review article metaboloepigenetic regulation of

Review ArticleMetaboloepigenetic Regulation of Pluripotent Stem Cells

Alexandra J Harvey12 Joy Rathjen123 and David K Gardner12

1Stem Cells Australia Parkville VIC 3010 Australia2School of BioSciences The University of Melbourne Parkville VIC 3010 Australia3School of Medicine University of Tasmania Hobart TAS 7000 Australia

Correspondence should be addressed to Alexandra J Harvey ajharveyunimelbeduau

Received 11 May 2015 Accepted 29 September 2015

Academic Editor Libera Berghella

Copyright copy 2016 Alexandra J Harvey et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The differentiation of pluripotent stem cells is associated with extensive changes in metabolism as well as widespread remodelingof the epigenetic landscape Epigenetic regulation is essential for the modulation of differentiation being responsible for celltype specific gene expression patterns through the modification of DNA and histones thereby establishing cell identity Each celltype has its own idiosyncratic pattern regarding the use of specific metabolic pathways Rather than simply being perceived asa means of generating ATP and building blocks for cell growth and division cellular metabolism can directly influence cellularregulation and the epigenome Consequently the significance of nutrients and metabolites as regulators of differentiation is centralto understanding how cells interact with their immediate environment This review serves to integrate studies on pluripotent stemcell metabolism and the regulation of DNAmethylation and acetylation and identifies areas in which current knowledge is limited

1 Introduction

Resurgence in metabolic research has revealed metabolismto be at the heart of cell-sensing mechanisms Not only doesmetabolism provide ATP to maintain homeostasis and cellreplication and intermediates that form the basic buildingblocks for cell proliferation but also metabolic processes andproducts can modulate signalling pathways transcriptionfactor activity and gene expression Metabolites can inducelong-term changes to the cell through the regulation of theepigenome a phenomenon referred to as metaboloepigenet-ics Every cell type has a unique metabolic phenotype and aunique epigenetic profile reflecting their cellular niche andfunction It is hypothesized that not only does the pattern ofmetabolism observed in different cell types serve to fulfil thatcellrsquos specific functions but also metabolism is involved inestablishing the epigenome of the cell during developmentThis implies that the intra- and extracellular metabolicenvironment in which cells reside either in vivo or in vitrocan have a profound effect on cellular phenotype Further theability of cells themselves to modify their own environmentin order to facilitate their function warrants consideration

The pluripotent epigenome must maintain transcriptionof pluripotency-related genes while being poised for rapidlineage-specific gene activation upon differentiation [1ndash3]Concomitantly cells constantly modulate their metabolicstate in response to extracellular signals including nutrientavailability [4] Significant changes in metabolism accom-pany the transition from the early embryo through differ-entiation [5 6] The availability and activity of metaboliccofactors and enzyme substrates generated through cel-lular metabolism can impact the regulation of transcrip-tion through modulation of epigenetic processes includinghistone methylation and acetylation Metabolism is conse-quently emerging as a central player in the regulation ofepigenetics and gene expression

Here we review recent advances in our understandingof the roles of metabolites and cofactors in modulating thepluripotent stem cell epigenome We discuss how stem cellmetabolism and chromatinmodifications are interconnectedhow their interactions can impact stem cell state and differ-entiation how culture conditions have the potential to induce(erasegenerate) epigenetic marks how these processes couldsignificantly impact the utility of cells and the potential

Hindawi Publishing CorporationStem Cells InternationalVolume 2016 Article ID 1816525 15 pageshttpdxdoiorg10115520161816525

2 Stem Cells International

for metabolic alterations to induce epigenetic deregulationWe refer the reader to existing reviews on mitochondrialcharacteristics of pluripotent stem cells [7ndash9]

2 Defining Pluripotent Stem Cell States

In the embryo and in culture pluripotent cells have beenshown to comprise a lineage of temporally distinct cell states(reviewed in [10]) Pluripotent stem cells either embryonic(derived from the inner cell mass (ICM) of the blastocyststage preimplantation embryo ES cells) or reprogrammedfrom a somatic cell to an embryonic stem cell-like state(induced pluripotent stem cells iPS cells) are defined bytheir ability to self-renew (to proliferate indefinitely) and bypluripotency as shown by the ability to act as a founder cellpopulation for all the cells of the embryo and adult Theseproperties underpin the potential use of these cells as a sourceof clinically relevant cells for therapeutics and drug discoveryMany studies have focused on defining themolecular proper-ties of ES cells but only recently have we begun to investigatethe physiology and metabolism of these cells

Mouse and human ES cells differ in their growth factorrequirements in vitro a consequence of their origins fromdifferent developmental stages Mouse ES cells isolated fromthe ICM are reliant on leukemia inhibitory factor (LIF) forongoing propagation while also requiring serum [11] Alter-natively mouse ES cells can be isolated in medium supple-mented with inhibitors of MekErk and GSK3 activation [12]Human ES cells are derived from a later stage pluripotent cellpopulation more similar to postimplantation epiblast [13]and are dependent on activinnodal and fibroblast growthfactor (FGF) signaling for self-renewal and pluripotency [14ndash16]The tissue of origin and gene expression profile of humanES cells suggest that they are representative of a later stagepluripotent cell state Pluripotent cells have been isolatedfrom the postimplantation epiblast or primitive ectoderm ofthe mouse Like human ES cells epiblast stem cells (EpiSC)require FGF and activin A for self-renewal and pluripotency[17ndash19] In culture these cells adopt the phenotype of theanterior primitive ectoderm from the late gastrula stageembryo [17]

Mouse ES cells can be cultured with inhibitors of FgfMekErk and Gsk3 to form a naıve ES cell state repre-sentative of the early inner cell mass [20ndash22] Inclusion ofthe GSK3 inhibitor minimizes the negative regulation ofbiosynthetic pathways [12] thereby modulating proliferativecapacity Alternatively mouse ES cells can be cultured inmedium supplementedwith the amino acid l-proline to formearly primitive ectoderm-like (EPL) cells that represent astage of pluripotency intermediate to ES cells and EpiSC [23]

Each population isolated or cultured represents a stemcell state within the continuum of the pluripotent lineage

3 Framework of Pluripotent StemCell Metabolism

Pluripotent stem cells and the pluripotent cells of the ICMfrom which they are derived exhibit a metabolism character-ized by high levels of glucose consumption combined with

the production of lactate (reviewed by [9 24] Figure 1) Thispattern of metabolism is maintained in conditions of oxygensufficiency distinguishing it from anaerobic glycolysis andhas therefore been termed aerobic glycolysis Warburg [25]first described aerobic glycolysis in cancer cells in culturewhich produce large amounts of lactate even in the presenceof sufficient oxygen for the complete oxidation of glucose (theWarburg effect) Initially it was thought that aerobic glycolysiswas specific to cancers but it has since been shown that thismetabolic trait occurs in other proliferating cells types suchas lymphocytes [26] Gardner [27 28] identified similaritiesbetween cancers and blastocysts and the significance of theWarburg effect for the development of the late stage embryo

What are the cellular advantages of aerobic glycolysis topluripotent stem cells Glucose is typically considered in itscapacity as an energy source Pluripotent cells are highlyproliferative with reports of cell cycle rates reducing to aslittle as 5ndash7 hours in the developing embryo [29] Tomaintainthis growth rate these cells will have to generate buildingblocks for proteins nucleic acids lipids and carbohydratesThe glycolytic metabolism of glucose may not generate asmuch ATP as oxidative phosphorylation per mol of glucosebut it can readily create equal amounts of ATPby an increasedflux of glucose Further enhanced glycolytic rate plays asignificant role in the generation of metabolic intermediatesfor biosynthesis The carbon in glucose is utilized in thesynthesis of triacylglycerols and phospholipids Glucose isprecursor for complex sugars of mucopolysaccharides andglycoproteins [27 28] Metabolism of glucose through thepentose phosphate pathway (PPP) generates ribose moietiesrequired for DNA and RNA synthesis and the NADPHrequired for the biosynthesis of lipids and other complexmolecules [30 31] Aerobic glycolysis is therefore the mecha-nism that ensures sufficient carbon flux through biosyntheticpathways in rapidly dividing cells [27 30 32 33]

The preferentialmetabolism of glucose through glycolysisreduces the use of oxidative metabolism in pluripotent cellswith a concomitant decrease in the formation of reactiveoxygen species (ROS) associated with oxidative phosphory-lation [34] From a developmental perspective this reducesoxidative stress within the cell thereby reducing the riskof DNA damage Given that pluripotent cells in vivo andin vitro act as founders for all cell types of the embryoand adult a metabolism that promotes genetic stabilitywould represent an evolutionary adaptation for successfuland faithful propagation

4 Key Metabolites Define the In VivoPluripotent Stem Cell Niche

Maintenance of pluripotency relies on a balance of com-plex cellular and acellular signals within the surround-ing microenvironment High levels of aerobic glycolysis inpluripotent cells form a localized area or niche character-ized by relatively high concentrations of lactate and lowextracellular pH surrounding the blastocyst (and potentiallyaround cell colonies in culture) The blastocyst uses thismicroenvironment to facilitate the implantation process [24]

Stem Cells International 3

NAD+

NADHPKM2

2ATP + H+

PPPG-6-P

F-6-P

LDH

Glutaminolysis

CitrateACL

JMJ Vitamin C

TET

NAD+

SIRT

HATHMTVitamin C

Glucose

acme

5hmC5mC

Nucleus

GlycolysisGlucose

2NADP+2NADPH

Pyruvate

NAD+

NADHNAD+ NADH

UCP2Pyruvate

PDK

PDH

Acetyl-CoA Citrate

120572KGTCAcycle

Mitochondrion

NAD+

NADH

Glu

OXPHOS

CO2

34ATPO2

GlutamineGlutamate

Lactate

HBPOGT O-GlcNAc

Acetyl-CoA

Acetyl-CoATHF

HCY

Met

SAH

SAM

5-CH3THF

Methioninecycle

Folatecycle

Glycine +Tdh

Thr

Methionine(human)

Threonine(mouse)

Figure 1 Metabolic regulation of the pluripotent epigenetic landscape Pluripotent stem cells are characterized by spherical electron-poormitochondria which contain few cristaeThese cells rely heavily on glycolysis for ATP generation (thick black arrows) resulting in significantlactate production through the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) Consequently oxidative phosphorylation(OXPHOS) contributes minimally to total ATP Glucose metabolized by the pentose phosphate pathway (PPP) generates the ribose moietiesrequired for DNA and RNA synthesis and the NADPH required for the biosynthesis of lipids and other complex molecules Intermediatemetabolites generated via metabolic pathways act as cofactors for epigenetic modifying enzymes Threonine and methionine metabolism isrequired for S-adenosyl methionine (SAM) generation via the folate and SAM cycles in mouse and human pluripotent stem cells respectively[60 61] SAM acts as a methyl donor for histone methyltransferases (HMT) as well as DNA methyltransferases Demethylation of SAMyields S-adenosylhomocysteine (SAH) which is in turn hydrolyzed to homocysteine Transfer of a methyl group to homocysteine from thefolate pathway regenerates methionine Acetyl coenzymeA (Acetyl-CoA) acts as a cofactor for histone acetyltransferases (HAT) Acetyl-CoAgenerated from glucose derived pyruvate modulates human pluripotent stem cell histone acetylation [76] although pyruvate dehydrogenasekinase (PDK) activity may limit the conversion of pyruvate to acetyl-CoA [79] Similarly uncoupling protein 2 (UCP2) functions to shuntpyruvate away from the tricarboxylic acid (TCA) cycle facilitating lactate production [79] Acetyl-CoA can also be generated from threoninecatabolism [62] Alpha-ketoglutarate (120572KG) [65] or supplementation with vitamin C in culture [122] reduces histone and DNA methylationin human pluripotent stem cells respectively These metabolites modulate histone and DNA demethylation reactions catalyzed by Jumonji(JMJ) and Ten-Eleven Translocation (TET) demethylases respectively [101] The hexosamine biosynthetic pathway (HBP) is an alternativeroute of glucose utilization that generates the coenzymeUDP-GlcNAc which together withO-linkedN-acetylglucosamine transferase (OGT)leads to histone O-GlcNAcylation [82] Flux through glycolysis and oxidative phosphorylation determines the NAD+ NADH ratio knownto regulate the activity of the NAD+-dependent histone deacetylases sirtuins (SIRT [90]) In addition a proline-dependent mechanism ofepigenetic regulation has been reported in pluripotent stem cells [74] however it is unclear how the metabolism of proline interacts withthese pathways Metabolic regulators of chromatin-modifying enzymes are highlighted in red

This environment assists in extracellular matrix degradationangiogenesis and immune-modulation of the mother at theimplantation site Lactate as it would appear is a very impor-tant signalling molecule that elicits numerous effects in thecell of origin and surrounding tissues Some of these effectscould be modulated through lactate-responsive transcriptionfactors Many cancers appear to recreate an embryonic-likephenotype and coopt embryonic pathways Cancers likeblastocysts generate a microenvironment characterized by

high lactate and reduced external pH created through aer-obic glycolysis to facilitate tissue invasion angiogenesis andimmunomodulationThe role of such amicroenvironment inin vitro stem cell culture has not been considered though it islikely to have a profound effect on pluripotent stem cells andthose cells surrounding them

Low oxygen is a characteristic of the stem cell niche invivo where the oxygen concentrationwithin the reproductivetract approximates 2ndash8 [35 36] Within the oviduct early


embryo development takes place in an oxygen concentrationfrom 5 to 85 (35ndash60mmHg) in the rabbit hamsterand rhesus monkey [35] Around the time of compactioncoinciding with the first lineage specification event theembryo traverses into the uterus which has a lower oxygenconcentration of 15ndash2 oxygen in the rhesus monkey 35in the rabbit 5 in the hamster [35] and 4 in the rat[36] Decreased oxygen in the uterus is particularly evidentat the time of implantation when in rabbits and hamsters adecrease from 53 to 35 oxygen is seen [35] Thereforeembryos appear to encounter a decreasing oxygen concen-tration gradient as they progress from the oviduct to theuterus Furthermore during the time of early implantationhypoxia and even anoxic conditions confront the invadingtrophectoderm (reviewed by [37]) At concentrations of 7oxygen and lower the activation of hypoxia-inducible factors(HIFs [38] reviewed by [39]) occurs within cells HIFsmodulate cellular homeostasis through the regulation ofglucose metabolism pH angiogenesis and iron metabolismsupporting a high rate of glycolysis

5 Regulation of Epigenetic Modifiers byMetabolic Cofactors

Cell state transitions are characterized by global changes inthe epigenetic landscape [40 41] As differentiation proceedsepigenetic modifications progressively restrict gene expres-sion silencing pluripotency genes and activating lineage-specific genes [42] Underlying pluripotency ES cells arecharacterized by an open and highly dynamic chromatinlandscape (reviewed by [43]) Progression of the pluripotentlineage and early events in differentiation are accompaniedby changes in the genomic architecture This is evidenced inchanges in mean replication timing (MRT) at loci across thegenome Changes inMRT provide evidence of changes in thegenomic organization that underpin the establishment of cellidentity [40] Large changes in MRT accompany the globalgenome reorganization also known as autosomal lyoniza-tion andoccur as EPL cells differentiate to cells representativeof a later primitive ectoderm (EpiSC and EBM6 [44]) andthe germ lineage progenitors Coincident with autosomallyonization are changes in the nuclear architecture and theformation and accumulation of late-replicating heterochro-matin at the nuclear periphery [44]

Stable modifications to DNA are catalyzed by DNAmethyltransferases (DNMTs) In general DNA methylationcan modify chromatin architecture and prevent transcrip-tion factor binding within gene promoters restricting geneexpression Methylation of lysine and arginine residueswithin histones H3 and H4 is catalyzed by residue-specificmethyltransferases (HMT) and can be associated with eithertranscriptional repression or activation Establishment ofmethylation patterns such as H3K4 di- or trimethylation(H3K4me2me3) andH3K27 trimethylation (H3K27me3) aregenerally associated with transcriptional activation whileH3K9me23 and H3K27me23 are associated with transcrip-tional repression S-Adenosyl methionine (SAM) acts as theprimary methyl donor for DNA and histone methylation

generated through one-carbon metabolism This pathwayintegrates the folate and methionine cycles (Figure 1) thelatter having metabolic inputs from methionine serine andglycine metabolism ES cells are characterized by elevatedglobal transcriptional activity [45] Repressive marks such asH3K9me3 are low in ES cells compared with differentiatedcells [46] Bivalent methylation marked by a combinationof active H3K4me3 and repressive H3K27me3 at a subset ofdevelopmental regulators has been proposed to establish aprimed epigenetic state ready for activation prior to ES celldifferentiation [2] and to safeguard differentiation [3]

DNA demethylation takes place either passively oractively Passive demethylation occurs with DNA repli-cation in the absence of maintenance methyltransferasesThe process of active demethylation is catalyzed by Ten-Eleven Translocation (TET) dioxygenases responsible forthe conversion of 5-methylcytosine (5mC) to 5-hydroxym-ethylcytosine (5hmC) [47ndash49] TET activity is dynamicallyregulated by alpha-ketoglutarate (120572KG) a product of theTCA cycle and succinate [50] Tet1 and Tet2 are highlyexpressed in mouse ES cells [51] with Tet1 also enriched inthe inner cell mass of mouse blastocysts [51] High 5hmClevels are present in mouse ES cells and decrease significantlyafter differentiation [49 52] In a similar manner Jumonjidemethylases are regulated by 120572KG [53] Jmjd1a and Jmjd2cknockdown leads to mouse ES cell differentiation regulatingpluripotent gene expression [54]

Histone acetylation participates in multiple chromatin-dependent processes including gene regulation DNA repli-cation and DNA damage repair Acetylation is generallyassociated with a more open chromatin configuration per-missive to transcription while deacetylation is associatedwith condensed compact chromatin leading to transcrip-tional repression Acetylation is catalyzed by histone acetyl-transferases (HATs) which transfer an acetyl group fromacetyl coenzyme A (acetyl-CoA) to lysine residues withthe concomitant production of CoA Cellular acetyl-CoAlevels fluctuate in response to various physiological cuesincluding nutrient availability andmetabolic activity Amajorsource of acetyl-CoA in cells is the conversion of citrate viaATP citrate lyase (ACL) siRNA-mediated silencing of ACLsignificantly decreases histone H2B H3 and H4 acetylationin HCT116 colorectal cancer cells [55] Glycolysis plausiblyhas a significant role in modulating acetyl-CoA levels andglucose availability can affect histone acetylation in an ACL-dependent manner [55]

Histone deacetylation is catalyzed byNAD+-independentor NAD+-dependent deacetylases (HDACs) Class I and IIHDACs are dependent on zinc while the activities of thesirtuin family of histone deacetylases (class III) are relianton NAD+ for their catalytic activity Sirtuins (SIRTs) act assensors of environmental stimuli and deacetylate histone andnonhistone substrates In addition they have roles in theregulation of a number of metabolite pathways includingglycolysis the TCA cycle and fatty acid oxidation telomeremaintenance tolerance to oxidative stress and DNA repairHigh rates of glycolysis establish a high NADHNAD+ ratiowhich downregulates sirtuin activity Studies of pluripo-tent stem cell histone acetylation have largely focused on


the regulation of deacetylation by class I and II HDACs andtheir inhibitors in the context of differentiation A genome-wide reduction of H3K9 acetylation (H3K9ac) is requiredfor mouse and human ES cell differentiation [56 57] Partialinhibition of ES cell HDAC activity has been shown topromote ES cell self-renewal [58 59] Therefore acetylationmaintains a highly dynamic configuration permissive totranscriptional activation

6 Linking Pluripotent Stem Cell Metabolismwith Epigenetics and Cell State

61 The Role of Essential Amino Acids in Modulating Pluripo-tent Stem Cell Methylation Pivotal studies in mouse ES cellshighlight the role of specific amino acids in pluripotent cellregulation Depletion of individual amino acids from ES cellculture identified threonine as a critical regulator of pluripo-tency Threonine catabolism supports mouse ES cell self-renewal while elimination of threonine fromculturemediumresults in slowed proliferation and increased differentiation[60 61] Threonine catabolism contributes to cellular glycineand acetyl-CoA levels the former being required for SAMsynthesis through the SAM cycle (Figure 1) Depletion ofthreonine from the culture medium or knockdown of thre-onine dehydrogenase (Tdh) in mouse ES cells decreasedSAM accumulation [60] and altered differentiation potential[62] Analysis of 13C-labelled threonine demonstrated thatthreonine contributes significantly to the acetyl-CoA poolin mouse ES cells and that glycine derived from 13C-Thrcontributed to SAM synthesis Removal of threonine leads tothe loss of methylated histone H3 Threonine reduction notsufficient to induce cell death was accompanied by a decreasein H3K4me3 [62] suggestive of a more repressive epigeneticlandscape Loss ofH3K4me3 could be rescued by supplemen-tationwith threonine orwith glycine and pyruvate associatedwith an increase in the SAMSAH ratio [62]

Human ES cells also require SAM but generated throughan alternative metabolic pathway a consequence of theirlack of a functional TDH [63] Systematic elimination ofamino acids from culture medium identified methionine asa critical amino acid Methionine deprivation resulted ina reduction in cell number within 5 hours attributable toincreased cell death and was associated with a reductionin SAM levels and NANOG expression [64] Knockdown ofthe methionine adenosyltransferases MAT2A and MAT2Bthat catalyze the conversion of methionine to SAM similarlydecreased cell numbers after 48 h suggesting that SAMrather than methionine was essential for cell survival Theearly reduction in cell numbers on methionine depletion isreversible but later impacts on cell proliferation followingprolonged methionine deprivation are not [64] We interpretthis to mean that maintenance of SAM levels is criticalfor stem cell survival and reductions in the metaboliteinterfaces with apoptosis machinery Short (5 h) and long(24 h) term methionine deprivation led to a rapid decreasein H3K4me3 accompanied by a modest reduction in globalDNAmethylation the effects of short term deprivation couldbe abrogated through supplementation with SAM [64]

These approaches identify SAM as a major methyl donorwithin pluripotent cells and show that reduction of SAMreduces histone methylation The processes used to reduceSAM within the cells likely reduce levels below physiologicalranges and impact on the pools of other important metabo-lites Shyh-Chang et al [62] detected reduced NADHNAD+and glycine and increased ATP glucose-6-phosphate andfructose-6-phosphate within the first 6 hours of threoninedepletion suggesting that culture media are rapidly depletedof other nutrients essentially starving cells This alone mayaccount for the increased cell death observed in both studiesparticularly as human ES cells were shown to replenish SAMwithin 24 hours through recycling of homocysteine [64]These data suggest that threonine and potentially methionineare critical to maintain metabolic balance and therefore cellsurvival within pluripotent stem cells in roles independentof SAM generation The question remains whether themodulation of SAM concentrations within cells within aphysiological range provides a mechanism for the cells tolink their metabolome with their epigenome These datasuggest that homocysteine may be important in modulatingcell survival The methionine pathway is reliant on transferof a methyl group to homocysteine from the folate pathwayto regenerate methionine The ability of glycine in thepresence of pyruvate to restore cell survival after shorttermmethionine withdrawal implicates the folate pathway inmodulating pluripotent cell survival

Shiraki et al [64] noted that short termmethionine depri-vation potentiated subsequent cell differentiation with morecells exhibiting lineage-specific marker expression on day 4when induced to differentiate with known differentiation-inducing conditions It will be important to understandwhether methionine deprivation potentiates differentiationby poising cells in a more primed state relative to methioninesupplemented conditions or whethermethionine deprivationselects a population of cells more receptive to differentiation-inducing conditions

62 Glutamine Regulates ES Cell Methylation Glutamine hasbeen shown to regulate pluripotency and histone methy-lation Most proliferating mammalian cells rely on thecatabolism of two molecules glucose and glutamine tofulfill their energy carbon and nitrogen requirements [4]As expected naıve and primed cells consume glucose andglutamine although steady-state levels of TCA cycle inter-mediates were lower in naıve ES cells [65] Neither naıvenor primed mouse ES cells were able to proliferate in theabsence of glucose [65] demonstrating an absolute require-ment for this metabolite These cell states could howeverbe distinguished by their ability to proliferate in the absenceof glutamine with naıve but not primed ES cells able toproliferate albeit at a reduced rate in glutamine deficientmedium [65]Theproliferation of naıve ES cells in glutamine-deficient medium was supported by an increase in glutamateproduction from glucose while the addition of precursorsof glutamine synthesis to the medium of primed ES cellsenabled proliferation in glutamine-depleted medium [65]


This suggests that the transition fromnaıve to primed ES cellsestablishes a reliance on glutamine and TCA cycle activity tosupport proliferation

Naıve ES cells exhibited an increased 120572KG succinateratio (Carey et al 2015) where elevated120572KG could impact theepigenome through modulation of Jumonji and TET activ-ity Following glutamine deprivation increased H3K9me3H3K27me3 H3K26me3 andH4K20me3 levels were detectedin naıve ES cells which could be reversed through mediumsupplementation with cell permeable 120572KG [65]These resultssuggest that the high levels of intracellular 120572KG found innaıve ES cells sustained through glucose-dependent gluta-mate production maintain an epigenetic landscape charac-terized by low levels of histone methylation As cells progressto the primed state their metabolic phenotype changesglucose-dependent glutamate production and intracellular120572KG levels decrease and a concomitant increase of histonemethylation accumulates consistent with the higher levels ofmethylation seen in these ES cells

63 l-Proline Metabolism Induces Changes in the EpigenomeThat Reflect Pluripotent Cell Identity Pluripotent early prim-itive ectoderm-like (EPL) cells can be formed from primedES cells in culture [66] The loss of ICM- and ES cell-specificmarker gene expression coupled with increased expressionof the primitive ectoderm markers [23 66ndash68] increasedproliferation rate [23] and a restricted ability to form cellpopulations characteristic of the primitive endoderm lineage[69 70] show EPL cells to be distinct from ES cells and alignthem with the embryonic primitive ectoderm The aminoacid l-proline has been shown to induce the differentiationof ES cells to EPL cells [23 71ndash73] l-Proline activity isfacilitated by uptake via the amino acid transporter SNAT2inhibition of l-proline uptake through SNAT2 prevents EPLcell formation [71] l-Proline activity is reliant on intra-cellular l-proline concentration and l-proline metabolismInhibition of proline dehydrogenase prevents the formationof EPL cells ([72] Rathjen unpublished) and removal of l-proline from EPL cells leads to the reestablishment of theES cell phenotype Suppression of l-proline biosynthesis andcreation of a shortage within the cell has been hypothesizedto safeguard ES cell identity and prevent autoregulation ofdifferentiation [73]

The addition of l-proline to ES cells induces changesto the epigenome and transcriptome Analysis of the grossgenomic organization of ES and EPL cells has shown thesecells to be similar but repeatable changes in MRT do occurwith EPL cell formation and these can be used to distinguishthe two cell types [40] It follows that the metabolism of l-proline and the cell identity changes induced by this processare manifest in changes to genome architecture Analysis ofhistone methylation patterns in proline-treated cells showedthat epigenetic remodelling in part regulated changes tothe transcriptome The addition of l-proline increased themethylation of H3 at lysines 9 and 36 and induced a repro-gramming of H3K9 and H3K36methylation status across thegenome [74] Changes inmethylation correlatedwith loci thatwere regulated by l-proline The epigenetic changes inducedby l-proline were suppressed when ascorbic acid was added

to the cells to date the method of ascorbic acid action isnot known The availability of l-proline to the ES cell whichrepresents the balance of l-proline availability and synthesisin the cell and subsequent metabolism regulates pluripotentcell identity with low availability enforcing an ES cell stateand increased levels of l-proline inducing EPL cell formation[23 71 73] As part of this process l-proline induces changesto the epigenome characteristic of the EPL cell state

64 Glucose Regulation of the Pluripotent Stem Cell EpigeneticLandscape A high glycolytic rate drives citrate synthesisleading to the production of cytosolic acetyl-CoA In turnacetyl-CoA can act as a cofactor for histone acetylation [75]Moussaieff et al [76] have shown that acetyl-CoA levels inhuman ES cells were twofold higher than those found in theirdifferentiating counterparts In ES cells glucose flux throughglycolysis was the primary contributor to the acetyl-CoApool as cells differentiated the ability to generate acetyl-CoAthrough this pathway was lost [76] Acetyl-CoA levels reducesignificantly upon mouse ES cell differentiation although inthese cells this was a result of reduced threonine catabolism[61]The addition of acetate a precursor of acetyl-CoA to dif-ferentiating human ES cells delayed cell differentiation [76]

The initiation of human ES cell differentiation led to a lossof H3K9K27 acetylation (H3K9H3K27ac) marks that areassociated with transcriptional repression while the additionof acetate to differentiating cells blocked this loss Inhibitionof glycolysis by 2-deoxyglucose previously associated withdifferentiation [77] similarly decreased H3K9H3K27ac aneffect that could be reversed with the addition of acetate [76]The study by Moussaieff et al suggests that the generationof acetyl-CoA from glucose-derived pyruvate is required tosupport maintenance of a pluripotent epigenetic landscapeOthers have questioned the use of pyruvate by pluripotentstem cell mitochondria [78 79] Direct quantification ofacetyl-CoAflux frompyruvate is needed to clarify the activityof this pathway

These data support a link between metabolites metabo-lism and epigenetic regulation (summarized in Table 1) butit is still difficult to define this relationship in pluripotentcells There remains a need to understand more fully theregulation of pluripotent stem cell metabolism the relativeactivity of metabolic pathways and the impact of nutrientavailability on pathway activity The study by Moussaieff etal (2015) has identified glucose as a source for acetyl-CoAcorrelated acetate availability with the level of histone acety-lation in pluripotent stem cells and differentiated derivativesCoincident with changes in metabolism with differentiation[76] lower levels ofH3K9ac have been observed following theinitiation of differentiation compared with ES cells [56 80]along with other global changes in the epigenetic landscape[43 44] Conceivably modulation of the metabolic interme-diate pool may serve to facilitate these dynamics and thewidespread nature of epigenetic change with the initiation ofdifferentiation

65 Other Potential Pathways Modifying Epigenetic RegulatorActivity Pluripotent cells in the embryo and in culture arepoised to undergo the most extensive epigenetic regulation


Table 1 Summary of metabolites linked with epigenetic modifications in pluripotent stem cells

Metabolite Epigenetic intermediate Metabolic pathway Epigenetic target References

Threonine SAM (via glycine) FolateSAM cycles H3K4me3 [60ndash62]Acetyl-CoA

Methionine SAM SAM cycle H3K4me3 [64]

Glutamine 120572KG TCA

H3K9me3[65]H3K27me3

H3K26m3H4K20me3

l-Proline TBD TBDMRT

[74]H3K9meH3K36me

Glucose Acetyl-CoA (glycolysis-derived) H3K9K27ac [76](from citrate) Pyruvate oxidation

TBD TBD O-GlcNAc meTETs [83]TBD NAD+ TBD acsirtuins mdash

event that will occur within the organismrsquos lifespan or duringES cell differentiation respectively Not surprisingly withinthese cells a number of metabolic pathways appear to bepoised to change activity potentially to provide or limit thepools of modification donors such as SAM and acetyl-CoAand to respond to the processes with metabolic regulatorycues Our knowledge of the metabolic positioning of apluripotent cell is however remarkably sparse It is highlylikely that similar roles for other metabolic pathways inpluripotency cell state transitions and lineage specificationwill be shown Pathways that are still to be examined includebiosynthetic pathways like the hexosamine biosynthesispathway and many amino acid synthetic pathways nutrientsensing mechanisms and metabolic regulatory mechanismssuch as the sirtuin family of histone deacetylases

The O-GlcNAc transferase (Ogt) is essential for ES cellviability and loss of the gene disrupts embryogenesis [81] andES cell self-renewal [82] More recently Ogt has been shownto associate preferentially with transcriptional start sites ofa number of genes in ES cells and regulate gene expressionof genes involved in metabolic and signaling pathways Ogtassociates with Tet1 in a complex in these cells and Tet1promotesDNAbinding ofOgt [83] It is tempting to speculatethat Ogt is affecting gene regulation in association withTet1 and through localized epigenetic modifications in genepromotersThe activity of Ogt would appear to be pleiotropicwithin the cell and alternative mechanisms of gene activationand repression may account for Ogt activity Further thecolocalization ofOgt Tet1 andH3K4me3 at hypomethylatedCpG-rich gene promotersmay act tomaintain these areas freeof methylation and modulate the epigenome to maintain thepluripotent state

It is known that SIRT1 a member of the sirtuin histonedeacetylase family is expressed in the preimplantationembryo and in ES cells [84ndash86] Inhibition of SIRTs in theembryo negatively impacts blastocyst development and

increases ROS production [87 88] In pluripotent cellsSIRT1 levels are downregulated as the cells commencedifferentiation [84 89] These proteins have been shown toplay key roles in linking the metabolome with the epigenomeby acting as sensors of environmental stimuli regulatinga number of metabolic pathways and modulating theacetylation of histone and nonhistone substrates (reviewedby [90]) It remains to be determined if any of the rolesSIRT1 plays in the early embryo and pluripotent cells areattributable to SIRT1-mediated modulation of histoneacetylation and the epigenome

7 Replicating the In Vivo Environment andCell States In Vitro

Within the niche the metabolism of pluripotent cells is finelybalanced configured to supply the metabolites required forgrowth and for maintenance of DNA and yet poised torespond to the challenges that will be placed on the cell asit differentiates and specifically the requirements of the mas-sive DNA remodeling that accompanies the early events ofdifferentiationOptimization of in vitro culture conditions forpluripotent cells has largely centered on propagating cells thatare pluripotent differentiation competent and grossly kary-otypically normal It has been assumed that in vitro cultureconditions based on commonly used tissue culture mediaand developed with a focus on growth factor regulation willsustain the metabolism of the cell It is known that commonculture media are not suitable for embryo culture andsignificant increases in embryo viability have been achievedby developing media that recapitulate the physiological envi-ronment [91] Our greater understanding of how metabolitescan impact not only cell physiology but also epigeneticsraises questions about the impact of mediummetabolite con-centrations on the pluripotent epigenome and on cell state


The composition of the culture medium can significantlyinfluence metabolic pathway use in pluripotent cells andvariations in culture conditions could underlie much ofthe variability that exists in studies elucidating pluripotentmetabolism We have shown that serum supplementationand supplementation with serum replacer of human ES cellculture changed the production and consumption rates ofamino acids and increased glucose uptake by the cells [92]In contrast to others however we were unable to showalterations of metabolism on the induction of differentiationa difference we ascribe to the maintenance of base mediumcomposition throughout differentiation in our experiments[92] In a similar way metabolic differences between naıveand primed ES cellsmaymerely reflect basemedium variabil-ity Zhou et al (2012) attributed metabolic change with cellstate without recognizing the potential influence of changingbase media [93] In contrast Carey et al (2015) showedthat glutamine independence could be established in primedcells independent of base media Clearly there are metabolicdifferences between cell states however it is difficult todistinguish true metabolic differences when base mediumcomposition is not maintained

Anumber of protocols for establishing naıve pluripotencyhave recently been developed for human cells [94ndash97] Eachof these methodologies used distinct culture conditions thatwill establish inherent differences in underlying metaboliteuse The consequence of these changes in metabolism maynot impact pluripotency differentiation capacity or grosskaryotype but may result in significant alterations to theepigenome Epigenetic codes will likely be perpetuated dur-ing self-renewal and differentiation potentially influencingfuture cell events We have advocated the need to understandthe interplay between metabolism and the culture mediumto enable true optimization of development in the embryo[98 99] and in pluripotent stem cells [9]

Abnormalities in cellular metabolism have been linkedwith alterations in the epigenetic landscape contributing tonumerous diseases including cancer [100 101] It will there-fore be important to establish the metabolic mechanismsregulating pluripotent stem cell epigenetics that underliepluripotency and differentiation and examine the impact ofmetabolic perturbations on epigenetic control to ensure thesecells and their differentiated derivatives exhibit a normalphysiology and are not predisposed to disease states

71 Oxygen The Forgotten Metabolite The majority of alltissue culture including the majority of pluripotent cellculture is performed in the presence of atmospheric oxy-gen (sim20) The in vivo environment in which prevas-cularisation embryos develop constitutes a relatively lowoxygen environment (2ndash8) Departure from a physiolog-ical oxygen concentration in culture significantly impactspreimplantation embryo development While embryos arecapable of developing under 20 oxygen this has beenassociated with increased DNA fragmentation [102ndash104]altered genomic [105 106] and proteomic profiles [107] andperturbed metabolic activity [91 108] The changes inducedby atmospheric oxygen are not consistent with the viabilityof the blastocyst [99] Despite the embryonic requirement for

physiological oxygen and the negative impact of atmosphericoxygen on embryo viability cell derivatives of the embryoincluding pluripotent cells are routinely cultured and char-acterized in 20 oxygen

We have documented oxygen-dependent changes inpluripotent cell metabolism that occur in the absence ofovert changes in standardmeasures of self-renewal in humanES cells [109] Others have described similar changes inmetabolite use in response to oxygen [110ndash112] consistentwith a conserved cellular response to oxygen availability [39]Further the availability of oxygen can significantly impact thepluripotent epigenome Increased 51015840 methylcytosine stainingin response to high oxygen has been described in preimplan-tation embryos [113] with increased expression of SIRT1 andTET1 also reported [114] Maintenance of blastocyst integrityunder low oxygen is likely mediated by HIF2120572 [105] HIF2120572has been shown to be responsible for long-term adaptationto hypoxia in human ES cells [115] binding directly to OCT4NANOG and SOX2 proximal promoters in human ES cellscultured at low oxygen concentrations [116] Methylation oftheOCT4hypoxia response element (HRE) in humanES cellsis marked by a significant increase in H3K36me3 a marker oftranscriptional activation under 5 oxygen conditions com-pared with atmospheric oxygen

Within the NANOG and

SOX2 HREs human ES cells maintained under atmosphericconditions exhibited high H3K9me3 levels representing amarker of transcriptional silencing and significantly reducedH3K4me3 and H3K36me3 compared to cells cultured underhypoxic conditions [116] consistent with a more closedconformational chromatin with atmospheric oxygen culture

The oxygen concentration used to isolate human ES cellshas been shown to impact X inactivation status with thosecultured under physiological oxygen able to maintain bothactiveX chromosomes [117] In contrast those cultured underatmospheric conditions readily inactivate an X chromosomeSIRT activity is responsive to alterations in cellular redoxincluding oxidative stress (reviewed by [118]) and may beone mechanism by which oxygen-regulated changes to theepigenome are established Alternatively HIF activation hasbeen shown to upregulateMAT2A transcription in hepatomacells reducing SAM levels and leading toDNAdemethylation[119] Whether a similar relationship exists in pluripotentstem cells remains to be determined

72 Regulation of Demethylation by Vitamin C Vitamin C iscommonly added to culture as an antioxidant yet it has beenshown to regulate DNAmethylation dynamics in human andmouse pluripotent stem cells acting as a key regulator ofTETs [120] and the Jumonji family of histone demethylases[121]The culture of human ES cells without added vitamin Cincreased DNA methylation while the presence of vitaminC promotes DNA demethylation [122] In mouse ES cellssupplementation with vitaminC leads to a rapid increase in5hmC dependent on Tet activity [123] Addition of vitamin Cto culture establishes an epigenetic landscape more similar tothe inner cell mass of the embryo [123] potentially revealinga role for vitamin C in culture independent of its antioxidantcapacity


8 The Significance of Metaboloepigenetics toPluripotent Stem Cell Biology

It is clear that metabolism can drive cell state transitionsthrough interacting with the signalling machinery and moresubtly through modification of the epigenome What is lessclear is how perturbations in metabolism impact subsequentpotency and cell function A consequence of perturbingmetabolism is that heritable changes to DNA are passed onto daughter cells

In vitro embryo culture has been found to be associatedwith alterations in DNA methylation and the expression ofimprinted loci [124ndash128] Assisted reproductive technologiesthat involve the culture of the preimplantation embryo havebeen associated with the early onset of metabolic disease(reviewed by [129]) and an increased frequency of epigeneticdisorders [130] in offspring Imprinting abnormalities follow-ing in vitro culture have been described in embryos culturedwith different media formulations [131] A key difference inthese media formulations is the provision of amino acidsproviding a correlation between metabolite concentration inthe environment of the preimplantation embryo and lifelongimpacts on the resulting child The importance of estab-lishing correct metabolic regulation in embryos in culturecannot be underestimated There is still much work to do inmedium optimization Studies on the impact of the variouscommercially available media formulations on human ES cellepigenetics are essentially lacking

Independently derived human ES cells display rela-tively stable methylation patterns [132] and share equivalentgenomic arrangements [40] These analyses do not encom-pass the entirety of the epigenetic landscape and smallerdifferences may exist between cell lines and between whatis considered the norm of cells in culture versus cells inthe embryo A heavy reliance on glycolysis may by defaultactivate the major pathways that regulate the epigeneticlandscape providing sufficient intermediates to enable themaintenance of pluripotency However subtle differencesin medium formulation may impact less well-characterizedmodifications The process of ES cell isolation must by itsvery nature place selective pressure on cells that is likelyto be resolved in part through heritable modificationsto the epigenome that embed changes in gene expressionSignificant differences in the transcriptome of human EScell and ICM cells have been shown demonstrating thatthe process of ES cell derivation significantly alters geneexpression [133] and providing evidence for this selectiveprocess Adaptation may also involve modification to themetabolome of cells ES cells pluripotent by all standardmeasures can display disparatemetabolic profiles suggestingmetabolic adaptation can occur It will be important to assesswhether these changes in themetabolome impact in turn thepluripotent epigenome eliciting changes in differentiationpotential andor cell functionThese studies are not completeand next-generation sequencing is required to establish acomprehensive characterization of the ES cell epigenomein culture to identify the impact of culture adaptation onepigenetic integrity

81The Transition fromDifferentiated to Pluripotent Cell StateIs Accompanied by Changes in Metabolism The introductionof pluripotency transcription factors to somatic cells bringsabout progressive loss of the somatic phenotype and theacquisition of a pluripotent-like cell state (induced pluripo-tent stem iPS cells) Reprogramming to a pluripotent-likestate requires the remodelling of both metabolism (reviewedby [9]) and chromatin organization (reviewed by [134])Genome-wide chromatin remodeling is initiated in responseto reprogramming factor expression [135] establishing anepigenetic profile similar to that of embryonic stem cellswhere key developmental genes remain poised in a bivalent(repressed but activatable) state [136 137] Acquisition of apluripotent-like state necessitates the upregulation of glycol-ysis and downregulation of oxidative phosphorylation [7778 138] Appropriately modulating metabolism is essential toestablish the pluripotent cell state evidenced by the abilityto enhance or reduce reprogramming efficiency throughmetabolite modulation including through modulation ofoxygen concentrations [77 139 140] Despite the capacity toacquire a number of pluripotent characteristics iPS cells arenot physiologically equivalent to their ES cell counterparts

Differences in metabolism between ES and iPS cells havebeen described Increased levels of SAMpathwaymetabolitesand differences in unsaturated fatty acids are seen in iPS cells[141] suggesting that the reprogramming of metabolism isincomplete or perturbed in these cells We have documentedalterations in the capacity of iPS cells to regulate metabolismin response to oxygen (Harvey et al unpublished) iPS cellshave been shown to retain an epigenetic memory of theirsomatic origin [142ndash145] which is perpetuated through dif-ferentiation [144ndash146] Conceivably the higher level of globalDNAmethylation in iPS cells is evidence of the inappropriateregulation of metabolism during reprogramming Severalfactors used to reprogram somatic cells are known regulatorsof metabolism For example Lin28a has been shown toenhance the translation of mRNAs for several metabolicenzymes and thereby to regulate glycolysis and OXPHOS[147] These findings raise questions on the potential rolesthese factors will play if any in modulating pluripotent stemcell metabolism and how their impact on iPS cell metabolismis reflected in the epigenome Epigenetic modifications couldconceivably impact the physiology of iPS disease models orthe utility of these cells in drug discovery or regeneration

9 Conclusions

Many factors impact the relative activity of metabolic path-ways and the composition of metabolite pools within thecell including the extracellular milieu the regulation ofthe cell environment interface cell identity and functionand the stress imposed on the cell by extracellular andintracellular regulators Metabolites such as acetyl-CoA andSAM connect metabolism to signaling and gene expressionThe availability of these compounds also impacts epigeneticmodifications in the cell with low levels resulting in reduc-tions in acetylation and methylation respectively Evidenceas reviewed here suggests that the metabolome plays adefining role in the epigenetic regulation of the cell including


cells of the pluripotent lineageWhat is equally clear from theavailable evidence is that there is much more work neededto describe the role of metabolism in the epigenome andthen to understand the biological programs regulated bymetabolically controlled epigenetic mechanisms Integrationof how metabolism changes with cell states is needed as themajority of studies to date fail to delineate between cell statesor more specifically address the transitions between them

In culture the ability of a cell to adapt to its environmentmay be reflected in changes to the epigenomeThese changesare selected to promote survival in an environment definedby nutrient availability and effected through the activityof metabolic pathways and are perpetuated within the cellpopulation Despite this knowledge the impact of differentmedia used for pluripotent stem cell maintenance and iPS cellgeneration is poorly appreciated where the quality of cellswithin a medium is generally evaluated without metabolicanalysis As with the blastocystinner cell mass an inappro-priate nutrient composition for the culture of pluripotent cellsmay compromise ldquometabolic fidelityrdquo and have significantdownstream impacts on development and viability It islikely that these impacts are mediated through epigeneticregulation and in culture alterations will be perpetuatedwith cell division Selected changes in metabolism couldlimit the availability of cofactors like SAM and acetyl-CoAwith long term heritable alterations to the epigenomeThesecould impact the identity of daughter cells bias differentia-tion potential or compromise the function of differentiatedderivatives potentially in subtle but important ways

Elucidating the dynamics of and mechanisms that con-trol cellular responses to metabolite availability will provideopportunities to manipulate cell fate Establishing the appro-priate balance of nutrients to support ongoing developmentrequires a clearer understanding of the regulation of thepathways modulating metabolic control and identificationof mechanisms that are perturbed by specific environmentalconditions Future studies should address the impact ofmetabolic adaptations of pluripotent stem cells to variousculture conditions in the absence of changes to the baseformulation the metabolic regulation of differentiation andhow differences in metabolism impact cell function As epi-genetic landscapes can impact disease states including cancerand neurodegenerative disorders the appropriate regulationof these enzymes established through metabolic pathwayswill rely on the establishment of physiologically relevantconditions to support the continuum of pluripotent stemcell states The impact of culture protocols on downstreamepigenetic profiles and differentiated cell function will needto be investigated to inform of any deleterious conditions thatnegatively alter cell physiology

Abbreviations

ac AcetylationACL ATP citrate lyase120572KG Alpha-ketoglutarateATP Adenosine triphosphateF-6-P Fructose-6-phosphateG-6-P Glucose-6-phosphate

G6PD Glucose-6-phosphate dehydrogenaseGlu GlutamateGSH GlutathioneHATs Histone acetyltransferasesHMT Histone methyltransferasesHCY HomocysteineJMJ Jumonji demethylases5mC Methylcytosine5hmC HydroxymethylcytosineLDH Lactate dehydrogenaseme MethylationMet MethionineNAD+ Nicotinamide adenine dinucleotideNADH Reduced form of NAD+NADP+ Nicotinamide adenine dinucleotide phosphateNADPH Reduced form of NADP+O2 Oxygen

O-GlcNAc O-linked119873-acetyl glycosylationOXPHOS Oxidative phosphorylationPDH Pyruvate dehydrogenasePKM2 Pyruvate kinase M2PPP Pentose phosphate pathwayPro ProlineSAH S-AdenosylhomocysteineSAM S-Adenosyl methionineSIRTs SirtuinsTCA Tricarboxylic acid cycleTET Ten-Eleven Translocation demethylasesTdh Threonine dehydrogenaseTHF Tetrahydrofolate5-CH

3-THF 5-Methyltetrahydrofolate

Thr Threonine

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

References

[1] V Azuara P Perry S Sauer et al ldquoChromatin signatures ofpluripotent cell linesrdquoNature Cell Biology vol 8 no 5 pp 532ndash538 2006

[2] B E Bernstein T S Mikkelsen X Xie et al ldquoA bivalent chro-matin structure marks key developmental genes in embryonicstem cellsrdquo Cell vol 125 no 2 pp 315ndash326 2006

[3] P Voigt W-W Tee and D Reinberg ldquoA double take on bivalentpromotersrdquo Genes and Development vol 27 no 12 pp 1318ndash1338 2013

[4] M G Vander Heiden L C Cantley and C B ThompsonldquoUnderstanding the warburg effect themetabolic requirementsof cell proliferationrdquo Science vol 324 no 5930 pp 1029ndash10332009

[5] D K Gardner and A J Harvey ldquoBlastocyst metabolismrdquoReproduction Fertility and Development 2015

[6] N Shyh-Chang G Q Daley and L C Cantley ldquoStem cellmetabolism in tissue development and agingrdquo Developmentvol 140 no 12 pp 2535ndash2547 2013


[7] R Bukowiecki J Adjaye and A Prigione ldquoMitochondrialfunction in pluripotent stem cells and cellular reprogrammingrdquoGerontology vol 60 no 2 pp 174ndash182 2014

[8] X Xu S Duan F Yi A Ocampo G-H Liu and J C IBelmonte ldquoMitochondrial regulation in pluripotent stem cellsrdquoCell Metabolism vol 18 no 3 pp 325ndash332 2013

[9] A J Harvey J Rathjen and D K Gardner ldquoThe metabolicframework of pluripotent stem cells and potential mechanismsof regulationrdquo in StemCells in ReproductiveMedicine C SimonA Pellicer and R Reijo Pera Eds pp 164ndash179 CambridgeUniversity Press New York NY USA 3rd edition 2013

[10] J Rathjen ldquoThe states of pluripotency pluripotent lineagedevelopment in the embryo and in the dishrdquo ISRN Stem Cellsvol 2014 Article ID 208067 19 pages 2014

[11] F A Brook and R L Gardner ldquoThe origin and efficientderivation of embryonic stem cells in themouserdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 94 no 11 pp 5709ndash5712 1997

[12] Q-L Ying J Wray J Nichols et al ldquoThe ground state ofembryonic stem cell self-renewalrdquoNature vol 453 no 7194 pp519ndash523 2008

[13] T OrsquoLeary B Heindryckx S Lierman et al ldquoTracking theprogression of the human inner cell mass during embryonicstem cell derivationrdquo Nature Biotechnology vol 30 no 3 pp278ndash282 2012

[14] J AThomson J Itskovitz-Eldor S S Shapiro et al ldquoEmbryonicstem cell lines derived from human blastocystsrdquo Science vol282 no 5391 pp 1145ndash1147 1998

[15] D James A J Levine D Besser and A Hemmati-BrivanlouldquoTGF120573activinnodal signaling is necessary for the mainte-nance of pluripotency in human embryonic stem cellsrdquo Devel-opment vol 132 no 6 pp 1273ndash1282 2005

[16] LVallierMAlexander andRA Pedersen ldquoActivinNodal andFGF pathways cooperate to maintain pluripotency of humanembryonic stem cellsrdquo Journal of Cell Science vol 118 no 19pp 4495ndash4509 2005

[17] Y Kojima K Kaufman-Francis J B Studdert et al ldquoThetranscriptional and functional properties of mouse epiblaststem cells resemble the anterior primitive streakrdquoCell StemCellvol 14 no 1 pp 107ndash120 2014

[18] I GM Brons L E SmithersMW B Trotter et al ldquoDerivationof pluripotent epiblast stem cells from mammalian embryosrdquoNature vol 448 no 7150 pp 191ndash195 2007

[19] P J Tesar J G Chenoweth F A Brook et al ldquoNew celllines from mouse epiblast share defining features with humanembryonic stem cellsrdquo Nature vol 448 no 7150 pp 196ndash1992007

[20] J Silva O Barrandon J Nichols J Kawaguchi T W Theunis-sen and A Smith ldquoPromotion of reprogramming to groundstate pluripotency by signal inhibitionrdquo PLoS Biology vol 6article e253 2008

[21] J Nichols and A Smith ldquoThe origin and identity of embryonicstem cellsrdquo Development vol 138 no 1 pp 3ndash8 2011

[22] T Boroviak R Loos P Bertone A Smith and J Nichols ldquoTheability of inner-cell-mass cells to self-renew as embryonic stemcells is acquired following epiblast specificationrdquo Nature CellBiology vol 16 no 6 pp 516ndash528 2014

[23] JMWashington J Rathjen F Felquer et al ldquoL-Proline inducesdifferentiation of ES cells a novel role for an amino acid in theregulation of pluripotent cells in culturerdquoTheAmerican Journalof PhysiologymdashCell Physiology vol 298 no 5 pp C982ndashC9922010

[24] D K Gardner ldquoLactate production by the mammalian blasto-cyst manipulating themicroenvironment for uterine implanta-tion and invasionrdquo BioEssays vol 37 pp 364ndash371 2015

[25] O Warburg ldquoOn respiratory impairment in cancer cellsrdquoScience vol 124 no 3215 pp 269ndash270 1956

[26] T Wang C Marquardt and J Foker ldquoAerobic glycolysis duringlymphocyte proliferationrdquo Nature vol 261 no 5562 pp 702ndash705 1976

[27] D K Gardner ldquoEmbryo development and culture techniquesrdquoinAnimal Breeding Technology for the 21st Century J Clark Edpp 13ndash46 Harwood Academic London UK 1998

[28] D K Gardner ldquoChanges in requirements and utilization ofnutrients duringmammalian preimplantation embryo develop-ment and their significance in embryo culturerdquoTheriogenologyvol 49 no 1 pp 83ndash102 1998

[29] M H L Snow ldquoGastrulation in the mouse growth andregionalization of the epiblastrdquo Journal of Embryology andExperimental Morphology vol 42 pp 293ndash303 1977

[30] M JMorgan andP Faik ldquoCarbohydratemetabolism in culturedanimal cellsrdquo Bioscience Reports vol 1 no 9 pp 669ndash686 1981

[31] L J Reitzer B M Wice and D Kennell ldquoThe pentosecycle Control and essential function in HeLa cell nucleic acidsynthesisrdquo The Journal of Biological Chemistry vol 255 no 12pp 5616ndash5626 1980

[32] D A Hume and M J Weidemann ldquoRole and regulationof glucose metabolism in proliferating cellsrdquo Journal of theNational Cancer Institute vol 62 no 1 pp 3ndash8 1979

[33] L J Mandel ldquoEnergymetabolism of cellular activation growthand transformationrdquo in Current Topics in Membranes andTransport vol 27 chapter 8 pp 261ndash291 Elsevier 1986

[34] K M Holmstrom and T Finkel ldquoCellular mechanisms andphysiological consequences of redox-dependent signallingrdquoNature ReviewsMolecular Cell Biology vol 15 no 6 pp 411ndash4212014

[35] B Fischer and B D Bavister ldquoOxygen tension in the oviductand uterus of rhesus monkeys hamsters and rabbitsrdquo Journal ofReproduction amp Fertility vol 99 no 2 pp 673ndash679 1993

[36] J A Mitchell and J M Yochim ldquoMeasurement of intrauterineoxygen tension in the rat and its regulation by ovarian steroidhormonesrdquo Endocrinology vol 83 no 4 pp 691ndash700 1968

[37] H J Leese ldquoMetabolic control during preimplantation mam-malian developmentrdquo Human Reproduction Update vol 1 no1 pp 63ndash72 1995

[38] B-H Jiang G L Semenza C Bauer and H H MartildquoHypoxia-inducible factor 1 levels vary exponentially over aphysiologically relevant range of O2 tensionrdquo American Journalof PhysiologymdashCell Physiology vol 271 no 4 pp C1172ndashC11801996

[39] G L Semenza ldquoHypoxia-inducible factors in physiology andmedicinerdquo Cell vol 148 no 3 pp 399ndash408 2012

[40] T Ryba I Hiratani T Sasaki et al ldquoReplication timing a fin-gerprint for cell identity and pluripotencyrdquo PLoS ComputationalBiology vol 7 no 10 Article ID e1002225 2011

[41] H Julienne B Audit and A Arneodo ldquoEmbryonic stem cellspecific lsquomasterrsquo replication origins at the heart of the loss ofpluripotencyrdquo PLoS Computational Biology vol 11 no 2 ArticleID e1003969 2015

[42] T Chen and S Y R Dent ldquoChromatin modifiers and remod-ellers regulators of cellular differentiationrdquo Nature ReviewsGenetics vol 15 no 2 pp 93ndash106 2014


[43] M J Boland K LNazor and J F Loring ldquoEpigenetic regulationof pluripotency and differentiationrdquo Circulation Research vol115 no 2 pp 311ndash324 2014

[44] I Hiratani T Ryba M Itoh et al ldquoGenome-wide dynamicsof replication timing revealed by in vitro models of mouseembryogenesisrdquo Genome Research vol 20 no 2 pp 155ndash1692010

[45] S Efroni R Duttagupta J Cheng et al ldquoGlobal transcriptionin pluripotent embryonic stem cellsrdquo Cell Stem Cell vol 2 no5 pp 437ndash447 2008

[46] R D Hawkins G C Hon L K Lee et al ldquoDistinct epigenomiclandscapes of pluripotent and lineage-committed human cellsrdquoCell Stem Cell vol 6 no 5 pp 479ndash491 2010

[47] B Delatte R Deplus and F Fuks ldquoPlaying TETris with DNAmodificationsrdquoThe EMBO Journal vol 33 no 11 pp 1198ndash12112014

[48] H Wu and Y Zhang ldquoReversing DNA methylation mecha-nisms genomics and biological functionsrdquoCell vol 156 no 1-2pp 45ndash68 2014

[49] M Tahiliani K P Koh Y Shen et al ldquoConversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalianDNAbyMLLpartner TET1rdquo Science vol 324 no 5929 pp 930ndash935 2009

[50] M Xiao H Yang W Xu et al ldquoInhibition of 120572-KG-dependenthistone and DNA demethylases by fumarate and succinatethat are accumulated in mutations of FH and SDH tumorsuppressorsrdquo Genes and Development vol 26 no 12 pp 1326ndash1338 2012

[51] K P Koh A Yabuuchi S Rao et al ldquoTet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specifica-tion in mouse embryonic stem cellsrdquo Cell Stem Cell vol 8 no2 pp 200ndash213 2011

[52] A Szwagierczak S Bultmann C S Schmidt F Spadaand H Leonhardt ldquoSensitive enzymatic quantification of5-hydroxymethylcytosine in genomic DNArdquo Nucleic AcidsResearch vol 38 article e181 2010

[53] Y-I Tsukada J Fang H Erdjument-Bromage et al ldquoHistonedemethylation by a family of JmjC domain-containing pro-teinsrdquo Nature vol 439 no 7078 pp 811ndash816 2006

[54] Y-H LohW Zhang X Chen J George andH-H Ng ldquoJmjd1aand Jmjd2c histoneH3 Lys 9 demethylases regulate self-renewalin embryonic stem cellsrdquo Genes amp Development vol 21 no 20pp 2545ndash2557 2007

[55] K E Wellen G Hatzivassiliou U M Sachdeva T V Bui JR Cross and C B Thompson ldquoATP-citrate lyase links cellularmetabolism to histone acetylationrdquo Science vol 324 no 5930pp 1076ndash1080 2009

[56] J Krejcı R Uhlırova G Galiova S Kozubek J Smigova and EBartova ldquoGenome-wide reduction in H3K9 acetylation duringhuman embryonic stem cell differentiationrdquo Journal of CellularPhysiology vol 219 no 3 pp 677ndash687 2009

[57] J-H Lee S R L Hart and D G Skalnik ldquoHistone deacetylaseactivity is required for embryonic stem cell differentiationrdquoGenesis vol 38 no 1 pp 32ndash38 2004

[58] H Hezroni B S Sailaja and E Meshorer ldquoPluripotency-related valproic acid (VPA)-induced genome-wide histone H3lysine 9 (H3K9) acetylation patterns in embryonic stem cellsrdquoJournal of Biological Chemistry vol 286 no 41 pp 35977ndash35988 2011

[59] C B Ware L Wang B H Mecham et al ldquoHistone deacetylaseinhibition elicits an evolutionarily conserved self-renewal pro-gram in embryonic stem cellsrdquo Cell Stem Cell vol 4 no 4 pp359ndash369 2009

[60] P B Alexander J Wang and S L McKnight ldquoTargeted killingof a mammalian cell based upon its specializedmetabolic staterdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 38 pp 15828ndash15833 2011

[61] J Wang P Alexander L Wu R Hammer O Cleaver and SL McKnight ldquoDependence of mouse embryonic stem cells onthreonine catabolismrdquo Science vol 325 no 5939 pp 435ndash4392009

[62] N Shyh-Chang J W Locasale C A Lyssiotis et al ldquoInfluenceof threoninemetabolism on S-adenosylmethionine and histonemethylationrdquo Science vol 339 no 6116 pp 222ndash226 2013

[63] A J Edgar ldquoThe human L-threonine 3-dehydrogenase gene isan expressed pseudogenerdquoBMCGenetics vol 3 article 18 2002

[64] N Shiraki Y Shiraki T Tsuyama et al ldquoMethioninemetabolism regulates maintenance and differentiation ofhuman pluripotent stem cellsrdquo Cell Metabolism vol 19 no 5pp 780ndash794 2014

[65] B W Carey L W S Finley J R Cross C D Allis andC B Thompson ldquoIntracellular 120572-ketoglutarate maintains thepluripotency of embryonic stem cellsrdquoNature vol 518 no 7539pp 413ndash416 2015

[66] J Rathjen J-A Lake M D Bettess J MWashington G Chap-man and P D Rathjen ldquoFormation of a primitive ectodermlike cell population EPL cells from ES cells in response tobiologically derived factorsrdquo Journal of Cell Science vol 112 part5 pp 601ndash612 1999

[67] J N Hughes N Dodge P D Rathjen and J Rathjen ldquoA novelrole for gamma-secretase in the formation of primitive streak-like intermediates from ES cells in culturerdquo STEM CELLS vol27 no 12 pp 2941ndash2951 2009

[68] T A Pelton S Sharma T C Schulz J Rathjen and P DRathjen ldquoTransient pluripotent cell populations during prim-itive ectoderm formation correlation of in vivo and in vitropluripotent cell developmentrdquo Journal of Cell Science vol 115no 2 pp 329ndash339 2002

[69] J-A Lake J Rathjen J Remiszewski and P D RathjenldquoReversible programming of pluripotent cell differentiationrdquoJournal of Cell Science vol 113 part 3 pp 555ndash566 2000

[70] C Yap H N Goh M Familari P D Rathjen and J RathjenldquoThe formation of proximal and distal definitive endodermpopulations in culture requires p38 MAPK activityrdquo Journal ofCell Science vol 127 no 10 pp 2204ndash2216 2014

[71] B S N Tan A Lonic M B Morris P D Rathjen andJ Rathjen ldquoThe amino acid transporter SNAT2 mediates l-proline-induced differentiation of ES cellsrdquo American Journalof PhysiologymdashCell Physiology vol 300 no 6 pp C1270ndashC12792011

[72] L Casalino S ComesG Lambazzi et al ldquoControl of embryonicstem cell metastability by L-proline catabolismrdquo Journal ofMolecular Cell Biology vol 3 no 2 pp 108ndash122 2011

[73] C DrsquoAniello A Fico L Casalino et al ldquoA novel autoregulatoryloop between theGcn2-Atf4 pathway andL-Prolinemetabolismcontrols stem cell identityrdquo Cell Death amp Differentiation vol 22no 7 p 1234 2015

[74] S Comes M Gagliardi N Laprano et al ldquoL-proline induces amesenchymal-like invasive program in embryonic stem cells byremodeling H3K9 and H3K36 methylationrdquo Stem Cell Reportsvol 1 no 4 pp 307ndash321 2013


[75] C Choudhary B T Weinert Y Nishida E Verdin and MMann ldquoThe growing landscape of lysine acetylation linksmetabolism and cell signallingrdquo Nature Reviews Molecular CellBiology vol 15 no 8 pp 536ndash550 2014

[76] A Moussaieff M Rouleau D Kitsberg et al ldquoGlycolysis-mediated changes in acetyl-CoA and histone acetylation con-trol the early differentiation of embryonic stem cellsrdquo CellMetabolism vol 21 no 3 pp 392ndash402 2015

[77] C D L Folmes T J Nelson A Martinez-Fernandez et alldquoSomatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogrammingrdquo CellMetabolism vol 14 no 2 pp 264ndash271 2011

[78] S Varum A S Rodrigues M B Moura et al ldquoEnergymetabolism in human pluripotent stem cells and their differen-tiated counterpartsrdquo PLoS ONE vol 6 no 6 Article ID e209142011

[79] J Zhang I Khvorostov J S Hong et al ldquoUCP2 regulates energymetabolism and differentiation potential of human pluripotentstem cellsrdquo The EMBO Journal vol 30 no 24 pp 4860ndash48732011

[80] Y Qiao R Wang X Yang K Tang and N Jing ldquoDual roles ofhistone H3 lysine 9 acetylation in human embryonic stem cellpluripotency and neural differentiationrdquo Journal of BiologicalChemistry vol 290 no 4 pp 2508ndash2520 2015

[81] R Shafi S P N Iyer L G Ellies et al ldquoThe O-GlcNAc trans-ferase gene resides on the X chromosome and is essential forembryonic stem cell viability and mouse ontogenyrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 97 no 11 pp 5735ndash5739 2000

[82] H Jang T W Kim S Yoon et al ldquoO-GlcNAc regulatespluripotency and reprogramming by directly acting on corecomponents of the pluripotency networkrdquo Cell Stem Cell vol11 no 1 pp 62ndash74 2012

[83] P Vella A Scelfo S Jammula et al ldquoTet proteins connect theO-linked N-acetylglucosamine transferase Ogt to chromatin inembryonic stem cellsrdquo Molecular Cell vol 49 no 4 pp 645ndash656 2013

[84] V Calvanese E Lara B Suarez-Alvarez et al ldquoSirtuin 1regulation of developmental genes during differentiation ofstem cellsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 107 no 31 pp 13736ndash137412010

[85] M W McBurney X Yang K Jardine M Bieman J Thrsquong andM Lemieux ldquoThe absence of SIR2120572 protein has no effect onglobal gene silencing inmouse embryonic stem cellsrdquoMolecularCancer Research vol 1 no 5 pp 402ndash409 2003

[86] M W McBurney X Yang K Jardine et al ldquoThe mammalianSIR2alpha protein has a role in embryogenesis and gametoge-nesisrdquo Molecular and Cellular Biology vol 23 no 1 pp 38ndash542003

[87] Y Kawamura Y Uchijima N Horike et al ldquoSirt3 protectsin vitromdashfertilized mouse preimplantation embryos againstoxidative stressmdashInduced p53-mediated developmental arrestrdquoThe Journal of Clinical Investigation vol 120 no 8 pp 2817ndash2828 2010

[88] J M Campbell M B Nottle I Vassiliev M Mitchell and MLane ldquoInsulin increases epiblast cell number of in vitro culturedmouse embryos via the PI3KGSK3p53 pathwayrdquo Stem Cellsand Development vol 21 no 13 pp 2430ndash2441 2012

[89] Y L Lee Q Peng S W Fong et al ldquoSirtuin 1 facilitatesgeneration of induced pluripotent stem cells from mouse

embryonic fibroblasts through the miR-34a and p53 pathwaysrdquoPLoS ONE vol 7 no 9 Article ID e45633 2012

[90] M CHaigis andD A Sinclair ldquoMammalian sirtuins biologicalinsights and disease relevancerdquo Annual Review of PathologyMechanisms of Disease vol 5 pp 253ndash295 2010

[91] M Lane and D K Gardner ldquoUnderstanding cellular disrup-tions during early embryo development that perturb viabilityand fetal developmentrdquo Reproduction Fertility and Develop-ment vol 17 no 3 pp 371ndash378 2005

[92] J Rathjen C Yeo C Yap B S N Tan P D Rathjen and D KGardner ldquoCulture environment regulates amino acid turnoverand glucose utilisation in human ES cellsrdquo Reproduction Fertil-ity and Development vol 26 no 5 pp 703ndash716 2013

[93] WZhouMChoiDMargineantu et al ldquoHIF1120572 induced switchfrom bivalent to exclusively glycolytic metabolism during ESC-to-EpiSChESC transitionrdquoTheEMBO Journal vol 31 no 9 pp2103ndash2116 2012

[94] Y-S Chan J Goke J-H Ng et al ldquoInduction of a humanpluripotent state with distinct regulatory circuitry that resem-bles preimplantation epiblastrdquo Cell Stem Cell vol 13 no 6 pp663ndash675 2013

[95] O Gafni L Weinberger A A Mansour et al ldquoDerivation ofnovel human ground state naive pluripotent stem cellsrdquoNaturevol 504 no 7479 pp 282ndash286 2013

[96] TWTheunissen B E Powell HWang et al ldquoSystematic iden-tification of culture conditions for induction and maintenanceof naive human pluripotencyrdquo Cell Stem Cell vol 15 no 4 pp471ndash487 2014

[97] C B Ware A M Nelson B Mecham et al ldquoDerivation ofnaıve human embryonic stem cellsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 111 no12 pp 4484ndash4489 2014

[98] D K Gardner T B Pool and M Lane ldquoEmbryo nutrition andenergy metabolism and its relationship to embryo growth dif-ferentiation and viabilityrdquo Seminars in Reproductive Medicinevol 18 no 2 pp 205ndash218 2000

[99] D K Gardner and P L Wale ldquoAnalysis of metabolism to selectviable human embryos for transferrdquo Fertility and Sterility vol99 no 4 pp 1062ndash1072 2013

[100] M J Hitchler and F E Domann ldquoMetabolic defects provide aspark for the epigenetic switch in cancerrdquo Free Radical Biologyand Medicine vol 47 no 2 pp 115ndash127 2009

[101] W G Kaelin Jr and S L McKnight ldquoInfluence of metabolismon epigenetics and diseaserdquo Cell vol 153 no 1 pp 56ndash69 2013

[102] Y Goto Y Noda T Mori and M Nakano ldquoIncreased gener-ation of reactive oxygen species in embryos cultured in vitrordquoFree Radical Biology andMedicine vol 15 no 1 pp 69ndash75 1993

[103] Y Kitagawa K Suzuki A Yoneda and T Watanabe ldquoEffectsof oxygen concentration and antioxidants on the in vitro devel-opmental ability production of reactive oxygen species (ROS)and DNA fragmentation in porcine embryosrdquo Theriogenologyvol 62 no 7 pp 1186ndash1197 2004

[104] H-C Kwon H-W Yang K-J Hwang et al ldquoEffects of lowoxygen condition on the generation of reactive oxygen speciesand the development in mouse embryos cultured in vitrordquoJournal of Obstetrics and Gynaecology Research vol 25 no 5pp 359ndash366 1999

[105] A J Harvey K L Kind M Pantaleon D T Armstrong andJ G Thompson ldquoOxygen-regulated gene expression in bovineblastocystsrdquo Biology of Reproduction vol 71 no 4 pp 1108ndash11192004


[106] P F Rinaudo G Giritharan S Talbi A T Dobson and RM Schultz ldquoEffects of oxygen tension on gene expression inpreimplantation mouse embryosrdquo Fertility and Sterility vol 86no 4 supplement pp 1265e1ndash1265e36 2006

[107] M G Katz-Jaffe D W Linck W B Schoolcraft and D KGardner ldquoA proteomic analysis of mammalian preimplantationembryonic developmentrdquoReproduction vol 130 no 6 pp 899ndash905 2005

[108] P L Wale and D K Gardner ldquoOxygen regulates amino acidturnover and carbohydrate uptake during the preimplantationperiod of mouse embryo developmentrdquo Biology of Reproduc-tion vol 87 article 24 pp 21ndash28 2012

[109] A J Harvey J Rathjen L J Yu and D K Gardner ldquoOxygenmodulates human embryonic stem cell metabolism in theabsence of changes in self-renewalrdquo Reproduction Fertility andDevelopment 2014

[110] D R Christensen P C Calder and F D Houghton ldquoEffectof oxygen tension on the amino acid utilisation of humanembryonic stemcellsrdquoCellular Physiology andBiochemistry vol33 no 1 pp 237ndash246 2014

[111] C E Forristal D R Christensen F E Chinnery et al ldquoEnvi-ronmental oxygen tension regulates the energymetabolism andself-renewal of human embryonic stem cellsrdquo PLoS ONE vol 8no 5 Article ID e62507 2013

[112] J Turner L-E Quek D Titmarsh et al ldquoMetabolic profilingand flux analysis of MEL-2 human embryonic stem cells duringexponential growth at physiological and atmospheric oxygenconcentrationsrdquo PLoS ONE vol 9 no 11 Article ID e1127572014

[113] W Li K Goossens M Van Poucke et al ldquoHigh oxygentension increases global methylation in bovine 4-cell embryosand blastocysts but does not affect general retrotransposonexpressionrdquo Reproduction Fertility and Development 2014

[114] C A Burroughs G L Williamson M C Golding and CR Long ldquo3 oxidative stress induced changes in epigeneticmodifying gene mRNA in pre-implantation in vitro bovineembryosrdquo Reproduction Fertility and Development vol 25 no1 p 149 2012

[115] C E Forristal K LWright N A Hanley R O C Oreffo and FD Houghton ldquoHypoxia inducible factors regulate pluripotencyand proliferation in human embryonic stem cells cultured atreduced oxygen tensionsrdquo Reproduction vol 139 no 1 pp 85ndash97 2010

[116] R Petruzzelli D R Christensen K L Parry T Sanchez-Elsnerand F D Houghton ldquoHIF-2120572 regulates NANOG expressionin human embryonic stem cells following hypoxia and reoxy-genation through the interaction with an oct-sox cis regulatoryelementrdquo PLoS ONE vol 9 no 10 Article ID e108309 2014

[117] C J Lengner A A Gimelbrant J A Erwin et al ldquoDerivationof pre-X inactivation human embryonic stem cells underphysiological oxygen concentrationsrdquo Cell vol 141 no 5 pp872ndash883 2010

[118] J-W Hwang H Yao S Caito I K Sundar and I RahmanldquoRedox regulation of SIRT1 in inflammation and cellular senes-cencerdquo Free Radical Biology and Medicine vol 61 pp 95ndash1102013

[119] Q Liu L Liu Y Zhao et al ldquoHypoxia induces genomicDNA demethylation through the activation of HIF-1alpha andtranscriptional upregulation of MAT2A in hepatoma cellsrdquoMolecular CancerTherapeutics vol 10 no 6 pp 1113ndash1123 2011

[120] E A Minor B L Court J I Young and G Wang ldquoAscor-bate induces ten-eleven translocation (Tet) methylcytosine

dioxygenase-mediated generation of 5-hydroxymethylcyto-sinerdquo The Journal of Biological Chemistry vol 288 no 19 pp13669ndash13674 2013

[121] T Wang K Chen X Zeng et al ldquoThe histone demethylasesJhdm1a1b enhance somatic cell reprogramming in a vitamin-C-dependent mannerrdquo Cell Stem Cell vol 9 no 6 pp 575ndash5872011

[122] T-L Chung R M Brena G Kolle et al ldquoVitamin C promoteswidespread yet specific DNA demethylation of the epigenomein human embryonic stem cellsrdquo Stem Cells vol 28 no 10 pp1848ndash1855 2010

[123] K Blaschke K T EbataMMKarimi et al ldquoVitaminC inducesTet-dependent DNA demethylation and a blastocyst-like statein ES cellsrdquo Nature vol 500 no 7461 pp 222ndash226 2013

[124] A S DohertyM RWMann KD TremblayM S Bartolomeiand R M Schultz ldquoDifferential effects of culture on imprintedH19 expression in the preimplantation mouse embryordquo Biologyof Reproduction vol 62 no 6 pp 1526ndash1535 2000

[125] S Khosla W Dean D Brown W Reik and R Feil ldquoCulture ofpreimplantation mouse embryos affects fetal development andthe expression of imprinted genesrdquo Biology of Reproduction vol64 no 3 pp 918ndash926 2001

[126] T Li T H Vu G A Ulaner et al ldquoIVF results in de novo DNAmethylation and histone methylation at an Igf2-H19 imprintingepigenetic switchrdquo Molecular Human Reproduction vol 11 no9 pp 631ndash640 2005

[127] M R W Mann S S Lee A S Doherty et al ldquoSelectiveloss of imprinting in the placenta following preimplantationdevelopment in culturerdquoDevelopment vol 131 no 15 pp 3727ndash3735 2004

[128] B A Market Velker M M Denomme and M R W MannldquoLoss of genomic imprinting in mouse embryos with fastrates of preimplantation development in culturerdquo Biology ofReproduction vol 86 no 5 article 143 pp 141ndash116 2012

[129] A J Watkins T Papenbrock and T P Fleming ldquoThe preim-plantation embryo handle with carerdquo Seminars in ReproductiveMedicine vol 26 no 2 pp 175ndash185 2008

[130] L N Odom and J Segars ldquoImprinting disorders and assistedreproductive technologyrdquo Current Opinion in EndocrinologyDiabetes and Obesity vol 17 no 6 pp 517ndash522 2010

[131] B A Market-Velker A D Fernandes and M R W MannldquoSide-by-side comparison of five commercial media systemsin a mouse model suboptimal in vitro culture interferes withimprint maintenancerdquo Biology of Reproduction vol 83 no 6pp 938ndash950 2010

[132] O Adewumi B Aflatoonian L Ahrlund-Richter et al ldquoChar-acterization of human embryonic stem cell lines by the Interna-tional Stem Cell Initiativerdquo Nature Biotechnology vol 25 no 7pp 803ndash816 2007

[133] A Galan P Diaz-Gimeno M E Poo et al ldquoDefining thegenomic signature of totipotency and pluripotency during earlyhumandevelopmentrdquoPLoSONE vol 8 no 4Article ID e621352013

[134] B Papp andK Plath ldquoEpigenetics of reprogramming to inducedpluripotencyrdquo Cell vol 152 no 6 pp 1324ndash1343 2013

[135] R P Koche Z D Smith M Adli et al ldquoReprogramming factorexpression initiates widespread targeted chromatin remodel-ingrdquo Cell Stem Cell vol 8 no 1 pp 96ndash105 2011

[136] T S Mikkelsen J Hanna X Zhang et al ldquoDissecting directreprogramming through integrative genomic analysisrdquo Naturevol 454 no 7200 pp 49ndash55 2008


[137] MWernig AMeissner R Foreman et al ldquoIn vitro reprogram-ming of fibroblasts into a pluripotent ES-cell-like staterdquoNaturevol 448 no 7151 pp 318ndash324 2007

[138] A Prigione and J Adjaye ldquoModulation of mitochondrialbiogenesis and bioenergetic metabolism upon in vitro and invivo differentiation of human ES and iPS cellsrdquo InternationalJournal of Developmental Biology vol 54 pp 1729ndash1741 2011

[139] Y YoshidaK Takahashi KOkita T Ichisaka and S YamanakaldquoHypoxia enhances the generation of induced pluripotent stemcellsrdquo Cell Stem Cell vol 5 no 3 pp 237ndash241 2009

[140] S Zhu W Li H Zhou et al ldquoReprogramming of humanprimary somatic cells by OCT4 and chemical compoundsrdquo CellStem Cell vol 7 no 6 pp 651ndash655 2010

[141] A D Panopoulos O Yanes S Ruiz et al ldquoThe metabolomeof induced pluripotent stem cells reveals metabolic changesoccurring in somatic cell reprogrammingrdquo Cell Research vol22 no 1 pp 168ndash177 2012

[142] J Deng R Shoemaker B Xie et al ldquoTargeted bisulfite sequenc-ing reveals changes inDNAmethylation associatedwith nuclearreprogrammingrdquo Nature Biotechnology vol 27 no 4 pp 353ndash360 2009

[143] H Ma R Morey R C OrsquoNeil et al ldquoAbnormalities in humanpluripotent cells due to reprogramming mechanismsrdquo Naturevol 511 no 7508 pp 177ndash183 2014

[144] K Kim A Doi B Wen et al ldquoEpigenetic memory in inducedpluripotent stem cellsrdquo Nature vol 467 no 7313 pp 285ndash2902010

[145] J M Polo S Liu M E Figueroa et al ldquoCell type of origininfluences the molecular and functional properties of mouseinduced pluripotent stem cellsrdquo Nature Biotechnology vol 28no 8 pp 848ndash855 2010

[146] R Lister M Pelizzola Y S Kida et al ldquoHotspots of aberrantepigenomic reprogramming in human induced pluripotentstem cellsrdquo Nature vol 471 no 7336 pp 68ndash73 2011

[147] N Shyh-Chang H Zhu T Yvanka de Soysa et al ldquoXLin28enhances tissue repair by reprogramming cellular metabolismrdquoCell vol 155 no 4 pp 778ndash792 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


for metabolic alterations to induce epigenetic deregulationWe refer the reader to existing reviews on mitochondrialcharacteristics of pluripotent stem cells [7ndash9]

2 Defining Pluripotent Stem Cell States

In the embryo and in culture pluripotent cells have beenshown to comprise a lineage of temporally distinct cell states(reviewed in [10]) Pluripotent stem cells either embryonic(derived from the inner cell mass (ICM) of the blastocyststage preimplantation embryo ES cells) or reprogrammedfrom a somatic cell to an embryonic stem cell-like state(induced pluripotent stem cells iPS cells) are defined bytheir ability to self-renew (to proliferate indefinitely) and bypluripotency as shown by the ability to act as a founder cellpopulation for all the cells of the embryo and adult Theseproperties underpin the potential use of these cells as a sourceof clinically relevant cells for therapeutics and drug discoveryMany studies have focused on defining themolecular proper-ties of ES cells but only recently have we begun to investigatethe physiology and metabolism of these cells

Mouse and human ES cells differ in their growth factorrequirements in vitro a consequence of their origins fromdifferent developmental stages Mouse ES cells isolated fromthe ICM are reliant on leukemia inhibitory factor (LIF) forongoing propagation while also requiring serum [11] Alter-natively mouse ES cells can be isolated in medium supple-mented with inhibitors of MekErk and GSK3 activation [12]Human ES cells are derived from a later stage pluripotent cellpopulation more similar to postimplantation epiblast [13]and are dependent on activinnodal and fibroblast growthfactor (FGF) signaling for self-renewal and pluripotency [14ndash16]The tissue of origin and gene expression profile of humanES cells suggest that they are representative of a later stagepluripotent cell state Pluripotent cells have been isolatedfrom the postimplantation epiblast or primitive ectoderm ofthe mouse Like human ES cells epiblast stem cells (EpiSC)require FGF and activin A for self-renewal and pluripotency[17ndash19] In culture these cells adopt the phenotype of theanterior primitive ectoderm from the late gastrula stageembryo [17]

Mouse ES cells can be cultured with inhibitors of FgfMekErk and Gsk3 to form a naıve ES cell state repre-sentative of the early inner cell mass [20ndash22] Inclusion ofthe GSK3 inhibitor minimizes the negative regulation ofbiosynthetic pathways [12] thereby modulating proliferativecapacity Alternatively mouse ES cells can be cultured inmedium supplementedwith the amino acid l-proline to formearly primitive ectoderm-like (EPL) cells that represent astage of pluripotency intermediate to ES cells and EpiSC [23]

Each population isolated or cultured represents a stemcell state within the continuum of the pluripotent lineage

3 Framework of Pluripotent StemCell Metabolism

Pluripotent stem cells and the pluripotent cells of the ICMfrom which they are derived exhibit a metabolism character-ized by high levels of glucose consumption combined with

the production of lactate (reviewed by [9 24] Figure 1) Thispattern of metabolism is maintained in conditions of oxygensufficiency distinguishing it from anaerobic glycolysis andhas therefore been termed aerobic glycolysis Warburg [25]first described aerobic glycolysis in cancer cells in culturewhich produce large amounts of lactate even in the presenceof sufficient oxygen for the complete oxidation of glucose (theWarburg effect) Initially it was thought that aerobic glycolysiswas specific to cancers but it has since been shown that thismetabolic trait occurs in other proliferating cells types suchas lymphocytes [26] Gardner [27 28] identified similaritiesbetween cancers and blastocysts and the significance of theWarburg effect for the development of the late stage embryo

What are the cellular advantages of aerobic glycolysis topluripotent stem cells Glucose is typically considered in itscapacity as an energy source Pluripotent cells are highlyproliferative with reports of cell cycle rates reducing to aslittle as 5ndash7 hours in the developing embryo [29] Tomaintainthis growth rate these cells will have to generate buildingblocks for proteins nucleic acids lipids and carbohydratesThe glycolytic metabolism of glucose may not generate asmuch ATP as oxidative phosphorylation per mol of glucosebut it can readily create equal amounts of ATPby an increasedflux of glucose Further enhanced glycolytic rate plays asignificant role in the generation of metabolic intermediatesfor biosynthesis The carbon in glucose is utilized in thesynthesis of triacylglycerols and phospholipids Glucose isprecursor for complex sugars of mucopolysaccharides andglycoproteins [27 28] Metabolism of glucose through thepentose phosphate pathway (PPP) generates ribose moietiesrequired for DNA and RNA synthesis and the NADPHrequired for the biosynthesis of lipids and other complexmolecules [30 31] Aerobic glycolysis is therefore the mecha-nism that ensures sufficient carbon flux through biosyntheticpathways in rapidly dividing cells [27 30 32 33]

The preferentialmetabolism of glucose through glycolysisreduces the use of oxidative metabolism in pluripotent cellswith a concomitant decrease in the formation of reactiveoxygen species (ROS) associated with oxidative phosphory-lation [34] From a developmental perspective this reducesoxidative stress within the cell thereby reducing the riskof DNA damage Given that pluripotent cells in vivo andin vitro act as founders for all cell types of the embryoand adult a metabolism that promotes genetic stabilitywould represent an evolutionary adaptation for successfuland faithful propagation

4 Key Metabolites Define the In VivoPluripotent Stem Cell Niche

Maintenance of pluripotency relies on a balance of com-plex cellular and acellular signals within the surround-ing microenvironment High levels of aerobic glycolysis inpluripotent cells form a localized area or niche character-ized by relatively high concentrations of lactate and lowextracellular pH surrounding the blastocyst (and potentiallyaround cell colonies in culture) The blastocyst uses thismicroenvironment to facilitate the implantation process [24]


NAD+

NADHPKM2

2ATP + H+

PPPG-6-P

F-6-P

LDH

Glutaminolysis

CitrateACL

JMJ Vitamin C

TET

NAD+

SIRT

HATHMTVitamin C

Glucose

acme

5hmC5mC

Nucleus

GlycolysisGlucose

2NADP+2NADPH

Pyruvate

NAD+

NADHNAD+ NADH

UCP2Pyruvate

PDK

PDH

Acetyl-CoA Citrate

120572KGTCAcycle

Mitochondrion

NAD+

NADH

Glu

OXPHOS

CO2

34ATPO2

GlutamineGlutamate

Lactate

HBPOGT O-GlcNAc

Acetyl-CoA

Acetyl-CoATHF

HCY

Met

SAH

SAM

5-CH3THF

Methioninecycle

Folatecycle

Glycine +Tdh

Thr

Methionine(human)

Threonine(mouse)






































H3K9me3[65]H3K27me3

H3K26m3H4K20me3


[74]H3K9meH3K36me



























9 Conclusions






Abbreviations





Thr Threonine



References


































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


NAD+

NADHPKM2

2ATP + H+

PPPG-6-P

F-6-P

LDH

Glutaminolysis

CitrateACL

JMJ Vitamin C

TET

NAD+

SIRT

HATHMTVitamin C

Glucose

acme

5hmC5mC

Nucleus

GlycolysisGlucose

2NADP+2NADPH

Pyruvate

NAD+

NADHNAD+ NADH

UCP2Pyruvate

PDK

PDH

Acetyl-CoA Citrate

120572KGTCAcycle

Mitochondrion

NAD+

NADH

Glu

OXPHOS

CO2

34ATPO2

GlutamineGlutamate

Lactate

HBPOGT O-GlcNAc

Acetyl-CoA

Acetyl-CoATHF

HCY

Met

SAH

SAM

5-CH3THF

Methioninecycle

Folatecycle

Glycine +Tdh

Thr

Methionine(human)

Threonine(mouse)






































H3K9me3[65]H3K27me3

H3K26m3H4K20me3


[74]H3K9meH3K36me



























9 Conclusions






Abbreviations





Thr Threonine



References


































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


































H3K9me3[65]H3K27me3

H3K26m3H4K20me3


[74]H3K9meH3K36me



























9 Conclusions






Abbreviations





Thr Threonine



References


































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















9 Conclusions






Abbreviations





Thr Threonine



References


































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Abbreviations





Thr Threonine



References


































































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

review article metaboloepigenetic regulation of

Documents