Results from previous studies suggesting that cholangiocyte undergo EMT are inconclusive because they entirely rely on double immunofluorescence staining for cholangiocyte markers and surrogate markers for mesenchymal cells including FSP-1.20–22 As pointed out above, it remains unknown if FSP-1-positive cells express collagen and therefore also contribute to the ECM-producing cells in vivo. Cell fate tracking for cholangiocytes has not been performed so far and genetic evidence that cholangiocytes lose their epithelial characteristics and acquire a mesenchymal phenotype check details and start to

synthesize ECM is therefore still missing. Our current study did not address the role of cholangiocytes in EMT. To analyze if hepatocyte-derived cells indeed contribute to ECM production in liver fibrosis, we utilized a reporter mouse in which GFP is expressed under the murine collagen α1(I) promoter/enhancer in combination with a cell fate tracing technique used

in the previous study by Zeisberg et al.6 Our in vitro results using primary cultured hepatocytes initially appeared to support the concept of EMT. Hepatocytes not only exhibited fibroblast-like morphological changes, but also expressed collagen α1(I) in response to TGFβ-1. Cell fate tracing technique using ROSA26 stop β-gal and Alb Cre mice excluded the possibility that GFP-expressing cells were contaminating mesenchymal cells. However, the GFP-expressing hepatocytes did not express mesenchymal markers such as FSP-1 or α-SMA. A small number of cells that became positive for FSP-1 or α-SMA in hepatocyte culture click here were not positive for β-gal and were rare contaminating MCE公司 cells. Thus, our observation that hepatocytes express collagen α1(I) with a “fibroblast-like” morphological change does not satisfy the definition of EMT, as it

is not associated with mesenchymal marker expression. A number of studies have demonstrated that cultured hepatocytes express mesenchymal markers in response to TGFβ-1.13, 23 However, these studies failed to show that cells positive for mesenchymal markers were truly hepatocyte-derived cells and do not represent contaminating cells. Only Zeisberg et al.6 employed the cell fate tracing technique to demonstrate that hepatocyte-derived cells express a mesenchymal marker (FSP-1). The reliability of the immunostaining (FSP-1 and β-gal) is open to question (discussed below). The fact that we (Supporting Fig. S7) as well as Kaimori et al.13 observed no increase in FSP-1 mRNA levels in hepatocytes treated with TGFβ-1 challenges the observation of Zeisberg et al. Taken together, our study and previous reports do not provide evidence that primary cultured hepatocytes undergo EMT and acquire expression of FSP-1. More important, our in vivo experiments detected no hepatocyte-derived collagen type I-expressing cells.