Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), the most common chronic disease of joint cartilage. A potential mechanism of MSC-based therapies has been attributed to the paracrine secretion of trophic factors, where extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor sources compete are optimal with respect their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSCs) may represent a promising cellular source for the manufacture of EV therapeutics. In this study, we isolated and tested the efficacy of EVs secreted by MSCs and by iMSC for treatment of OA using an in vitro model.
To obtain high-quality EVs, we optimized the culture conditions for MSCs and iMSCs, the supernatant collection time, and EV extraction methods. MSCs and iMSCs were cultured in vitro in serum-free clinical grade condition. The cells were characterized for surface expression pattern, proliferation ability, senescence rate and differentiation capacity during long term-expansion. The culture media were collected continuously during the cell expansion, and EVs were isolated using an FPLC-anion exchange chromatography (AEX) approach. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blots as well as non-conventional flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on IL-1αtreated human chondrocytes, to mimic the OA environment.
We observed that the use of a serum-free, chemically defined medium for isolation and culture of hMSCs allowed us to expand a population with a stable phenotype from early to late passages. It is already well known that MSC proliferation, differentiation and function decline with passaging, in fact, after 3 passages we observed a drastic impact on cell growth and differentiation. Paracrine activity of hMSCs during long-term expansion was also evaluated. The number and size of vesicles released by hMSCs increased proportionally with their age in culture. EVs collected during hMSC long-term expansion retained tetraspanin (CD9, CD63 and CD81) expression and did not vary with parental cells age. Anti-inflammatory activity of MSC-EVs were evaluated in an in vitro model using osteoarthritic chondrocytes; administration of hMSC-EVs showed positive effects for early passages-derived vesicles only. The expression of IL-6 and IL-8 was significantly reduced after treatment with hMSC-derived EV at passage3. Over time in culture, the dimension of the vesicles increased while their anti-inflammatory effect was reduced. Concurrently, the expansion of iMSCs in serum-free conditions in vitro was optimized to define the best culture conditions to maintain the cells and to define the best time window in which to isolate EVs with maximum biological activity.
Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. The focus of our study was directed on determining the optimal range of time in which MSCs and iMSC are biological functionally with respect to production of EVs in a serum-free culture system. This paracrine application may represent a novel therapeutic approach for the treatment of OA.