Chronological Aging Stiffens Cells, Alters Mitochondria, and Impairs Mitochondrial Mechanical Responsiveness in Mesenchymal Stem Cells

Abstract

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes, play a critical role for maintaining bone homeostasis. Although, aging impairs MSC function and contributes to several complications, the effects of aging on subcellular structure and related gene expression need further investigation. Here we established an in vitro system to study MSCs isolated from bones of 5, 12, and 24 months old mice which showed significantly decreased bone volume and exercise performance with age. RNA sequencing revealed downregulation of genes related to cell-matrix interactions, cell metabolism, and division. Functionally MSCs extracted from 24mo showed significantly increased adipogenesis and reduced osteogenesis compared to 5mo, which was accompanied by reduced levels of cell proliferative marker Ki67 and increased expression of senescence protein p16. Data-driven segmentation of F-actin architecture revealed no cell-wide or nuclei-associated alteration, while 24mo MSCs showed decreased nuclear volume and increased spreading and higher nuclear stiffness compared to 5mo. Mitochondria from 12mo and 24mo showed increased length and volume of fibers when compared to 5mo, which was accompanied by gene expression changes associated with mitochondrial inflammation and oxidative phosphorylation. To test the functional consequences, MSCs were subjected to mechanical stress for 72 hours using 90Hz, 07g low-intensity vibration (LIV). While 5mo MSCs showed a robust fusing of individual mitochondrial fibers, LIV response of 12mo and 24mo MSCs were progressively less, indicating an already stressed mitochondria compromised to mechanical challenge. In summary, this study has established an in vitro assay system, revealing age-associated impairments in differentiation, mitochondrial function, and mechanotransduction capacity.