Single-Cell RNA-Seq Analysis Reveals Four Stages of Replicative Senescence in Human Umbilical Vein Endothelial Cells

Cellular senescence is a phenomenon marked by an irreversible growth arrest with altered physiological properties. Many studies have focused on the characteristics of cells that have already entered a senescent state. However, to elucidate the mechanisms of cellular aging, it is essential to investigate the gradual transition of proliferative cells into senescent cells. We assumed that cellular senescence is a complex and multi-step process, and each stage exhibits unique traits. To test this hypothesis, we utilized publicly available single-cell RNA-Seq (scRNA-Seq) data from human umbilical vein endothelial cells (HUVECs) undergoing replicative senescence. We employed Seurat and Monocle 3 to capture the transition from proliferating to senescent states in HUVECs. Four clusters were identified, and each cluster displayed distinct expression patterns of cellular senescence markers and the senescence-associated secretory phenotypes (SASPs). We also employed SCENIC to identify the expression patterns of core transcription factors (TFs) during replicative senescence. While the majority of TFs exhibited a linear trend, HMGB1, FOSL1, SMC3, RAD21, SOX4, and XBP1 showed fluctuating expression patterns during replicative senescence. Furthermore, the expression patterns of these TFs exhibited slight differences in the ionizing radiation (IR) model of senescence. Overall, our study unveils the distinct characteristics of each phase during replicative senescence and identifies expression trends in TFs that may play pivotal roles in this process. These findings highlight the intricate nature of cellular senescence and provide new insights into the process of cellular aging.