This project was funded by the Deutsche Forschungsgemeinscha

This project was funded by the Deutsche Forschungsgemeinschaft within FOR 2251 (project grants EB 285/2-1 and WI 3272/3-1) and in part by additional contributions from the Bundesministerium für Bildung und Forschung (D.I.S award Nos. BIOSCAT [05K12YE1]) and the Horizon 2020 programme of the European Union, iNEXT [653706; D.I.S].
Introduction Hepatocytes possess de-differentiation and re-differentiation potential to achieve liver regeneration (Tarlow et al., 2014). Such findings have challenged the long-standing belief that differentiation is an irreversible process in mammals (Jopling et al., 2011, Metcalf, 2007). However, to date detailed mechanisms of how somatic 3555 undergo de-differentiation and subsequent re-differentiation remain undefined, and whether lineage reversibility exists in adult stem cells remains controversial (Zhau et al., 2011). One useful model for studying lineage reversibility is the multi-potent mesenchymal stromal cells (MSCs) (Makino et al., 1999, Pittenger et al., 1999, Wislet-Gendebien et al., 2005), which can differentiate into hepatocyte-like cells (dHeps) (Jiang et al., 2014, Kuo et al., 2008, Lee et al., 2004) that possess therapeutic potentials for liver diseases. Currently, whether and how differentiated hepatic progenies from MSCs can be de-differentiated is unknown. Studies do show, however, DNA methylation plays an important role in controlling MSC differentiation (Tsai et al., 2012). DNA methyltransferases (DNMTs) control gene transcription and cellular phenotypic changes during liver organogenesis (Snykers et al., 2009). It has been reported that inhibition of DNMTs increases liver-specific gene expression to maintain a hepatic fate; a concomitant decrease of DNMT1 and increase of DNMT3 expression is associated with hepatic maturation in mouse hepatoblasts (Gailhouste et al., 2013). DNMT1 is the key to methylation maintenance, while DNMT3 is responsible for the initiation of de novo methylation (Li, 2002). However, the function roles of each DNMT in hepatic lineage commitment of MSCs are unknown. The purpose of this study is to investigate the hepatic lineage reversibility in MSCs as well as the underlying molecular mechanisms. We hypothesize that alteration of culture conditions may achieve hepatic lineage reversibility between MSCs and their differentiated hepatic progenies.
Results
Discussion In this study, we show that hepatic lineages are reversible between adult MSCs and hepatocytes. Such reversibility is regulated by the TGFβ1-DNMT axis. To the best of our knowledge, this is also the demonstration that hepatocytes can be converted into multi-potent MLCs without ectopic gene delivery. Our findings support the occurrence of lineage reversibility in mammalian cells, indicating that such a phenomenon is not restricted to plants and non-mammalian vertebrates (Brockes, 1997, Brockes and Kumar, 2002, Weigel and Jurgens, 2002). Our findings demonstrate a role of DNMTs in hepatic lineage reversibility of MSCs, with individual DNMTs exerting opposite effects; DNMT1 repressed HD and promoted dHD; while DNMT3a promoted HD and repressed dHD. The two enzymes seem to have both unique and overlapping target cytosine-phosphate-guanine (CpG) sites, as shown by the promotion of all hepatogenic-specific genes and hepatic functions via DNMT1 silencing, but a more specific repression of the CYP family via DNMT3a silencing (Figure 4). In addition, the inhibitory effect of DNMT1 appears to be more predominant than the activating effect of DNMT3a, because a pan-DNMT inhibitor resulted in the promotion of HD (Figure 4). Although DNMT3b expression is also strongly associated with HD and responded to TGFβ1 stimulation, DNMT3b knockdown fails to affect hepatic gene expression and function. While more data are necessary to clarify the function of DNMT3b in HD, we speculate that the enzyme may cooperate with other chromatin modifiers. Epigenetic effects exerted by these modifiers may have been sufficient for HD regulation, explaining why we do not observe significant changes resulting from DNMT3b knockdown (Baubec et al., 2015, Morselli et al., 2015).