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

Date Published: January 31, 2019

Publisher: Public Library of Science

Author(s): Eliane Antonioli, Natália Torres, Mario Ferretti, Carla de Azevedo Piccinato, Andrea Laurato Sertie, Johnson Rajasingh.


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.

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.

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.

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.

Partial Text

In recent years, there has been increasing evidence that persistent activation of the growth-promoting mammalian target of rapamycin (mTOR) pathway plays a central role in cellular senescence and organismal aging [1–3], representing a key molecular driver of stem cell depletion and reduced tissue regenerative capacity [4–6]. Importantly, attenuation of mTOR signaling with rapamycin seems to preserve the clonogenic ability and function, besides delaying the activation of senescence mechanisms, in mouse and human stem cells from various tissues, including hematopoietic [5, 7, 8], epithelial [6, 9], spermatogonial [10] and mesenchymal stem cells [11].

It is well established that MSC cultures from a wide variety of healthy tissues have finite replicative lifespan and cease proliferating after a certain number of divisions [12]. The need for extensive MSC expansion for cellular therapy applications led to the search for new approaches to obtain sufficient number of cells before replicative senescence is reached. mTOR specific inhibitors, such as rapamycin, have emerged as potential adjuvant candidates for retarding the process of replicative senescence [15, 16]. Despite not yet being used for cellular therapy purposes, rapamycin has already been approved by the U.S. Food and Drug Administration (FDA) for a variety of clinical applications, including immunosuppressive and anticancer treatments [17].




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