Senescence did not alter the chondroprotective effect of extracellular vesicles from adipose mesenchymal stem cells in osteoarthritis

Abstract

Purpose: Age is the most important risk factor in degenerative osteoarthritis (OA) and is associated with the accumulation of senescent cells that contribute to functional decline of joint. We previously demonstrated that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) largely mediate the therapeutic effect of parental cells in OA. Here, we assessed the impact of senescence on the characteristics of EVs from adipose tissue-derived MSCs (ASC-EVs) and their properties in an in vitro model of OA Methods: ASCs were induced to senescence using 25μM etoposide for 24 hours. Senescence was assessed at day 12 by quantifying proliferation rate (Trypan blue counting, BrdU incorporation), SA-βGal activity (immunostaining and dedicated quantitative assay), nuclear γH2AX foci number (immunofluorescence), stress fiber visualization (phalloidin staining) and expression of cyclin dependent kinase inhibitors (CDKI) (RT-qPCR). ASC-EVs were isolated by differential ultracentrifugation and characterized by size, concentration, total protein content, structure (cryo-TEM) and immunophenotype (CD9, CD63, CD81, Alix). In vitro OA model used human chondrocytes isolated from OA patients, which were stimulated with IL1β for 48h before culture with ASCs or ASC-EVs for 7 days. Expression of chondrocytic and inflammatory markers was quantified by RT-qPCR and SASP factors were quantified by ELISA in supernatants Results: After 12 days, senescence-induced ASCs experienced growth arrest and increase of SA-βGal staining, of p21 CDKI expression, of nuclear γH2AX foci, of stress fibers and of several SASP factors (IL6, IL8, MMP3) confirming expression of all main features of senescence. Senescent ASCs produced 4-fold more EVs than healthy ASCs and senescent ASC-EVs were larger and contained slightly less total protein amounts. In the in vitro model of OA chondrocytes, both healthy and senescent ASCs decreased fibrotic markers (type III COLLAGEN), catabolic and hypertrophic markers (MMP3, MMP13, AP) and increased COX2. By contrast, healthy ASCs decreased the expression of IL6 while senescent ASCs highly increased IL6 expression in OA chondrocytes. Looking at the role of ASC-EVs on OA chondrocytes, we found out that both healthy and senescent ASC-EVs were able to increase the expression of AGG and type II COLLAGEN while they decreased the expression of MMP13, AP, type X COLLAGEN, HMOX1 and IL6. Finally, healthy and senescent ASC-EVs decreased the number of SA-βGal positive OA chondrocytes but did not impact the expression of p21 in these IL1β-induced chondrocytes. Conclusions: Our Results indicated a chondroprotective effect of ASC-EVs, independently of the senescent state of parental cells and suggested that EVs might act through different mechanisms than ASCs, which warrants further investigation