Specific age-associated DNA methylation changes in human dermal fibroblasts

Abstract

Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (<23 years) and elderly donors (>60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner.

Figure 1. Molecular characterization of fibroblasts and MSC.

Dermal fibroblast and mesenchymal stromal cells from bone marrow display a very similar immunophenotype (A). Adipogenic differentiation could not be induced in fibroblasts whereas fat droplet formation was observed in about 40% of the MSC upon differentiation (B,E; blue: DAPI; green: BODIPY; scale bar: 100 µm). Osteogenic differentiation was monitored by Alizarin Red staining of calcium phosphate precipitates and this could only be induced in MSC but not in fibroblasts (C,F; scale bar: 100 µm). Chondrogenic differentiation was analyzed by Alcian Blue staining of glucosaminoglycans in micromass culture and this was again only observed in MSC but not in fibroblasts (D; scale bar: 50 µm).

Figure 2. Relationship of DNA methylation profiles.

DNA methylation profiles of fibroblasts and MSC were analyzed with the HumanMethylation27 BeadChip that facilitates simultaneous analysis of 27,578 unique CpG sites. Unsupervised hierarchical clustering (Euclidian distance) of all CpG sites separated fibroblasts and MSC in two different groups. Furthermore, cell preparations from the same anatomical site clustered together (A). Alternatively, we used principal components analysis (PCA) to visualize the relationship of fibroblasts from different dermal regions (B).

Figure 3. Age-associated methylation changes in fibroblasts and MSC.

Mean DNA methylation of 27,578 CpG sites was compared between fibroblasts from young donors (<23 years, eight samples) and from elderly donors (>60 years; six samples) by scatter plot analysis. 75 CpG sites revealed more than 15% age-associated differential methylation (indicated as black spots; A). Scatterplot analysis of MSC from young (<50 years, four samples) and elderly donors (>50 years, four samples) demonstrated that 1060 CpG have more than 15% age-associated DNA methylation changes (B). Comparison of fibroblasts revealed that 30 CpG sites were overlapping differentially methylated upon aging in MSC and fibroblasts (black spots; C). This is highly significantly more than expected by chance alone but differential methylation often occurred in opposite directions.

Figure 4. Age-associated DNA methylation changes in CDKN2B.

Cyclin-dependent kinase inhibitor 2B (CDKN2B) belongs to the INK4A/ARF/INK4B locus and six CpG sites within the second exon are represented by the microarray (A). All of these were hyper-methylated upon aging in fibroblasts (B) and they were all hypo-methylated in MSC (C). Pyrosequencing of five neighbouring CpG sites in this region verified significant hyper-methylation upon aging in fibroblasts (D) and hypo-methylation in MSC (E). * P<0.05; ** P<0.01; *** P<0.001).

Figure 5. Long-term culture-associated methylation changes in fibroblasts and MSC.

Differential methylation between passage 3 and passage 21 of fibroblasts (6 year old donor) was plotted against differential DNA methylation data upon long-term culture of MSC that has been described before (A). Black spots demonstrate CpG sites with differential methylation of more than 15% upon long-term culture of both cell types and these showed a very good correlation in long-term culture associated changes in fibroblasts and MSC (R = 0.764). We have also compared long-term culture-associated DNA methylation changes and age-associated changes in fibroblasts and they display only moderate correlation (R = 0.337; B).