Retroviral-mediated gene transfer in human hepatocytes.
BACKGROUND
The ability to modify human hepatocytes genetically is an essential first step in the development of liver-directed ex vivo gene therapy for inherited metabolic disease. The purpose of these studies was to prove that the genome of human hepatocytes can be altered successfully to express foreign genetic material.


METHODS
Human hepatocytes were plated at 2 or 4 x 10(6) cells/10 cm Primaria (Falcon, Oxnard, Calif.) plates. Fresh virus from the amphotropic viral producer cell line BAG, containing the Escherichia coli beta-galactosidase gene lacZ, was placed directly onto hepatocyte cultures and quantitative analysis of cells staining positive for the lacZ gene was undertaken. In a different human liver, a variety of viruses from producer cell lines containing clones of the human low-density lipoprotein (LDL) receptor were plated directly on cultures of human hepatocytes, and gene transfer was demonstrated by increased uptake of fluorescent-labeled LDL.


RESULTS
Beta-galactosidase production in hepatocytes was assayed histochemically with the chromogenic substrate X-gal. The highest percentage of cells staining positive for expression of enzyme was seen at 4 x 10(6) cells/plate (43.66% +/- 1.02% vs 27.99% +/- 2.31%). Gene transfer was also documented by the uptake of fluorescent-labeled LDL with a variety of different vectors containing the human LDL receptor.


CONCLUSIONS
(1) Human hepatocytes can be cultured in vitro and are susceptible to retroviral infection, (2) functional gene transfer is demonstrated by intracellular function of foreign genes, and (3) the level of expression appears dependent on plating density. We conclude that human hepatocytes are suitable targets for genetic manipulation and may play an important role in human gene therapy trials.
Raper
S E
SE
Department of Surgery, University of Michigan Medical School, Ann Arbor.
Wilson
J M
JM
Grossman
M
M
D002232:Carbocyanines; D002469:Cell Separation; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D006651:Histocytochemistry; D006801:Humans; D008077:Lipoproteins, LDL / Q000493:pharmacokinetics; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008875:Middle Aged; D012190:Retroviridae / Q000502:physiology*; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; C024286:3,3'-dioctadecylindocarbocyanine; D001616:beta-Galactosidase