A sequential EMT-MET mechanism drives the differentiation of human embryonic stem cells towards hepatocytes

ARTICLE Received 24 Dec 2016 | Accepted 3 Mar 2017 | Published 3 May 2017 DOI: 10.1038/ncomms15166 OPEN A sequential EMT-MET mechanism drives the differentiation of human embryonic stem cells towards hepatocytes Qiuhong Li1,2,3,*, Andrew P. Hutchins1,*, Yong Chen1, Shengbiao Li1, Yongli Shan1, Baojian Liao1, Dejin Zheng1, Xi Shi1, Yinxiong Li1, Wai-Yee Chan1,4, Guangjin Pan1, Shicheng Wei2,3, Xiaodong Shu1 & Duanqing Pei1 Reprogramming has been shown to involve EMT–MET; however, its role in cell differentiation is unclear. We report here that in vitro differentiation of hESCs to hepatic lineage undergoes a sequential EMT–MET with an obligatory intermediate mesenchymal phase. Gene expression analysis reveals that Activin A-induced formation of definitive endoderm (DE) accompanies a synchronous EMT mediated by autocrine TGFb signalling followed by a MET process. Pharmacological inhibition of TGFb signalling blocks the EMT as well as DE formation. We then identify SNAI1 as the key EMT transcriptional factor required for the specification of DE. Genetic ablation of SNAI1 in hESCs does not affect the maintenance of pluripotency or neural differentiation, but completely disrupts the formation of DE. These results reveal a critical mesenchymal phase during the acquisition of DE, highlighting a role for sequential EMT–METs in both differentiation and reprogramming. 1 CAS Key laboratory of Regenerative Biology, Guangdong Key laboratory of Stem Cell and Regenerative Medicine and CUHK-GIBH Joint Research Laboratory on Stem Cells and Regenerative Medicine, Guangzhou Institute of Biomedicine and Health-Guangzhou Medical University Joint School of Biological Sciences, South China Institute of Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, 190 Kai Yuan Avenue, Science Park, Guangzhou 510530, China. 2 Central Laboratory, School and Hospital of Stomatology, Peking University, Beijing 100871, China. 3 Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China. 4 CUHK-GIBH Joint Research Laboratory on Stem Cells and Regenerative Medicine, School of Biomedical Sciences, Faculty of Medicine, the Chinese University of Hong Kong, Hong Kong, China. * These authors contributed equally to this work. Correspondence and requests for materials should be addressed to X.S. (email: ) or to D.P. (email: ). NATURE COMMUNICATIONS | 8:15166 | DOI: 10.1038/ncomms15166 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15166 R eprogramming of somatic cells into pluripotent ones with defined factors not only provides a new way to generate functional cells for regenerative medicine, but also establishes a new paradigm for cell fate decisions. For the latter, a cell at a terminally differentiated state can be restored back to pluripotency under well-defined conditions fully observable through molecular and cellular tools. Indeed, the reprogramming process has been analysed in great detail to reveal novel insights into the mechanism of cell fate changes1–3. Of particular interest is the acquisition of epithelial characteristics from mesenchymal mouse embryonic fibroblasts (MEFs) commonly employed as starting cells in reprogramming experiments4. Termed the mesenchymal to epithelial transition (MET), we and others have described the MET as marking the earliest cellular change upon the simultaneous transduction of reprogramming factors POU5F1 (OCT4), SOX2, KLF4 and MYC or OSKM into MEFs5,6. However, when delivered sequentially as OK þ M þ S, they initiate a sequential epithelial to mesenchymal transition (EMT)-MET process that drives reprogramming more efficiently than the simultaneous approach7, suggesting that the switching between mesenchymal and epithelial fates underlies the reprogramming process, that is, the acquisition of pluripotency. We then speculated that such a sequential EMT–MET process might underlie cell fate decisions in other situations such as differentiation, generally viewed as the reversal of reprogramming with the loss of pluripotency. Herein, we report that a similar epithelial–mesenchymal–epithelial transition drives the differentiation of human embryonic stem cells (hESCs) towards hepatocytes. A synchronous EMT occurs during the formation of DE and DE cells are in a typical mesenchymal-like status, while further differentiation of DE to hepatocyte-like cells is accompanied by a MET. We reveal that the intermediate mesenchymal DE cells is induced by an autocrine TGFb signalling and mediated by SNAI1. On the other hand, the neural differentiation of hESCs is not dependent on TGFb signalling or SNAI1. Thus, EMT-related transcriptional factor such as SNAI1 participates in lineage-specific cell fate changes. Results A sequential EMT–MET connects hESCs to hepatocytes. Human embryonic stem cells robustly express E-cadherin (CDH1) and are epithelial cells in a pluripotent state. Conversely, hepatocytes are also epithelial cells, but are somatic and fully differentiated. Naively it seems possible that epithelial hESCs could move directly to hepatocytes with the gradual loss of pluripotency and gain of hepatic characteristics, without the necessity to pass through a mesenchymal state. To map the cell fate changes along the differentiation pathway between hESCs and hepatocytes, we adopted a serum-free, chemically defined protocol of hepatic differentiation of hESCs based on the stepwise addition of Activin A, FGF4/BMP2, HGF/KGF and then Oncostatin M8,9. As shown in Fig. 1a, there were distinct stages marked by POU5F1/NANOG (pluripotency), SOX17/FOXA2 (definitive endoderm, DE), HNF4A/AFP (hepatoblast) and albumin (ALB)/TTR (hepatocyte-like cell) at days 0, 3, 13 and 21, respectively. The cells at day 21 showed typical metabolic activities of hepatocytes such as ALB secretion, synthesis of glycogen or urea, uptake of low-density lipoprotein (LDL) and so on (Supplementary Fig. 1), indicating the effectiveness of the protocol. We characterized the molecular signature of this process first by performing RNA-seq analysis of a time course from days 0 to 21, and compared it with the RNA-seq data of primary human hepatocytes and liver10–12. Principal component (PC) analysis indicated that the cells transitioned from pluripotent 2 stem cell to DE then to hepatocyte-like state (Fig. 1b), based on the gene loading for the respective PCs (Supplementary Fig. 2). In addition, we noticed that PC2 and PC3 contain many EMT-related genes that were dynamically regulated during the hepatic differentiation of hESCs ( Fig. 1c; Supplementary Fig. 2). We next performed real-time RT-polymerase chain reaction (PCR) analysis which confirmed the induction of mesenchymal genes at the DE and hepatoblast stages of hepatic differentiation (Fig. 1d). For example, the mesenchymal gene CDH2, VIM and SNAI1 were all upregulated from D3 (...truncated)