Pharmacogenetic selection of transplanted human hepatocytes in immunocompetent rats

Abstract

To introduce a genetic survival advantage for transplanted human hepatocytes over host cells in rats. Green fluorescent protein (GFP) was introduced into Huh-7 human hepatoma cells to create fluorescent GFP-Huh-7 cells. mRNA of CYP2E1, the enzyme that converts acetaminophen (APA) into hepatotoxic intermediates, was quantified by real-time polymerase chain reaction (PCR). The effects of APA on GFP-Huh-7 and control Huh-7 cells were determined in a cell culture. Immunological tolerance was induced by the injection of GFP-Huh-7 cells into fetal rats in utero. The GFP-Huh-7 cells were transplanted after birth of the rats into tolerant rats followed by APA treatment. Serum alanine aminotransferase (ALT) levels and liver histological data were obtained. GFP-Huh-7 cells were detected by quantitive PCR and microscopy. CYP2E1 mRNA levels in the GFP-Huh-7 cells were 2.7% of parental Huh-7 cells. In 1 mmol/L APA, parental Huh-7 cells decreased by 60% while GFP-Huh-7 cells increased to within 95% of untreated controls after 5 days. In rats in which GFP-Huh-7 cells were transplanted and treated with APA, serum ALT increased to a peak of 200 U/L on day 1 and returned to normal levels by day 3. Fluorescence microscopy of liver specimens from rats transplanted with GFP-Huh-7 cells showed substantial increases in GFP-Huh-7, but not Huh-7 cells by day 7 after APA treatment. Real-time PCR confirmed a 10-fold increase of GFP mRNA in APA-treated rats, but not in those without APA treatment. The difference in CYP2E1 gene expression between GFP-Huh-7 and rat hepatocytes provides a convenient means for the enrichment of transplanted human cells in rat liver.