The liver can regenerate itself through the progenitor cells it harbors. It was demonstrated the isolation of epithelial progenitor/stem cells from the fetal human liver, which contains a large number of hepatoblasts with very wide spectra of features and capabilities. Progenitor liver cells displayed clonogenic capacity, expressed genes observed in hepatocytes, bile duct cells and oval cells, and incorporated genes transferred by adenoviral or lentiviral vectors. Under culture conditions, progenitor cells proliferated for several months, with each cell undergoing more than forty divisions, but they retained normal karyotypes. Progenitor cells differentiated into mature hepatocytes in mice with severe combined immunodeficiency, both when in an ectopic location and when in the liver itself. Cells integrated in the liver parenchyma and proliferated following liver injury. An abundance of progenitor cells in the fetal human liver is consistent with models indicating depletion of progenitor/stem cells during aging and maturation of organs.

Fetal cells, properties and applications

The findings indicate that large numbers of highly viable progenitor epithelial cells can be isolated from fetal human liver. These cells exhibited 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. The presence of cells expressing both glycogen and CK-19 in cell cultures was in further agreement with bilineage gene expression in cells. 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 our cells as possessing progenitor phenotype. The major goal was to induce differentiation in these cells along the hepatocyte lineage for obvious implications in cell and gene therapy. Replication of progenitor cells in culture, with retention of differentiation capacity, would be helpful in a variety of biological studies. Progenitor cells were obviously not derived from single cells. Nonetheless, these cells showed 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, cells expressed telomerase, which could be beneficial for continued cell division. It was noteworthy that after ~50 population doublings, fetal cells showed decreasing proliferation though mature hepatocytes are difficult to maintain and expand in cell culture. No hepatic growth factors were incorporated (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, fetal derived 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; these observations were reflected in these culture conditions. 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 fetal progenitor cells to TGFα 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, fetal progenitor cells were genetically unperturbed despite more than 40 to 50 population doublings over 16-18 subpassages. Such proliferation capacity in these 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. Nevertheless scientific data show that significant proportions of transplanted cells are lost in mice shortly after transplantation. These findings are not surprising because a large fraction of transplanted cells sequestered in portal vein radicles and hepatic sinusoids undergoing 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 fetal cells 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 bioassays were 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, described here, 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.