Abstract
Articular cartilage is an avascular, alymphatic, and aneural system with very low regeneration potential because of its limited capacity for self-repair. Mesenchymal stem cells (MSCs) are the preferred choice for cell-based therapies. Glycogen synthase kinase 3 (GSK-3) inhibitors are compounds that can induce the Wnt signaling pathway, which is involved in chondrogenesis and cartilage development. Here, we investigated the influence of lithium chloride (LiCl) and SB216763 synergistically with TGF-β3 on chondrogenic differentiation in human mesenchymal stem cells derived from Wharton’s jelly tissue (hWJ-MSCs). hWJ-MSCs were cultured and chondrogenic differentiation was induced in monolayer and pellet experiments using chondrogenic medium, chondrogenic medium supplemented with LiCl, or SB216763 for 4 weeks. After in vitro differentiation, cultured cells were examined for the expression of Sox9, ACAN, Col2a1, and β-catenin markers. Glycosaminoglycan (GAG) accumulation was also examined by Alcian blue staining. The results indicated that SB216763 was more effective than LiCl as evidenced by a higher up-regulation of the expression of cartilage-specific markers, including Sox9, ACAN, Col2a1 as well as GAG accumulation. Moreover, collagen type II expression was strongly observed in cells cultured in the chondrogenic medium + SB216763 as evidenced by western blot analysis. Both treatments appeared to mediate the Wnt signaling pathway by up-regulating β-catenin gene expression. Further analyses showed that all treatments suppressed the progression of chondrocyte hypertrophy, determined by decreased expression of Col10a1 and Runx2. These results indicate that LiCl and SB216763 are potential candidates for further in vivo therapeutic trials and would be of great importance for cartilage regeneration.
Highlights
Articular cartilage is a highly specialized connective tissue of the synovial joints
We investigated the influence of lithium chloride (LiCl) and SB216763, which act synergistically with TGF-β3 on chondrogenic differentiation in hWJ-Mesenchymal stem cells (MSCs) by monolayer and pellet cultures experiments
The use of chondrogenically differentiated MSCs has been proposed as a therapeutic strategy for cartilage regeneration [30]
Summary
Articular cartilage is a highly specialized connective tissue of the synovial joints. Wharton’s jelly tissue (WJ) is an alternative source of MSCs, which show properties similar to MSCs from other sources It is a rich source of primitive cells [6,9]. The Wnt/β-catenin signaling pathway plays a crucial role in the hypertrophic maturation of chondrocytes during the endochondral ossification process [19]. Another key regulator of the Wnt signaling pathway is glycogen synthase kinase 3 (GSK-3) that mediates β-catenin phosphorylation [20]. Lithium chloride (LiCl) or SB216763 has the potential to be inhibitor of GSK-3, which inactivates the phosphorylation of β-catenin to initiates the Wnt signaling pathway [23,24]. The differentiated cells were characterized by GAG analysis, immunofluorescent staining, western blot, and gene expressions analysis
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