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Reversible conversion of epithelial and mesenchymal phenotypes in SV40 large T antigen-immortalized rat liver cell lines.

Takenouchi T, Yoshioka M, Yamanaka N, Kitani H - Cell Biol Int Rep (2010) (2010)

Bottom Line: This process is also essential for liver morphogenesis in embryonic development.To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes.The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 12, Tsukuba, Ibaraki 3058634, Japan.

ABSTRACT
EMT (epithelial-mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.

No MeSH data available.


Related in: MedlinePlus

Immunocytochemical characterization of RNPC cellsThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RNPC cells were plated in eight-well chamber slides in DMEM-based medium, then fixed and stained with specific antibodies against CK18, αSMA and desmin (A). The cells were also cultured for 9 days after the replacement of the medium with a DF-based medium, then fixed and stained with specific antibodies against CK18 and αSMA (B). Scale bar = 100 μm.
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Figure 5: Immunocytochemical characterization of RNPC cellsThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RNPC cells were plated in eight-well chamber slides in DMEM-based medium, then fixed and stained with specific antibodies against CK18, αSMA and desmin (A). The cells were also cultured for 9 days after the replacement of the medium with a DF-based medium, then fixed and stained with specific antibodies against CK18 and αSMA (B). Scale bar = 100 μm.

Mentions: For comparison, we established SV40LT-immortalized cell lines from the non-parenchymal cell fraction of rat livers and examined their ability to undergo phenotypic conversion. After selection with G418 treatment, a single collected colony was expanded as a cell line, RNPC cells. RNPC cells exhibited fibroblastic morphology in DMEM-based medium and were completely negative for CK18 staining (Figure 5A, upper panels). They strongly expressed αSMA and also expressed desmin at a lower intensity (Figure 5A, lower panels). These results suggest that RNPC cells are characterized as activated HSCs, although they retained some characteristics of quiescent HSCs. When the RNPC cells were transferred to DF-based medium, neither morphological change nor the induction of CK18 expression was observed after 9 days of culture (Figure 5B), except for a marginal reduction in the number of αSMA-positive cells as observed in RL/DMEM cells (Figures 3 and 5).


Reversible conversion of epithelial and mesenchymal phenotypes in SV40 large T antigen-immortalized rat liver cell lines.

Takenouchi T, Yoshioka M, Yamanaka N, Kitani H - Cell Biol Int Rep (2010) (2010)

Immunocytochemical characterization of RNPC cellsThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RNPC cells were plated in eight-well chamber slides in DMEM-based medium, then fixed and stained with specific antibodies against CK18, αSMA and desmin (A). The cells were also cultured for 9 days after the replacement of the medium with a DF-based medium, then fixed and stained with specific antibodies against CK18 and αSMA (B). Scale bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475435&req=5

Figure 5: Immunocytochemical characterization of RNPC cellsThe morphology of the live cells was observed under a phase-contrast microscope [PH in (A)]. The RNPC cells were plated in eight-well chamber slides in DMEM-based medium, then fixed and stained with specific antibodies against CK18, αSMA and desmin (A). The cells were also cultured for 9 days after the replacement of the medium with a DF-based medium, then fixed and stained with specific antibodies against CK18 and αSMA (B). Scale bar = 100 μm.
Mentions: For comparison, we established SV40LT-immortalized cell lines from the non-parenchymal cell fraction of rat livers and examined their ability to undergo phenotypic conversion. After selection with G418 treatment, a single collected colony was expanded as a cell line, RNPC cells. RNPC cells exhibited fibroblastic morphology in DMEM-based medium and were completely negative for CK18 staining (Figure 5A, upper panels). They strongly expressed αSMA and also expressed desmin at a lower intensity (Figure 5A, lower panels). These results suggest that RNPC cells are characterized as activated HSCs, although they retained some characteristics of quiescent HSCs. When the RNPC cells were transferred to DF-based medium, neither morphological change nor the induction of CK18 expression was observed after 9 days of culture (Figure 5B), except for a marginal reduction in the number of αSMA-positive cells as observed in RL/DMEM cells (Figures 3 and 5).

Bottom Line: This process is also essential for liver morphogenesis in embryonic development.To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes.The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes.

View Article: PubMed Central - PubMed

Affiliation: Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Ohwashi 12, Tsukuba, Ibaraki 3058634, Japan.

ABSTRACT
EMT (epithelial-mesenchymal transition) is a key process in the development of liver fibrosis. This process is also essential for liver morphogenesis in embryonic development. To study the cellular and molecular basis of EMT, we established two phenotypically different SV40 large T antigen-immortalized cell lines from rat hepatocytes. The first cell line, which had an epithelial morphology and was established in DMEM (Dulbecco's modified Eagle's medium)/Ham's F-12 (DF)-based medium (RL/DF cells), expressed CK18 (cytokeratin 18), a marker of parenchymal hepatocytes. The other, a mesenchymal-like cell line established in DMEM-based medium (RL/DMEM cells), expressed αSMA (α-smooth muscle actin), a marker of hepatic myofibroblasts. Epithelial RL/DF cells underwent phenotypic changes, such as increased expression of αSMA, when the culture medium was switched to DMEM-based medium. In contrast, mesenchymal RL/DMEM cells were induced to express the epithelial marker CK18 with a concomitant decrease in αSMA expression when the culture medium was replaced with DF-based medium. These cell lines may provide novel in vitro models for the study of the conversion between epithelial and mesenchymal phenotypes during EMT in liver fibrosis and morphogenesis.

No MeSH data available.


Related in: MedlinePlus