Evidence for Mesenchymal2Epithelial Transition Associated with Mouse Hepatic Stem Cell Differentiation Bin Li . , Yun-Wen Zheng . , Yuuki Sano, Hideki Taniguchi* Department of Regenerative Medicine, Yokohama City University Graduate School of Medicine, Yokohama, Japan Abstract Mesenchymal2epithelial 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 mesenchymal2epithelial transition associated with stem cells is unclear. To determine whether mesenchymal2epithelial 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 mesenchymal2epithelial 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. Citation: Li B, Zheng Y-W, Sano Y, Taniguchi H (2011) Evidence for Mesenchymal2Epithelial Transition Associated with Mouse Hepatic Stem Cell Differentiation. PLoS ONE 6(2): e17092. doi:10.1371/journal.pone.0017092 Editor: Eliana Abdelhay, Instituto Nacional de Ca ˆncer, Brazil Received November 18, 2010; Accepted January 18, 2011; Published February 11, 2011 Copyright: ß 2011 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Supported by Grants-in-Aid (18591421, 19390340, 20591531 and 21249071) and the 21st Century Center of Excellence (COE) program of the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan and the grants from Strategic Promotion of Innovative Research and Development (S- innovation, 62890004) from Japan Science and Technology Agency (JST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rtanigu@med.yokohama-cu.ac.jp . These authors contributed equally to this work. Introduction Mesenchymal-epithelial transition (MET) events are defined as those in which mesenchymal cells lose their motile, migratory properties and acquire cell polarity and adhesion to epithelia. MET and the reverse process, epithelial mesenchymal transition (EMT), both occur in normal tissue, including gastrulating and regenerating tissue, as well as abnormal tissues of fibrotic organs or tumors [1,2]. Thus, it is necessary to reveal the relationship between EMT/MET and stem cells. Indeed, EMT drives mammary epithelial cells to de-differentiate into mammary stem cells and cancer stem cells which are mesenchymal-like [3]. Moreover, induced pluripotent stem cells (iPSCs) are derived from mouse embryonic fibroblasts (MEF) by MET at the early stage of reprogramming [4–6]. These results suggest the possibility that MET is associated with stem cell activities. Recent work reported that rat hepatic oval cells (hepatic progenitors) express mesenchymal markers; this result indicated but not fully confirmed that MET existed in the process of hepatic progenitor cell differentiation [7]. On the other hand, hepatocytes, cholangiocytes and liver are known to undergo EMT under sophisticated regulation [8–12]. Furthermore, according to the pathological progression from normal liver to hepatic cirrhosis and then to hepatic carcinoma, EMT may be associated [8,13–15]. Therefore, MET in hepatic stem cells is important to multiple processes, including liver development, regeneration, and chronic liver injury. In order to identify EMT/MET, vimentin is widely applied as a mesenchymal indicator [1,3,16–19]. Vimentin is an intermediate filament protein functionally involved in maintaining the structure of mesenchymal cells [20]. In addition to serving as a marker in EMT/MET, vimentin plays a versatile role in cancer cell motility. In prostate cancer cells, for example, vimentin links the motility and un-differentiated state of cells [21]. Notwithstanding, in breast cancer, vimentin mRNA expression is related to mesenchymal cell shape and motility [22]. In normal tissue injury, depletion of vimentin delays wound healing by mesenchymal fibroblasts trans- differentiating into epithelia [23]. Little is known about the relationship between vimentin expression and normal cell activation. To address these questions, we investigated the mesenchymal characteristics of fetal liver cells and observed MET in developing mouse liver. Furthermore, based on our previous work [24], we isolated hepatic stem cells with flow cytometry in order to study PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e17092