Differential analysis of genome-wide methylation and gene expression in mesenchymal stem cells of patients with fractures and osteoarthritis

ABSTRACT

Insufficient activity of the bone-forming osteoblasts leads to low bone mass and predisposes to fragility fractures. The functional capacity of human mesenchymal stem cells (hMSCs), the precursors of osteoblasts, may be compromised in elderly individuals, in relation with the epigenetic changes associated with aging. However, the role of hMSCs in the pathogenesis of osteoporosis is still unclear. Therefore, we aimed to characterize the genome-wide methylation and gene expression signatures and the differentiation capacity of hMSCs from patients with hip fractures. We obtained hMSCs from the femoral heads of women undergoing hip replacement due to hip fractures and controls with hip osteoarthritis. DNA methylation was explored with the Infinium 450K bead array. Transcriptome analysis was done by RNA sequencing. The genomic analyses revealed that most differentially methylated loci were situated in genomic regions with enhancer activity, distant from gene bodies and promoters. These regions were associated with differentially expressed genes enriched in pathways related to hMSC growth and osteoblast differentiation. hMSCs from patients with fractures showed enhanced proliferation and upregulation of the osteogenic drivers RUNX2/OSX. Also, they showed some signs of accelerated methylation aging. When cultured in osteogenic medium, hMSCs from patients with fractures showed an impaired differentiation capacity, with reduced alkaline phosphatase activity and poor accumulation of a mineralized matrix. Our results point to 2 areas of potential interest for discovering new therapeutic targets for low bone mass disorders and bone regeneration: the mechanisms stimulating MSCs proliferation after fracture and those impairing their terminal differentiation.

KEYWORDS:

• Epigenetics
• osteoarthritis
• osteoporosis
• stem cells
• transcription factors

Disclosure of potential conflicts of interest

WVC and MB are employees of NTX, the company that performed RNA sequencing. Other authors do not have conflicts of interest relevant to this paper.

Acknowledgment

We thank the skillful technical assistance of Jana Arozamena.

Funding

This study was supported by a grant from Instituto de Salud Carlos III (PI12/615), through a program potentially co-funded by FEDER Funds from the European Union. Carolina Sañudo was partially supported by IDIVAL. The methylation analysis service was performed at CEGEN-PRB2-ISCIII; it is supported by grant PT13/0001, ISCIII-SGEFI / FEDER

Author Contributions

Study design: JAR, FMPC; Clinical data Collection: CGI, MIPN, MAA. Experimental data collection: FMPC, AR, CS, WVC, MB, AFF. Data analysis and interpretation: WVC, MB, AR, FMPC, MFF, JAR. Drafting Manuscript: AR, FMPC, JAR; Revising Manuscript Content: AR, FMPC, JAR. Approving final version of manuscript: All authors. JAR takes responsibility for the integrity of the data analysis.