Remodeling of lipid metabolism orchestrates human adipose-derived mesenchymal stem cells osteodifferentiation

Mesenchymal stem cell (MSC) osteodifferentiation involves adaptations of lipid metabolism but the specific regulatory roles of lipid species remain underexplored. We use a global metabolomics approach, comprising LC-MS lipidomics and NMR metabotolomics, to identify mechanistic features of human adipose-derived MSC (hAMSC) osteodifferentiation and new markers of osteogenic progression. Results show that, upon differentiation, cellular metabolism progresses through an early osteocommitment phase (day 7) followed by active osteodifferentiation (day 21), with underlying proliferation contributing towards membrane remodeling, oxidative stress protection and lipid-supported energy/signaling processes. At day 7, differentiating cells exhibit coordinated lipid remodeling and activation of the phosphocreatine (PCr)-creatine (Cr) axis. This leads to accumulation of phosphocholine, PCr and sphingomyelin, in preparation for mineralization and extracellular vesicle formation. Concomitantly, calcium oscillatory signaling and active purinergic metabolism are predicted. Other early features persist until day 21, including increased polyunsaturated fatty acids (PUFAs)-rich plasmalogen levels, enhanced endogenous substrate mobilization and metabolic autonomy. At this later stage, accumulation of storage lipids occurs, likely contributing to ω-3/ω-6 balance. FA regulation seems to be accompanied by a shift from TCA/OXPHOS towards β-oxidation and PCr hydrolysis, as predominant energy sources. Late-stage osteodifferentiation is also characterized by inhibition of pathways associated with undifferentiated state maintenance, while several upstream regulators emerge as pro-osteogenic. The resulting metabolic framework enables actionable monitoring and rational optimization of bone tissue engineering strategies.