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Spatiotemporal hepatocyte heterogeneity impacts in vivo gene engineering [snRNA-Seq]

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, 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE267146 | GEO | 2024/05/15

REPOSITORIES: GEO

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Project description:Wild-type and AHR-KO rats harboring a 29-bp deletion mutation in exon 2 of the AHR gene were used in this study. Wild-type and AHR-KO rats were generated from heterozygous breeding stocks generated on a Sprague-Dawley outbred background and maintained at the Hamner Institutes for Health Sciences, Research Triangle Park, North Carolina Persistent activation of the aryl hydrocarbon receptor (AHR) is believed to play a key role in the mode-of-action for TCDD induced rat liver tumorigenesis. It has been hypothesized that the cellular responses of hepatocytes to AHR activation may differ across regions of the liver lobule and that zone-specific effects of AHR agonists may play a role in the pathogenesis of rat liver tumorigenesis. Dose-dependent changes in gene expression were observed in both populations of hepatocytes collected from WT rats which were consistent with activation of AHR signaling. No significant or dose-dependent changes in gene expression were observed in samples from AHR-KO rats. In addition, evidence of inflammatory signaling pathway activation was observed only in centrilobular hepatocytes. Evidence of cell adhesion pathway enrichment was observed only in periportial hepatocytes. Benchmark dose analysis demonstrated that dose-dependent changes in gene expression occurred at lower doses in centrilobular as compared to periportal hepatocytes. These results indicate zone-specific differences in the sensitivity and response of hepatocytes to persistent AHR activation. AHR-KO and WT rats (n = 10 rats/dose/genotype) were dosed by oral gavage (4-5 doses / week, 19 total doses) with varying concentrations of TCDD in corn oil (0, 3, 22, 100, 300, 1000 ng/kg/day). Cryopreserved liver slices collected at necropsy were thawed, cryosectioned (12 Âµm thickness), fixed with ethanol and âquick-stainedâ with hematoxylin. Centrilobular and periportal hepatocytes were collected using laser capture microdissection. Total RNA was extracted, amplified and labeled using low-yield techniques and global transcriptomic profiles were obtained using Affymetrix Gene-Titan peg arrays.

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Spatiotemporal hepatocyte heterogeneity impacts in vivo gene engineering [snRNA-Seq]

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