Spatio-temporal dynamics of the liver shapes hepatocytes heterogeneity and impacts in vivo gene transfer and editing

Abstract The liver is a central organ for metabolism and hepatocytes are the main cell type responsible for most of its functions. Several previous studies investigated the cell types involved in tissue homeostasis and regeneration 1–4 , however the mechanisms underlying post-natal liver growth and establishment of the mature hepatocyte phenotypes remain to be fully understood. Here we investigate liver tissue dynamics in mice during growth and adulthood, by spatial transcriptomics 5 , clonal analysis, and lineage tracing. We observe progressive establishment of metabolic zonation of hepatocytes following weaning, with specification of the centrilobular identity only in adults. We report that only a fraction of hepatocytes proliferates in the newborn liver, generating most of the adult tissue and that preferential genetic modification (either gene transfer or gene editing) of the more proliferating hepatocytes allows expansion of the genetically engineered liver area, stably maintained throughout tissue homeostasis. We also describe age-dependent differences in the efficiency and distribution of lentiviral in vivo gene delivery, with higher efficiency of gene transfer in young compared to adult animals and a skewed localization within the liver lobule. We identify high proteasome activity in the peri-central lobular area as the major determinant of the observed outcome and successfully revert it by proteasomal inhibition before gene transfer. Overall, our findings provide new insights into the spatio-temporal dynamics of the liver during post-natal growth and hepatocyte heterogeneity, which extends our understanding of liver biology and have important implications for therapeutic applications.