The self-renewal capacity of the liver is well established in models of hepatic resection, injury and carcinogenesis. Although proliferation of mature hepatocytes alone restores the liver after partial hepatectomy and hepatocytes can repopulate the liver repeatedly, under appropriate situations poorly differentiated 'oval cells' arise in the liver and can generate hepatic lineages. Although stem/progenitor cells from adult organs, for example, the pancreas and bone marrow, may also generate mature hepatocytes, such cells are rare in the adult liver. The engraftment of transplanted cells in the liver drew significant interest from the field of cell therapy. Transplanted cells regulate gene expression physiologically and can proliferate in the liver. However, despite the early promise of cell therapy, the scarcity of donor human livers, the absence of proliferation in cultured hepatocytes and the poor viability of hepatocytes after cryopreservation impose restrictions. It is possible that use of fetal cells will overcome these problems and help advance cell and gene therapy. Fetal liver cells are largely diploid, whereas maturing hepatocytes exhibit increasing polyploidy, which attenuates cell proliferation and eventually produces cell senescence. Progenitor cells from the fetal rat liver can generate both hepatocytes and bile duct cells in animals. These considerations suggested that the fetal human liver could be an appropriate source of stem/progenitor cells. The human liver arises from the foregut endoderm after four weeks of gestation and develops rapidly, such that bile is produced by 14 weeks. During this gestational period, hepatic cells express hepatocyte markers, for example, albumin, alphafetoprotein (AFP), a-1 microglobulin, glycogen, glucose-6-phosphatase (G-6-P) and Hep-Par-1, and biliary markers, for example, gamma glutamyl transpeptidase (GGT), dipeptidyl peptidase IV (DPPIV), cytokeratin (CK)-19 and Das-1-monoclonal antibody-reactive antigen. Human hepatoblasts express these markers throughout the second trimester (20-24 weeks), despite significant development of the fetal liver, which offered opportunities to isolate and study large numbers of progenitor cells.

The scientific findings indicate that large numbers of highly viable progenitor epithelial cells can be isolated from fetal human liver. These cells exhibit unique properties, including the capacity to replicate extensively, to express genes observed in hepatoblasts and oval cells, including AFP, GGT, CK-8, CK-19, CD34 and PAI-1, and to produce hepatocytes in immunodeficient mice. Taken together, this evidence of extensive replication capacity, presence of oval cell markers and coexpression of biliary and hepatic lineage markers indicate that it is possible to designate fetal cells as possessing progenitor phenotype. These cells show significant clonogenic capacity, which indicates that single-cell-derived colonies can possibly be expanded for further analysis. It is noteworthy that the normal adult liver, which contains replicatively quiescent cells, is devoid of telomerase activity. By contrast, fetal cells express telomerase, which could be beneficial for continued cell division. It was noteworthy that after ~50 population doublings, fetal cells showed decreasing proliferation. By contrast, mature hepatocytes are difficult to maintain and expand in cell culture. Thought when grown these cells do not require hepatic growth factors (except those present in fetal bovine serum), lipids and extracellular matrix components, which are required for culturing mature hepatocytes. Also, unlike murine embryonic liver cells, human fetal progenitor cells survived and proliferated without requiring feeder cells. Specific manipulations, including the release of cells with low trypsin/EDTA concentrations, were aimed at limiting cell membrane injury and selective removal of loosely adherent cells. It is noteworthy that hydrocortisone inhibits proliferation of fibroblasts and erythroid/ granulocyte-macrophage hematopoietic progenitor cells, whereas insulin promotes hepatocyte attachment; and this is true with fetal cells. Although mature hepatocytes dedifferentiate in culture with rapid loss of tissue specific genes, such as albumin, ASGR, etc., fetal epithelial cells expressed liver genes despite extensive culture. Moreover, these cells correctly regulated HBV enhancer/promoter, which requires the presence of multiple hepatic transcription factors. Furthermore, the mitogenic responsiveness of these progenitor cells to TGFa and EGF was in agreement with oval cell responses, as shown previously with F344 rat derived cells. Although indefinite cell replication has been induced in somatic cells by expressing the SV40 T antigen or the catalytic subunit of telomerase, it is unresolved whether genetic transformation will induce greater susceptibility for cancer. By contrast, these progenitor cells were genetically unperturbed despite more than 40 to 50 population doublings over 16-18 subpassages. Such proliferation capacity of cells indicates that cells isolated from a single fetal liver could potentially generate billions or even trillions of cells; whereas only 1-10 billion hepatocytes are required for treating an adult person and proportionately fewer cells will be necessary for treating a child. Therefore, expansion of progenitor cells in culture will facilitate novel clinical applications and help alleviate organ shortages. If highly efficient permanent gene transfer, such as those using lentiviral or retroviral vectors, were combined with effective strategies to repopulate the liver extensively, ex vivo liver gene therapy will once again become attractive. In this context, integration and differentiation of fetal liver cells in the parenchyma of the mouse liver indicate that use of such cells will be appropriate for liver repopulation. Scientific data indicate that significant proportions of transplanted cells were lost in mice shortly after transplantation. These findings were not surprising because a large fraction of transplanted cells sequestered in portal vein radicles and hepatic sinusoids undergoes phagocytotic clearance, even in syngeneic recipients. These cell losses constitute removal of approximately 70-80% of all transplanted cells. However, it is unclear at present whether human cells are at a survival disadvantage in the mouse liver compared with rodent cells. Interspecies differences in growth factors, extracellular matrix components, cell-cell interactions or other factors, could potentially regulate survival of human hepatocytes in the mouse liver. Of course, these findings do not exclude the possibility that fetal cells will show far superior engraftment in the human liver. Nonetheless, it will be of great interest to establish how engraftment of human cells in the mouse liver may be improved, because this will be relevant for developing novel models of human disease, as well as establishing reproducible bioassays to test the properties of human hepatocytes before use in cell or gene therapy. In addition, under suitable situations, transplanted hepatocytes proliferate significantly in rodents, and the mouse liver can be repopulated virtually completely with transplanted cells. In this respect, proliferation of fetal cells in the mouse liver following CCl4-induced hepatotoxicity was in agreement with the properties of rodent hepatocytes. Therefore, the clinical implications of scientific findings should be obvious for cell and gene therapy, especially when coupled with scientific data showing excellent recovery of cells following cryopreservation, which should greatly facilitate banking of cells for use at short notice. The intraperitoneal bioassay was effective in demonstrating differentiation of progenitor cells into hepatocytes. This in vivo assay should be helpful for analyzing progenitor cell subpopulations, including analyzing progenitor cells for quality controls prior to clinical use. The availability of human progenitor cells capable of extensive proliferation, will facilitate development of bioartificial liver (BAL) devices, which are being tested for liver failure, but are limited to porcine hepatocytes or less effective cell lines. Seeding of BAL devices with primary adult hepatocytes has been limited by their inability to proliferate. Additional applications of human progenitor liver cells concern development of novel models for pathophysiological studies, drug discovery systems and drug toxicity studies.