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. 2020 Jan 31;10(1):1622.
doi: 10.1038/s41598-020-58377-w.

Multi-platform NMR Study of Pluripotent Stem Cells Unveils Complementary Metabolic Signatures towards Differentiation

Bénédicte Elena-Herrmann  1   2 , Emilie Montellier  3 , Anne Fages  4 , Reut Bruck-Haimson  5 , Arieh Moussaieff  6
Affiliations

Multi-platform NMR Study of Pluripotent Stem Cells Unveils Complementary Metabolic Signatures towards Differentiation

Bénédicte Elena-Herrmann et al. Sci Rep. .

Abstract

Stem cells, poised to revolutionize current medicine, stand as major workhorses for monitoring changes in cell fate. Characterizing metabolic phenotypes is key to monitor in differentiating cells transcriptional and epigenetic shifts at a functional level and provides a non-genetic means to control cell specification. Expanding the arsenal of analytical tools for metabolic profiling of cell differentiation is therefore of importance. Here, we describe the metabolome of whole pluripotent stem cells (PSCs) using high-resolution magic angle spinning (HR-MAS), a non-destructive approach for Nuclear Magnetic Resonance (NMR) analysis. The integrated 1H NMR analysis results in detection of metabolites of various groups, including energy metabolites, amino acids, choline derivatives and short chain fatty acids. It unveils new metabolites that discriminate PSCs from differentiated counterparts and directly measures substrates and co-factors of histone modifying enzymes, suggesting that NMR stands as a strategic technique for deciphering metabolic regulations of histone post-translational modifications. HR-MAS NMR analysis of whole PSCs complements the much used solution NMR of cell extracts. Altogether, our multi-platform NMR investigation provides a consolidated picture of PSC metabolic signatures and of metabolic pathways involved in differentiation.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Representative 1H NMR metabolic fingerprints of CGR8 ESCs, as recorded (A) for whole cells by HR-MAS spectroscopy, and (B) for aqueous extracts by conventional solution NMR. GPC: glycerophosphocholine; PC: phosphocholine; FA: fatty acids; UFA: unsaturated fatty acids; PUFA: polyunsaturated FA; UDP-GlcNAc: UDP-N-acetylglucosamine; UDP-X: UDP-galactose/UDP-glucose/UDP-glucuronate/UDP-GlcNAc; *solvent trace: ethanol.
Figure 2
Figure 2
Multivariate statistical analyses of 1H NMR fingerprints discriminates PSC and differentiated (Diff) cells. HR-MAS investigation of whole cells (A,C,E) and solution NMR analysis of aqueous extracts (B,D,F): (A,B) Score plots from principal component analysis (PCA) (model A: N = 12, 4 components, R2X = 0.992, Q2 = 0.978; model B: N = 12, 2 components, R2X = 0.938, Q2 = 0.828). (C,D) Score plots and (E–F) associated loadings from OPLS discriminant analysis (model C: N = 12, 1 predictive +1 orthogonal components, R2X = 0.979, R2Y = 0.999, Q2 = 0.998; model D: N = 12, 1 predictive +1 orthogonal components, R2X = 0.923, R2Y = 0.984, Q2 = 0.963). GPC: glycerophosphocholine; PC: phosphocholine; UDP-GlcNAc: UDP-N-acetylglucosamine; UDP-X: UDP-galactose/UDP-glucose/UDP-glucuronate/UDP- GlcNAc; *solvent traces: methanol, acetone.
Figure 3
Figure 3
Volcano plot of metabolic alterations between differentiated and PSC cells as measured by (A) HR-MAS analysis of whole cells and (B) solution NMR of aqueous extracts. Red lines reflect the filtering criteria (|log2(FC)| = 1 and q = 0.05). q-values are corrected p-values with the Benjamini-Hochberg procedure to control the false discovery rate. GPC: glycerophosphocholine; PC: phosphocholine; UDP-GlcNAc: UDP-N-acetylglucosamine; UDP-X: UDP-galactose/UDP-glucose/UDP-glucuronate/UDP- GlcNAc; Ile: isoleucine; *acetone.
Figure 4
Figure 4
Schematic pathways for metabolic regulations of histones post-translational modifications: actetylation (A), methylation (B), O-glycosylation (C) and phosphorylation (D). Metabolites detected in PSC or differentiated cells by NMR are marked in red. Those detected by HR-MAS only in our experiments are additionally underlined. Abbrevations: 5-MTHF: 5-methyltetrahydrofolate; αKG: α-ketoglutarate; DMG: dimethylglycine; SAH: S-adenosyl-l-homocysteine; SAM: S-adenosyl-l-methionine; THF: tetrahydrofolate; UDP-GlcNAc: UDP-N-acetylglucosamine; AMPK: AMP-activated protein kinase; HAT: histone acetyltransferases; HDAC: histone deacetylases; HMT: histone methyltransferases; JMJC HDM: JmjC family of histone demethylases; LSD HMD: LSD family of histone demethylases; OGT: O-Linked N-Acetylglucosamine Transferase; SIRT HDAC: sirtuin family of histone deacetylases.
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