Spatiotemporal liver dynamics shape hepatocellular heterogeneity and impact in vivo gene engineering

Background and Aims

Hepatocytes are the primary cells of the liver, essential for metabolism and important targets for in vivo gene therapy, which has the potential to treat monogenic diseases caused by defects in their functions. Integrating the transgene into the target cell’s genome is crucial for long-term expression following a single dose administered early in life, achievable through integrating vectors or genome editing. To ensure the persistence of the genetic modification throughout liver growth and turnover, it is also necessary to target the cells involved in these processes. Although previous research has focused on liver homeostasis and regeneration, the growth and maturation of hepatocytes remain not fully understood. Here, we explore the evolution of hepatocyte heterogeneity during liver growth and evaluate its implications for in vivo gene engineering.

Methods

We performed clonal tracing, single-cell and spatial transcriptomics on mouse livers of various ages. We evaluated the efficiency, stability, and lobule distribution of lentiviral gene transfer and targeted transgene integration.

Results

We found that a subset of clonogenic hepatocytes (15-20%) in the newborn liver generates >90% of the adult tissue and co-localizes with hematopoietic islands within a spatial niche. Preferential gene editing of these clonogenic hepatocytes resulted in an increased proportion of the gene-engineered liver area, supporting their role in liver growth. Age-dependent hepatocellular heterogeneity affected the efficiency of lentiviral gene delivery in vivo and its distribution throughout the hepatic lobule. The gradual establishment of metabolic zonation after weaning and elevated proteasome activity in the peri-central area in adults influenced the observed age-related outcomes.

Conclusion

These insights into spatiotemporal hepatocyte dynamics enhance our understanding of liver biology and have important implications for therapeutic strategies.

Impact and implications

We provide new insights into the spatiotemporal dynamics of the mouse liver during postnatal growth, highlighting both proliferative and transcriptomic heterogeneity among hepatocytes and their impact on the efficiency and distribution of in vivo lentiviral gene delivery and targeted gene editing. Understanding and manipulating the biological processes behind this heterogeneity can enhance gene transfer outcomes. We report that not all hepatocytes contribute equally to liver growth, indicating that effectively targeting clonogenic hepatocytes in the newborn liver is crucial for the long-term maintenance of therapeutic genetic modifications. Furthermore, this phenomenon can be leveraged to expand the pool of genetically corrected cells, as illustrated here by a targeted gene editing strategy. Finally, we reveal the existence of a tissue niche that supports the proliferation of both clonogenic hepatocytes and hematopoietic progenitors in neonatal livers. Gaining a deeper understanding of this niche and its signals could be beneficial for regenerative purposes.