Transcriptional Profiling at Single-Cell Resolution Reveals Diversity and Regulatory Networks of Primary and Secondary Senescent Cells.

Senescent cells accumulate with age following stress-induced cell cycle arrest triggered by DNA damage, oncogene activation, and replicative exhaustion. While they contribute to tissue repair and tumor suppression, their persistent senescence-associated secretory phenotypes (SASPs) drive age-related diseases. The heterogeneity of senescent cell populations, particularly the distinction between primary and secondary senescence, remains incompletely understood at single-cell resolution. Here, we established models of primary senescence by X-ray irradiation of human renal epithelial cells and secondary senescence by exposing proliferating cells to conditioned media from primary senescent cells. Single-cell RNA sequencing revealed structured transcriptional trajectories culminating in distinct terminal clusters in primary (C5, C6, and C8) and secondary (C3, C5, and C7) senescence. Primary senescence preferentially converged on extracellular matrix- and fibrosis-associated programs, whereas secondary senescence exhibited more inflammatory and signaling-responsive programs, while both contexts shared a partially overlapping transcriptional module enriched in stress-response and cytokine-related transcriptional modules. We identified subtype-associated genes distinguishing primary from secondary senescent cells, as well as candidate transcriptional regulators-such as HMGA1, NFKB1, and JUNB-associated with conserved and context-specific senescence programs. This study provides a single-cell-resolved transcriptional map of divergent and shared molecular features relevant to renal aging and disease.