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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Narcisi, Roberto (Erasmus MC)


Mesenchymal stem/stromal cells (MSCs) are promising for cell-based cartilage tissue engineering strategies as they can differentiate into the chondrogenic lineage. Although the inflammatory environment often present in the diseased joint can negatively influence cartilage MSC differentiation and cartilage maintenance, we previously demonstrated that the administration of the inflammatory factor TNFα enhanced chondrogenesis of MSC when added prior chondrogenic induction. Interestingly, the master regulator of cellular metabolism sirtuin1 (SIRT1) has also been reported to enhance chondrogenesis in MSC, but it can be inhibited by TNFα. Here we aimed to investigate the combinatory effect of the TNF-α treatment with a SIRT1 activator, in order to evaluate the ability of this co-treatment to further enhance chondrogenic differentiation of MSC. Our hypothesis is that the inhibition of SIRT1 is a potential side effect of the TNFα treatment, and it can be reverted by actively keeping high levels of SIRT1 using a specific activator.
Human MSCs (N=5) were isolated and expanded for 2 passages in a 10% fetal calf serum containing media supplemented with 1 ng/ml FGF-2 (basic expansion media). After, cells were further expanded for one additional passage (4-5 days) divided in 4 conditions consisting in the basic expansion media supplemented with (1) vehicle; (2) 50 ng/ml TNFα; (3) 1 µM SRT1720, a SIRT1 activator; (4) 50 ng/ml TNFα + 1 µM SRT1720. Concentration of TNFα was based on our previous publication, and concentration of SRT1720 was based on a screening evaluating specificity and toxicity of the compound. Chondrogenesis was induced by 3D pellet cultures using a TGFβ-based media for 21 days (same protocol for all the expansion conditions), and it was evaluated by (immune)histochemistry (Collagen type-2 and thionine for glycosaminoglycans) and RT-PCR (AGN and COL2A1). Prior chondrogenesis, monolayer cells were evaluated for bioenergetics parameters (XF-Seahorse Analyzer, Bioscience) and β-catenin pathway activation (western blot).
After chondrogenic induction, TNFα-expanded cells resulted to be the most chondrogenic, while the TNFα + SRT1720-expanded cells were the least chondrogenic, often with no visible staining for either Collagen type-2 or thionine. Interestingly, an in-depth bioenergetics analysis revealed that the MSC stimulated with TNFα + SRT1720 had the higher levels of basal and maximal oxygen consumption rate (OCR), and of the extracellular acidification rate (ECAR), indicating an overall metabolic enhancement. Analysis of the WNT/β-catenin signaling on those cells, on one hand confirmed our previous observation that TNFα-stimulated cells have enhanced β-catenin activity, on the other hand it showed that further administration of SRT1720 on TNFα-stimulated MSC abolished this effect.
Contrary to our initial hypothesis, co-stimulation with TNFα and the SIRT1 activator SRT1720 during expansion is detrimental for chondrogenic capacity of MSC. This is possibly due to an hyper- metabolic activation in these cells and a negative effect on the WNT signaling pathway, parameters known to sustain MSC differentiation.

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