Generation of hepatocytes from oval cell precursors in culture

Although there is experimental evidence supporting the involvement of hepatic stem cells in the pathogenesis of liver cancers, the detection and isolation of these cells remains elusive. A logical approach to detecting these cells would take advantage of their ability to differentiate (or to give rise to cells that differentiate) into hepatocytes. This approach requires an assay system that is conducive to hepatocytic differentiation. Here, we report the development of an in vitro system consisting of a three-dimensional collagen gel matrix and a fibroblast feeder layer that supports hepatocytic differentiation from precursor epithelial (oval) cell lines. The LE/2 and LE/6 oval cell lines used in this study are nontumorigenic cells that are derived from the livers of adult rats fed a choline-deficient diet containing 0.1% ethionine for 2 and 6 weeks, respectively. These lines consist of small cells that are phenotypically immature with few cytoplasmic organelles and a high nuclear-to-cytoplasmic ratio. After 4 weeks in the three-dimensional culture system, these cells acquired typical hepatocytic morphology. By electron microscopy, the cells formed canalicular structures that are typical of hepatocytes and were organelle rich, displaying peroxisomes, abundant mitochondria, and rough endoplasmic reticulum. The cells produced albumin and displayed a cytokeratin (CK) pattern typical of hepatocytes (CK 8 and CK 18-positive and CK 19-negative). The presence of a mesenchymal cell feeder layer was essential for supporting hepatocytic differentiation. Without a feeder layer but in the presence of hepatocyte growth factor and/or keratinocyte growth factor, the precursor cells formed ductal structures, suggestive of differentiation along the bile duct lineage. The three-dimensional system described provides direct proof of the lineage generation capacity of oval cells. It offers a model to study factors that may be important for hepatocytic differentiation from precursor cells and a means to assay cell populations for their ability to give rise to normal and transformed hepatocytes.