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Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells

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Abstract

The role of cellular metabolism in regulating cell proliferation and differentiation remains poorly understood1. For example, most mammalian cells cannot proliferate without exogenous glutamine supplementation even though glutamine is a non-essential amino acid1,2. Here we show that mouse embryonic stem (ES) cells grown under conditions that maintain naive pluripotency3 are capable of proliferation in the absence of exogenous glutamine. Despite this, ES cells consume high levels of exogenous glutamine when the metabolite is available. In comparison to more differentiated cells, naive ES cells utilize both glucose and glutamine catabolism to maintain a high level of intracellular α-ketoglutarate (αKG). Consequently, naive ES cells exhibit an elevated αKG to succinate ratio that promotes histone/DNA demethylation and maintains pluripotency. Direct manipulation of the intracellular αKG/succinate ratio is sufficient to regulate multiple chromatin modifications, including H3K27me3 and ten-eleven translocation (Tet)-dependent DNA demethylation, which contribute to the regulation of pluripotency-associated gene expression. In vitro, supplementation with cell-permeable αKG directly supports ES-cell self-renewal while cell-permeable succinate promotes differentiation. This work reveals that intracellular αKG/succinate levels can contribute to the maintenance of cellular identity and have a mechanistic role in the transcriptional and epigenetic state of stem cells.

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Figure 1: 2i is necessary and sufficient to confer glutamine independence.
Figure 2: 2i/L alters glucose and glutamine utilization.
Figure 3: Histone demethylation is regulated by intracellular αKG in ES cells.
Figure 4: αKG promotes the maintenance of pluripotency.

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Acknowledgements

B.W.C. is a Howard Hughes Medical Institute fellow of the Jane Coffin Childs Memorial Research Fund. L.W.S.F. is the Jack Sorrell Fellow of the Damon Runyon Cancer Research Foundation (DRG-2144-13). This work was funded by grants from the National Institutes of Health/National Institute of General Medical Sciences (C.D.A.) and from the National Cancer Institute (C.B.T.). We thank C. Li for assistance with FACS analysis and M. Dawlaty, D. Faddah and R. Jaenisch for sharing cell lines used in this study.

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Authors and Affiliations

Authors

Contributions

B.W.C. and L.W.S.F. designed and performed all experiments in the study under the guidance of C.D.A. and C.B.T. J.R.C. contributed material support. B.W.C., L.W.S.F., C.D.A. and C.B.T. wrote the manuscript.

Corresponding authors

Correspondence to C. David Allis or Craig B. Thompson.

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Competing interests

C.B.T. is a co-founder of Agios Pharmaceuticals and a member of the board directors of Merck. C.D.A. is a co-founder of Chroma Therapeutics and Constellation Pharmaceuticals.

Extended data figures and tables

Extended Data Figure 1 Pluripotent stem cells can proliferate in the absence of glutamine when cultured in 2i/LIF medium.

a, Doubling time of ESC-V19 cells cultured in S/L or 2i/L. b, Growth curve of ESC-1 cells cultured in S/L or S/L/2i medium devoid of glucose. c, Samples of S/L (left) and 2i/L (right) media with and without glutamine were analysed by GC–MS. Representative chromatograms of the total ion count reveal a clear glutamine (Q) peak in +Q media (grey) and no detectable glutamine in −Q media (red). m, minutes. d, Teratoma formation from ES cells grown in 2i/L medium without glutamine for 3 days. Representative images of haematoxylin and eosin staining reveal neural tissue (ectoderm), hepatocytes and pancreatic acinar cells (endoderm) and smooth muscle (mesoderm). Scale bar, 200 μm. e, Growth curve of ESC-1 cells grown in glutamine-free 2i/L or 2i medium. f, Gene expression analysis confirms that EpiSCs, which represent post-implantation pluripotency, were generated from ESC-1 cells by culture with Fgf and activin A. Transcript levels were assessed by quantitative real-time polymerase chain reaction with reverse transcription (qRT–PCR), normalized to Gapdh and expressed as a ratio of values of mouse ES cells cultured in 2i/L medium. g, Growth curve of EpiSCs cultured in serum-free epiblast medium (serum-free medium containing FGF and activin A (Fgf/ActA)) with or without glutamine. h, Growth curve of an iPSC line derived from fibroblasts using Oct3/4 (O), Klf4 (K) and Sox2 (S) cultured in glutamine-free S/L or 2i/L medium. i, Doubling time of ESC-1 cells cultured in 2i/L medium in the presence and absence of glutamine. j, Growth curve of ESC-V19 cells cultured in glutamine-free 2i/L medium in the presence or absence of 1 mM methylsulphoxide (MSO). k, ESC-V19 cells grown in glutamine-free S/L (left) or 2i/L (right) medium with or without 4 mM DM-αKG. For growth curve experiments, cells were seeded on day 0 in complete medium and then were changed to experimental medium on day 1 (indicated by red arrow). Data are presented as the mean ± s.d. of triplicate wells from a representative experiment.

Extended Data Figure 2 Mouse ES cells cultured with 2i demonstrate altered glucose and glutamine utilization.

a, 2i enables glutamate synthesis from glucose-derived carbons. ESC-1 cells cultured in S/L, S/L/2i or 2i/L medium were incubated with medium containing [U-13C]glucose for 4 h and the fraction of glutamate containing glucose-derived carbons is shown. b, ESC-1 cells were cultured for 4 h in glutamine-free S/L or 2i/L medium containing [U-13C]glucose and the total amount of glutamate labelled by glucose-derived carbons is shown. c, Incorporation of 14C derived from [U-14C]glucose (14C-glc) (left) or derived from [U-14C]glutamine (14C-gln) (right) into total cellular protein after 48 h incubation. P < 0.05 for 14C-glc, P = 0.1 for 14C-gln, calculated by unpaired two-tailed Student’s t-test. Data are presented as the mean ± s.d. of triplicate wells (a, b) or ± s.e.m. of quadruplicate wells (c) from a representative experiment.

Extended Data Figure 3 Regulation of histone methylation in 2i/LIF cells.

a, Western blot analysis of ESC-1 cells grown in glutamine-free 2i/L medium for 24 h with supplementation as indicated. b, c, H3K27me3 ChIP-qPCR of ESC-1 cells cultured in S/L (b) or 2i/L (c) medium with or without 30 μM GSK-J4 for 5 h. Data are presented as the mean ± s.e.m. of triplicate samples. *P < 0.05 by unpaired Student’s two-tailed t-test.

Extended Data Figure 4 Generation of JMJD3 mutant cells.

a, Schematic of targeting strategy for guide RNAs (gRNAs) to mouse Jmjd3 exon 17. gRNA sequences are highlighted in blue. b, Representative sequences from two clones used in this study. Sanger sequencing revealed indels as shown in schematic. Red dashes, deleted bases; red bases, insertions. gRNA is highlighted in blue and protospacer adjacent motif (PAM) sequences are identified in green. Predicted cut site is indicated by red triangle. Location of in-frame downstream stop is indicated on the right. c, An example chromatogram for clone JMJD3Δ/Δ-2 showing single-base-pair insertions at the predicted Cas9 cleavage site.

Extended Data Figure 5 αKG increases Tet activity in mouse ES cells.

a, Simplified schematic of the reaction mechanism of Tet1/2 enzymes. b, Relative per cent 5-methylcytosine (% 5-mC) in ESC-1 cells cultured in S/L medium with or without DM-αKG for 24 h. Each data point represents a sample from triplicate wells of a representative experiment. c, Gene expression in ESC-1 cells cultured with DM-αKG or DM-succinate for 3 days. d, Gene expression in ESC-1 cells cultured in S/L medium with or without DM-αKG for four passages. e, Gene expression in wild-type or Tet1/Tet2 double-knockout (KO) mouse ES cells cultured with DM-αKG or DM-succinate for 72 h. qRT–PCR data (ce) was normalized to actin or Gapdh and samples were normalized to the control group. Oct3/4 is not expected to change and is included as a control. Data are presented as the mean ± s.e.m. of triplicate wells.

Extended Data Figure 6 αKG increases Nanog expression.

a, Representative histogram of GFP intensity of Nanog–GFP cells treated with or without DM-αKG for 3 days. Grey represents background staining. b, ESC-1 cells were cultured in S/L medium with DM-αKG for four passages and then switched to medium containing the indicated amounts of DM-αKG (0.5–4 mM) or vehicle control (S/L) for 3 days. GFP expression (M.F.I.) was determined by fluorescence-activated cell sorting (FACS). Data are presented as the mean ± s.d. of triplicate wells from a representative experiment.

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Carey, B., Finley, L., Cross, J. et al. Intracellular α-ketoglutarate maintains the pluripotency of embryonic stem cells. Nature 518, 413–416 (2015). https://doi.org/10.1038/nature13981

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