The role of cellular metabolism in regulating cell proliferation and differentiation
remains poorly understood
. For example, most mammalian cells cannot proliferate without exogenous glutamine
supplementation even though glutamine is a non-essential amino acid
. Here we show that mouse embryonic stem cells (ESCs) grown under conditions that
maintain naïve pluripotency
are capable of proliferation in the absence of exogenous glutamine. Despite this,
ESCs consume high levels of exogenous glutamine when the metabolite is available.
In comparison to more differentiated cells, naïve ESCs utilize both glucose and glutamine
catabolism to maintain a high level of intracellular α–ketoglutarate (αKG). Consequently,
naïve ESCs exhibit an elevated αKG/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 that contribute to
the regulation of pluripotency-associated gene expression. In vitro, supplementation
with cell-permeable αKG directly supports ESC 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 play a mechanistic role
in the transcriptional and epigenetic state of stem cells.
Mouse ESCs can be maintained in two medium formulations: a serum-free medium reported
to support a cellular phenotype that mimics “naïve” epiblast cells of the inner cell
mass (ICM) (2i/LIF or 2i/L) or a serum-based medium that supports the proliferation
of a more committed ESC phenotype (serum/LIF or S/L)
. To characterize ESC metabolism, we investigated whether cells cultured in these
two media have different requirements for glucose and/or glutamine. ESCs cultured
in either medium proliferated at equivalent rates when glucose and glutamine were
abundant and cells cultured with or without 2i were unable to proliferate in the absence
of glucose (Extended Data Fig. 1a,b). In contrast, cells cultured in 2i/L, but not
S/L, proliferated robustly in the absence of exogenous glutamine (Fig. 1a and Extended
Data Fig. 1c). Likewise, four newly-derived ESC lines (ESC-1-4) exhibited convincing
glutamine-independent proliferation in 2i/L medium while retaining features of pluripotent
cells, including ESC-like morphology, reactivity to alkaline phosphatase (AP) and
the ability to form teratomas (Fig. 1b,c, Extended Data Fig. 1d). Cells cultured in
2i medium alone could also proliferate in the absence of exogenous glutamine (Extended
Data Fig. 1e).
This effect was not due to differences in medium nutrient formulations as supplementing
S/L medium with the GSK3β and ERK inhibitors present in 2i also enabled glutamine-independent
proliferation while maintaining ESC morphology and markers of pluripotency (Fig. 1d,e).
An alternative ESC medium containing BMP4 and LIF added to the same serum-free formulation
as in 2i/L
failed to support glutamine-independent growth (Fig. 1f). Likewise, epiblast stem
cells (EpiSCs) could not proliferate in the absence of exogenous glutamine (Extended
Data Fig. 1f,g). However, the ability to undertake glutamine-independent growth was
not limited to embryonic pluripotency; fibroblast-derived induced pluripotent cells
(iPSCs) were also able to proliferate in glutamine-free 2i/L medium (Extended Data
Fig. 1h). These results indicate that the GSK3β and ERK inhibitors in 2i-containing
medium are both necessary and sufficient to enable proliferation of pluripotent cells
in the absence of exogenous glutamine.
The fact that cells proliferated in the absence of exogenous glutamine in 2i/L medium,
albeit at a slower rate than cells cultured in glutamine-replete medium (Extended
Data Fig. 1i), indicates that these cells must be capable of de novo glutamine synthesis.
Indeed, chemical inhibition of glutamine synthase was sufficient to block proliferation
of cells in glutamine-free 2i/L medium (Extended Data Fig. 1j). Likewise, addition
of cell-permeable dimethyl-α-ketoglutarate (DM-αKG), a precursor for glutamine synthesis,
was sufficient to enable glutamine-independent proliferation in both S/L and 2i/L
conditions (Extended Data Fig. 1k), suggesting that the supply of precursors for glutamine
synthesis determines the ability of ESCs to proliferate in the absence of glutamine.
In support of this model, cells cultured in 2i/L preserved larger intracellular pools
of glutamate following glutamine withdrawal than cells cultured in S/L (Fig. 1g).
These results suggest that 2i/L cells can generate glutamate (and glutamine) from
carbon sources other than glutamine itself.
Despite their different growth requirements, cells cultured in both S/L and 2i/L consumed
high levels of glucose and glutamine, while excreting similar levels of lactate, consistent
with the metabolic profile of most proliferating cells, including cancer cells and
pluripotent cells (Fig. 2a)
. Oxidation of glucose and glutamine via the mitochondrial TCA cycle provides a critical
source of the biosynthetic precursors required for cell proliferation. With the exception
of α-ketoglutarate (αKG), steady-state levels of TCA cycle metabolites were reproducibly
diminished in ESCs cultured in 2i/L (Fig. 2b).
In most cells, glutamine is catabolized to αKG to support TCA cycle anaplerosis (Fig.
2c). ESCs grown in S/L medium exhibited high levels of TCA cycle intermediates and
virtually all intracellular glutamate, αKG and malate were rapidly labeled following
addition of [U-13C]glutamine (Fig. 2d). In contrast, a substantial fraction of these
metabolites failed to label with glutamine in ESCs grown in 2i/L. Instead, there was
a rapid labeling of these three metabolite pools from [U-13C]glucose (Fig. 2e). Quantitation
of metabolite fluxes revealed that although the flux of glutamine-derived carbons
through αKG was similar in both conditions, glutamine flux through malate was significantly
diminished in cells cultured in 2i/L, indicating that the entry of glutamine-derived
αKG into the TCA cycle is repressed by culture in 2i/L (Fig. 2f). Instead, when cells
are cultured in 2i/L, a substantial amount of both αKG and malate was produced from
glucose (Fig. 2g).
Consistent with these results, cells cultured with 2i inhibitors demonstrated substantial
glucose-dependent glutamate production (Extended Data Fig. 2a). Consequently, during
conditions of glutamine depletion, cells cultured in 2i/L medium were able to use
glucose-derived carbons to maintain elevated glutamate pools sufficient to support
cell growth (Extended Data Fig. 2b). Moreover, in comparison to their S/L counterparts,
2i/L cells utilized more glucose-derived carbon and relatively less glutamine-derived
carbon to support protein synthesis (Extended Data Fig. 2c), confirming that 2i promotes
increased glucose-dependent amino acid synthesis.
Diminished glutamine entry into the TCA cycle, coupled with the observed efflux of
glucose-derived carbons from the TCA cycle as glutamate, suggested that cells cultured
in 2i/L might not be oxidizing all the αKG produced from glutamine in the mitochondria.
Indeed, the αKG/succinate ratio was robustly elevated by 2i/L in every ESC line tested
(Fig. 3a). Cellular αKG/succinate ratios have been implicated in the regulation of
the large family of αKG-dependent dioxygenases
. As Jumonji-domain containing histone demethylases and the Tet family of DNA demethylases
comprise a major subset of these enzymes, the elevated ratio of αKG/succinate observed
in cells grown in 2i/L medium could have important implications for the regulation
of chromatin structure.
Since αKG was largely derived from glutamine metabolism (Fig. 2d), we tested whether
glutamine deprivation affected histone lysine methylations known to be regulated in
part by αKG-dependent demethylases
. Cells cultured in glutamine-free medium exhibited increases in tri-methylation and
decreases in mono-methylation on H3K9, H3K27, H3K36 and H4K20 while H3K4 methylations
remained unchanged (Fig. 3b). DM-αKG reversed the increase in H3K27me3 and H4K20me3
observed in glutamine deficient medium (Extended Data Fig. 3a), confirming that these
changes could be accounted for by the decline in glutamine-dependent αKG. Treatment
, a cell-permeable inhibitor that preferentially inhibits UTX and JMJD3, the two H3K27me3-specific
JmjC-family histone demethylases (Fig. 3c), induced a dose-dependent increase in H3K27me3
with a concomitant reduction of H3K27me1 that was comparable in magnitude to the difference
observed when cells were cultured in the presence or absence of glutamine (Fig. 3b,d).
The above data suggest that the methylations of certain histone lysines, including
H3K27, are actively suppressed by αKG-dependent histone demethylases in ESCs maintained
in 2i/L medium.
In ESCs “bivalent domains” are developmentally regulated genomic regions characterized
by the co-localization of H3K4me3 and H3K27me3
. Recent genome-wide analysis of H3K27me3 in S/L and 2i/L cultured ESCs reported that
H3K27me3 was specifically depleted at bivalent domain gene promoters in 2i/L cultured
. The present data suggest that the observed increase in αKG might promote αKG-dependent
H3K27me3 demethylation in 2i/L ESCs. Indeed, cells cultured in 2i/L exhibited a greater
increase in H3K27me3 at bivalent domain promoters when incubated with the H3K27me3
demethylase inhibitor GSK-J4 than cells cultured in S/L (Fig. 3e and Extended Data
Fig. 3b,c). The average fold-change across 14 bivalent promoters tested showed a highly
significant increase in 2i/L-cultured ESCs compared to S/L-cultured ESCs (Fig. 3e).
Similarly, two independent cell lines with mutations in the Jumonji domain of the
H3K27me3 demethylase JMJD3 (JMJD3Δ/Δ-1 and JMJD3Δ/Δ-2) (Extended Data Fig. 4a–c) demonstrated
increases in H3K27me3 levels relative to control lines that were significantly elevated
in cells cultured in 2i/L, reflecting enhanced demethylation at these loci in ESCs
cultured in 2i/L (Fig. 3f). Furthermore, treatment with GSK-J4, but not the inactive
isomer GSK-J5, increased the αKG/succinate ratio in cells cultured in 2i/L (Fig. 3g).
These results indicate that 2i/L rewires glutamine metabolism to maintain αKG pools
favoring active demethylation of a variety of histone marks.
In addition to reduced H3K27me3 at bivalent domain promoters, cells cultured in 2i/L
exhibit DNA hypomethylation
. Incubating cells with ascorbic acid, a cofactor for αKG-dependent dioxygenases,
activates Tet-dependent gene expression and promotes DNA demethylation
. Therefore, we tested whether αKG treatment could exert similar effects (Extended
Data Fig. 5a). Total DNA methylation was reduced in cells cultured with cell-permeable
αKG (Extended Data Fig. 5b) and treatment with αKG, but not succinate, induced expression
of ICM and germline-associated genes previously identified as targets of Tet-mediated
activation (Extended Data Fig. 5c)
. The effects of αKG persisted upon extended passaging (Extended Data Fig. 5d) and
were largely abrogated in Tet1/Tet2 double knockout ESCs (Extended Data Fig. 5e).
These results suggest that intracellular αKG production may stimulate the activity
of multiple αKG-dependent dioxygenases in order to coordinately regulate the epigenetic
marks characteristic of naïve pluripotency.
To test whether modulation of the αKG/succinate ratio can influence pluripotent cell
fate decisions, we performed colony-forming assays with S/L-cultured ESCs in the presence
of αKG or succinate. S/L+DM-αKG colonies had brighter AP staining and retained the
compact colony morphology typical of undifferentiated ESCs (Fig. 4a). While the total
number of colonies was similar in all three conditions, the S/L+DM-αKG wells contained
more than double the number of fully undifferentiated colonies compared to S/L and
S/L+DM-succinate (Fig. 4b). As a further test of the ability of αKG to promote maintenance
of ESCs, we utilized a knock-in Nanog-GFP reporter line
and found that αKG was sufficient to enhance Nanog expression in a dose-dependent
manner (Figure 4c and Extended Data Fig. 6). These results support the conclusion
that αKG promotes the self-renewal of ESCs in vitro.
The above data demonstrate that the cellular αKG/succinate ratio contributes to the
ability of ESCs to suppress differentiation. The rewiring of cellular metabolism by
inhibitors of GSK3β and MAPK/ERK signaling results in a reprogramming of glucose and
glutamine metabolism that leads to accumulation of αKG and favors demethylation of
repressive chromatin marks such as DNA methylation and H3K9me3, H3K27me3, and H4K20me3
(see Supplementary Discussion). Future studies will investigate the mechanisms through
which these inhibitors influence the nuclear/cytosolic accumulation of αKG derived
from glucose and glutamine. While we cannot rule out chromatin-independent effects
of αKG supplementation on ESCs, our results support the notion that chromatin in pluripotent
ESCs is responsive to alterations in intracellular metabolism. Indeed, recent clonal
analysis of pluripotent cells revealed that DNA methylation is highly dynamic, balancing
the antagonistic processes of removal and addition
. Together, these results suggest that continued elucidation of the interconnections
between signal transduction and cellular metabolism will shed important light on stem
cell biology, organismal development and cellular differentiation.
ESC1-4 lines are V6.5 ESCs derived from C57BL6 X 129S4/SvJae F1 embryos in 2i/L medium.
Cells were derived from E3.5 blastocysts following standard ESC isolation procedures
. Flushed blastocysts were plated onto laminin-coated dishes (20 μg/ml, Stemgent 06-0002)
in 2i/L medium. Mice were purchased from Jackson Labs, Bar Harbor, ME (C57BL/6 JAX
000664 and 129S4/SvJae JAX 009104). Tet1/2 double knockout ESCs
, V19 ESCs (ESC-V19) and OKS iPS cells
were a kind gift from Rudolf Jaenisch (MIT/Whitehead Institute Cambridge, MA). All
cells were routinely tested for mycoplasma contamination.
Maintenance media for ESCs were as follows: serum/LIF (S/L) maintenance medium contained
Knockout DMEM (Gibco) supplemented with 15% ESC-qualified FBS (Gemini), penicillin/streptomycin
(Life Technologies), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine (Life Technologies)
and leukemia inhibitory factor (LIF) plated onto irradiated feeder mouse embryonic
fibroblasts (MEFs); 2i/LIF (2i/L) maintenance conditions used a base medium made from
a 1:1 mix of DMEM/F12 (Life Technologies 11302-033) and Neurobasal (Life Technologies
21103-049) containing N2 and B27 supplements (Life Technologies 17502-048 and 17504-044,
1:100 dilutions), penicillin/streptromycin, 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine,
LIF, CHIR99021 at 3 μM (Stemgent) and PD0325901 at 1 μM (Stemgent). Experimental media
utilized for all experiments (except growth curves without glucose, 13C isotope tracing
experiments and 14C labeling experiments) contained 1:1 mix of glutamine-free DMEM
(Life Technologies 11960-051) and Neurobasal (Life Technologies 21103-049) with or
without 2 mM glutamine. With the exception of 15% dialyzed FBS (Gemini 100-108) in
S/L experimental medium, all other supplements were equivalent to maintenance media
(S/L or 2i/L). For growth curves without glucose, 13C isotope tracing experiments
and 14C labeling experiments, medium contained 1:1 mix of glutamine- and glucose-free
DMEM (Invitrogen A14430-01) and glutamine- and glucose-free Neurobasal (Invitrogen
0050128DJ) containing either 20 mM [U-13C]glucose or 2 mM [U-13C]glutamine (Cambridge
Isotope Labs) and either 20 mM unlabeled glucose or 2 mM unlabeled glutamine as necessary;
all supplements were the same as experimental media described above (S/L or 2i/L).
All experiments were performed using feeder-free conditions. ESC-1 EpiSCs were cultured
feeder-free on fibronectin (Sigma) coated plates in EpiSC maintenance medium including
DMEM/F12, N2 and B27 supplements, penicillin/streptromycin, 0.1 mM 2-mercaptoethanol,
L-glutamine, 75 μg/ml BSA (Gibco) supplemented with human activin A (20 ng/ml; Peprotech)
and bFgf (10 ng/ml; Invitrogen). EpiSCs were passaged 1:2 or 1:4 using Accutase every
other day. For ESC to EpiSC differentiation, ESC-1 cells were plated onto fibronectcin-coated
dishes. Twenty-four hours after plating the medium was changed to EpiSC maintenance
medium supplemented with 6 μM JAK inhibitor (Calbiochem) for five passages. Analysis
was performed on passage 7 EpiSCs. GSK-J4 and GSK-J5 were purchased from Tocris Bioscience.
ESC-1 cells were plated in maintenance medium at a concentration of 2.5 × 105 cells
per T25 dish. The following day medium was changed to 2i/L experimental medium with
or without glutamine. 72 hours later, 1 × 106 cells were harvested from each group
and mixed 1:1 with experimental medium (without glutamine) plus Matrigel Basement
Membrane Matrix (BD) or experimental medium alone and injected into the flanks of
recipient SCID mice aged 8–12 weeks (NOD scid gamma JAX 005557 purchased from Jackson
Labs, Bar Harbor, ME). All conditions produced tumors in 4–8 weeks. Mice were euthanized
before tumor size exceeded 1.5 cm in diameter. Tumors were excised and fixed in 4%
paraformaldehyde overnight at 4°C. Tumors were paraffin-embedded and sections were
stained with hematoxylin and eosin according to standard procedures by Histoserv Inc.
All animal procedures were designed following NIH guidelines and approved by the Institutional
Animal Care and Use Committee (IACUC) at The Rockefeller University.
Glucose, glutamine and lactate measurements
Glucose, glutamine and lactate levels in culture medium were measured using a YSI
7100 multichannel biochemistry analyzer (YSI Life Sciences). Fresh medium was added
to 12-well plates of sub-confluent cells and harvested 48 hours later. Changes in
metabolite concentrations relative to fresh media were normalized to protein content
of each well. These experiments were performed independently at least two times.
For all metabolite experiments, cells were seeded in their standard culture medium
in 6-well plates and the next day were changed into experimental medium. Medium was
changed again at the indicated time before harvest (usually 1–24 hours). Metabolites
were extracted with 1 mL ice-cold 80% methanol supplemented with 20 μM deuterated
2-hydroxyglutarate (D-2-hydroxyglutaric-2,3,3,4,4-d5 acid, d5-2HG) as an internal
standard. After overnight incubation at −80°C, lysates were harvested and centrifuged
at 21,000g for 20 minutes to remove protein. Extracts were dried in an evaporator
(Genevac EZ-2 Elite) and resuspended by incubation at 30°C for 2 hours in 50 μL of
40 mg/mL methoxyamine hydrochloride in pyridine. Metabolites were further derivatized
by addition of 80 μL of MSTFA + 1% TCMS (Thermo Scientific) and 70 μL ethyl acetate
(Sigma) and incubated at 37°C for 30 minutes. Samples were analyzed using an Agilent
7890A GC coupled to Agilent 5975C mass selective detector. The GC was operated in
splitless mode with constant helium gas flow at 1 mL/min. 1 μL of derivatized metabolites
was injected onto an HP-5MS column and the GC oven temperature ramped from 60°C to
290°C over 25 minutes. Peaks representing compounds of interest were extracted and
integrated using MassHunter software (Agilent Technologies) and then normalized to
both the internal standard (d5-2HG) peak area and protein content of duplicate samples
as determined by BCA protein assay (Thermo Scientific). Ions used for quantification
of metabolite levels are as follows: d5-2HG m/z 354; αKG, m/z 304; aspartate, m/z
334; glutamate, m/z 363; malate, m/z 335 and succinate, m/z 247. All peaks were manually
inspected and verified relative to known spectra for each metabolite. For isotope
tracing studies, experiments were set up as described above using glucose- and glutamine-free
DMEM:NB media base supplemented with 12C-glucose (Sigma) and 12C-glutamine (Gibco)
or the 13C versions of each metabolite, [U-13C]glucose or [U-13C]glutamine (Cambridge
Isotope Labs). Enrichment of 13C was assessed by quantifying the abundance of the
following ions: αKG, m/z 304-315; aspartate, m/z 334-346; glutamate, m/z 363-377 and
malate, m/z 335-347. Correction for natural isotope abundance was performed using
. Flux was calculated as the product of the first order rate constant of the kinetic
labeling curve and relative metabolite pool size (normalized to mean S/L values for
. The flux from glucose- and glutamine-derived carbons was calculated for each of
three independent experiments and the average flux for each metabolite was shown.
Flux experiments represent the average of three independent experiments; all other
experiments were performed independently at least twice and a representative experiment
ESCs were plated at 7.5 × 105 per 6-well plate into experimental medium (S/L or 2i/L)
containing 0.01% unenriched D-[U-14C]-glucose (Perkin Elmer NEC042V250UC) or L-[U-14C]-glutamine
(Perkin Elmer NEC451050UC). 48 hours later, cells were washed with PBS, scraped and
pelleted at 4°C. Protein pellets devoid of lipid fractions were isolated according
to the Bligh-Dyer method
. Briefly, pellets were resuspended in 200 μL dH20, 265 μL 100% methanol and 730 μL
of chloroform. Samples were vortexed for 1 hour at 4°C. The organic phase was removed
and the remaining sample washed with 1x volume of methanol and spun at 14,200g for
5 minutes. The supernatant was discarded and the pellet was resuspended in 6 M guanidine
hydrochloride at 65°C for 30–45 minutes. Samples were quantified using Beckman LS
60001C instrument. Values represent the average from four wells normalized to protein
of duplicate samples. Labeling experiments were performed twice.
ESC or EpiSCs were plated in maintenance medium at a concentration of 375,000 cells
per 12-well plate. The following day cells were washed with PBS and media were changed
to experimental media (for S/L conditions this included dialyzed FBS) with or without
individual metabolites. Cells were counted each day using a Beckman Coulter Multisizer
4. All growth curves were performed independently at least two times.
Native ChIP assays (histones) were performed with approximately 6 × 106 ESCs per experiment.
Cells were subject to hypotonic lysis and treated with micrococcal nuclease to recover
mono- to tri-nucleosomes. Nuclei were lysed by brief sonication and dialyzed into
N-ChIP buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton
X-100) for 2 hr at 4°C. Soluble material was incubated overnight at 4°C following
addition of 0.5–1 μg of antibody bound to 25 μL protein A Dynal magnetic beads (Invitrogen),
with 5% kept as input DNA. Magnetic beads were washed, chromatin was eluted and ChIP
DNA was dissolved in 10 mM Tris pH 8 for quantitative PCR reactions (see below). Three
separate ChIP experiments were performed on replicate biological samples. The data
shown are the average qRT-PCR values (n=3).
Primers are listed below. All qPCR was performed using an Applied Biosystems StepOnePlus
system and Power SYBR Green PCR master mix. ChIP samples were diluted 1:100 in H20
and 5 μL used per reaction. ChIP-qPCR signals were calculated as percent input.
Forward primer 5′ - 3′
Reverse primer 5′ - 3′
RNA was isolated using the RNeasy kit (Qiagen). After DNase treatment, 1–2 μg RNA
was used for cDNA synthesis using the First-Strand Synthesis kit (Invitrogen). Quantitative
RT-PCR analysis was performed in biological triplicate using an ABI Prism 7000 (Applied
Biosystems) with Platinum SYBR green. All data were generated using cDNA from three
wells for each condition.
Forward primer 5′ - 3′
Reverse primer 5′ - 3′
Genomic DNA was extracted from ESC samples using Puregene Core Kit A (Sigma). DNA
methylation was measured using the colorimetric MethylFlash Methylated DNA quantification
kit (Epigentek) according to manufacturer instructions. ELISA experiments were performed
independently two times.
A Cas9-2A-PURO plasmid was purchased from Addgene (Addgene plasmid 48139)
. Two gRNAs targeting exon 17 of mouse JMJD3 were designed using the online software
(crispr.mit.edu) resource from the Zhang Laboratory (MIT Cambridge, MA) and were cloned
into Cas9-2A-Puro using the BbsI restriction enzyme sites. ESC-1 cells cultured in
2i/L medium were transfected with either Cas9-2A-Puro control or JMJD3 gRNA-containing
plasmids using Lipofectamine 2000 (Life Technologies). After 24 hours, cells were
changed to fresh medium containing 1 μg/ml puromycin for 48 hours. Following selection,
cells were cultured for 24 hours in 2i/L medium and then split to clonal density.
After approximately 7 days, colonies were picked and expanded for analysis. Genomic
DNA was purified from individual clones and used for PCR amplification of regions
surrounding each gRNA target site. gRNA #1 product is 367 bp and gRNA #2 is 317 bp.
Cloning of PCR products was performed using pGEM-T Easy (Promega). Mutants were identified
by Sanger sequencing (Genewiz Inc.).
Forward primer 5′ - 3′
Reverse primer 5′ - 3′
Jmjd3 gRNA #1
Jmjd3 gRNA #2
Forward primer 5′ - 3′
Reverse primer 5′ - 3′
Jmjd3 gRNA #1
Jmjd3 gRNA #2
were cultured in S/L experimental medium for three passages and 2.5 × 104 cells were
plated into a 6-well plate. Twenty-four hours later media was changed to S/L medium
containing vehicle control or DM-αKG. Media were subsequently changed 48 hours later
and cells harvested the following day. FACS analysis was performed at The Rockefeller
University Flow Cytometry Resource Center using a BD LSR II. Data were generated using
FlowJo. Experiments were performed two independent times and a representative experiment
depicting triplicate biological wells is shown.
Western blot analysis
Lysates were extracted in 1X laemmli buffer, separated by SDS–PAGE and transferred
to Immobilon PVDF (Millipore) membranes. Membranes were blocked in 5% milk prepared
in phosphate-buffered saline (PBS) plus 0.1% Tween 20 (PBS-T), incubated with primary
antibodies overnight at 4°C and HRP-conjugated secondary antibodies for 1_h the following
day. After ECL application (Millipore), imaging was performed using Lumimager LAS-3000
(FujiFilm). The following antibodies were used for Western blotting: H3 (Abcam 1791),
H3K4me3 (Active Motif 39159), H3K4me1 (Millipore 07-436), H3K9me1 (kind gift of T.
Jenuwein), H3K9me3 (Active Motif 39161), H4 (Abcam 0158), H4K20me1 (Abcam 9051), H4K20me3
(Millipore 07-463), H3K27me1 (Millipore 07-448), H3K27me3 (Millipore 07-449), H3K36me3
(Abcam 9050) and H3K36me1 (Millipore 07-548). All antibodies were used at a dilution
of 1:1000. H3K27me3 antibody used for ChIP-qPCR, Cell Signaling 9733BF.
ESCs free of feeder MEFs were plated at 100 cells per well in 6-well plates coated
with 20 μg/mL mouse laminin (Stemgent 06-0002) in maintenance S/L medium. The following
day media was changed to S/L experimental medium containing dimethyl-α-ketoglutarate
(4mM, Sigma 349631), dimethyl-succinate (4mM, Sigma W239607) or DMSO vehicle control.
Four days later cells were washed with PBS and stained for alkaline phosphatase using
Vector Red Alkaline Phosphatase Kit (Vector Labs) according to manufacturer’s instructions.
Self-renewal assays were performed independently at least two times.
Comparisons were made using unpaired two-tailed Student’s t-tests or 2-way ANOVA with
appropriate post-test (determined using GraphPad Prism) as indicated. Experiments
were performed with three or four replicates as is the standard in the field. Variation
is shown as s.d., s.e.m. or 95% confidence intervals as indicated in figure legends.
Extended Data Figure 1
Pluripotent stem cells can proliferate in the absence of glutamine when cultured in
a, Doubling time of ESC-V19 cells cultured in serum/LIF (S/L) or 2i/LIF (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 analyzed
by gas chromatography-mass spectrometry. 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 ESCs grown in 2i/L medium
without glutamine for three 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 qRT-PCR,
normalized to Gapdh and expressed as a ratio of values of mESCs cultured in 2i/L medium.
g, Growth curve of epiblast stem cells (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 induced pluripotent (iPS) cell 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 ESC-V19 cells cultured in glutamine-free 2i/L medium
in the presence or absence of 1 μM methyl-sulfoxide (MSO). k, ESC-V19 cells grown
glutamine free S/L (left) or 2i/L (right) medium with or without 4 mM dimethyl-α-ketoglutarate
(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
mESCs 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 four hours and the fraction of glutamate containing glucose-derived carbons is
shown. b, ESC-1 cells were cultured for four hours in glutamine-free S/L or 2i/L medium
containing [U-13C]glucose and the total amount of glutamate labeled 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 hour incubations. 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
hours with supplementation as indicated (DM-αKG, dimethyl-α-ketoglutarate). b,c, H3K27me3
ChIP-PCR of ESC-1 cells cultured in S/L (b) or 2i/L (c) medium with or without 30
μM UTX/JMJD3 inhibitor GSK-J4 for five hours. 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 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 identified in green. Predicted cut site 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 predicted Cas9 cleavage
Extended Data Figure 5
αKG increases Tet activity in mESCs
a, Simplified schematic of the reaction mechanism of TET1/2 enzymes. b, Relative percent
5-methylcytosine (% 5-mC) in ESC-1 cells cultured in S/L medium with or without DM-αKG
for 24 hours. 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 three 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 knock
out (KO) mESCs cultured with DM-αKG or DM-succinate for 72 hours. qRT-PCR data (c–e)
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 Fig. 6
αKG increases Nanog expression
a, Representative histogram of GFP intensity of Nanog-GFP cells treated with or without
DM-αKG for three 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 three days.
GFP expression (mean fluorescence intensity, M.F.I.) was determined by FACS. Data
are presented as the mean ± s.d. of triplicate wells from a representative experiment.