BACKGROUND: Maintenance of liver-specific functions has been shown to be stabilized by co-cultivation of hepatocytes with hepatic stellate cells (HSC). Because the limited lifespan of human HSC is a major hurdle to their use, the authors report here the amplification of human HSC populations in vitro by retroviral transfer of human telomerase reverse transcriptase (hTERT). METHODS: Human HSC strain LI 90 cells were transduced with a retroviral vector SSR#197 expressing hTERT and green fluorescent protein (GFP) cDNA flanked by a pair of loxP. TWNT-1, one of SSR#197-immortalized HSC, was characterized. Differentiated liver functions were evaluated in an immortalized human hepatocyte NKNT-3-TWNT-1 co-culture system. RESULTS: TWNT-1 cells showed differential functions of HSC, including uptake of acetylated low-density lipoproteins and synthesis of collagen type I and hepatocyte growth factor. Efficient excision of the retrovirally transferred hTERT and GFP cDNAs was achieved by TAT-mediated expression of the Cre recombinase and subsequent GFP-negative cell sorting. When co-cultured with TWNT-1 cells, NKNT-3 increased protein expression of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9 and urea synthesis. CONCLUSIONS: TWNT-1 cells could be valuable in the study of integrated liver functions and contribute to the optimization of liver cell therapies and bioartificial livers.
BACKGROUND: Maintenance of liver-specific functions has been shown to be stabilized by co-cultivation of hepatocytes with hepatic stellate cells (HSC). Because the limited lifespan of human HSC is a major hurdle to their use, the authors report here the amplification of human HSC populations in vitro by retroviral transfer of humantelomerase reverse transcriptase (hTERT). METHODS:Human HSC strain LI 90 cells were transduced with a retroviral vector SSR#197 expressing hTERT and green fluorescent protein (GFP) cDNA flanked by a pair of loxP. TWNT-1, one of SSR#197-immortalized HSC, was characterized. Differentiated liver functions were evaluated in an immortalized human hepatocyte NKNT-3-TWNT-1 co-culture system. RESULTS: TWNT-1 cells showed differential functions of HSC, including uptake of acetylated low-density lipoproteins and synthesis of collagen type I and hepatocyte growth factor. Efficient excision of the retrovirally transferred hTERT and GFP cDNAs was achieved by TAT-mediated expression of the Cre recombinase and subsequent GFP-negative cell sorting. When co-cultured with TWNT-1 cells, NKNT-3 increased protein expression of the detoxifying cytochrome P450-associated protein isoenzymes 3A4 and 2C9 and urea synthesis. CONCLUSIONS: TWNT-1 cells could be valuable in the study of integrated liver functions and contribute to the optimization of liver cell therapies and bioartificial livers.
Authors: Alice A Chen; David K Thomas; Luvena L Ong; Robert E Schwartz; Todd R Golub; Sangeeta N Bhatia Journal: Proc Natl Acad Sci U S A Date: 2011-07-11 Impact factor: 11.205
Authors: Rebecca Y Klinger; Juliana L Blum; Bevin Hearn; Benjamin Lebow; Laura E Niklason Journal: Proc Natl Acad Sci U S A Date: 2006-02-13 Impact factor: 11.205