Growth and Differentiation Potential of Human Hepatocytes

Chise Tateno¹ and Katsutoshi Yoshizato^{1, 2}

      Tissues and organ are considered to contain stem cells even in adults. However, studies have not been available that show the presence of hepatic stem cells in adults. We have demonstrated that small sized hepatocytes (17 m in diameter) isolated from rats by centrifugations and FACS have a high growth potential in vitro and in vivo. We also showed the presence of such highly proliferative small hepatocytes in adult humans. Small percentage (<0.1 %) of isolated human hepatocytes grew to form colonies, the colony-forming efficiency being decreased with the age of donors. Colony-forming hepatocytes could be subcultured 4 to 7 times, thereafter terminated their growth. They showed a liver epithelial cell-like morphology and expressed marker proteins of both hepatocytes and bile duct cells in a low-density culture. In contrast, the cells took on a typical mature hepatocyte-like morphology and expressed matured hepatocyte markers, but did not express bile duct cell markers in a high-density culture. These hepatocytes in spheroidal aggregates expressed a variety of cytchrome P450 mRNAs at a level as high as isolated donor hepatocytes. The results suggest that these proliferative small hepatocytes have liver progenitor cell-like characteristics.

      To test the growth potential of human small hepatocytes in vivo, we developed a method to yield humanized mice whose liver is replaced with human hepatocytes. Other investigators also developed such chimeric mice with respect to human hepatocytes. However, it has not been determined first whether we can produce a mouse whose liver is completely replaced with human hepatocytes and second whether the human heptocytes propagated in mice function in metabolizing chemicals and drugs in a manner identical to those in the human body. We transplanted human hepatocytes into urokinase plasminogen activator-transgenic SCID mice. Human hepatocytes progressively repopulated the host liver. However, the recipients eventually died when human albumin (hAb) in blood exceeded 3 mg/ml. The hosts (chimeric mice) survived beyond 3 mg/ml hAb when treated with an anti-complement drug, and developed livers with repopulation indexes of as high as 90%. Small hepatocytes showed a significantly higher initial repopulation rate than parenchymal hepatocytes. The liver expressed mRNAs of a variety of human cytochrome P450 (hCYPs) in a manner similar to the donor liver. hCYP3A4 mRNA was induced in the liver when the mice were treated with rifampicin. The chimeric mouse developed in the present study suffices to meet human hepatocyte-related medical and pharmacological needs.

¹Yoshizato Project, CLUSTER
²Developmental Biology Laboratory, Department of Biological Science, Graduate School of Science, Hiroshima University, Japan