Usage of Adenovirus Expressing Thymidine Kinase Mediated Hepatocellular Damage for Enabling Mouse Liver Repopulation with Allogenic or Xenogenic Hepatocytes

Abstract

It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2-/- γc-/- mice using an adenovirus encoding herpes virus thymidine kinase (AdTk) and two consecutive doses of ganciclovir (GCV). We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy

“…We then investigated whether cold-preserved hepatocytes in IGL-1 and HTS-FRS solutions could be used for cell transplantation. For this purpose, we used a simplified mouse model of liver injury (32) that relies on the rapid induction of liver damage in a temporally controlled manner following a single intraperitoneal injection of GCV at high dose in immunodeficient mice infected with AdTk. The experimental protocol is described in Figure 3A.…”

“…We used, with some minor modifications, a recently described mouse model in which, following drug-induced liver damage, the liver can be efficiently repopulated by xenogeneic hepatocytes (3,32). Male Balb/C Rag2 tm1Fwa Cd3g tm1Amk mice (4-6 weeks old, bred at CIMA, Pamplona, Spain) were intravenously inoculated with 5 × 10 9 PFU of recombinant adenoviruses expressing herpes simplex virus thymidine kinase (AdTk).…”

“…In these mice, liver damage is characterized by inflammation accompanied by increased hepatocyte size and enlarged nuclei and provides a growth advantage for transplanted human hepatocytes. The AdTk-GCV effect is maintained for 10 weeks and then is progressively resolved, as indicated by regenerative nodules (32). As soon as the human liver perfusion was planned, a single dose of 50 mg/kg ganciclovir (GCV; Roche) was intraperitoneally administered to induce subacute liver damage.…”

“…Freshly isolated hepatocytes were not available. As cultured human hepatocytes were previously shown to be a suitable source of cells for the generation of chimeric livers in this model (32), hepatocytes from the same donor were cultured in long-term medium for 7 days and were transplanted in a group of mice used as controls.…”

“…To our knowledge, the engraftment and repopulation of a damaged liver by cold-stored human hepatocytes has never been tested. We thus studied the impact of hypothermic preservation in three clinically safe (i.e., drug-agency approved) and animal product-free preservative solutions on the viability and in vitro functionality of human hepatocytes and also after transplantation in mice with liver damage (32). Hypothermic preservation slows down cell metabolism and leads to structural membrane damage and reduction of the ATP intracellular pool (16,30,46); therefore, different solutions have been elaborated to counteract these problems.…”