Narcisi, Roberto (Erasmus MC )


Mesenchymal stem/stomal cells (MSCs) are often studied for their possible tissue engineering applications. During in vitro expansion, however, MSCs enter a state of permanent growth arrest while remaining metabolically active; this phenomena is known as cellular senescence. Senescence can negatively affect tissue homeostasis and an increased number of senescence cells can be found in pathological tissues (e.g. osteoarthritic cartilage). Moreover, It has been shown that cellular senescence may alters the differentiation capacity of MSCs towards the osteogenic and adipogenic lineage, while little is known about the influence of cellular senescence on chondrogenesis. Therefore, the aim of this study was to determine the effect of senescence on chondrogenic differentiation capacity of MSCs.
Cellular senescence was induced in MSCs (N=4) either in monolayer prior chondrogenic differentiation, or at different time points during chondrogenic pellet culture (day-7 or day-14) using a 20 Gy gamma irradiation protocol. Senescence markers P16, P21 and IL16, and the β-galactosidase staining were used to confirm irradiation-induced senescence.
Chondrogenic differentiation capacity was induced by a standard TGFβ-based protocol for 21 days using a 3D pellet culture system, and evaluated by (immuno)histochemistry and RT-PCR. To investigate the paracrine effect of senescent cells on recipient cells, we treated chondrogenic pellets using 2-day conditioned media from senescent cells and treat chondrogenic pellets for 24h, which were then analyzed for the expression of chondrogenic (SOX9, COL2A1 and AGCN) and catabolic (MMP-1, MMP-3, MMP-13 and ADAMTS4) markers. Western blot analysis on phosphorylated SMAD2 (P-Smad2 monoclonal antybody) was performed to identify TGFβ signaling activation. Non senescent cells or conditioned media from non-senescent cells was used as control.
When cellular senescence was induced prior differentiation, it abolished the chondrogenic capacity of MSCs with more than 95% reduction of GAG and Collagen type-2 deposition, as well as for all the chondrogenic markers measured by RT-PCR, in all the donor tested. A similar trend but with a less significant reduction was observed when senescence was induced at day-7 of differentiation. Interestingly, no effect on chondrogenic differentiation was detected when irradiation-induced senescent was applied at day-14 of differentiation. Moreover, medium conditioned by pellets cultures made of senescent cells had no significant effect on the expression of catabolic and anabolic markers measured by RT-PCR in recipient chondrogenic pellets. This suggests the negligible paracrine effect of senescent cells in our model.
In order to better understand how senescence was able to interfere with the chondrogenic process, we analyzed the ability of senescent MSCs to respond to TGFβ, the main pro-chondrogenic factor for MSCs. Upon stimulation with TGFβ1, phosphorylated-SMAD2 levels (an intracellular TGFβ effectors) were strongly reduced in senescent MSCs compared to control.

In this study we showed that cellular senescence reduced the chondrogenic differentiation capacity of MSC, but only when senescence occurs early during differentiation, and likely by negatively impacting the ability of the cells to respond to the pro-chondrogenic factor TGFβ1. This is a step forward in the understanding of the molecular mechanisms governing cellular senescence in MSC, and towards better optimizing the use of MSC for tissue engineering applications.


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