ESTABLISHMENT OF IMMORTALIZED HUMAN HEPATIC STELLATE SCAVENGER CELLS TO DEVELOP 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 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

Optimized Lentiviral Transduction of Mouse Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have attracted much attention as potential platforms for transgene delivery and cell-based therapy for human disease. MSCs have the capability to self-renew and retain multipotency after extensive expansion in vitro, making them attractive targets for ex vivo modification and autologous transplantation. Viral vectors, including lentiviral vectors, provide an efficient means for transgene delivery into human MSCs. In contrast, mouse MSCs have proven more difficult to transduce with lentiviral vectors than their human counterparts, and because many studies use mouse models of human disease, an improved method of transduction would facilitate studies using ex vivo-modified mouse MSCs. We have worked toward improving the production of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors and optimizing transduction conditions for mouse MSCs using lentivirus vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G), the ecotropic murine leukemia virus envelope glycoprotein (MLV-E), and the glycoproteins derived from the Armstrong and WE strains of lymphocytic choriomeningitis virus (LCMV-Arm, LCMV-WE). Mouse MSCs were readily transduced following overnight incubation using a multiplicity of infection of at least 40. Alternatively, mouse MSCs in suspension were readily transduced after a 1-h exposure to lentiviral pseudotypes immediately following trypsin treatment or retrieval from storage in liquid nitrogen. LCMV-WE pseudotypes resulted in efficient transduction of mouse MSCs with less toxicity than VSV-G pseudotypes. In conclusion, our improved production and transduction conditions for lentiviral vectors resulted in efficient transduction of mouse MSCs, and these improvements should facilitate the application of such cells in the context of mouse models of human disease