DKK1 AND FRZB are necessary for chondrocyte (RE)differentiation and prevention of cell hypertrophy in 3D cultures of human chondrocytes and human mesenchymal stem cells

Abstract

Purpose: Autologous chondrocytes and mesenchymal stem cells (MSCs) in either monoculture or co-culture, for the enhancement of chondrogenesis, show great promise for treatment of cartilage disorders. However, maintaining the chondrogenic phenotype and avoiding hypertrophy of chondrogenic differentiating MSCs remain a big challenge in these cell-based strategies. Previously, we identified Dickkopf 1 homolog (DKK1) and frizzled-related protein (FRZB) as key factors in controlling the articular chondrocyte phenotype. DKK1 and FRZB are naturally occurring antagonists of the WNT signaling pathway. The expression of DKK1 and FRZB is low in cultured human mesenchymal stem cells (hMSCs) and high in cultured human articular chondrocytes (hChs). We have previously shown that addition of either DKK1 or FRZB blocks hypertrophy in chondrogenically differentiating hMSCs and that expression of DKK1 and FRZB is lost in OA. We hypothesized that DKK1 and FRZB are necessary for chondrogenesis and for preventing cell hypertrophy in chondrocyte terminal differentiation. Methods: To block DKK1 and FRZB the variable domain of single chain heavy chain only antibodies (VHH) was used to neutralize DKK1 and FRZB. We tested our hypothesis using 3D pellet cultures of three relevant human cell based systems: isolated hChs, isolated hMSCs and co-culture of hChs with hMSCs. The effects were analyzed using real-time quantitative polymerase chain reaction (qPCR), histology, and immunohistochemistry (IHC). Results: In the presence of DKK1 and FRZB neutralizing VHH, glycosaminoglycan (GAG) deposition and Collagen type II staining were significantly reduced in redifferentiating chondrocytes (Figure 1 A and B) and in chondrogenic differentiating hMSCs, indicating loss of chondrogenic potential. Upon neutralization of DKK1 and FRZB in co-cultures, the cells in pellets showed hypertrophic differentiation as indicated by matrix mineralization, apoptosis and significantly increased expression of the hypertrophic markers, COL10A1, MMP13 and RUNX2. This is indicative of terminal differentiation. Conclusions: DKK1 and FRZB are necessary for multiple steps during chondrogenesis: first DKK1 and FRZB are necessary for the initial steps of chondrogenic differentiation of hMSCs and in redifferentiation of cultured chondrocytes, and secondly in preventing chondrocyte hypertrophy in terminal differentiation of articular chondrocytes (Figure 2).Figure 2. DKK1 and FRZB are necessary for multiple steps during chondrogenesis to ensure chondrogenesis and to prevent cell hypertrophy in terminal differentiation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)