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.
Highlights
The limited ability of articular cartilage, bone and tendon to regenerate has prompted the development of cell-based tissue engineering techniques
To evaluate chondrogenesis markers of chondrocytes were assessed; Alcian Blue staining for glycosaminoglycans and aggrecan gene expression
Tenogenic differentiation was evaluated histologically using Masson’s Trichrome staining indicating areas of organised and disorganised collagen fibril formation within the tissues which were confirmed with Transmission electron microscopy (TEM) and gene expression of COL1A1 (Fig 1E and 1F), Serpin peptidase inhibitor F (SERPINF1) (Fig 1G) and thrombospondin 4 (THBS4) (Fig 1H) [41]
Summary
The limited ability of articular cartilage, bone and tendon to regenerate has prompted the development of cell-based tissue engineering techniques. One cell therapy option is mesenchymal stem cells (MSC); a heterogeneous population of multi-potent cells with the ability to differentiation into tissues including cartilage, bone and tendon, accommodating tissue repair and homeostasis. The principles of tissue engineering involve a multifarious interaction of factors, and knowledge of the extent MSC phenotype and differentiation capacity alter with ageing is limited. MSCs have been utilised in clinical trials of cell therapies for cartilage repair and osteoarthritis (reviewed [1]), bone fracture treatment [2] and in a limited number of tendon therapies [3]. The therapeutic efficiency of MSCs for clinical applications remains limited, possibly due to the attenuation of their regenerative potential in aged patients with chronic diseases
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
