Metabolic oxidation regulates embryonic stem cell differentiation

Abstract

Metabolites offer an important unexplored complementary approach to understanding the pluripotency of stem cells. Using MS-based metabolomics, we show that embryonic stem cells are characterized by abundant metabolites with highly unsaturated structures whose levels decrease upon differentiation. By monitoring the reduced and oxidized glutathione ratio as well as ascorbic acid levels, we demonstrate that the stem cell redox status is regulated during differentiation. On the basis of the oxidative biochemistry of the unsaturated metabolites, we experimentally manipulated specific pathways in embryonic stem cells while monitoring the effects on differentiation. Inhibition of the eicosanoid signaling pathway promoted pluripotency and maintained levels of unsaturated fatty acids. In contrast, downstream oxidized metabolites (for example, neuroprotectin D1) and substrates of pro-oxidative reactions (for example, acyl-carnitines), promoted neuronal and cardiac differentiation. We postulate that the highly unsaturated metabolome sustained by stem cells allows them to differentiate in response to in vivo oxidative processes such as inflammation.

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Figure 1: Embryonic stem cells are characterized by abundant metabolites with highly unsaturated structures.
Figure 2: GSH/GSSG ratio and ascorbic acid levels are inversely related in response to ESC differentiation.
Figure 3: Inhibition of the eicosanoid signaling pathway promotes the pluripotent state of embryonic stem cells.
Figure 4: Metabolites that undergo mitochondrial β-oxidation, and neuroprotectin D1, promote cardiac and neuronal differentiation.

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Acknowledgements

We thank G.G. Haraldsson and C.D. Magnusson (University of Iceland) for providing us with selachyl alcohol; C.T. McMurray and J.W. Trauger for helpful comments; and S. Hilcove, S. Ku and D. Watry for technical assistance. We gratefully acknowledge financial support from the California Institute for Regenerative Medicine, US Department of Energy, US National Science Foundation, US National Cancer Institute and US National Institutes of Health. A.S.-R. thanks Fundación Ramón Areces for his postdoctoral fellowship.

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Contributions

O.Y. and J.C. contributed equally to this work. O.Y. and J.C. designed and performed experiments, analyzed data and wrote the manuscript. G.J.P. analyzed data and wrote the manuscript. D.M.W. and H.P.B. analyzed data. A.S.-R. performed organic synthesis. S.A.T. and C.D. performed experiments. S.D. and G.S. oversaw the project, assisted in data analysis, wrote the manuscript and approved all intellectual content.

Corresponding authors

Correspondence to Sheng Ding or Gary Siuzdak.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Figures 1–11 and Supplementary Table 2 (PDF 906 kb)

Supplementary Video 1

Beating cardiac cell clusters are visible in all wells treated with acyl-carnitines at day 16 of differentiation. (MOV 2889 kb)

Supplementary Table 1

Mass/charge and the corresponding possible glycerophosphocholines (GPCho) isomers as stated by the database Lipid Maps. (XLS 8390 kb)

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Yanes, O., Clark, J., Wong, D. et al. Metabolic oxidation regulates embryonic stem cell differentiation. Nat Chem Biol 6, 411–417 (2010). https://doi.org/10.1038/nchembio.364

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