Genome-Wide Methylation Changes Associated with Replicative Senescence and Differentiation in Endothelial and Bone Marrow Mesenchymal Stromal Cells

Abstract

Bone marrow mesenchymal stromal cells (BMSCs) are multipotent cells able to self-renew and differentiate, depending on the microenvironment, into adipocytes and osteoblasts. These cells have a limited number of replications and enter replicative senescence during in vitro expansion. The role of DNA methylation (DNAm) assumes importance in cell function and commitment; however, its exact contribution to BMSC differentiation and replicative senescence is still unclear. We performed a genome-wide DNAm analysis on BMSCs cultured in vitro at early passages and induced to differentiate into adipocytes and osteoblasts, and on replicative senescent BMSCs and HUVECs, to identify DNAm patterns of senescence and differentiation. We also compared BMSCs and HUVECs in replicative senescence and found that, in both cellular systems, genome-wide hypomethylation was accompanied by a higher-than-expected overlap of differentially methylated positions (DMPs) and concordance in terms of direction of the change. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on lineage-independent senescence-associated DMPs revealed 16 common pathways, including Insulin resistance, Molecule adhesion, and Wnt/β-catenin signaling. In both adipogenesis and osteogenesis, we observed a general demethylation of CpG sites compared with undifferentiated BMSCs with a higher number of DMPs in osteogenesis. KEGG analysis resulted in 30 pathways enriched in osteoblasts and only 2 in adipocytes when compared to undifferentiated cells. When comparing differentiated BMSCs with senescent ones, osteogenesis exhibited a greater overlap with senescence in terms of number of DMPs and direction of methylation change compared to adipogenesis. In conclusion, this study may be useful for future research on general mechanisms that occur in replicative senescence and furthermore to identify trajectories of BMSC differentiation and common aspects of differentiated and senescent cells.

Keywords: DNA methylation; adipocytes; bone-marrow mesenchymal stromal cells; cellular senescence; endothelial cells; osteoblasts.

Figure 1

Experimental design and characterization of cellular models used for genome-wide DNAm analysis. (A) Experimental design: early-passage HUVECs and BMSCs were cultured until they reached replicative senescence (Sen); BMSCs were induced to differentiate into adipocytes (AD) and osteoblasts (OS) for 15 days. (B) Growth curve showing Cumulative Population Doublings (CPDs) of BMSCs and HUVECs. (C) Representative images and quantification of the SA β-Gal staining positivity in early (SA β-Gal < 10%) and senescent (SA β-Gal > 80%) HUVECs and BMSCs. (D) p16(INK4a) relative expression in arbitrary units (a.u.) in early and senescent cells obtained through Real Time PCR. Data were normalized using β-actin as internal control. (E) Runx2, Osteocalcin (OCN), and Bone morphogenetic protein 2 (BMP-2) relative expression (a.u.) and Alizarin red staining were used to verify the osteogenic differentiation (OS). (F) PPARγ, Adiponectin (ADIPOQ) and Fatty Acid Binding Protein 4 (FABP4) mRNA relative expression and Oil Red O staining were used to verify the adipogenic differentiation (AD). Data were normalized using β-actin as internal control. Data are the mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001 for paired t-test.

Figure 2

Principal Component Analysis (PCA) of DNAm values obtained by genome-wide analysis (A) in early and senescent HUVEC and in early, senescent, and differentiated BMSC samples, and (B) in early, senescent, and differentiated BMSC samples (n = three replicates per condition). The percentage of variance explained by the first and the second principal components is reported in X and Y axes, respectively.

Figure 3

(A,B) Volcano plot showing differentially methylated CpGs (FDR < 0.05) in (A) Senescent (Sen) vs. early BMSCs and (B) Sen vs. early HUVECs. Differences in DNAm values are plotted against −log₁₀(q-values). DMPs with an absolute DNAm difference >0.20 are highlighted in red (hypermethylated) and dark green (hypomethylated). (C) Boxplots showing the distribution of differences in the DNAm values of the CpGs between senescent and early BMSCs and HUVECs. p for Wilcoxon signed-rank tests. (D) Scatter plot comparing DNAm differences between Sen and early HUVECs and BMSCs. DMPs (FDR < 0.05) with concordant senescence-associated hypo- and hyper-methylation are indicated in green and red, respectively; DMPs with an absolute DNAm difference >0.20 are highlighted with darker green and red colors. (E) KEGG pathways significantly enriched in genes containing DMPs that are common to both cellular lineages in senescence. Pathways are ranked according to the significance of enrichment (grey bars, upper y-axis). Ratios referring to the proportion of targeted genes related to the total number of genes in each pathway are displayed (red line graph, bottom y-axis).

Figure 4

(A,B,D) Volcano plot showing differentially methylated CpGs (FDR < 0.05) in (A) osteoblasts (OS) vs. early BMSCs, (B) adipocytes (AD) vs. early BMSCs, (D) AD vs. OS. Differences in DNAm scores are plotted against −log₁₀(q-values). DMPs with an absolute DNAm difference are highlighted in dark red (hypermethylated) and dark green (hypomethylated). (C) Scatter plot comparing DNAm differences associated to adipogenic differentiation (x-axis) and osteogenic one (y-axis). DMPs (FDR < 0.05) with hypo- and hyper-methylation in both the comparisons are indicated in green and red, respectively; DMPs with an absolute DNAm difference >0.20 are highlighted with darker green and red colors.

Figure 5

(A,B) Scatterplots comparing DNAm differences induced by (A) osteogenic (OS) and (B) adipogenic (AD) commitment vs. replicative senescence (Sen) of BMSCs. DMPs (FDR < 0.05) with hypo- and hyper-methylation in both the comparisons are indicated in green and red, respectively; DMPs with an absolute DNAm difference >0.20 are highlighted with darker green and red colors. (C) KEGG pathways significantly enriched in genes containing DMPs that are common between BMSC osteogenic (upper panel), adipogenic (lower panel) commitment, and replicative senescence. Pathways are ranked according to the significance of enrichment (grey bars, upper y-axis). Ratios referring to the proportion of targeted genes related to the total number of genes in each pathway are displayed (red line graph, bottom y-axis).