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.

Figure 1.

Methylation analysis of hMSCs. (A) Volcano plot of DNA methylation differences in enhancer regions of hMSCs obtained from fractures and OA. In green, sites with a FDR<0.05 and absolute β differences larger than 0.1 (B) Heat-map showing β values of enhancer regions. In red more methylated and in green less methylated. Samples are named with a laboratory identifier code (JAR).

Figure 2.

Relationship between methylation and gene expression signatures. (A) Venn diagram summarizing the association between differential DNA methylation and differential gene expression (comparisons of hMSCs from fractures over hMSCs from controls). (B) Pathways enrichment analysis of genes with hypomethylated enhancers that were upregulated in fractures.

Figure 3.

Epigenetic aging of hMSCs. Left: Comparison of epigenetic and chronological age. Regression lines for each patient group are shown. Right: Deviation from the overall regression line with the 2 groups combined (mean and SE residuals in each patient group).

Figure 4.

Proliferation capacity and expression of selected genes by hMSCs from patients with fractures (FRX) and osteoarthritis (OA). (A) Proliferation assessed by Ki67 staining. (B) Proliferation by a MTT assay. (C) Expression of osteogenic markers by hMSCs from FRX and OA. (D) Expression of adipogenic markers by hMSCs.

Figure 5.

Differentiation capacity of hMSCs. (A) Osteogenic and adipogenic differentiation of hMSCs, as revealed by Alizarin red staining and Oil red staining, respectively. (B) Osteogenic differentiation of hMSCs from patients with fractures (FRX) and with osteoarthritis (OA), semiquantitative analysis. (C) Alkaline phosphatase activity in hMSC maintained in osteogenic medium. (D) Expression of the osteoblastic differentiation drivers OSX and RUNX2 in hMSCs from patients with FRX and OA maintained in osteogenic medium.