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In Vivo Pharmacological Selection Increases by 50-fold Transplanted Human Hepatocyte Survival in Immunocompetent Rats

Purpose: We have shown that transplanted human hepatocytes can survive in immunocompetant rats when hosts are rendered tolerant by intrafetal inoculation of those cells [Gastroenterol. 2006 128:1416-1423]. However, the fraction of human cells in this model did not exceed 1%. To increase the ratio of human to rat liver cells, human hepatocytes with a survival advantage over host cells were transplanted and hepatotoxic doses of a selection agent administered in situ. Methods: siRNAs were prepared to down-regulate CYP2E1 which converts acetaminophen (APAP) into toxic metabolites. However, GFP-Huh 7 cells without siRNA transfection had low levels of CYP2E1 RNA, and this conferred protection against APAP in cell culture compared to rat hepatocytes. Fetal rats were tolerized with GFP-Huh7 cells, and 5 x 106 GFP-Huh 7 cells were transplanted 5 days after birth by splenic injection. Rats were treated with APAP 1250 mg/kg (n=13) or normal saline (n=2) by intraperitoneal injection on day 19. ALT was measured 1 day prior and on days 1-7 after injection. BrdU was injected 1 day prior to sacrifice. Livers from treated and control rats were stained with anti-BrdU, anti-human albumin, DAPI, and examined by histology, and immunohistochemistry visualized by fluorescence microscopy. Results: As expected, ALT peaked 1-2 days after APAP injection with means of 180 U/l, while saline-injected controls had means of 75 U/l. See 'Human hepatocyte number, GFP positive cells in APAP-treated rats' table. On day 1, 2-3 GFP (+) cells were noted in each field. However, on day 7, 40-50 GFP (+) cells in clusters were seen in each field. There were no GFP (+) cells in the control non-APAP-treated rats. Human albumin was present in GFP (+) cells, and the number of dividing cells in APAP-treated rats was greatly increased compared to controls on day 7. Conclusions: Low CYP2E1 gene expression in Huh-7 cells provides an effective survival advantage in the presence of APAP in vitro. That difference between Huh-7 and host hepatocytes provides an efficient means for pharmacological selection resulting in >50-fold enrichment in rats. Down regulation of CYPs by siRNA may permit application of the selection to other liver cell lines. Transplanted Huh-7 cells respond to regenerative stimuli from APAP-induced liver damage with substantial proliferation in rats.

liver ; hepatocytes ; immunocompetant rats ; siRNAs ; GFP-Huh 7 cells ; CYP2E1 gene

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