Abstract
Tissues within an organism and even cell types within a tissue can age with different velocities. However, it is unclear whether cells of one type experience different aging trajectories within a tissue depending on their spatial location. Here, we used spatial transcriptomics in combination with single-cell ATAC-seq and RNA-seq, lipidomics and functional assays to address how cells in the male murine liver are affected by age-related changes in the microenvironment. Integration of the datasets revealed zonation-specific and age-related changes in metabolic states, the epigenome and transcriptome. The epigenome changed in a zonation-dependent manner and functionally, periportal hepatocytes were characterized by decreased mitochondrial fitness, whereas pericentral hepatocytes accumulated large lipid droplets. Together, we provide evidence that changing microenvironments within a tissue exert strong influences on their resident cells that can shape epigenetic, metabolic and phenotypic outputs.
Main
Aging is characterized by a general physiological decline that is accompanied by metabolic, epigenetic and transcriptional changes1. A common attribute for these alterations is an increased inter-individual heterogeneity as observed in large cohorts. Even on an organismal level within populations of genetically identical individuals, variability seems intrinsically interconnected with aging. For example, in cohorts of Caenorhabditis elegans or mice, some individuals die much earlier than others2.
It is largely appreciated that transcriptional variability increases with age3,4,5. Although whole-tissue omics approaches have been important to get an insight into the uniform changes that occur on the organ level during aging, such methods cannot investigate heterogeneity on a cellular level. It is therefore unresolved whether all cells of the same cell type in a tissue age in the same way or whether the location of the cells within a tissue matters in this context. The development of single-cell and spatial omics methods renders it now possible to obtain (spatially resolved) molecular profiles at close to single-cell resolution, thus providing promising tools for deciphering the multifaceted process of aging6.
The liver is a heterogeneous tissue that consists of hepatocytes arranged in repeating units of hexagonally shaped lobules. Blood flows into the lobule from portal veins and hepatic arteries at the corners of the lobules to the central veins. This architecture creates gradients of oxygen, nutrients and hormones7. This gradual change in the lobule’s microenvironment is also referred to as liver zonation8, and the resulting spatial division of labor is essential for the optimal function of the liver. For example, the outer highly oxygenated periportal lobule layers perform mitochondrial-dependent metabolic tasks such as β-oxidation whereas the low oxygen concentrations at the pericentral areas will drive glycolysis7. As hepatocytes are the primary cells that perform these metabolic processes and their metabolic characteristics depend on location, the liver is an attractive tissue to address the impact of location and metabolic state on the aging trajectory within a dedicated cell type.
Here, we used spatial transcriptomics, single-cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) in combination with lipidomics and functional assays for mitochondrial activity to reveal zonation-specific patterns of hepatocyte aging. An obvious phenotypic difference between the young and aging liver is the deposition of fat, which is concentrated mainly around the central vein. Using spatial transcriptomics, we gain insight into this phenotype’s molecular underpinnings by identifying genes involved in lipid biosynthetic pathways. On the other hand, the most substantial age-related changes in the periportal region are associated with mitochondrial dysfunction. Zonation and age are important axes of separation in single-cell ATAC data, indicating that location and organismal aging are major drivers of epigenomic changes in the liver. Using scRNA-seq on sorted hepatocytes, we show that transcriptional noise is buffered by an increase in ploidy. The data presented here shed light on age-related changes in liver tissue microenvironments and will serve as a resource for the hepatic and aging community.
Results
Spatial transcriptomics gives insights into zonation-specific and age-related metabolic rearrangements
Transcriptional profiling of the mouse liver has revealed alterations in metabolic pathways9,10,11, with a major fraction of genes contributing to alterations in lipid metabolism (Extended Data Fig. 1a,b and Supplementary Table 1). Changes in lipid metabolism have been described to occur during aging and recently lipidomics started to identify corresponding changes in lipid profiles12. Liver pathologies that involve fat deposition, such as nonalcoholic fatty liver disease (NAFLD) show a tendency towards zonated lipid deposition around the central area13, but we were not aware of any dataset investigating lipid deposition in the aging liver with respect to the specific zones. To assess the lipid deposition around the main zones, we performed RNAScope for pericentral (Cyp2e1, Glul) and periportal markers (Albumin, Cyp2f2) (ref. 14) combined with hematoxylin and eosin (H&E) staining in liver isolated from young (3–4 months) and old (18–22 months) mice (Fig. 1a and Extended Data Fig. 1c). Importantly, Sirius red staining showed no profound increase in liver fibrosis in old livers (Extended Data Fig. 1c). On the contrary, oil red O (O-R-O) staining (Extended Data Fig. 1d, upper panel) and immunohistochemical (IHC) staining for PLIN2 (Extended Data Fig. 1d, lower panel), a protein known to be enriched at the outer membrane of lipid droplets (LDs)15, showed that large LDs accumulate around the central vein in aged livers.
