Decoding the Regulatory Landscape of Ageing in Musculoskeletal Engineered Tissues Using Genome-Wide DNA Methylation and RNASeq.

Abstract

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based regenerative medicine and tissue engineering. Epigenetic mechanisms, like DNA methylation, contribute to the changes in gene expression in ageing. However there was a lack of sufficient knowledge of the role that differential methylation plays during chondrogenic, osteogenic and tenogenic differentiation from ageing MSCs. This study undertook genome level determination of the effects of DNA methylation on expression in engineered tissues from chronologically aged MSCs. We compiled unique DNA methylation signatures from chondrogenic, osteogenic, and tenogenic engineered tissues derived from young; n = 4 (21.8 years ± 2.4 SD) and old; n = 4 (65.5 years±8.3SD) human MSCs donors using the Illumina HumanMethylation 450 Beadchip arrays and compared these to gene expression by RNA sequencing. Unique and common signatures of global DNA methylation were identified. There were 201, 67 and 32 chondrogenic, osteogenic and tenogenic age-related DE protein-coding genes respectively. Findings inferred the nature of the transcript networks was predominantly for 'cell death and survival', 'cell morphology', and 'cell growth and proliferation'. Further studies are required to validate if this gene expression effect translates to cell events. Alternative splicing (AS) was dysregulated in ageing with 119, 21 and 9 differential splicing events identified in chondrogenic, osteogenic and tenogenic respectively, and enrichment in genes associated principally with metabolic processes. Gene ontology analysis of differentially methylated loci indicated age-related enrichment for all engineered tissue types in 'skeletal system morphogenesis', 'regulation of cell proliferation' and 'regulation of transcription' suggesting that dynamic epigenetic modifications may occur in genes associated with shared and distinct pathways dependent upon engineered tissue type. An altered phenotype in engineered tissues was observed with ageing at numerous levels. These changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients. In addition we have identified a number of tissue-dependant pathways, which warrant further studies.