Hepatic progenitor populations in embryonic, neonatal, and adult liver

Oval cells, small cells with oval-shaped nuclei, are induced to proliferate in the livers of animals treated with carcinogens and are thought to be related to liver stem cells and/or committed liver progenitor cell populations. We have developed protocols for identifying and isolating antigenically related cell populations present in normal tissues using monoclonal antibodies to oval cell antigens and fluorescence-activated cell sorting. We have isolated oval cell-antigen-positive (OCAP) cells from embryonic, neonatal, and adult rat livers and have identified culture conditions permitting their growth in culture. The requirements for growth of the OCAP cells included substrata of type IV collagen mixed with laminin, basal medium with complex lipids and low calcium, specific growth factors (most potently, insulin-like growth factor II and granulocyte-macrophage colony-stimulating factor), and co-cultures of embryonic, liver-specific stroma, strongly suggesting paracrine signaling between hepatic and hemopoietic precursor cells. The growing OCAP cultures proved to be uniformly expressing oval cell markers but were nevertheless a mixture of hepatic and hemopoietic precursor cells. To separate the hepatic and hemopoietic subpopulations of OCAP cells, we surveyed known antibodies and found ones that uniquely identify either hepatic or hemopoietic cells. Several of these antibodies were used in panning procedures and fluorescence-activated cell sorting to eliminate contaminant cell populations, particularly hemopoietic and endothelial cells. Using specific flow cytometric parameters, three cellular subpopulations could be isolated separately that were identified by immunochemistry and molecular hybridization assays as probable: (i) committed progenitors to hepatocytes; (ii) committed progenitors to bile ducts; or (iii) a mixed population of hemopoietic cells that contained a small percentage of hepatic blasts that are possibly pluripotent. The hepatic precursor cells have been characterized using immunochemistry, flow cytometry, and molecular hybridization assays. The hepatic blasts are small (7-10 microns) cells with high nuclear to cytoplasmic ratios and with minimal complexity of the cytoplasm. Cultures of the committed progenitors were found to differentiate into cells with recognizable parenchymal cell fates. We discuss our studies in the context of our model of the liver as stem cell and lineage system and suggest that a slow, unidirectional, terminal differentiation process, paralleling more rapid ones in the skin or gut, occurs at all times in the liver and is thought to vary primarily in kinetics during quiescent versus regenerative states.(ABSTRACT TRUNCATED AT 400 WORDS)