Comprehensive bulk and single-cell RNA sequencing uncovers senescence-associated biomarkers in therapeutic mesenchymal stem cells

Pekker, Emese and Qorri, Erda and Enyedi, Márton Zs. and Szukacsov, Valéria and Ayaydin, Ferhan and Szabó-Kriston, Éva and Csányi, Bernadett and Mórocz, Mónika and Sükösd, Farkas and Kiss-Tóth, Endre and Haracska, Lajos (2026) Comprehensive bulk and single-cell RNA sequencing uncovers senescence-associated biomarkers in therapeutic mesenchymal stem cells. SCIENTIFIC REPORTS, 16 (1). No. 999. ISSN 2045-2322

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Abstract

Mesenchymal stem cells (MSCs) hold great promise in cell therapy, but their effectiveness declines with repeated cell divisions due to senescence. Canines, sharing aging characteristics with humans, serve as a valuable model to study this process in a translational context. In the present study, we performed an in-depth characterization of senescence in canine MSCs using a combination of morphological, molecular, and transcriptomic analyses. Early (P2) and late-passage (P6) canine MSCs were characterized using a combination of senescence-associated beta-galactosidase staining, cell cycle profiling, and both bulk and single-cell RNA sequencing to capture global transcriptional changes. By employing a passage-based in vitro approach, the present study demonstrates that late-passage cells (P6) compared to early-passage cells (P2) exhibit hallmark features of senescence, including morphological alterations, elevated SA-beta-galactosidase activity, and considerable transcriptional changes. These changes were represented by significant upregulation of established senescence marker genes, alongside potential novel candidates and downregulation of genes associated with cell cycle progression and proliferation. Moreover, single-cell RNA sequencing uncovered heterogeneous distribution of senescent subpopulations, upregulation of SASP-related genes and reduced proliferation markers. Our findings demonstrate that combining classical markers with bulk and single-cell RNA sequencing facilitates senescent cell identification while improving quality control for clinical MSC samples.

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