Transgenic Pig Expressing Red Fluorescent Protein, Kusabila-Orange as a Novel Tool for Preclinical Research of Hepatocyte Transplantation

Abstract

Hepatocyte transplantation is hoped to be an alternative or a supplementary treatment of liver transplantation. To approach clinical trial, we have to establish the confidence in mechanism and safety by experiment with larger-size animals comparable to human, such as pigs. This study shows the usefulness of transgenic pigs expressing red fluorescence to investigate the distribution, survival, tumorgenecity, etc. of transplanted cells. Materials and methods: Donor hepatocytes were isolated from transgenic Kusabira-Orange expressing pigs (41 days old, weighing 10kg) born by in vitro fertilization with sperms of transgenic-cloned Kusabira-Orange pig originally established by Matsunari et al (Cloning and Stem Cells 10;313, 2008) and ova of domestic pig. Recipients were non-transgenic Kusabira-Orange negative littermates born by the method described above. Under enflurane anesthesia, recipient pigs received a minimal abdominal midline incision and a double lumen cannula was inserted into supramesenteric vein to hepatic portal region. Kusabira-Orange positive donor hepatocytes were isolated by collagenase perfusion from the transgenic pig and parenchymal hepatocytes were obtained by low speed centrifugation. Hepatocytes (1×109 suspended in 100mL of saline containing 5unit/mL heparin) were transplanted though the cannula. To estimate allogeneic immnogenesity, full thickness skin (3×3cm) from the same donor was grafted orthotopically on the neck region of a non-transgenic recipient pig. No immunosuppressive treatment was done. Recipients were sacrificed 7 and 39 days after transplantation and harvested blood and organs such as lung, heart, liver, pancreas, kidney for examination. Results and discussion: Strong red fluorescence was detected in both parenchymal and non-parenchymal hepatocytes of transgenic donor pig by fluorescent microscope. Transplanted cells were detectable in the recipient liver and lung 7 days after, while skin graft was rejected within 7 days. The hepatocytes remained in recipient liver and lung on day 39 with slight reduction in number compared to day 7. Slightly more cells were remained in the liver. Although skin was rejected acutely, hepatocytes remained as long as for 39 days. As for antigenecity, Kusabira-Orange is xenogenic and somatic cells allogeneic between present donor recipient combination. Because the former is intracellular protein, the xenogenic antigenecity may not accelerate the normal allogeneic rejection. Immunosuppressive treatment will induce longer survival. One major problem in clinical application of hepatocyte transplantation is the low survival rate of donor cells. Quick disappearance of the cells is proposed to be due to non-specific inflammatory response or instant blood-mediated inflammatory reaction (IBMIR) but precise mechanisms still remain to be clarified. The transgenic pig presented here will be a helpful and conclusive research tool to overcome the deficit and to improve the procedure of cell transplantation. In addition, identical clones of non-transgenic and transgenic littermates obtained by nuclear transfer methodology will create a more simple research model to focus on the IBMIR research. Conclusion: Transgenic pigs expressing fluorescent protein, Kusabira-Orange provide a useful model of cell transplantation preclinical studies.