Comprehensive Bulk and Single-Cell RNA Sequencing Uncovers Senescence-Associated Biomarkers in Therapeutic Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) hold great promise in cell therapy but face diminished efficacy due to replicative senescence. This study presents a comprehensive transcriptional analysis of senescence in canine MSCs, a physiologically relevant model for human aging research. Employing this in vitro model, the present study demonstrates that late-passage cells exhibit hallmark features of senescence, including pronounced morphological alterations, elevated SA-beta-galactosidase activity, and considerable transcriptional changes. Key senescence marker genes, such as CDKN1A, CDKN2A, and CCND1 were upregulated, while critical regulators of cell proliferation and cell cycle progression genes showed downregulation in late-passage cells. GO enrichment analyses linked these gene expression changes to important senescence pathways, such as ERK1/2 signaling and hypoxia response, which were reflected in distinct transcriptional profiles across single-cell clusters. These changes were further explored through single-cell RNA sequencing, revealing heterogeneous distribution of senescent cell populations, including clusters enriched with senescence and SASP-related genes, such as IGFBP2, IGFBP7, and PAPPA, alongside reduced proliferation markers. By combining established marker genes such as CCND1, IGFBP2, CDKN1A, TGFB2 with a panel of senescence-associated genes comprising both previously identified and novel candidates from this study, such as CDKN2A, IGFBP7, CRYAB, ITGA2, PTN, NDUFA4L2, COL11A1, we observed a considerable higher proportion of senescent cells in late-passage clusters, underscoring the potential of this approach for accurate senescent cell identification and reinforcing the value of dog as a suitable model for studying human senescence mechanisms.