Multipotential differentiation of adult liver stem cells in culture

Various approaches have been used to examine the differentiation potential of cultured rat liver stem cells. These include modification of culture media to contain differentiation-promoting agents and the use of combina-

Fig. 5. Endocardial differentiation of WB-F344 cells following transplantation into the heart. A cartilaginous structure in the ventricular chamber of the heart (A, C) is lined by flat cells that express P-galactosidase (C, D) and von Willebrand factor (B). Cells on the surface of the structure, as well as cells in the mass, contain a Y chromosome (arrows, E).

Fig. 5. Endocardial differentiation of WB-F344 cells following transplantation into the heart. A cartilaginous structure in the ventricular chamber of the heart (A, C) is lined by flat cells that express P-galactosidase (C, D) and von Willebrand factor (B). Cells on the surface of the structure, as well as cells in the mass, contain a Y chromosome (arrows, E).

tions of extracellular matrix materials as culture substrates. These studies have yielded evidence that cultured stemlike epithelial cells can be induced to express characteristics of differentiated liver cell types in culture. In the following sections, we review the results of our studies with WB-F344 rat liver epithelial cells as well as some studies from the literature on the differentiation of oval cells in culture.

6.1. Hepatocytic Differentiation of Oval Cells In Vitro

Oval cell cultures established from rats treated with 3'-methyl-4-dimethylaminoazobenzene exhibit typical epithelial morphology in culture, express various cytokeratins, vimentin, y-glutamyltranspeptidase, and BDS7 antigen (226). The phenotype of these cells can be modified by culturing them on fibronectin-coated dishes in medium containing various differentiation-promoting agents. Inclusion of sodium butyrate in the growth medium inhibits cellular proliferation and produces dramatic morphological alterations in the cultured oval cells (226). In the presence of sodium butyrate alone or in combination with dexamethasone, cultured oval cells synthesize albumin and express tyrosine aminotransferase activity (226).

Pack and coworkers have also examined the effects of differentiation-promoting chemicals on the phenotypic characteristics of cultured oval cell lines (191). Oval cell lines (OC/CDE) were established from cells isolated from rats maintained on a choline-deficient diet supplemented with ethion-ine (191). Exposure of OC/CDE cell lines to either sodium butyrate or dim-ethylsulfoxide resulted in cessation of cell proliferation, increased cell size, expression of albumin (in 35-40% of cells), and enhanced glucose-6-phos-phatase, y-glutamyltranspeptidase, and alkaline phosphatase activities (191). Tyrosine aminotransferase activity was not detected in OC/CDE cell cultures treated with either sodium butyrate or dimethylsulfoxide (191). These studies combined demonstrate that cultured oval cells can be induced to express some characteristics of differentiated hepatocytes.

Lazaro and colleagues (227) developed a three-dimensional cell culture system that supports the hepatocytic differentiation of oval cell lines. In this system, LE/2 and LE/6 oval cells were cultured in a collagen I gel matrix, supported by a fibroblast feeder layer. After several weeks in culture, these oval cells acquired a phenotype characterized by typical hepatocyte morphology and ultrastructure, expression of a hepatocytic cytokeratin pattern (CK8+, CK18+, CK19 ), and production of albumin (227). In the absence of a fibro-blast feeder layer, oval cells cultured in this model system with defined growth factors HGF or keratinocyte growth factor (KGF) produced ductal structures, suggesting differentiation toward the biliary epithelial cell lin eage (227). This study showed that cultured oval cells are bipotent, and that their differentiation fate is influenced by soluble factors (growth factors and others) produced by stromal cells.

6.2. Hepatocytic Differentiation by RLE-13 Rat Liver Epithelial Cells in Culture

A number of different simple epithelial cell lines have been established in culture from normal rat livers (reviewed in ref. 15), including RLE-13, which was established from a normal adult Fischer 344 rat (228). Schroeder and colleagues induced hepatocytic differentiation of RLE-13 cells by treatment with 5-aza deoxycytidine, followed by culture in defined growth medium containing FGF1/2, oncostatin M, HGF, and dexamethasone (229). Culture of the RLE-13 cells under these conditions resulted in significant enlargement of cell size, increased organelle complexity, and decreased proliferation. Concurrent with the morphological alterations, differentiating RLE-13 cells expressed hepatocyte-specific markers, including tyrosine aminotrans-ferase, and liver-enriched transcription factors, including HNF4 (229). These results combine to suggest that RLE-13 cells acquire a hepatocytic pheno-type when maintained in culture under defined conditions.

6.3. Hepatocytic Differentiation of WB-F344 Rat Liver Stem Cells in Culture

Exposure to sodium butyrate in culture inhibits proliferation, alters normal cellular morphology (increased cell size and decreased nuclear/cyto-plasmic ratio), and dramatically increases cellular protein synthesis in WB-F344 rat liver epithelial cells (230). Ultrastructurally, WB-F344 cells treated with sodium butyrate demonstrate complex cytoplasm with extensive rough endoplasmic reticulum, numerous mitochondria, and large numbers of primary and secondary lysosomes. These cells also express dexamethasone-inducible tyrosine aminotransferase enzyme activity (230), which is a marker of hepatocyte differentiation (231,232). The dexametha-sone-inducible tyrosine aminotransferase activity developing in these WB-F344 cells treated with sodium butyrate responds to the modulating effects of insulin and l-tyrosine in a manner that closely resembles that of cultured hepatocytes and hepatoma cell lines (233,234). These studies demonstrated that WB-F344 cells can be induced to express traits of differentiated hepatocytes in vitro.

Using a similar cell culture system, Couchie and colleagues evaluated the ability of WB-F344 cells to differentiate into the biliary epithelial cell lineage (235). WB-F344 cells cultured on dishes coated with laminin-rich

Matrigel in the presence of growth medium containing sodium butyrate demonstrated reduced proliferation and formed cordlike structures between islets of cells (235). WB-F344 cells in the cordlike structures were elongated, and expressed several biliary markers, including BDS7, CK19, and y-glutamyl transpeptidase (235). These results suggest that WB-F344 cells adopt a biliary epithelial cell phenotype in response to specific factors/signals in cell culture.

6.4. WB-F344 Cells Differentiate Into Cardiac Myocytes in Cell Culture

To examine the capacity for WB-F344 cells to differentiate into myocytes in culture, a cardiac tissue microenvironment was established ex vivo using primary cultures of neonatal rat cardiac myocytes. After 3-4 d, these cultures were seeded with WB-F344 cells (carrying genes for E. coli P-galactosidase and green fluorescent protein). The P-galactosidase-positive or fluorescent WB-F344-derived myocytes were identified in these cocultures 6-9 d later (236). WB-F344-derived myocytes were binucleated and demonstrated nascent myofibrils, sarcomeres, and sarcoplasmic reti-culum by electron microscopy. The cardiac phenotype of these cells was verified by immunodetection of cardiac-specific proteins. These WB-F344-derived myocytes demonstrated calcium transients and were electrically coupled with the established neonatal-derived myocytes, as evidenced by fluorescence recovery after photobleaching (FRAP) experiments (236). Evidence of fusion between WB-F344 cells and cultured cardiac myocytes was not found. These results suggest that WB-F344 cells can be induced to differentiate into cardiac myocytes in culture, and that a myocyte lineage can be modeled ex vivo, allowing for systematic investigation of the mechanistic basis for this differentiation event.

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