The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that intravenous injection of adult bone marrow cells in the FAH(-/-) mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.

Evidence suggests that haematopoietic stem cells might have unexpected developmental plasticity, highlighting therapeutic potential. For example, bone-marrow-derived hepatocytes can repopulate the liver of mice with fumarylacetoacetate hydrolase deficiency and correct their liver disease. To determine the underlying mechanism in this murine model, we performed serial transplantation of bone-marrow-derived hepatocytes. Here we show by Southern blot analysis that the repopulating hepatocytes in the liver were heterozygous for alleles unique to the donor marrow, in contrast to the original homozygous donor cells. Furthermore, cytogenetic analysis of hepatocytes transplanted from female donor mice into male recipients demonstrated 80,XXXY (diploid to diploid fusion) and 120,XXXXYY (diploid to tetraploid fusion) karyotypes, indicative of fusion between donor and host cells. We conclude that hepatocytes derived form bone marrow arise from cell fusion and not by differentiation of haematopoietic stem cells.

Mice that could be highly repopulated with human hepatocytes would have many potential uses in drug development and research applications. The best available model of liver humanization, the uroplasminogen-activator transgenic model, has major practical limitations. To provide a broadly useful hepatic xenorepopulation system, we generated severely immunodeficient, fumarylacetoacetate hydrolase (Fah)-deficient mice. After pretreatment with a urokinaseexpressing adenovirus, these animals could be highly engrafted (up to 90%) with human hepatocytes from multiple sources, including liver biopsies. Furthermore, human cells could be serially transplanted from primary donors and repopulate the liver for at least four sequential rounds. The expanded cells displayed typical human drug metabolism. This system provides a robust platform to produce high-quality human hepatocytes for tissue culture. It may also be useful for testing the toxicity of drug metabolites and for evaluating pathogens dependent on human liver cells for replication.The liver is the principal site for the metabolism of xenobiotic compounds, including medical drugs. Because many hepatic enzymes are species specific, it is necessary to evaluate the metabolism of candidate pharmaceuticals using cultured primary human hepatocytes 1,2 . Today, hepatocytes are isolated primarily from cadaveric organs and then shipped to the location where testing will be performed. The condition (viability and state of differentiation) of hepatocytes from cadaveric sources is highly variable, and many cell preparations are of marginal quality. The availability of high-quality human hepatocytes is further hampered by the fact that they cannot be substantially expanded in tissue culture 3,4 . Hepatocytes from readily available mammalian species, such as the mouse, are not suitable for drug testing because they have a different complement of metabolic enzymes and

Current strategies for hepatic gene therapy are either quantitatively inefficient or suffer from lack of permanent gene expression. We have utilized an animal model of hereditary tyrosinaemia type I (HT1), a recessive liver disease caused by deficiency of fumarylacetoacetate hydrolase (FAH), to determine whether in vivo selection of corrected hepatocytes could improve the efficiency of liver gene transfer. As few as 1,000 transplanted wild-type hepatocytes were able to repopulate mutant liver, demonstrating their strong competitive growth advantage. Mutant hepatocytes corrected in situ by retroviral gene transfer were also positively selected. In mutant animals treated by multiple retrovirus injections >90% of hepatocytes became FAH positive and liver function was restored to normal. Our results demonstrate that in vivo selection is a useful strategy for hepatic gene therapy and may lead to effective treatment of human HT1 by retroviral gene transfer.

The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor͞stem cells. To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and a method for their isolation was developed. Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver repopulation. In competitive repopulation experiments, hepatic oval cells were at least as efficient as mature hepatocytes in repopulating the liver. In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver nonparenchymal cells. This finding supports a model in which intrahepatic progenitors differentiate into hepatocytes irreversibly. To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for 8 months and oval cells were then serially transferred into Fah mutants. The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor. We conclude that hepatic oval cells do not originate in bone marrow but in the liver itself, and that they have valuable properties for therapeutic liver repopulation. In mammals, hepatic cells, especially hepatocytes can rapidly proliferate after acute liver injury to repair liver damage (1). Usually, undifferentiated liver progenitors do not take part in such acute liver regeneration. However, hepatocyte progenitors are still required in some chronic injury responses, especially when the ability of differentiated hepatocytes to divide is impaired (reviewed in refs. 2 and 3). The existence of endogenous stem cells inside mammalian liver was first suggested by the observation that treatment with carcinogens results in the emergence of oval cells in the portal region of the hepatic lobule (4-9). Oval cells are Ϸ10 m in size, have a high nuclear͞ cytoplasmic ratio, and express markers of immature liver cells such as ␣-fetoprotein (AFP) (10, 11). In addition, oval cells express markers of both the biliary epithelium (CK19) and hepatocyte lineages (albumin). In vitro, these cells can be differentiated into both of these hepatic epithelial lineages under appropriate conditions.Although the anatomical location of the oval cell proliferation suggests the existence of a hepatocyte progenitor in the Canal of Hering (12), the precise origin of oval cells remains uncertain. Most studies imply the existence of an undifferentiated oval cell precursor in this location that proliferates and gives rise to oval cells (13)(14)(15)(16). In analogy to the hematopoietic system, oval cells would then represent a committed bipotential progenitor. Recent studies in both rat and human have suggested that oval cells could be derived from precursors in the bone marrow (17). Others, however, have postulated that oval cells may arise by dedifferentiation of hepatocytes,...