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Skeletal homeostasis is an exquisitely regulated process most directly influenced by bone resorbing osteoclasts, bone forming osteoblasts, and the mechano-sensing osteocytes. These cells work together to constantly remodel bone as a mechanism to prevent from skeletal fragility. As such, when an individual experiences a disconnect in these tightly coupled processes, fracture incidence increases, such as during ageing, gonadal hormone deficiency, weightlessness, and diabetes. While therapeutic options have significantly aided in the treatment of low bone mineral density (BMD) or osteoporosis, limited options remain for anabolic or bone forming agents. Therefore, it is of interest to continue to understand how osteoblasts regulate their metabolism to support the energy expensive process of bone formation.    Objective  The current project sought to rigorously characterize the distinct metabolic processes and intracellular metabolite profiles in stromal cells throughout osteoblast differentiation using untargeted metabolomics.    Methods  Primary, murine bone marrow stromal cells (BMSCs) were characterized throughout osteoblast differentiation using standard staining protocols, Seahorse XFe metabolic flux analyses, and untargeted metabolomics.    Results  We demonstrate here that the metabolic footprint of stromal cells undergoing osteoblast differentiation are distinct, and while oxidative phosphorylation drives adenosine triphosphate (ATP) generation early in the differentiation process, mature osteoblasts depend on glycolysis. Importantly, the intracellular metabolite profile supports these findings while also suggesting additional pathways critical for proper osteoblast function.    Conclusion  These data are the first of their kind to characterize these metabolites in conjunction with the bioenergetic profile in primary, murine stromal cells throughout osteoblast differentiation and provide provocative targets for future investigation.    Supplementary Information  The online version contains supplementary material available at 10.1007/s11306-021-01829-9.

Untargeted metabolomics in primary murine bone marrow stromal cells reveals distinct profile throughout osteoblast differentiation