The Aging Landscape by <scp>scRNAseq</scp> of Mesenchymal Lineage Cells in Mouse Bone

A decrease in osteoblast number and bone formation are seminal contributors to age-related osteoporosis. However, the aging-associated molecular mechanisms that impact osteoblast precursors, osteoblasts, osteocytes, and other bone mesenchymal cell types remain unclear. We performed single-cell RNA-sequencing of mesenchymal cells present at the endosteum and periosteum of young and old C57BL/6 mice of both sexes. Osteoblast precursors and osteoblasts from female endosteum exhibited the greatest changes with aging. Transcriptional changes revealed decreased matrix protein production and autophagy, as well as increased senescence, phosphorylation, and hypoxia. Because deficient macroautophagy in osteoblast lineage cells decreases bone formation, we contrasted the transcriptional changes caused by autophagy inactivation in Atg7^f/f; Osx1-Cre mice with those caused by aging and found a causal link between autophagy deficiency and increased senescence in osteoblastic cells. Overall, these findings reveal distinct features of aging in males and females and molecular pathways that might be implicated in the development of intracortical porosity in the female skeleton. The transcriptional changes in periosteal cells indicate mechanisms that might contribute to the decreased response to mechanical loading and delayed fracture healing in the old skeleton. Our data provides a comprehensive resource and serves as a reference for understanding how future genetic and pharmacological interventions impact molecular mechanisms of aging in osteoblasts and other mesenchymal cells in the skeleton.