Fluorescence-Guided Spatial Mapping of p21-Expressing Senescent Cells in Aged Liver at Single-Cell Resolution

Aging is a regulated process marked by the accumulation of senescent cells, which remain viable but no longer divide. Senescent cells contribute to age-associated phenotypes and diseases, including osteoarthritis, dementia, and cancer, but their scarcity and heterogeneity have limited study. Here, we developed a fluorescence-guided high-resolution spatial transcriptomic profiling approach to precisely locate and profile p21-reporter-positive cells in aged liver. This method enabled unbiased detection of a p21-associated, senescence-enriched cell population and revealed its diverse cellular identities, including hepatocytes, macrophages, neutrophils, and plasma cells. Our analysis further showed that activated macrophages and hepatic stellate cells were more likely to exhibit a p21 positive (p21⁺) state than their resting counterparts. Transcriptomic profiling of p21-expressing cells indicated heterogeneous senescence-associated secretory phenotype (SASP) programs, with distinct inflammatory and remodeling signatures across cell identities and their spatial positions. In parallel, we identified an aggregation of interferon-stimulated gene (ISG)-expressing cells with limited overlap with p21 positivity, suggesting a distinct aging-associated stress program. Taken together, our fluorescence-guided spatial transcriptomic framework enables high-resolution, single-cell mapping of senescence in situ, delineating both senescent cell type specificity and cell identity-independent senescence programs, thereby advancing a more comprehensive understanding of regulatory mechanisms underlying aging.