BIOARTIFICIAL LIVERS FROM HEPATIC PROGENITOR CELLS

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Rodent hepatic progenitor cells, purified by multiparametri fluorescence activated cell sorting, will be seeded into a hollow fiber bioreactor under defined ex vivo culture conditions to establish a bioartifical liver. all current forms of bioartifical livers are derived from cell lines that permit stability and longevity in the bioreactors (weeks to months) but have minimal differentiated function, or are derived from mature liver cells that yield a bioartificial liver that is short-lived (typically one week) but have a broad range of tissue- specific functions. A stable bioartificial liver with a broad rang of differentiated function should be achievable if hepatic progenitor cells are use, since purified populations of these cells are capable ex vivo of maturing to produce all the cell types of the liver. Stability will be achieved also by using ex vivo expansion conditions that re as defined as possible and include little or no serum; defined and purified extracelluar matrix components as substratum; and defined and purified soluble signals (hormones, growth factors, nutrients). The hypothesis will be tested using rodent ells, and if successful, a similar approach will be used to establish a human bioartificial liver. The proposal is based in numerous studies on rat liver stem cell biology demonstrating that rat liver, at all ages at least to 1 year of life, contains progenitor cells located by each of the portal triads. These progenitor cells produce daughter cells that mature through a unidirectional, differentiation process ending at the central vein. Thus, the plates of parenchymal cells within each acinus in vivo are, thus, lineages of maturing liver cells with age-dependent size, ploidy, growth and differentiative potential. Protocols have been developed, and antigenic profiles defined by which to identify and isolate three subpopulations of hepatic progenitors and two subpopulations of mature parencymal cells using a combination of panning and multiparametric fluorescence activated cell sorting (FACS): 1) hepatoblasts, pluripotent hepatic progenitors; 2) committed bile duct progenitors; 3) committed hepatocyte progenitors; 4) periportal hepatocyes (presumptive young hepatocytes); and 5) pericentral hepatocytes (presumptive old hepatocytes). In addition, we have developed in vivo bioassays for fate studies and ex vivo conditions that permit cell expansion and others that drive differentiation of each of the three subpopulations. The rate bioartificial liver will be established from each of the 5 subpopulations of parenchymal cells by seeding them into commercially available hollow fiber bioreactors and under appropriate ex vivo expansion conditions. For the hepatic progenitor subpopulations, their requisite feeder cells of ACSs-purified hemopoietic OCAP cells (myeloid cells that bear an oval cell antigen3+) and age-and liver-specific stormal feeder cells (from E14- E16 livers) will be put into separate bioreactors, and then the bioreactors with the feeder cells will be coupled in tandem with the one containing the hepatic progenitor cells. In parallel, we will try to identify the factors that are part of the paracrine signaling between these three embryonic hepatic cell types (progenitors of parenchymal, hemopoietic and mesenchymal cells) to enable us to eliminate one or both of the fewer cells as components of the vioartificial liver system. If the factors are novel, we will attempt to isolate and clone them through subtractive hybridization methods using biologically active feeders, the embryonic cells, versus inactive feeders derived from the adult counterparts to the cells. the bioreactors derived from progenitor cells will be characterized for fetal and adult liver-specific functions, and, as controls, for hemopoietic markers, by means of immunochemistry, biochemical assays, and molecular hybiridizaion assays. The fates of the cells in he bioreactors will be compared with those identified from in vivo bioassays.