Generation of hepatocytes from oval cell precursors in culture

Although there is experimental evidence supporting the involvement of hepat ic stem cells in the pathogenesis of liver cancers, the detection and isola tion of these cells remains elusive A logical approach to detecting these c ells would take advantage of their ability to differentiate (or to give ris e to cells that differentiate) into hepatocytes, This approach requires an assay system that is conducive to hepatocytic differentiation. Here, we rep ort the development of an in vitro system consisting of a three-dimensional collagen gel matrix and a fibroblast feeder layer that supports hepatocyti c 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 containi ng 0.1% ethionine for 2 and 6 weeks, respectively. These Lines consist of s mall cells that are phenotypically immature with few cytoplasmic organelles and a high nuclear-to-cytoplasmic ratio. After 4 weeks in the three-dimens ional culture system, these cells acquired typical hepatocytic morphology, By electron microscopy, the cells formed canalicular structures that are ty pical of hepatocytes and were organelle rich, displaying peroxisomes, abund ant mitochondria, and rough endoplasmic reticulum, The cells produced album in and displayed a cytokeratin (CK) pattern typical of hepatocytes (CK 8 an d CK 18-positive and CK 19-negative). The presence of a mesenchymal cell fe eder layer was essential for supporting hepatocytic differentiation. Withou t a feeder layer but in the presence of hepatocyte growth Factor and/or ker atinocyte growth factor, the precursor cells formed ductal structures, sugg estive of differentiation along the bile duct lineage. The three-dimensiona l system described provides direct proof of the lineage generation capacity of oval cells. it offers a model to study factors that may be important Fo r hepatocytic differentiation from precursor cells and a means to assay cel l populations for their ability to give rise to normal and transformed hepa tocytes.