Correlation of COL10A1 induction during chondrogenesis of mesenchymal stem cells with demethylation of two CpG sites in the COL10A1 promoter

Objective. Human articular chondrocytes do not express COL10A1 and do not undergo hypertrophy except in close vicinity to subchondral bone. In contrast, chondrocytes produced in vitro from mesenchymal stem cells (MSCs) show premature COL10A1 expression and cannot form stable ectopic cartilage transplants, which indicates that they may be phenotypically unstable and not suitable for treatment of articular cartilage lesions. CpG methylation established during natural development may play a role in suppression of COL10A1 expression and hypertrophy in human articular chondrocytes. This study was undertaken to compare gene methylation patterns and expression of COL10A1 and COL2A1 in chondrocyte and MSC populations, in order to determine whether failed genomic methylation patterns correlate with an unstable chondrocyte phenotype after chondrogenesis of MSCs. Methods. COL10A1 and COL2A1 regulatory gene regions were computationally searched for CpG-rich regions. CpG methylation of genomic DNA from human articular chondrocytes, MSCs, and MSC-derived chondrocytes was analyzed by Combined Bisulfite Restriction Analysis and by sequencing of polymerase chain reaction fragments amplified from bisulfite-treated genomic DNA. Results. The CpG island around the transcription start site of COL2A1 was unmethylated in all cell groups independent of COL2A1 expression, while 9 tested CpG sites in the sparse CpG promoter of COL10A1 were consistently methylated in human articular chondrocytes. Induction of COL10A1 expression during chondrogenesis of MSCs correlated with demethylation of 2 CpG sites in the COL10A1 promoter. Conclusion. Our findings indicate that methylation-based COL10A1 gene silencing is established in cartilage tissue and human articular chondrocytes. Altered methylation levels at 2 CpG sites of COL10A1 in MSCs and their demethylation during chondrogenesis may facilitate induction of COL10A1 as observed during in vitro chondrogenesis of MSCs.