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In addition to demonstrating the importance of DNMTs
In addition to demonstrating the importance of DNMTs, we show that TGFβ1 differentially regulated DNMTs, and that TGFβ1 is both necessary and sufficient to induce lineage conversion of hepatocytes into MLCs with phenotypes and multipotency resembling MSCs. Upon withdrawal of TGFβ1 on day 28, we observed that Hep-converted MLCs possessed MSC-like DNMTs expression patterns (Figures 6N and 6O) due to the induction of endogenous TGFβ1 expression (Figures 6H and 6L). This outcome supports the existence of an MSC-specific methylome that maintains MLC phenotypes and multipotency.
α-Fetoprotein (AFP) is a marker indicating early hepatic fate. Although AFP is not detectable in hepatocytes in vivo, expression of AFP may be detected in the in vitro culture conditions owing to a loss of contact (Gleiberman et al., 1989). Moreover, TGFβ1 downregulated albumin and AFP expression and induced fibroblast morphology and mesenchymal marker expression (mesenchymal fate) in Hep3B transferase (Caja et al., 2011). Therefore, AFP may serve as a marker to distinguish hepatic fate from mesenchymal fate and allows us to track cellular conversion from the former to the latter.
Fibrosis is often a consequence of various chronic liver diseases and leads to hepatic failure as well as cancer. TGFβ1, a master inducer of EMT, orchestrates fibrogenesis. Persistent TGFβ1 stimulation converts both hepatocytes and MSCs into myofibroblasts and participates in liver fibrosis, and even contributes to the development of tumor (Sekiya et al., 2016). While blocking TGFβ signaling in hepatocytes alone attenuates liver fibrosis (Dooley et al., 2008) and facilitates liver regeneration (Karkampouna et al., 2016), MLCs may be at an intermediate state during the transition to myofibroblasts. Therefore, inhibition of the TGFβ1-DNMT axis in vivo may convert Hep-derived myofibroblasts back into MLCs or hepatocytes, and may attenuate liver fibrosis and facilitate liver regeneration. Our previous studies established a protocol for functional dHeps with therapeutic potentials when transplanted in vivo (Jiang et al., 2014, Kuo et al., 2008, Lee et al., 2004). However, dHeps required 28 days of in vitro differentiation, a lengthy duration that hampers their clinical application. In this study, we successfully accelerate differentiation to 14 days by silencing DNMT1 (Figure 4). Transplantation of dHeps with TGFβ receptor inhibitor or DNMT1 antagonist may stabilize hepatic characteristics of transplanted dHeps. On the other hand, although transplanted dHeps may de-differentiate into ddHeps, ddHeps may promote liver regeneration like MSCs through immunomodulation and trophic support (Christ et al., 2015). Hep-converted MLCs have proliferation ability and higher hepatic lineage potential (Figure 7B). Therefore, these cells may also serve as an alternative cell source for liver regeneration (Du et al., 2014, Takeuchi et al., 2015).
Experimental Procedures
Author Contributions
Acknowledgments
This work was supported in part by the Novel Bioengineering and Technological Approaches to Solve Two Major Health Problems in Taiwan sponsored by the Taiwan Ministry of Science and Technology Academic Excellence Program under grant number MOST 105-2633-B-009-003. The authors acknowledge financial support from the Ministry of Science and Technology, Taiwan (MOST103-2314-B-010-053-MY3, MOST 106-2321-B-010-008, MOST 106-2911-I-010-502, and MOST 105-2633-B-010-002). This study was also supported by Aiming for the Top University Plan, a grant from Ministry of Education. The authors also acknowledge the technical support provided by Flow Cytometry Core Facility of National Yang Ming University and Imaging Core Facility of Nanotechnology of the UST-YMU. We acknowledge Prof. Ting-Fen Tsai from National Yang-Ming University, Taiwan for the kind gift of E12.5 fetal livers and thank Prof. Chi-Chang Juan from National Yang-Ming University, Taiwan for the kind gift of FL83B cells.