Single-cell transcriptomic analysis uncovers diverse and dynamic senescent cell populations

Senescence is a state of enduring growth arrest triggered by sublethal cell damage. Given that senescent cells actively secrete proinflammatory and matrix-remodeling proteins, their accumulation in tissues of older persons has been linked to many diseases of aging. Despite intense interest in identifying robust markers of senescence, the highly heterogenous and dynamic nature of the senescent phenotype has made this task difficult. Here, we set out to comprehensively analyze the senescent transcriptome of human diploid fibroblasts at the individual-cell scale by using single-cell RNA-sequencing analysis through two approaches. First, we characterized the different cell states in cultures undergoing senescence triggered by different stresses; we found distinct cell subpopulations that expressed mRNAs with roles in growth arrest, survival, and the secretory phenotype. Second, we characterized the dynamic changes in the transcriptomes of cells as they developed etoposide-induced senescence; by tracking cell transitions across this process, we found two different senescence programs that developed divergently, one in which cells expressed traditional senescence markers and one in which cells expressed long noncoding RNAs and splicing was dysregulated. Finally, we obtained evidence that the proliferation status at the time of senescence initiation affected the path of senescence, as determined based on the expressed RNAs. We propose that a deeper understanding the transcriptomes of different senescent cell programs will help develop more effective interventions directed at this detrimental cell population. Overall design: WI-38 proliferating (PDL 24, CTRL) fibroblasts were grown until their replicative potential was exhausted (PDL 57) and they reached replicative senescence (RS), exposed to 10 Gray of Ionizing Radiation (IR) then cultured 10 days, treated with 50 µM Etoposide (ETO) for 10 days. Additionally, WI-38 cells were either treated with DMSO or 50 µM etoposide and collected at 0 (untreated) 1, 2, 4, 7, and 10 days after treatment. Then, cells were analyzed using single-cell RNA sequencing.