TRANSPLANTATION ASSAY FOR LIVER REPOPULATING CELLS

Oregon Health And Science University, Portland OR

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Abstract

Tyrosinemia type I (HT) is caused by deficiency of fumarylacetoacetate hydrolase (FAH). Its main clinical features include progressive liver failure, renal tubular damage and liver cancer. Recently we have created a murine model of FAH deficiency by gene knock-out in embryonic stem cells. Affected animals display a complex phenotype of hepatic dysfunction and neonatal death. The drug NTBC corrects liver dysfunction and neonatal lethality and withdrawal of this medication at a later age gives rise to a phenotype similar to human HT 1. FAH deficiency induces progressive hepatocellular damage and we have recently shown that metabolically corrected hepatocytes have a strong selective growth advantage over deficient cells. Because of this in vivo selection, transplantation of wild-type hepatocytes can lead to a complete repopulation of FAH deficient liver. This repopulation is clonal and nodular and can be affected by as few as 1,000 donor cells. We will use this repopulation assay to determine the nature of the repopulating cells (number, size, differentiation phenotype) in mouse liver. The effects of liver injury and developmental stages (fetal, newborn, adult) on the repopulating cells will be determined. FAH deficient hepatocytes, which have been transduced with a FAH expressing retrovirus have the same selective advantage as transplanted wild-type cells. We will use retroviral marking to trace the lineage and regenerative potential of infected liver cells. By crossbreeding to SCID mice, we will also create an immune deficient FAH mutant mouse. These animals will be used to develop an analogous liver repopulation assay for xenogeneic donor hepatocytes from rat, primates and humans.