detected in liver and spleen samples of 1/3 and 2/3 transplanted mice, respectively.
Conclusion Pig proislets carry and express PERV sequences. Following xenotransplantation of pig proislets to fetal lambs and NOD-scid mice, there was no clear evidence for (i) the transmission of PERV to host tissues and (ii) PERV integration into host genomes, despite long-term graft survival and intragraft PERV expression in mouse recipients and despite evidence for micro-chimerism in both recipient lamb and mouse tissues.