Transcriptomic and epigenetic mechanisms controlling cholangiocyte transdifferentiation into hepatocytes

BACKGROUND & AIMS: The liver's ability to regenerate is well established, yet the mechanisms involved in chronic liver disease remain to be fully uncovered. Recent animal and human studies showed that transdifferentiation between cholangiocytes and hepatocytes could play a role in this process. Here, we uncover the molecular mechanisms that drive this cellular plasticity in human patients. METHODS: We derived intrahepatic cholangiocyte organoids from the three liver lobes of patients with end-stage metabolic dysfunction-associated steatohepatitis. The resulting organoid lines were differentiated into biphenotypic cells expressing hepatocyte markers mimicking the transdifferentiation process occurring during chronic injury in vivo. We then combined single-nuclei RNA sequencing and single-nuclei ATAC sequencing to uncover molecular pathways and epigenetic regulations required for the in vitro conversion of cholangiocytes into hepatocytes. These analyses led to the identification of transcription factors regulating this process. RESULTS: Our analyses suggest that cholangiocyte plasticity is independent of their liver location and that specific lobes are unlikely to be more regenerative than others. Single-nuclei ATAC sequencing analyses identify the opening of chromatin in intrahepatic cholangiocyte organoids as a mechanism of plasticity, while functional validations reveal that the transcription factor HNF4G could play a key role in the induction of hepatocyte markers. The relevance of these findings was validated against single-nuclei RNA sequencing data from liver biopsies of patients with end-stage metabolic dysfunction-associated steatohepatitis. CONCLUSIONS: Overall, our results confirm that cholangiocytes can transdifferentiate into hepatocytes. This process is promoted by HNF4G, it involves epigenetic remodelling, and is consistent across the three liver lobes. This knowledge paves the way for future therapies aiming to enhance liver regeneration by increasing cholangiocyte plasticity. IMPACT AND IMPLICATIONS: Recent reports proposed the transdifferentiation of cholangiocytes into hepatocytes as the main regenerative process occurring during chronic liver diseases. However, the mechanisms involved remain to be fully uncovered. Our study addresses this knowledge gap in humans. We first showed that cholangiocyte organoids derived from different liver lobes have equivalent capacity to produce cells expressing hepatocyte markers. Furthermore, detailed single-nucleus analyses of transcriptomic and epigenetic signatures in cholangiocytes transdifferentiating into hepatocytes in vitro revealed that this process involves genome-wide chromatin reorganization, confirming that cellular plasticity is closely linked to epigenetic regulation. Finally, these experiments indicated the expression of HNF4G in transdifferentiating cholangiocytes, while functional studies established that HNF4G upregulation is sufficient to increase the expression of hepatocyte markers in cholangiocytes. Thus, manipulating HNF4G expression can act as a potential therapeutic target for promoting liver plasticity and regeneration in end-stage liver disease.