Gas chromatography–mass spectrometry analysis of human mesenchymal stem cell metabolism during proliferation and osteogenic differentiation under different oxygen tensions

• GC–MS was used to investigate glucose metabolism in hMSC and hMSC-derived osteoblasts (hMSC-OS) cultured at 2% or 20% O2.
• hMSC and hMSC-OS exhibited different metabolic responses to low oxygen tension.
• Hypoxia-induced inhibition of PDH was more pronounced in hMSC-OS compared to hMSC.
• Hypoxia increased the apparent activity of the malate aspartate shuttle in hMSC, but not in hMSC-OS.

Bone marrow derived human mesenchymal stem cells (hMSC) are the primary cell type in bone tissue engineering, and their life span during osteogenic differentiation is associated with changes in oxygen tension. As a ubiquitous regulator of cellular metabolic activity, oxygen tension influences the profiles of metabolites in the entire metabolic network and plays an important role in hMSC survival, function, and osteogenic differentiation. In the current study, we hypothesize that hMSC have a metabolic phenotype that supports growth in low oxygen environments and that this phenotype changes upon differentiation, leading to differential responses to oxygen tension. We developed a gas chromatography–mass spectrometry (GC–MS) based metabolic profiling approach to analyze the metabolic fate of 13C-glucose in glycolysis and the tricarboxylic acid cycle (TCA) in undifferentiated hMSC and hMSC-derived osteoblasts (hMSC-OS) in response to perturbation in oxygen tension; specifically we compared changes induced by culture under 20% vs. 2% O2. The isotope enrichments in the metabolites were calculated and used to infer activities of specific metabolic enzymes and the associated pathways. The results revealed contrasting metabolic profiles for hMSC and the hMSC-OS in both 20% and 2% O2 states, with the most significant differences involving coupling of glycolysis to the TCA cycle, glutaminolysis, and the malate-aspartate shuttle. The results have important implications in defining the optimal culture conditions for hMSC expansion and osteogenic differentiation.