Chondrocyte methylome signatures relevant to passage number in prolonged ML culture

We focused on the relevance of DNA methylation on the genome scale of equine chondrocytes expanded in prolonged ML culture. We performed methylome analysis of equine chondrocyte cell lines stemming from different passages in ML culture. In the applied RRBS approach we investigated differential methylation of the enriched CpG fraction of the genome of equine cartilaginous cells derived from 2, 6, 11, 13 and 14 passage. We have also undertaken the efforts to estimate the interrelations between detected methylation variability and altered expression of genes relevant to dedifferentiation events present in horse cartilaginous cells. NGS analysis of bisulfite converted genomic DNA of cartilage cells revealed 1880 hypomethylated and 2256 hypermethylated CpG sites in the coding regions of the genome of chondrocytes from lower passage numbers in relation to the higher ones. Certain genes associated with passage-related differential methylation were important for the processes of cellular interactions, chondrocyte maturity, extracellular matrix (ECM) turnover and the immunological pathways involved in pathology of articular cartilage. Four of differentially methylated genes displayed also chondrocyte passage-specific expression patterns providing the crosstalk between DNA methylation and gene activity in the processes relevant to prolonged ML based culture of cartilage cells.