Single-cell RNA sequencing to detect age-associated genes that identify senescent cells in the liver of aged mice

Senescent cells are predicted to occur and increase in animal tissues with aging. However, senescent cells in the tissues of aged animals remain to be identified. We refer to the marker genes to identify senescent cells in tissues as “age-associated genes”. In this study, we searched for age-associated genes to identify senescent cells in the livers of aged animals. We performed single-cell RNA sequencing (scRNA-seq) to screen candidates for age-associated genes using young and aged rat primary hepatocytes. To remove animal species specificity, gene expression analyses in mouse livers were performed, confirming age-associated increases in the mRNA expression levels of Glipr1, Clec12a, and Phlda3. Moreover, the mRNA expression levels of Glipr1 and Phlda3 were increased by stress-induced premature senescence using doxorubicin in primary hepatocytes and livers of young mice. Transcriptome data of aged rat hepatocytes suggested that Glipr1, Clec12a, and Phlda3 were expressed in almost identical cells. Fluorescence in situ hybridization (FISH) confirmed the presence of cells with abundant Glipr1, Clec12a, and Phlda3 mRNA in 27-month-old mouse primary hepatocytes, which are considered to be senescent cells. This study is the first to identify Glipr1, Clec12a, and Phlda3 as age-associated genes in the mouse liver.