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

Spagnoli FM, Amicone L, Tripodi M, Weiss MC - J. Cell Biol. (1998)

Bottom Line: Palmate cells show none of these properties.Derivation of epithelial from palmate cells is confirmed by characterization of the progeny of individually fished cells.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.

View Article: PubMed Central - PubMed

Affiliation: Unité de Génétique de la Différenciation, URA 1773 du Centre National de la Recherche Scientifique, Institut Pasteur, 75724 Paris Cedex 15, France.

ABSTRACT
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.

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Spontaneous phenotypic transition of the palmate cells toward an epithelial-hepatocytic phenotype.  (A) Phase-contrast micrographs showing the morphology of the palmate cells at intermediate (p11) and late  passages (p30). (B) Appropriate re-organization of the  epithelial polarity markers  (E-cad., ZO-1, and CK) in  the palmate cells at passage  30. The phase-contrast and  immunofluorescence micrographs of the ZO-1 visualization is of the same field. (C)  Reexpression of hepatic  functions after environmental treatments in the palmate  cells late passage. Northern  blot analysis of RNA extracted from the E14 parental line (E14), the palmate  clone (pal.) at early (p4) and  late (p30) passages as well as  control Fao rat hepatoma cell line (c). Cultured cells were examined in the usual growth conditions (−), and 1 wk after addition of  DMSO or gelatin. Each lane was loaded with 20 μg of total RNA. Probes used are indicated on the left. (D) Activation of LETF  (HNF1α and HNF4) expression in palmate cells continuously kept in culture. The gel shift assays were performed with labeled oligonucleotides corresponding to the HNF1 site PE56, the HNF4 site C3P and the HNF3 site of the mouse TTR promoter, as detailed in Materials and Methods. The displacement of the protein-DNA complexes was obtained with anti-HNF1α or with an excess of unlabeled oligonucleotides for HNF4 and HNF3. Nuclear extracts of FGC4, as positive control (c), of E14 parental line (E14), of the epithelial (ep.)  and palmate (pal.) clones at early (p3) and late (p30) passage were examined. Bar, 20 μm.
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Figure 7: Spontaneous phenotypic transition of the palmate cells toward an epithelial-hepatocytic phenotype. (A) Phase-contrast micrographs showing the morphology of the palmate cells at intermediate (p11) and late passages (p30). (B) Appropriate re-organization of the epithelial polarity markers (E-cad., ZO-1, and CK) in the palmate cells at passage 30. The phase-contrast and immunofluorescence micrographs of the ZO-1 visualization is of the same field. (C) Reexpression of hepatic functions after environmental treatments in the palmate cells late passage. Northern blot analysis of RNA extracted from the E14 parental line (E14), the palmate clone (pal.) at early (p4) and late (p30) passages as well as control Fao rat hepatoma cell line (c). Cultured cells were examined in the usual growth conditions (−), and 1 wk after addition of DMSO or gelatin. Each lane was loaded with 20 μg of total RNA. Probes used are indicated on the left. (D) Activation of LETF (HNF1α and HNF4) expression in palmate cells continuously kept in culture. The gel shift assays were performed with labeled oligonucleotides corresponding to the HNF1 site PE56, the HNF4 site C3P and the HNF3 site of the mouse TTR promoter, as detailed in Materials and Methods. The displacement of the protein-DNA complexes was obtained with anti-HNF1α or with an excess of unlabeled oligonucleotides for HNF4 and HNF3. Nuclear extracts of FGC4, as positive control (c), of E14 parental line (E14), of the epithelial (ep.) and palmate (pal.) clones at early (p3) and late (p30) passage were examined. Bar, 20 μm.

Mentions: As described in the preceding paragraph, the palmate cells can evolve towards an epithelial phenotype after modification of the growth conditions. In addition, these cells undergo spontaneously the same morphological changes when maintained in continuous culture. As illustrated in Fig. 7 A, at 11 passages the E14 palmate clone showed both cell morphologies: among the palmate cells tightly packed epithelial islands appear. At 30 passages, cultures were essentially composed of cells of regular and polygonal shape. Concomitantly a well-organized compartmentalization of ZO-1 and E-cadherin can be observed (Fig. 7 B). Furthermore, in this clone after 30 passages treatment with DMSO or growth on gelatin-coated dishes is sufficient to lead to expression of albumin and TTR (Fig. 7 C).


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

Spagnoli FM, Amicone L, Tripodi M, Weiss MC - J. Cell Biol. (1998)

Spontaneous phenotypic transition of the palmate cells toward an epithelial-hepatocytic phenotype.  (A) Phase-contrast micrographs showing the morphology of the palmate cells at intermediate (p11) and late  passages (p30). (B) Appropriate re-organization of the  epithelial polarity markers  (E-cad., ZO-1, and CK) in  the palmate cells at passage  30. The phase-contrast and  immunofluorescence micrographs of the ZO-1 visualization is of the same field. (C)  Reexpression of hepatic  functions after environmental treatments in the palmate  cells late passage. Northern  blot analysis of RNA extracted from the E14 parental line (E14), the palmate  clone (pal.) at early (p4) and  late (p30) passages as well as  control Fao rat hepatoma cell line (c). Cultured cells were examined in the usual growth conditions (−), and 1 wk after addition of  DMSO or gelatin. Each lane was loaded with 20 μg of total RNA. Probes used are indicated on the left. (D) Activation of LETF  (HNF1α and HNF4) expression in palmate cells continuously kept in culture. The gel shift assays were performed with labeled oligonucleotides corresponding to the HNF1 site PE56, the HNF4 site C3P and the HNF3 site of the mouse TTR promoter, as detailed in Materials and Methods. The displacement of the protein-DNA complexes was obtained with anti-HNF1α or with an excess of unlabeled oligonucleotides for HNF4 and HNF3. Nuclear extracts of FGC4, as positive control (c), of E14 parental line (E14), of the epithelial (ep.)  and palmate (pal.) clones at early (p3) and late (p30) passage were examined. Bar, 20 μm.
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Figure 7: Spontaneous phenotypic transition of the palmate cells toward an epithelial-hepatocytic phenotype. (A) Phase-contrast micrographs showing the morphology of the palmate cells at intermediate (p11) and late passages (p30). (B) Appropriate re-organization of the epithelial polarity markers (E-cad., ZO-1, and CK) in the palmate cells at passage 30. The phase-contrast and immunofluorescence micrographs of the ZO-1 visualization is of the same field. (C) Reexpression of hepatic functions after environmental treatments in the palmate cells late passage. Northern blot analysis of RNA extracted from the E14 parental line (E14), the palmate clone (pal.) at early (p4) and late (p30) passages as well as control Fao rat hepatoma cell line (c). Cultured cells were examined in the usual growth conditions (−), and 1 wk after addition of DMSO or gelatin. Each lane was loaded with 20 μg of total RNA. Probes used are indicated on the left. (D) Activation of LETF (HNF1α and HNF4) expression in palmate cells continuously kept in culture. The gel shift assays were performed with labeled oligonucleotides corresponding to the HNF1 site PE56, the HNF4 site C3P and the HNF3 site of the mouse TTR promoter, as detailed in Materials and Methods. The displacement of the protein-DNA complexes was obtained with anti-HNF1α or with an excess of unlabeled oligonucleotides for HNF4 and HNF3. Nuclear extracts of FGC4, as positive control (c), of E14 parental line (E14), of the epithelial (ep.) and palmate (pal.) clones at early (p3) and late (p30) passage were examined. Bar, 20 μm.
Mentions: As described in the preceding paragraph, the palmate cells can evolve towards an epithelial phenotype after modification of the growth conditions. In addition, these cells undergo spontaneously the same morphological changes when maintained in continuous culture. As illustrated in Fig. 7 A, at 11 passages the E14 palmate clone showed both cell morphologies: among the palmate cells tightly packed epithelial islands appear. At 30 passages, cultures were essentially composed of cells of regular and polygonal shape. Concomitantly a well-organized compartmentalization of ZO-1 and E-cadherin can be observed (Fig. 7 B). Furthermore, in this clone after 30 passages treatment with DMSO or growth on gelatin-coated dishes is sufficient to lead to expression of albumin and TTR (Fig. 7 C).

Bottom Line: Palmate cells show none of these properties.Derivation of epithelial from palmate cells is confirmed by characterization of the progeny of individually fished cells.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.

View Article: PubMed Central - PubMed

Affiliation: Unité de Génétique de la Différenciation, URA 1773 du Centre National de la Recherche Scientifique, Institut Pasteur, 75724 Paris Cedex 15, France.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus