2017-Choi-Prolonged Mek1_2 suppression impairs.pdf

1 0 a u g u s t 2 0 1 7|V O L 5 4 8|N a t u R E|2 1 9LEttERdoi:10.1038/nature23274Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cellsJiho Choi1,2,3,4*,aaron J.Huebner1,2,3,4*,Kendell Clement3,4,5,Ryan M.Walsh1,2,3,4,andrej savol1,Kaixuan Lin6,Hongcang gu5,Bruno Di stefano1,2,3,4,Justin Brumbaugh1,2,3,4,sang-Yong Kim7,Jafar sharif8,Christopher M.Rose9,arman Mohammad5,Junko Odajima2,Jean Charron10,toshi shioda2,andreas gnirke5,steven gygi9,Haruhiko Koseki8,Ruslan I.sadreyev1,andrew Xiao6,alexander Meissner3,4,5&Konrad Hochedlinger1,2,3,4Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors(2i)in the presence of leukaemia inhibitory factor(LIF)(hereafter termed 2i/L)induces a naive state in mouse embryonic stem(ES)cells that resembles the inner cell mass(ICM)of the pre-implantation embryo1.Since the ICM exists only transiently in vivo,it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality.Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential.Furthermore,we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L.Mechanistically,we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects,in part through the downregulation of DNA methyltransferases and their cofactors.Finally,we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells.Taken together,our data suggest that,although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state,prolonged suppression results in irreversible changes that compromise their developmental potential.Dysregulation of DNA methylation as well as of Wnt and Mapk signalling has been linked to cellular transformation and chromosomal instability in ES cells25.Therefore,we sought to determine whether sustained perturbation of the Wnt/Mapk pathway and associated DNA hypomethylation during 2i/L culture affects the stability and function-ality of ES cells.Specifically,we used three isogenic 129S6 C57B6 F1 male ES cell lines(J34,J35 and J37),which were derived in 2i/L medium and then cultured in serum plus LIF(S/L)medium for four additional passages(Fig.1a).Each ES cell line was subsequently passaged onto a feeder layer of irradiated mouse embryonic fibroblasts(MEFs)in either 2i/L or S/L and propagated for an additional 6 or 16 passages(final passages p10 or p20,respectively).To assess the reversibility of any observed changes,we also switched the p20 2i/L-cultured ES cell lines back into S/L for an additional 3 or 10 passages.We assessed global DNA methylation patterns in our cultured ES cells using reduced representation bisulfite sequencing(RRBS).Similar to previous reports68,global methylation levels of 2i/L-cultured ES cells were lower than those of S/L-cultured ES cells and became remethylated when switched back to S/L(Extended Data Fig.1ac).This demethylation and subsequent remethylation occurs across most methylated features including CpG islands,shores,short and long inter-spersed nuclear elements(SINEs and LINEs,respectively)and long terminal repeats(LTRs)(Extended Data Fig.1d,e).By contrast,2i/L culture caused a progressive and irreversible erosion of DNA methyla-tion at most imprinting control regions(ICRs),resulting in the biallelic expression of the imprinted gene,Impact(Fig.1bd and Extended Data Fig.1f,g).We confirmed biallelic expression of additional imprinted genes using a Mus musculus Mus spretus F1 stem-cell line cultured in 2i/L for six passages(Extended Data Fig.1h).Female ES cells cultured in S/L exhibit attenuated Mapk signalling,increased Wnt signalling,and upregulation of transcription factors associated with a naive-like state when compared to male ES cells cultured in S/L9.Furthermore,female ES cells cultured in S/L were reported to be hypomethylated at imprinted and non-imprinted loci6,10,11,although the extent of hypomethylation seems to be variable12.We therefore compared the methylation status of our male ES cells with three isogenic(129S6 C57BL6 F1)female ES cell lines that were cultured in S/L for six passages.At the time of analysis,our female ES cell lines retained both X chromosomes,were hypomethyl-ated globally and at ICRs,and biallelically expressed Impact(Fig.1df and Extended Data Fig.1i).Unsupervised clustering based on global methylation levels revealed that S/L-cultured female ES cells clustered with ICM cells and 2i/L-cultured male ES cells(Fig.1e).However,when the same samples were clustered based on ICR methylation levels,female ES cells clustered with 2i/L-cultured male ES cells but apart from ICM cells and S/L-cultured male ES cells(Fig.1f).Moreover,we noticed a substantial overlap between differentially methylated regions that distinguish male from female ES cells grown in S/L,and those that distinguish male ES cells grown in S/L from those grown in 2i/L(Fig.1g).Thus,female ES cells cultured in S/L resemble male ES cells cultured in 2i/L with respect to imprinted and non-imprinted methylation patterns.The genomic distribution pattern of the histone variant H2A.X in pluripotent stem cells has been shown to predict their developmental potential13.Because 2i/L changes the chromatin landscape of ES cells relative to S/L14,we performed chromatin-immunoprecipitation followed by sequencing(ChIPseq)for H2A.X on 2i/L-cultured male ES cells and S/L-cultured female ES cells.We found that 2i/L-cultured male ES cells and S/L-cultured female ES cells lack H2A.X binding at 38,925 and 51,442 regions,respectively,relative to S/L-cultured male ES cells(Extended Data Fig.2ad).Moreover,genomic regions that lost H2A.X binding in both 2i/L-cultured male ES cells and S/L-cultured female ES cells(12,179 regions)were near genes associated with gastrulation,organ development and formation of the germ layer(Extended Data Fig.2ce).Of note,switching 2i/L-cultured male ES 1Massachusetts General Hospital Department of Molecular Biology,Boston,Massachusetts 02114,USA.2Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine,Boston,Massachusetts 02114,USA.3Department of Stem Cell and Regenerative Biology,Harvard University,Cambridge,Massachusetts 02138,USA.4Harvard Stem Cell Institute,1350 Massachusetts Avenue,Cambridge,Massachusetts 02138,USA.5Broad Institute of MIT and Harvard,Cambridge,Massachusetts 02142,USA.6Department of Genetics,Yale University School of Medicine,10 Amistad Street,New Haven,Connecticut 06519,USA.7New York University Langone Medical Center,New York 10016,USA.8Center for Integrative Medical Sciences,RIKEN National Research and Development Agency,1-7-22 Suehiuro-cho,Tsurumi-ku,Yokohama-shi,Kanagawa-ken 230-0045,Japan.9Department of Cell Biology,Harvard Medical School,240 Longwood Avenue,Boston,Massachusetts 02115,USA.10Centre de recherche sur le cancer de lUniversit Laval,CRCHU de Qubec,LHtel-Dieu de Qubec,9,rue McMahon,Quebec G1R 2J6,Canada.*These authors contributed equally to this work.2017 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.letterreSeArCH2 2 0|N a t u R E|V O L 5 4 8|1 0 a u g u s t 2 0 1 7cells to S/L did not restore H2A.X binding at most loci,suggesting that H2A.X depletion is irreversible at many sites,similar to ICR methyla-tion(Extended Data Fig.2a,b).Considering that proper genomic imprinting and H2A.X deposi-tion are essential for development13,15,we injected our different ES cell lines into 2n or 4n host blastocysts to determine their developmental potential(Fig.2a and Supplementary Tables 1,2).We found that 2i/L-cultured male ES cells and S/L-cultured female ES cells gave rise to fewer pups than S/L-cultured male ES cells after 2n blastocyst injections (Fig.2b),and several of the pups we obtained displayed phenotypes associated with aberrant imprinting15(Fig.2b,inset,and Extended Data Fig.3a).Moreover,adult chimaeras derived from 2i/L-cultured male ES cells or S/L-cultured female ES cells had far less agouti coat colour,indicating a poor contribution to adult tissues(Fig.2c).Nevertheless,some of these low-grade chimaeras produced ES-cell-derived offspring,demonstrating their ability to produce functional germ cells(Extended Data Fig.3b).When 4n blastocyst injections were performed,2i/L-cultured male ES cells and S/L-cultured female ES cells were 56 times less efficient than S/L-cultured male ES cells at producing entirely ES cell-derived(all-ES cell)pups(Fig.2d and Extended Data Fig.3ce).These results are consistent with a previous report,which showed that early passage female ES cells are slightly less effective at produc-ing all-ES cell pups compared to male ES cells16.Notably,none of the all-ES cell pups generated from 2i/L-cultured male or S/L-cultured female ES cells survived beyond birth(Fig.2d and Extended Data Fig.3d).Furthermore,we found that one of our male ES cell lines(J37)was unable to generate full-term all-ES cell pups when passaged in 2i/L for 16 passages(Supplementary Table 2).This ES cell line also failed to produce well-differentiated teratomas,suggesting that muta-tions acquired in this line compromised its differentiation potential(Extended Data Fig.4a,b).On the basis of these data,we conclude that continual passaging of male ES cells in 2i/L or female ES cells in S/L results in a progressive impairment of their developmental potential.Perturbation of Wnt/Mapk signalling2,3,DNA hypomethylation4,5 and H2A.X loss17 have been shown to affect genomic stability.We therefore performed karyotype analysis(Supplementary Table 3)and array comparative genomic hybridization(aCGH)on our ES cell lines cultured in 2i/L or S/L.2i/L-cultured ES cells at p10 and S/L-cultured ES cells at p20 had mostly diploid chromosome counts(40,XY)(Fig.3a and Extended Data Fig.5a).However,2i/L-cultured ES cell lines at p20 exhibited recurrent chromosomal aberrations including trisomy 6,8 and 19,as well as loss of the Y chromosome in many,if not all,cells analysed(Fig.3ac and Extended Data Fig.5a).To exclude the possi-bility that these abnormalities are specific to our cell lines,we analysed a Rex1GFP ES cell line18 that had been independently maintained in 2i/L conditions and found several chromosomal aberrations including trisomy 6 and 8(Extended Data Fig.5b).Although previous reports demonstrated that irradiated MEFs do not affect ES cells in a co-culture system19,we also passaged one of our ES cell lines in 2i/L without feeders for 16 passages.As expected,every cell karyotyped acquired trisomy 6 and 8,confirming that the 2i/L-derived chromosomal aberrations in ES cells were not due to the presence of MEFs(Extended Data Fig.5c).Considering these unexpected chromosomal abnormalities,we per-formed aCGH on DNA isolated from a rare full-term all-ES cell pup that was generated using p20 2i/L-cultured ES cells.We did not find evidence for gross copy number abnormalities in this pup(Extended Data Fig.5d),even though the parental bulk cultures had chromosomal aberrations in every cell counted(Fig.3a,middle,3b).We surmise that rare euploid ES cells present in the culture experienced a strong positive selection during development,yet succumbed postnatally owing to the a2i/Lon MEFsS/Lon MEFsSwitch to S/L on MEFsp23&30p10p4DNA methylationGene expressionH2AX depositionDevelopmental potentialGenomic stabilityp0J34,J35,J37 lines(all XY)ICM outgrowthcultured in 2i/L on MEFs for 7 daysMaintained in S/L on MEFs129S6 C57B6 F1BlastocystbcdICR methylation(n=18)ICR methylation(n=18)XY S/LXY 2i/L to S/LICMXX S/LXY 2i/LGlobal methylation levels(1-kb tiles n=127,671)ICR methylation(n=17)J34J35J37S/L p10J34J35J372i/L p10Dnmt1KO01Mcts2Peg10ImpactZrsr1NnatMeg3Rasgrf1Peg13Peg3Inpp5fMestNap1l5Plagl1Grb10Kcnq1SnrpnAirnNespasXY S/LXY 2i/LXY 2i/Lto S/LXX S/L101Impact expressionAllelic asymmetry between129S6 and C57B613,2141,40239,112Hypomethylated inXY 2i/L vs XY S/LHypomethylated in XX S/L vs XY S/LDifferentially methylated regionsICMXY S/LXY 2i/LXX S/LXY 2i/L to S/LMeg3Inpp5fGrb10Nap1l5Zrsr1ImpactKcnq1Peg3NnatNespasPeg13Mcts2Peg10AirnSnrpnPlagl1Mestefgp20101J34J35J37S/L p20J34J35J372i/L p20J34J35J372i/L to S/L p23Figure 1|Erosion of genomic imprints in 2i/L-cultured male ES cells and S/L-cultured female ES cells.a,Schematic of experimental design.p,passage.b,c,ICR methylation levels in ES cells at p10(b)and p20 and p23(c).KO,knockout.d,Allelic expression of the imprinted gene Impact.e,f,Global(e)and ICR(f)methylation levels in male ES cells at p20 and p23(n=3 biological replicates)and female ES cells at p6(n=3 biological replicates).g,Overlap of differentially methylated regions.a4n 1-cell embryoElectrofusionSurrogate motherDelivery4n placenta2n all-ES cell pup4n blastocystSurrogate motherDelivery2n chimaeric pup2-cellembryo2n blastocystEmbryo transferInjection of 2n ES cells2n blastocyst injection4n blastocyst injectionCleavageCleavageInjection of 2n ES cellsEmbryo transferb0%90100%30%5070%J34J352n blastocyst injections(p10)XY S/L4n blastocyst injections(p10)ResorptionBreathingChimaera01020304050Transferred embryos(%)Transferred embryos(%)050100Chimaerism(%agouti coat colour)Chimaerism(%agouti coat colour)J34J30Full-termBreathingAdultJ31J35XY 2i/LXX S/Lc01020304050d27/6061/15017/6015/150N/D20/148XY S/LXY 2i/LXX S/L20/1487/148XY S/LXY 2i/LXX S/L13/6310/636/639/2250/2255/1570/1579/2255/157Figure 2|Prolonged 2i/L culture impairs the developmental potential of ES cells.a,Schematic of blastocyst injection protocol.b,2n blastocyst injections.Numbers of animals obtained per total number of transferred embryos are shown.White arrow indicates the protruding tongue of a female pup.N/D,not determined.See Supplementary Table 1 for details.c,Top,quantification of coat colour chimaerism of the chimaeras from b.Bottom,representative images of the varying degrees of chimaerism.d,4n blastocyst injections.Numbers of animals obtained per total number of transferred embryos are shown.See Supplementary Table 2 for details.2017 Macmillan Publishers Limited,part of Springer Nature.All rights reserved.letter reSeArCH1 0 a u g u s t 2 0 1 7|V O L 5 4 8|N a t u R E|2 2 1epigenetic aberrations present.To confirm this,we performed RRBS on explanted keratinocytes derived from chimaeric mice produced with 2i/L-cultured ES cells and found that they were indeed aberrantly hypo-methylated at several imprinted loci(Extended Data Fig.5e).Female ES cells tend to lose one of their two X chromosomes with culture,thus becoming XO10.The loss of an X chromosome in XX ES cells has been associated with downregulation of naive-like genes and increased DNA methylation,similar to male ES cells cultured in S/L9,11.We hypothesized that the naive-like state of XX female ES cells partic