Recapitulation of mesenchymal condensation enhances in vitro chondrogenesis of human mesenchymal stem cells

Mesenchymal condensation is a critical transitional stage that precedes cartilage formation during embryonic development. We hypothesized that "priming" hMSCs to recapitulate mesenchymal condensation events prior to inducing differentiation would enhance their subsequent chondrogenic properties. Our prior studies have suggested that exposing hMSCs to hypoxia (2% O₂) induces condensation-like effects. We therefore assessed the effect of preconditioning for different time periods on the expression of condensation specific genes by growing hMSCs in expansion medium under different normoxic (20% O₂) and hypoxic conditions for up to 2 weeks, and subsequently induced chondrogenesis of preconditioned hMSCs. The total cultivation time for each group was 4 weeks and the chondrogenic properties were assessed using gene expression, biochemical analysis, and histological staining. Our results demonstrated the benefits of preconditioning were both time- and oxygen-dependent. Condensation specific genes, SOX-9 and NCAM, were significantly up-regulated in hypoxic conditions at the end of 1 week. COL X and MMP13 expression was also lower than the normoxic samples at this time point. However, this group did not exhibit more efficient chondrogenesis after 4 weeks. Instead, hMSCs preconditioned for 1 week and subsequently differentiated, both under 20% O₂, resulted in the most efficient chondrogenesis. Interestingly, while hypoxia appears to positively enhance expression of chondrogenic genes, this did not produce an enhanced matrix accumulation. The results of this study emphasize the significance of considering the timing of specific cues in developing protocols for stem cell-based therapies and underscore the complexity in regulating stem cell differentiation and tissue formation.