Lipid metabolic adaptations of multi-donor mesenchymal stem cells during osteodifferentiation

Mesenchymal stem cell (MSC) osteodifferentiation is accompanied by important lipid metabolic adaptations, which may reveal relevant biomarkers and potential osteoinductive species. However, high donor variability remains a challenge for biomarker identification. This work unveiled shared lipid features of human adipose-tissue MSC (hAMSC) for three independent donors, using an untargeted NMR spectroscopy methodology. The results showed that osteodifferentiation induced increases in esterified cholesterol preferentially enriched in shorter monounsaturated fatty acids (MUFA), and triacylglycerides containing longer fatty acids (FA), both consistent with increased lipid droplet formation in the cytosol. Membrane adaptations involved hydrolysis of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn), possibly to allow subsequent polyunsaturated FA incorporation (to enhance membrane fluidity) and facilitate removal of peroxidized FA, while originating inorganic phosphate (Pi) for mineralization. PtdCho levels seem closely linked to the creatine-phosphocreatine axis, reflecting a shared contribution to Pi generation. MUFA also appeared to serve as preferential substrates for β-oxidation, apparently in association with antioxidative mechanisms. The above metabolic effects were indicative of a common pathway modulation in the three donors, with predicted upregulation of ALP, collagen, antioxidant enzymes, LDL, and HSP27, and downregulation of ERK 1/2 (notably upregulated at day 7), arginase, and vacuolar H⁺-ATPase. Notably, the proposed donor-independent lipid signature enabled the detection of osteodifferentiating cells with nearly 90 % accuracy, highlighting the complex interplay among different lipid families, with esterified cholesterol, triacylglycerides, and phospholipids emerging as main players in osteodifferentiation.