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Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation.

Li B, Zheng YW, Sano Y, Taniguchi H - PLoS ONE (2011)

Bottom Line: However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear.The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased.On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions.

View Article: PubMed Central - PubMed

Affiliation: Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

ABSTRACT

Unlabelled: Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein.

Conclusion: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.

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Vimentin-positive mesenchymal fetal liver cells are highly proliferative in vivo.(A) Representative images of dual immunofluorescence of AFP and vimentin; (B) BrdU and vimentin in mice livers at different developmental stages. AFP and BrdU expressions decreased accompanied with vimentin reduction. (C) and (D) represented the relative quantitative assay of non-hematopoietic cells in (A) and (B) respectively. These results showed that AFP positive liver cells also expressed vimentin, and the vimentin+ cells are highly proliferative (BrdU+). Scale bars = 100 µm.
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pone-0017092-g006: Vimentin-positive mesenchymal fetal liver cells are highly proliferative in vivo.(A) Representative images of dual immunofluorescence of AFP and vimentin; (B) BrdU and vimentin in mice livers at different developmental stages. AFP and BrdU expressions decreased accompanied with vimentin reduction. (C) and (D) represented the relative quantitative assay of non-hematopoietic cells in (A) and (B) respectively. These results showed that AFP positive liver cells also expressed vimentin, and the vimentin+ cells are highly proliferative (BrdU+). Scale bars = 100 µm.

Mentions: According to the results described above (Figures 1, 2, 3, 4, 5), hepatic stem cells and a large population of fetal liver cells both cell types have the capacity to proliferate and differentiate into mature liver cells, and more importantly, express vimentin. AFP was first described as a hepatic lineage gene in embryonic gastrulation [26]. Therefore, to further investigate the correlation of vimentin and cell proliferative ability, we continued to characterize vimentin-positive fetal liver cells co-expressing AFP and BrdU, after removal of hematopoietic cells with CD45 and TER119 antibodies. As shown in Figure 6A, frequencies of vimentin and AFP were both very high at ED11.5 comparing with adult mouse liver (Figure 6A and 6C; Table 1). Almost all the vimentin-positive cells are AFP-positive at ED11.5 (100%) and ED13.5 (94.7±9.2%) mouse liver (Figure 6C), yet the percentage of AFP-positive in vimentin-expressing cells decreases dramatically at ED17.5 (15.0±10.2%) and disappears altogether in adult mouse liver (Figure 6C; Table 1). It is worth noticing that almost all the vimentin-positive cells are BrdU-positive in fetal mouse liver (ED11.5: 100%, ED13.5: 95.7±10.5%, ED17.5: 95.7±7.5%) (Figure 6D), as opposed to almost no BrdU-positive cells in adult mouse liver (0.5±0.9%). Thus, during the developing of mouse liver, the number of vimentin-positive cells decreases; meanwhile, the proportion of vimentin-positive cells that are also AFP- or BrdU-positive decreases as well (Table 1; Figure 6C and 6D). Taken together, these measurements provide an in vivo indication that in mouse liver of ED11.5 and ED13.5, hepatoblasts have mesenchymal character (vimentin+) with proliferative activation, whereas in ED17.5 and adult mouse liver, the number of activated cells decreases dramatically concomitant with vimentin reduction. Vimentin, on one hand, has high co-expression with AFP, suggesting that vimentin is associated with immaturity of the liver; on the other, vimentin is also linked to cell proliferation, in that the occurrence of vimentin is related to the liver tissue activity in the developing process.


Evidence for mesenchymal-epithelial transition associated with mouse hepatic stem cell differentiation.

Li B, Zheng YW, Sano Y, Taniguchi H - PLoS ONE (2011)

Vimentin-positive mesenchymal fetal liver cells are highly proliferative in vivo.(A) Representative images of dual immunofluorescence of AFP and vimentin; (B) BrdU and vimentin in mice livers at different developmental stages. AFP and BrdU expressions decreased accompanied with vimentin reduction. (C) and (D) represented the relative quantitative assay of non-hematopoietic cells in (A) and (B) respectively. These results showed that AFP positive liver cells also expressed vimentin, and the vimentin+ cells are highly proliferative (BrdU+). Scale bars = 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037942&req=5

pone-0017092-g006: Vimentin-positive mesenchymal fetal liver cells are highly proliferative in vivo.(A) Representative images of dual immunofluorescence of AFP and vimentin; (B) BrdU and vimentin in mice livers at different developmental stages. AFP and BrdU expressions decreased accompanied with vimentin reduction. (C) and (D) represented the relative quantitative assay of non-hematopoietic cells in (A) and (B) respectively. These results showed that AFP positive liver cells also expressed vimentin, and the vimentin+ cells are highly proliferative (BrdU+). Scale bars = 100 µm.
Mentions: According to the results described above (Figures 1, 2, 3, 4, 5), hepatic stem cells and a large population of fetal liver cells both cell types have the capacity to proliferate and differentiate into mature liver cells, and more importantly, express vimentin. AFP was first described as a hepatic lineage gene in embryonic gastrulation [26]. Therefore, to further investigate the correlation of vimentin and cell proliferative ability, we continued to characterize vimentin-positive fetal liver cells co-expressing AFP and BrdU, after removal of hematopoietic cells with CD45 and TER119 antibodies. As shown in Figure 6A, frequencies of vimentin and AFP were both very high at ED11.5 comparing with adult mouse liver (Figure 6A and 6C; Table 1). Almost all the vimentin-positive cells are AFP-positive at ED11.5 (100%) and ED13.5 (94.7±9.2%) mouse liver (Figure 6C), yet the percentage of AFP-positive in vimentin-expressing cells decreases dramatically at ED17.5 (15.0±10.2%) and disappears altogether in adult mouse liver (Figure 6C; Table 1). It is worth noticing that almost all the vimentin-positive cells are BrdU-positive in fetal mouse liver (ED11.5: 100%, ED13.5: 95.7±10.5%, ED17.5: 95.7±7.5%) (Figure 6D), as opposed to almost no BrdU-positive cells in adult mouse liver (0.5±0.9%). Thus, during the developing of mouse liver, the number of vimentin-positive cells decreases; meanwhile, the proportion of vimentin-positive cells that are also AFP- or BrdU-positive decreases as well (Table 1; Figure 6C and 6D). Taken together, these measurements provide an in vivo indication that in mouse liver of ED11.5 and ED13.5, hepatoblasts have mesenchymal character (vimentin+) with proliferative activation, whereas in ED17.5 and adult mouse liver, the number of activated cells decreases dramatically concomitant with vimentin reduction. Vimentin, on one hand, has high co-expression with AFP, suggesting that vimentin is associated with immaturity of the liver; on the other, vimentin is also linked to cell proliferation, in that the occurrence of vimentin is related to the liver tissue activity in the developing process.

Bottom Line: However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear.The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased.On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions.

View Article: PubMed Central - PubMed

Affiliation: Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

ABSTRACT

Unlabelled: Mesenchymal-epithelial transition events are related to embryonic development, tissue construction, and wound healing. Stem cells are involved in all of these processes, at least in part. However, the direct evidence of mesenchymal-epithelial transition associated with stem cells is unclear. To determine whether mesenchymal-epithelial transition occurs in liver development and/or the differentiation process of hepatic stem cells in vitro, we analyzed a variety of murine liver tissues from embryonic day 11.5 to adults and the colonies derived from hepatic stem/progenitor cells isolated with flow cytometry. The results of gene expression, immunohistochemistry and Western blot showed that as liver develops, the expression of epithelial markers such as Cytokeratin18 and E-cadherin increase, while expression of mesenchymal markers such as vimentin and N-cadherin decreased. On the other hand, in freshly isolated hepatic stem cells, the majority of cells (65.0%) co-express epithelial and mesenchymal markers; this proportion is significantly higher than observed in hematopoietic cells, non-hematopoietic cells and non-stem cell fractions. Likewise, in stem cell-derived colonies cultured over time, upregulation of epithelial genes (Cytokeratin-18 and E-cadherin) occurred simultaneously with downregulation of mesenchymal genes (vimentin and Snail1). Furthermore, in the fetal liver, vimentin-positive cells in the non-hematopoietic fraction had distinct proliferative activity and expressed early the hepatic lineage marker alpha-fetoprotein.

Conclusion: Hepatic stem cells co-express mesenchymal and epithelial markers; the mesenchymal-epithelial transition occurred in both liver development and differentiation of hepatic stem/progenitor cells in vitro. Besides as a mesenchymal marker, vimentin is a novel indicator for cell proliferative activity and undifferentiated status in liver cells.

Show MeSH