Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids

Abstract

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers.However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low-and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low-and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies. K E Y W O R D Smesenchymal stem/stromal cells, p16, senescence, spheroids, tunneling nanotubes

Investigating Tunneling Nanotubes in Cancer Cells: Guidelines for Structural and Functional Studies through Cell Imaging

“…This approach, coupling polymeric nanoparticles as a vector for anticancer therapy, would make it possible to homogenize the distribution of the drug to all the cellular components of the tumor, particularly in a hypoxic context [54] (iii) To use the ability of TNTs to promote cell survival as part of cellular therapies: the latter approach concerns cell therapies or stem cell transplantation, treatment of leukemia, and certain non-Hodgkin's lymphomas, whose main limitation is the senescence acquired by grafted cells limiting their effectiveness over time. One study demonstrated that the culture of these mesenchymal stem cells in threedimensional structures (spheroids) compared to two-dimensional culture favored the formation of TNTs, the intercellular exchange of cytoplasmic components, and modulated expression of senescence markers and ultimately improve the survival of these transplant cells [55].…”

Impact of 3D cell culture on bone regeneration potential of mesenchymal stromal cells

“…In this context, Cheng and colleagues have clearly demonstrated that 3D cultivation of human AD-MSCs as scaffold-free spheroids prolongs their replicative lifespan and decreases their senescence compared to 2D controls [ 93 ]. However, a recent report provided evidence that the number of senescent cells within MSC spheroids increases over time [ 94 ].…”

“…However, reports on the optimal osteoinductive stiffness are conflicting. While Hwang et al reported a stiffness of 4.47 kPa as osteoinductive [ 95 ], a much higher Young’s modulus ranging from 62 to 800 kPa has been suggested in studies from other laboratories [ 94 , 96 ]. Importantly, although stiffness can be manipulated in 2D to study MSC differentiation [ 121 , 123 ], it is important to consider that in vivo MSCs are exposed to the mechanophysical cues in all three dimensions of the surrounding niche.…”

Molecular Mechanisms Contributing to Mesenchymal Stromal Cell Aging

“…Tunneling nanotubes (TNTs) are thin protrusions of actin microfilaments able to transfer cytoplasm and organelles between connected cells. In MSC spheroids it was demonstrated that TNTs, by bridging cells, can shuttle components from low-passage to high passage MSCs thus modulating senescence [178].…”