Global transcriptional profiling using RNA sequencing and DNA methylation patterns in highly enriched mesenchymal...

Age-related bone loss in humans is associated with a decrease in bone formation relative to bone resorption, although the mechanisms for this impairment in bone formation with aging are not well understood. It is known that the precursors for the bone-forming osteoblasts reside in the mesenchymal cell population in bone marrow. Thus, in an effort to identify relevant genetic pathways that are altered with aging, we examined the gene expression and DNA methylation patterns from a highly enriched bone marrow mesenchymal cell population from young (mean age, 28.7 years) versus old (mean age, 73.3 years) women. Bone marrow mononuclear cells from these women were depleted of hematopoietic lineage (lin) and endothelial cells using a combination of magnetic- and fluorescence-activated cell sorting, yielding a previously characterized mesenchymal cell population (lin-/CD34-/CD31- cells) that is capable of osteoblast differentiation. Whole transcriptome RNA sequencing (RNAseq) of freshly isolated cells (without in vitro culture) identified 279 differentially expressed genes (p < 0.05, false discovery rate [q]< 0.10) between the young and old subjects. Pathway analysis revealed statistically significant (all p < 0.05) alterations in protein synthesis and degradation pathways, as well as mTOR, gap junction, calcium, melatonin and NFAT signaling pathways. Further, Reduced Representational Bisulphite sequencing (RRBS DNA methylation sequencing) revealed significant differences in methylation between the young and old subjects surrounding the promoters of 1528 target genes that also exhibited significant differences in gene expression by RNAseq. In summary, these studies provide novel insights into potential pathways affected by aging in a highly enriched human mesenchymal cell population analyzed without the confounding effects of in vitro culture. Specifically, our finding of alterations in several genes and pathways leading to impaired protein synthesis and turnover with aging in bone marrow mesenchymal cells points to the need for further studies examining how these changes, as well as the other alterations with aging that we identified, may contribute to the age-related impairment in osteoblast formation and/or function.