Gene transfer to mesenchymal stem cells constitutes a powerful approach to promote their differentiation into the appropriate cartilage phenotype. Although viral vectors represent gold standard vehicles, because of their high efficiency, their use is precluded by important concerns including an elevated immunogenicity and the possibility of insertional mutagenesis. Therefore, the development of new and efficient non-viral vectors is under active investigation. In the present study, we developed new non-viral carriers based on niosomes to promote the effective chondrogenesis of human MSCs. Two different niosome formulations were prepared by varying their composition on non-ionic surfactant, polysorbate 80 solely (P80), or combined with poloxamer 407 (P80PX). The best niosome formulation was proven to transfer a plasmid, encoding for the potent chondrogenic transcription factor SOX9 in hMSC aggregate cultures. Transfection of hMSC aggregates via nioplexes resulted in an increased chondrogenic differentiation with reduced hypertrophy. These results highlight the potential of niosome formulations for gene therapy approaches focused on cartilage repair.

Osteoarthritis (OA) is a prevalent joint disorder that lacks effective therapies to halt cartilage degeneration. Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) are being investigated as promising chondroprotective agents. Compared to primary MSCs, induced pluripotent stem cell (iPSC)-derived MSCs (MLCs) offer superior scalability and enhanced paracrine activity. The aim of this study was to explore the feasibility of using autologous MLC-derived sEVs as a potential therapeutic strategy for OA through the analysis of their protein cargo. iPSCs from an OA patient and a healthy donor were differentiated into MLCs. sEVs were isolated from these MLCs and characterized, with a particular focus on their protein cargo. Both iPSC lines were successfully differentiated into MLCs, which secreted sEVs with comparable size distributions and yields. The analysis of differentially expressed proteins revealed a high abundance of proteins associated with OA pathology and cartilage degradation in sEVs from OA MLCs compared to those from healthy MLCs. The persistence of OA-associated protein signatures in autologous MLC-derived sEVs may limit their therapeutic efficacy. These findings underscore the importance of carefully evaluating disease-specific protein profiles in sEVs for regenerative applications.