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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

Plots of cumulative population doublings of RL/DMEM and RL/DF cells against cumulative culture daysThe RL/DMEM and RL/DF cells were plated at 1×105 cells in 35 mm cell culture dishes in DMEM- and DF-based medium respectively. The cells were passaged at intervals of every 3 or 4 days just before reaching confluency. Then, the cells were harvested after trypsinization, and cell numbers were determined by haemocytometer counts. The proliferation rate of RL/DMEM cells was faster than that of the RL/DF cells (A). When the RL/DMEM cells were cultured in DF-based medium, the doubling time of these cells became gradually longer than that in DMEM-based medium (B). When the RL/DF cells were cultured in DMEM-based medium, the doubling time of these cells became gradually shorter than that in DF-based medium (C).
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Figure 1: Plots of cumulative population doublings of RL/DMEM and RL/DF cells against cumulative culture daysThe RL/DMEM and RL/DF cells were plated at 1×105 cells in 35 mm cell culture dishes in DMEM- and DF-based medium respectively. The cells were passaged at intervals of every 3 or 4 days just before reaching confluency. Then, the cells were harvested after trypsinization, and cell numbers were determined by haemocytometer counts. The proliferation rate of RL/DMEM cells was faster than that of the RL/DF cells (A). When the RL/DMEM cells were cultured in DF-based medium, the doubling time of these cells became gradually longer than that in DMEM-based medium (B). When the RL/DF cells were cultured in DMEM-based medium, the doubling time of these cells became gradually shorter than that in DF-based medium (C).

Mentions: After dissociation by collagenase perfusion, followed by sedimentation by low-speed centrifugation, typical binuclear hepatocytes were obtained at purities of more than 95% and cultured in either DMEM- or DF-based medium. After transfection of SV40LT followed by selection with G418, we obtained several G418-resistant colonies in both culture conditions. The number of G418-resistant colonies obtained from DF-based medium was higher than that obtained from DMEM-based medium, possibly due to the presence of several growth factors and supplements in the former medium. All of the G418-resistant colonies were composed of cells with very similar epithelial-like morphologies. Therefore we chose two representative cell lines, RL/DMEM and RL/DF cells (one from each culture condition) for the present study. As for proliferative ability, RL/DMEM cells proliferated at a doubling time of approx. 24 h and were stably passaged for more than 100 population doublings after the isolation of a single colony (Figure 1A), and RL/DF cells were similarly passaged for at least 100 population doublings, but they proliferated at a longer doubling time of approx. 30 h (Figure 1A).


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)

Plots of cumulative population doublings of RL/DMEM and RL/DF cells against cumulative culture daysThe RL/DMEM and RL/DF cells were plated at 1×105 cells in 35 mm cell culture dishes in DMEM- and DF-based medium respectively. The cells were passaged at intervals of every 3 or 4 days just before reaching confluency. Then, the cells were harvested after trypsinization, and cell numbers were determined by haemocytometer counts. The proliferation rate of RL/DMEM cells was faster than that of the RL/DF cells (A). When the RL/DMEM cells were cultured in DF-based medium, the doubling time of these cells became gradually longer than that in DMEM-based medium (B). When the RL/DF cells were cultured in DMEM-based medium, the doubling time of these cells became gradually shorter than that in DF-based medium (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Plots of cumulative population doublings of RL/DMEM and RL/DF cells against cumulative culture daysThe RL/DMEM and RL/DF cells were plated at 1×105 cells in 35 mm cell culture dishes in DMEM- and DF-based medium respectively. The cells were passaged at intervals of every 3 or 4 days just before reaching confluency. Then, the cells were harvested after trypsinization, and cell numbers were determined by haemocytometer counts. The proliferation rate of RL/DMEM cells was faster than that of the RL/DF cells (A). When the RL/DMEM cells were cultured in DF-based medium, the doubling time of these cells became gradually longer than that in DMEM-based medium (B). When the RL/DF cells were cultured in DMEM-based medium, the doubling time of these cells became gradually shorter than that in DF-based medium (C).
Mentions: After dissociation by collagenase perfusion, followed by sedimentation by low-speed centrifugation, typical binuclear hepatocytes were obtained at purities of more than 95% and cultured in either DMEM- or DF-based medium. After transfection of SV40LT followed by selection with G418, we obtained several G418-resistant colonies in both culture conditions. The number of G418-resistant colonies obtained from DF-based medium was higher than that obtained from DMEM-based medium, possibly due to the presence of several growth factors and supplements in the former medium. All of the G418-resistant colonies were composed of cells with very similar epithelial-like morphologies. Therefore we chose two representative cell lines, RL/DMEM and RL/DF cells (one from each culture condition) for the present study. As for proliferative ability, RL/DMEM cells proliferated at a doubling time of approx. 24 h and were stably passaged for more than 100 population doublings after the isolation of a single colony (Figure 1A), and RL/DF cells were similarly passaged for at least 100 population doublings, but they proliferated at a longer doubling time of approx. 30 h (Figure 1A).

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