In vivo reprogramming drives dedifferentiation and enhances liver regeneration

Mammalian tissues have a limited regenerative capacity. Previous studies showed that dedifferentiation contributes to tissue regeneration in non-mammalian vertebrate species such as zebrafish and newt. However, dedifferentiation is rarely observed in mammalian tissues even in the neonatal stage and therefore artificial induction of dedifferentiation might enhance regeneration in mammalian tissues. Here we demonstrate that short-term expression of Yamanaka 4 factors (4F) induces dedifferentiation and proliferation in the liver by using lineage-traceable, hepatocyte-specific 4F inducible mouse model. Global transcriptome analysis shows that 4F expression transiently reduces the expression of hepatic-lineage markers and induces the expression of a large set of proliferative markers and epigenetic modifiers along with global epigenetic changes as assessed by DNA-accessibility analysis. More importantly, lineage-tracing experiments showed that 4F-expressing hepatocytes acquire liver stem/progenitor cell markers, suggesting that 4F induces partial reprogramming. Moreover, 4F enhances MyoD-mediated transdifferentiation in the liver, suggesting that 4F endows hepatocytes with plasticity. Lastly, 4F expression attenuated liver injury associated with more proliferative capacity and better survival rate, indicating that 4F enhances liver regeneration. Taken together, these results demonstrate that liver-specific 4F expression induces dedifferentiation and promotes liver regeneration. Overall design: The Hep-4F mouse model carries Alb-Cre, tetO-4F and LoxP-STOP-LoxP-rtTA-IRES-GFP (Fig. 1a). In this mouse model, LoxP-STOP-LoxP cassette is excised by Alb-Cre, resulting in expression of rtTA together with GFP as a lineage-tracing marker, allowing for specific 4F induction in the liver when doxycycline (Dox) is administrated. We performed bulk RNA-Seq, bulk ATAC-Seq to determine the effect of 4F on liver tissue dedifferentiation and regeneration. We also performed single-cell RNA-Seq to identify the 4F responses within each cell population of liver tissues using +Dox and -Dox tissue samples. Regarding the bulk sequencing analysis, hte mice were treated with and without Dox in a time-series setup and then the liver tissues were collected for sequencing. Another set of controls with mice treated with carbon tetrachloride (CCl4) to induce liver injury and liver regeneration were also included in the bulk sequencing.