Identification of a bipotential precursor cell in hepatic cell lines derived from transgenic mice expressing cyto-Met in the liver.

Met murine hepatocyte (MMH) lines were established from livers of transgenic mice expressing constitutively active human Met. These lines harbor two cell types: epithelial cells resembling the parental populations and flattened cells with multiple projections and a dispersed growth habit that are designated palmate. Epithelial cells express the liver-enriched transcription factors HNF4 and HNF1alpha, and proteins associated with epithelial cell differentiation. Treatments that modulate their differentiation state, including acidic FGF, induce hepatic functions. Palmate cells show none of these properties. However, they can differentiate along the hepatic cell lineage, giving rise to: (a) epithelial cells that express hepatic transcription factors and are competent to express hepatic functions; (b) bile duct-like structures in three-dimensional Matrigel cultures. Derivation of epithelial from palmate cells is confirmed by characterization of the progeny of individually fished cells. Furthermore, karyotype analysis confirms the direction of the phenotypic transition: palmate cells are diploid and the epithelial cells are hypotetraploid. The clonal isolation of the palmate cell, an immortalized nontransformed bipotential cell that does not yet express the liver-enriched transcription factors and is a precursor of the epithelial-hepatocyte in MMH lines, provides a new tool for the study of mechanisms controlling liver development.

Temporal and tissue-specific expression of the MET ORF driven by the complete transcriptional unit of human A1AT gene in transgenic mice.

We inserted the sequence coding for the cytoplasmic portion of the human MET receptor into an 18-kb genomic fragment containing the entire human A1AT gene (encoding alpha-1-antitrypsin). Stringent control of gene expression, at the transcriptional, post-transcriptional and translational levels, was ensured by insertion of the MET open reading frame into A1AT, thus maintaining: (i) all the elements that confer tissue-specific transcription initiation, (ii) all the sequences involved in transcript processing and (iii) all the sequences which influence messenger stability and translational efficiency. The expression pattern of this vector in transgenic mice was identical to that of the human A1AT transgene, as well as to that of A1AT in humans with regard to both temporal and tissue-specific regulation.

From endoderm to pancreas: a multistep journey.

The formation of the vertebrate pancreas is a complex process that typifies the basic steps of embryonic development. It involves the establishment of competence, specification, signaling from neighboring tissues, morphogenesis, and the elaboration of tissue-specific genetic networks. A full analysis of this multistep process will help us to understand classic principles of embryonic development. Furthermore, this will provide the blueprint for experimental programming of pancreas formation from embryonic stem cells in the context of diabetes cell-therapy. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatogenesis, important gaps persist in our knowledge of early pancreas formation. This review will summarize the current understanding of the early mechanisms coming into play to pattern the "pre-pancreatic" region within the endoderm and, gradually, specify the pancreatic tissue.

Inhibition of MMH (Met murine hepatocyte) cell differentiation by TGF(beta) is abrogated by pre-treatment with the heritable differentiation effector FGF1.

MMH (Met murine hepatocyte) liver cells derived from transgenic mice expressing a truncated constitutively active form of human c-Met are non-transformed immortalized cell lines. We have previously shown that they harbor: (1) epithelial cells that express the liver-enriched transcription factors HNF4 and HNF1(alpha), and that can be stably induced by FGF1 to express liver functions, and (2) fibroblast-like bi-potential palmate cells that can differentiate into bile duct-like structures in Matrigel cultures, or into epithelial cells competent to express hepatic functions. Low concentrations of TGF(beta) have been found to inhibit growth and differentiation of MMH cells. The factor stabilized the palmate cell phenotype, and it provoked epithelial cells to acquire palmate-like morphological characteristics, in parallel with down-regulation of expression of HNF4 and HNF1(alpha) and activation of Snail transcripts. The effects of TGF(beta) were dominant if it was added with FGF1, but the effects on differentiation were abrogated if cells had been pre-treated with FGF1. This work identifies TGF(beta) as a factor that could be implicated in maintaining bi-potential precursor cells in the liver, FGF1 as one that could over-ride the TGF(beta) effects and Snail as a candidate for mediation of the signal.

Transgenic expression in the liver of truncated Met blocks apoptosis and permits immortalization of hepatocytes.

Hepatocyte growth factor induces proliferation, motility and differentiation of epithelial cells through the tyrosine kinase receptor encoded by the MET protooncogene. The cytoplasmic portion of Met (referred to as cyto-Met) is activated but only weakly transforming. In order to determine the effect of activated Met on hepatocytes, we have targeted truncated Met expression to the liver by incorporating the cDNA into a vector carrying the entire human alpha-1-antitrypsin transcriptional unit. Transgenic expression in the liver of truncated human Met, containing the regulatory and the catalytic cytoplasmic domains, renders hepatocytes constitutively resistant to apoptosis and reproducibly permits immortalization. The emerging stable cell lines are not transformed and maintain a highly differentiated phenotype judged by the retention of epithelial cell polarity and the expression of hepatocyte-enriched transcription factors as well as hepatic products.