Morocz, M. | bioRxiv Science

bioRxiv2025

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

BackgroundMesenchymal 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. MethodsIn 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. ResultsBy 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. ConclusionsOur 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.

cell biology↗

bioRxiv2023

The loss of DNA polymerase epsilon accessory subunits POLE3-POLE4 leads to BRCA1-independent PARP inhibitor sensitivity

The clinical success of PARP1/2 inhibitors prompts the expansion of their applicability beyond homologous recombination deficiency. Here, we demonstrate that the loss of the accessory subunits of DNA polymerase epsilon, POLE3 and POLE4, sensitizes cells to PARP inhibitors. We show that the sensitivity of POLE4 knockouts is not due to a compromised response to DNA damage or homologous recombination deficiency. Instead, POLE4 deletion generates replication stress with the accumulation of single-stranded DNA gaps upon PARP inhibitor treatment. In POLE4 knockouts, replication stress leads to elevated DNA-PK signaling revealing a role of POLE4 in regulating DNA-PK activation. Moreover, POLE4 knockouts show synergistic sensitivity to the co-inhibition of ATR and PARP. Finally, POLE4 loss enhances the sensitivity of BRCA1-deficient cells to PARP inhibitors and counteracts acquired resistance consecutive to restoration of homologous recombination. Altogether, our findings establish POLE4 as a promising target to improve PARP inhibitor driven therapies and hamper acquired PARP inhibitor resistance.

cell biology↗