Different Stages of Quiescence, Senescence, and Cell Stress Identified by Molecular Algorithm Based on the Expression of Ki67, RPS6, and Beta-Galactosidase Activity

Abstract

During their life span, cells have two possible states: a non-cycling, quiescent state (G0) and a cycling, activated state. Cells may enter a reversible G0 state of quiescence or, alternatively, they may undergo an irreversible G0 state. The latter may be a physiological differentiation or, following a stress event, a senescent status. Discrimination among the several G0 states represents a significant investigation, since quiescence, differentiation, and senescence are progressive phenomena with intermediate transitional stages. We used the expression of Ki67, RPS6, and beta-galactosidase to identify healthy cells that progressively enter and leave quiescence through G0-entry, G0 and G0-alert states. We then evaluated how cells may enter senescence following a genotoxic stressful event. We identified an initial stress stage with the expression of beta-galactosidase and Ki67 proliferation marker. Cells may recover from stress events or become senescent passing through early and late senescence states. Discrimination between quiescence and senescence was based on the expression of RPS6, a marker of active protein synthesis that is present in senescent cells but absent in quiescent cells. Even taking into account that fixed G0 states do not exist, our molecular algorithm may represent a method for identifying turning points of G0 transitional states that continuously change.

Keywords: cell cycle; mesenchymal stem cells; quiescence; senescence.

Figure 1

Follow-up of cell-cycle exit and cell-cycle re-entry. Panel (A): Graphic summary of the experimental procedure. MSCs at passage 3 (P3) were serum starved for 36 h and, at different time points (AP, S1, S2, S3, Q), cell samples were collected for analysis. Cells were then incubated in a medium containing serum and were cultivated for another 36 h. At different time points (T1, T2, T3, P), samples were collected for evaluation. Panel (B): Cell-cycle profiles of MSC samples collected at different time points during starvation and serum re-supplementation. The * indicates the statistical difference between AP, chosen as reference, and S1, S2, S3, and Q (* p < 0.05, ** p < 0.01, *** p < 0.001). The # indicates the statistical difference between Q, chosen as the reference, and T1, T2, T3, and P (# p < 0.05, ## p < 0.01, ### p < 0.001). Panel (C): Western blots of MSC samples collected at different time points during starvation and serum re-supplementation. The graphs show the quantification of Western blot bands performed by using GAPDH as the loading control. The * indicates the statistical difference between AP, chosen as the reference, and Q (* p < 0.05, ** p < 0.01, *** p < 0.001). The # indicates the statistical difference between Q, chosen as the reference, and P (# p < 0.05, ## p < 0.01).

Figure 2

Molecular characterization of cycling and quiescent cells. Panel (A): Representative micrographs of MSCs stained to identify nuclei (DAPI), pRPS6, and Ki67 and to evaluate SA-β-gal activity. See also Supplemental File S1. The scale bar corresponds to 100 microns. Panels (B–E): Graphs show the percentage of cycling, G0-entry, quiescent and G0-alert cells at different time points (AP, S1, S2, S3, Q, T1, T2, T3, P). Below each graph, the molecular algorithm we used to identify the different phenotypes is indicated. In each graph, the * indicates the statistical difference between AP, chosen as the reference, and S1, S2, S3, and Q (* p < 0.05, ** p < 0.01, *** p < 0.001). The # indicates the statistical difference between Q, chosen as the reference, and T1, T2, T3, and P (# p < 0.05, ## p < 0.01, ### p < 0.001).

Figure 3

Molecular characterization of stressed and senescent cells. Panel (A): Graphic summary of experimental procedure. MSCs at passage 3 (P3) were treated for 0.5 h with H₂O₂ and then incubated in cell culture medium for 64 h. At different time points (AP, SEN1, SEN2, SEN3, SEN4, SEN5), cell samples were collected for analysis. Panel (B): Representative micrographs of MSCs stained to identify nuclei (DAPI), pRPS6, and Ki67 and to evaluate SA-β-gal activity. The scale bar correspond to 100 microns. See also Supplemental File S3. Panels (C–G): Graphs show the percentage of cycling, stressed, pre-senescent and senescent cells at different time points (AP, SEN1, SEN2, SEN3, SEN4, SEN5). Below each graph, the molecular algorithm we used to identify the different phenotypes is indicated. In each graph, the * indicates the statistical difference between AP, chosen as the reference, and the other time points (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

Cell cycle and Western blot analysis of senescent cells. Panels (A,B): Cell-cycle profiles and western blot analysis of MSC samples collected at different time points following hydrogen peroxide treatment. The * indicates the statistical difference between AP, chosen as the reference, and the other time points (* p < 0.05, ** p < 0.01, *** p < 0.001). In panel (A), the graphs show the quantification of western blot bands that was performed by using GAPDH as the loading control.