Introduction: Hepatocyte transplantation (HTx) represents a promising alternative to liver transplantation for the treatment of metabolic liver disorders and liver failure. However, its clinical application has been limited by poor engraftment and repopulation efficiency of donor hepatocytes. In this study, we propose a novel approach to create a competitive growth advantage for donor hepatocytes by inducing senescence in host hepatocytes.

Methods: A novel murine model of HTx was established by inducing replicative senescence in host hepatocytes via inhibition of hepatocyte cytokinesis. An in vivo approach for inducing senescence in host hepatocyte was proposed through delivery of Cas9 mRNA and single-guide RNA (sgRNA) targeting cytokinesis, mediated by hepatocyte-selective lipid nanoparticles (LNPs). The long-term repopulation of HTx was evaluated in multiple liver injury models after inhibition of host hepatocyte cytokinesis.

Results: Inhibition of host hepatocyte cytokinesis induced characteristic senescent features, including cell cycle arrest, aging morphology, upregulation of senescence markers, and senescence-associated secretory phenotype. Under the condition of host hepatocytes cytokinesis inhibition, donor hepatocytes exhibited significantly enhanced long-term repopulation compared to controls (86.5% vs. 0%, 2 months post-HTx). The hepatocyte-selective LNPs achieved efficient in vivo targeting of hepatocyte, with a targeting efficiency exceeding 95%. Administration of LNP-Cas9-sgRNA targeting cytokinesis markedly enhanced the long-term repopulation of donor hepatocyte in normal livers, as well as in multiple liver injury models (e.g. CCL₄-induced fibrosis, DDC diet-induced cholestasis, high-fat diet-induced steatohepatitis). 

Conclusion: Host hepatocyte senescence is a key determinant for successful donor cell engraftment and repopulation in HTx. In vivo delivery of LNP-Cas9-sgRNA targeting cytokinesis provides a potent and translational strategy to enhance therapeutic efficacy of HTx, opening a new avenue for promoting hepatocyte therapy for liver diseases.