Effects of senolytic drugs on human mesenchymal stromal cells

Abstract

Background: Senolytic drugs are thought to target senescent cells and might thereby rejuvenate tissues. In fact, such compounds were suggested to increase health and lifespan in various murine aging models. So far, effects of senolytic drugs have not been analysed during replicative senescence of human mesenchymal stromal cells (MSCs).

Methods: In this study, we tested four potentially senolytic drugs: ABT-263 (navitoclax), quercetin, nicotinamide riboside, and danazol. The effects of these compounds were analysed during long-term expansion of MSCs, until replicative senescence. Furthermore, we determined the effect on molecular markers for replicative senescence, such as senescence-associated beta-galactosidase staining (SA-β-gal), telomere attrition, and senescence-associated DNA methylation changes.

Results: Co-culture experiments of fluorescently labelled early and late passages revealed that particularly ABT-263 had a significant but moderate senolytic effect. This was in line with reduced SA-β-gal staining in senescent MSCs upon treatment with ABT-263. However, none of the drugs had significant effects on the maximum number of population doublings, telomere length, or epigenetic senescence predictions.

Conclusions: Of the four tested drugs, only ABT-263 revealed a senolytic effect in human MSCs-and even treatment with this compound did not rejuvenate MSCs with regard to telomere length or epigenetic senescence signature. It will be important to identify more potent senolytic drugs to meet the high hopes for regenerative medicine.

Keywords: ABT-263; DNA methylation; Danazol; Mesenchymal stromal cells; Nicotinamide riboside; Quercetin; Senescence; Senolytic drugs; Telomere attrition.

Fig. 1

ABT-263 has senolytic effects in human MSCs. a Exemplary phase-contrast and fluorescence microscopic images of non-senescent cells (PKH67, green, passage 3) and senescent cells (PKH26, red, passage 12). w/o = without additional drug;  Scale bars = 100 μm. b Normalized fractions of cells determined by flow cytometric quantification of red (senescent) and green (non-senescent) cells indicate that particularly ABT-263 reduced senescent cells (normalized to untreated controls, n = 3, mean ± SD; *p ≤ 0.05). c Dose–response curves analysed after 3 days of treatment with drugs and viability estimated by flow-cytometric assays (normalized to untreated controls, n = 3, mean ± SD; individually assessed for early and late passage cells). d SA-β-gal staining performed either in non-senescent (passage 3) or senescent (passage 12) MSCs upon treatment with senolytic drugs for 3 days (exemplary images depicted, scale bars = 200 μm). NR nicotinamide riboside

Fig. 2

Treatment with senolytic drugs did not support long-term expansion or affect molecular markers of senescence. Long-term growth curves of cumulative population doublings (cPDs) a after initial pulse treatment for 3 days (d; period indicated by dashed lines) or b with continuous treatment with drugs (results exemplarily depicted for one donor). c Relative telomere length measured as telomere to single copy gene (T/S) ratio (monochrome multiplex qPCR) of non-senescent and senescent MSCs with and without senolytic drug treatment (n = 3, mean ± SD; **p ≤ 0.01). d Comparison of real and predicted passage numbers in non-senescent and senescent MSCs with and without senolytic drug treatment. e Average number of predicted passages directly compared for the different treatments (n = 3, mean ± SD). NR nicotinamide riboside