Individual response to mTOR inhibition in delaying replicative senescence of mesenchymal stromal cells

Abstract

Background aims: Delaying replicative senescence and extending lifespan of human mesenchymal stromal cells (MSCs) may enhance their potential for tissue engineering and cell based therapies. Accumulating evidence suggests that inhibitors of the mTOR signaling pathway, such as rapamycin, constitute promising pharmacological agents to retard senescence and extend stemness properties of various progenitor cell types. Here, we investigated whether the ability of rapamycin to postpone replicative senescence varies among bone marrow MSC samples (BM-MSCs) derived from different healthy donors, and explored the molecular mechanisms that drive rapamycin-mediated lifespan increment.

Methods: BM-MSCs at early passages were serially passaged either in absence or continuous presence of rapamycin and the number of cell population doublings until growth arrest was measured. The inhibition of mTOR signaling was assessed by the phosphorylation status of the downstream target RPS6. The expression levels of several senescence and pluripotency markers at early and late/senescent passages were analyzed by RT-qPCR, flow cytometry and western blot.

Results: We found that the lifespan extension in response to the continuous rapamycin treatment was highly variable among samples, but effective in most BM-MSCs. Despite all rapamycin-treated cells secreted significantly reduced levels of IL6, a major SASP cytokine, and expressed significantly higher levels of the pluripotency marker NANOG, the expression patterns of these markers were not correlated with the rapamycin-mediated increase in lifespan. Interestingly, rapamycin-mediated life-span extension was significantly associated only with repression of p16INK4A protein accumulation.

Conclusions: Taken together, our results suggest that some, but not all, BM-MSC samples would benefit from using rapamycin to postpone replicative arrest and reinforce a critical role of p16INK4A protein downregulation in this process.

Fig 1. Lifespan extension and growth kinetics in response to continuous mTOR inhibition varies among different BM-MSC samples.

(A) Cumulative population doubling (PD) curves of BM-MSC samples derived from 5 healthy young donors (BM09, BM12, BM13, BM16 and BM18) until replicative arrest in control conditions (DMSO) or in the continuous presence of rapamycin (RAPA). Each symbol represents a passage of rapamycin-treated (triangle) and untreated (dot) cells. The passage when untreated BM09, BM13, BM16 and BM18 samples entered replicative senescence while the corresponding rapamycin-treated cells continue to proliferate is referred to as the “deviation passage” and is indicated by an arrow. Since rapamycin had no impact on lifespan extension of BM12, no “deviation passage” was assigned for this sample. (B) PD time (PDT) of BM-MSC samples at each passage in control conditions (dot) or in the continuous presence of rapamycin (triangle). (C) Cumulative PD curves of BM09 in which rapamycin was removed (RAPA removal) until cells ceased growth and then replaced in culture medium (RAPA replacement). Each dot represents a passage of cells. Data shown in panels A to C are representative of results from at least two independent experiments.

Fig 2. The ability of BM-MSCs to respond to rapamycin continues along the entire replicative lifespan.

mTOR signaling inhibition is reflected by the phosphorylation status of RPS6, a downstream target of the mTOR pathway. Phosphorylated RPS6 (pRPS6) was quantified by western blot in untreated and rapamycin-treated BM09 and BM18 samples at passages when untreated cells entered replicative arrest (S-R = senescent passage without rapamycin; D+R = deviation passage with rapamycin), as well as when rapamycin-treated cells stop proliferating (S+R = senescent passage with rapamycin). Since rapamycin had no impact on lifespan extension of BM12, pRPS6 levels were measured in untreated and rapamycin-treated cells at the same senescent passage. Band intensities were densitometrically evaluated and bar graphs above bands represent the densitometric values of pRPS6 normalized to the loading control (β-actin). pRPS6 levels were greatly reduced by continuous rapamycin treatment. No consistent differences were seen in pRPS6 levels between rapamycin-treated BM09 and BM18 cells at the deviation passage (D+R) and at the final senescent passage (S+R). Data show representative blots from at least two independent experiments.

Fig 3. mTOR signaling inhibition leads to the regulation of molecular events often associated with stem cell senescence.

The expression of senescence- and pluripotency-related markers were analyzed in untreated and rapamycin-treated BM09, BM13, BM16 and BM18 samples at passages when untreated cells entered replicative senescence (deviation passage). Since rapamycin had no impact on lifespan extension of BM12, the expression levels of these markers were measured in untreated and rapamycin-treated cells at the same senescent passage. (A) p16^INK4A gene expression levels were analyzed by RT-qPCR. The bar graphs show relative gene expression levels after normalization to GAPDH. p16^INK4A protein expression levels were analyzed by western blot. Band intensities were densitometrically evaluated and bar graphs above bands represent the densitometric values of p16^INK4A normalized to the loading control (β-actin). These results are representative of at least two independent experiments. (B) The levels of IL6 and IL8 secretion were determined using a CBA proinflammatory kit. The bar graphs represent the obtained concentration values for IL6 and IL8 (pg/mL) normalized against cell numbers. These results are representative of two independent experiments performed in triplicate. (C) The gene expression levels of OCT4, SOX2 and NANOG were analyzed by RT-qPCR. The bar graphs show relative gene expression levels after normalization to GAPDH. These results are representative of two independent experiments performed in triplicate. S-R = senescent passage without rapamycin; D+R = deviation passage with rapamycin; S+R = senescent passage with rapamycin.

Fig 4. Downregulation of p16^INK4A expression is correlated with lifespan extension promoted by the continuous presence of rapamycin.

The fold reduction in p16^INK4A protein expression and in IL6 and IL8 secretion, and the fold increase in NANOG gene expression between untreated and rapamycin-treated BM09, BM13, BM16 and BM18 samples at passages when untreated cells entered replicative senescence (deviation passage), or in the case of BM12 at the same senescence passage, were plotted against the additional PD number obtained for the corresponding rapamycin-treated cells and statistically analyzed by Spearman correlation, as shown in the graphs.

Fig 5. p16^INK4A protein expression increased slightly in rapamycin-treated cells that stopped proliferating.

The protein expression levels of p16^INK4A were analyzed by western blot in rapamycin-treated BM-MSCs at the deviation passage with rapamycin (D+R) and at the senescent passage with rapamycin (S+R). Band intensities were densitometrically evaluated and bar graphs above bands represent the densitometric values of p16^INK4A normalized to the loading control (β-actin). The results obtained for BM12 is already showed in Fig 3a. Data show representative blots from at least two independent experiments.