Liver repopulation after cell transplantation in mice treated with retrorsine and carbon tetrachloride1

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Abstract
Efficiency of engraftment after liver cell transplantation is less than 1% under conventional conditions. Our aim was to develop a high-efficiency, nonsurgical, no-genetic-advantage mouse model of liver repopulation with transplanted cells. Mice were conditioned with nonlethal doses of a cell cycle inhibitor, retrorsine, 70 mg/kg, to irreversibly block proliferation of native hepatocytes. After the drug was eliminated, 2 million freshly isolated β-galactosidase–labeled liver cells were transplanted into the spleens of C57BL/6J recipient mice. To stimulate donor cell proliferation, three doses of carbon tetrachloride (CCl4), 0.5 ml/kg, were given. Several control groups were studied to evaluate the contribution of each treatment to liver repopulation. Repopulation, as measured by cell isolation from recipient livers 1–7 months after transplantation, was on average 20%. Repopulation was 10% if CCl4 was given only once, between 0.5% and 1% if only retrorsine or CCl4 were used, and 0.05% if no conditioning was used. Phenotypically, whole livers turned blue on exposure to X-gal staining, whereas negative (control) livers remained pale brown. More than 55% of liver repopulation resulted from clusters containing 21 or more cells, some of which contained more than 200 cells, suggesting seven or more rounds of cell division in a subset of transplanted cells. This murine study demonstrates high levels of repopulation after liver cell transplantation into nongenetically modified livers, using a cell cycle inhibitor and chemical liver injury to provide transplanted cells a proliferative advantage. Liver repop-ulation was effected mostly by a small fraction of transplanted cells. Analogous nonsurgical liver cell transplantation strategies, but with clinically applicable drugs, could be devised for the treatment of liver-based metabolic diseases.