Direct induction of induced hepatocytes (iHeps) from fibroblasts holds potential as a strategy for regenerative medicine but until now has only been shown in culture settings. Here, we describe in vivo iHep formation using transcription factor induction and genetic fate tracing in mouse models of chronic liver disease. We show that ectopic expression of the transcription factors FOXA3, GATA4, HNF1A, and HNF4A from a polycistronic lentiviral vector converts mouse myofibroblasts into cells with a hepatocyte phenotype. In vivo expression of the same set of transcription factors from a p75 neurotrophin receptor peptide (p75NTRp)-tagged adenovirus enabled the generation of hepatocyte-like cells from myofibroblasts in fibrotic mouse livers and reduced liver fibrosis. We have therefore been able to convert pro-fibrogenic myofibroblasts in the liver into hepatocyte-like cells with positive functional benefits. This direct in vivo reprogramming approach may open new avenues for the treatment of chronic liver disease.

Financial support statement: This study was supported by the Deutsche Krebshilfe (111147).Data availability: GEO accession numbers for miRNA expression array data (Exiqon) and gene expression profiling data (HZI) are NCBI GEO: GSE152920 and GSE152950. Authors contributions: A.B conceived the idea, designed the study, and provided the conceptual framework for the study. R-M.K, QH, and A.B performed all experiments and analyzed the data. L.vD and J.D helped with the cloning and the in vitro experiments. H-C.T helped with the xenograft experiments. Q.Y helped with animal experiments. O.O helped with the metabolic assays and luciferase reporter assays for the revised manuscript. AG provided us with the TRE-Myc, TRE-RAS, and LAP-tTA transgenic mouse models, which we bred and crossed to obtain the double transgenic LT2/MYC and LT2/RAS mice. A.B wrote the manuscript with the help of M.O, AD.S. and A.G. Y.X and R.Q helped with collection and analyses of human HCC tumor and matched non-tumor biopsies (China). A.B, M.P.M, M.O, and AD.S, provided conceptual evaluation of the project.

Background & Aims: Fibrosis, a cardinal feature of a dysfunctional liver, significantly contributes to the ever-increasing mortality due to end-stage chronic liver diseases. The cross-talk, between hepatocytes and hepatic stellate cells, is suggested to play a key role in fibrosis progression. While ample efforts have been devoted to elucidate hepatic stellate cells' functions during liver fibrosis, the regulatory functions of hepatocytes remain elusive. Methods: Using an unbiased functional microRNA screening, we investigated the ability of hepatocytes to regulate fibrosis by fine-tuning gene expression via microRNA modulation. The in vivo functional analyses were performed by inhibiting microRNA in hepatocytes using adeno-associated virus in carbon-tetrachloride-and 3,5-di diethoxycarbonyl-1,4-dihydrocollidine-induced liver fibrosis. Results: We discovered that blocking microRNA-221-3p function in hepatocytes during chronic liver injury facilitates recovery of the liver and faster resolution of the deposited extracellular matrix. Further, we demonstrate that reduced secretion of CC motif chemokine ligand 2, due to post-transcriptional regulation of G protein alpha inhibiting activity polypeptide 2 by microRNA-221-3p, mitigates liver fibrosis. Page%5%of%44% % % Conclusions: Collectively, microRNA modulation in hepatocytes, an easy-to-target cell type in the liver, may serve as a potential therapeutic approach for liver fibrosis.