iPSC-conditioned medium mitigates the adverse effects of osteoarthritic synovial fluid on chondrocyte cultures

PURPOSE: Knee osteoarthritis (OA) affects approximately 13-17 % of adults over 40 in Europe, making it the most prevalent musculoskeletal disorder. Cell-based therapies for OA but are limited by the pro-inflammatory OA environment, which may affect cell senescence, dedifferentiation, and fibrocartilage formation, which impact clinical outcomes. Conditioned medium (CM) from cells, particularly iPSCs, is a promising source due to their remarkable regenerative abilities. This study aims to explore the chondroprotective effects of iPSC-derived CM on chondrocytes cultured in an OA-like environment using synovial fluid (SF) from OA patients. METHODS: CM was collected from iPSC lines (GPCCi001-A and ND 41658∗H) the day before and on the day of passage, then centrifuged, and pooled to create a homogeneous environment. CMs were mixed 1:1 with standard chondrogenic medium and/or 10 % SF pooled from knee OA joints (n = 27, Kellgren-Lawrence 3 and 4), creating CM + SF mixtures. Controls included standard chondrogenic medium and SF alone. These media were added to a primary chondrocyte line culture. Cellular responses to treatment were assessed through proliferation, senescence, viability, oxidative stress, and expression of key cartilage and inflammation-related markers. RESULTS: CMs, alone or with SF, enhanced proliferation (ANOVA - p < 0.0001) and reduced the senescent cell percentage (Young: p = 0.0044; Senescent: p = 0.0021) compared to controls. Viability (Live: p = 0.5422; Dead: p = 0.3914) and cell cycle phase distribution (SubG1: p = 0.0246; G1/G0: p = 0.6380; S: p = 0.8555; G2/M: p = 0.5811) were not affected. ROS production was significantly lower in CM + SF cultures (p = 0.0070). CM and CM + SF have caused a considerable decline in COL1A1 (p = 0.1756), COMP (p = 0.0062), and COL10A1 (p = 0.0250) production and an increasing trend in ACAN (p = 0.2032), NFκB (p = 0.0402), Bax (p = 0.3340) and p21 (p = 0.0002) expression. CONCLUSIONS: iPSC-derived CM is non-toxic and may promote chondrocyte metabolic activity, reduce ROS levels, and mitigate the inflammatory effects of OA-derived SF.