Spatiotemporal hepatocyte heterogeneity impacts in vivo gene engineering
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, 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, clonal analysis, and lineage tracing. We observe the progressive establishment of metabolic zonation of hepatocytes following weaning, with the specification of the centrilobular identity only in adults. We report that only a fraction of hepatocytes proliferate in the newborn liver, generating most of the adult tissue, and that preferential gene editing of the more proliferating hepatocytes allows expansion of the genetically engineered liver area. 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 vector administration. Overall, our findings provide new insights into the spatiotemporal dynamics of hepatocytes during post-natal growth, which extend our understanding of liver biology and have important implications for therapeutic applications. Overall design: Liver samples from 3 mice per age (2 and 8 weeks old) were collected on ice. Single nuclei suspension was obtained from the livers by Singleron Biotechnologies and then equally pooled into a single sample for each age group to be processed for single nuclei RNA sequencing.