Mesenchymal stem cell (MSC) therapy offers significant potential for musculoskeletal regeneration. However, the low abundance of MSCs in bone marrow aspirates necessitates expansion in culture before their clinical application. Such ex vivo expansion leads to a loss of stem cell characteristics and thus, unpredictable therapeutic outcomes. Studies of the MSC secretome have highlighted the importance of endogenous FGF2 for their survival and maintenance of stemness. This seminar will describe studies related to MSC potency and therapeutic efficacy, and how these attributes can be enhanced by sustaining endogenous FGF2 signalling through supplementation with an affinity-selected heparan sulfate (HS) co-factor. Microfluidic assays show that HS improves the bioavailability of FGF2, so providing more distant MSCs with a source of active FGF2. This results in the sustained growth of multipotent MSC over extended passaging. When used to treat osteochondral injuries in small and large animal models, MSCs preconditioned in an HS-rich microenvironment show improved ICRS II and O’Driscoll healing scores compared with control. Moreover, MRI shows reduced osteochondral lesioning following treatment with MSCs supplemented with HS, with Instron biomechanical testing highlighting an associated improvement in biomechanical properties. Collectively, these data indicate that modulation of endogenous FGF2 signals by select HS glycosaminoglycans represents an effective strategy for the bioprocessing of MSCs with sustained therapeutic potency.