Single-cell RNA-seq analysis reveals the multi-step process of cellular senescence.

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 hypothesized that cellular senescence is a complex, multi-step process in which each stage is characterized by distinct cellular features and transcription factor expression patterns. 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 of these TFs exhibited different patterns 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 SASPs and TFs that may play pivotal roles in this process. Unlike previous bulk RNA-seq studies, this work uniquely integrates single-cell trajectory and transcription factor dynamics to decode phase-specific molecular signatures during replicative senescence. Here, we identify key transcription factors potentially involved in senescence induction and provide novel insights into the regulatory complexity of cellular aging.