1063RESEARCH REPORTINTRODUCTIONIn the mouse, pluripotent stem cell lines can be established from twodistinct phases of early development (Rossant, 2008). Embryonicstem (ES) cells are obtained from naïve epiblast in pre-implantationblastocysts (Batlle-Morera et al., 2008; Brook and Gardner, 1997;Evans and Kaufman, 1981; Martin, 1981). Epistem cells (EpiSCs)are derived from columnar epithelial epiblast of the early post-implantation embryo (Brons et al., 2007; Tesar et al., 2007). ES cellsretain the character of early epiblast and can be incorporated into thehost embryo when injected into blastocysts (Gardner and Rossant,1979). They subsequently contribute to all lineages of thedeveloping and adult mouse (Beddington and Robertson, 1989;Bradley et al., 1984; Gardner and Rossant, 1979; Nagy et al., 1993).By contrast, neither freshly isolated post-implantation epiblast cellsnor EpiSCs are capable of functional colonisation of a hostblastocyst (Rossant et al., 1978; Tesar et al., 2007).Both ES cells and EpiSCs are capable of multilineagedifferentiation in vitro and can form teratomas when grafted intoadult mice (Brons et al., 2007; Tesar et al., 2007). Both cell typesexpress the three transcriptional regulators, Oct4 (Pou5f1 – MouseGenome Informatics), Sox2 and Nanog, that are generallyconsidered to constitute the core pluripotency network (Boyer et al.,2005; Loh et al., 2006; Wang et al., 2006). However, there aresignificant differences in gene expression between ES cells andEpiSCs (Tesar et al., 2007). Furthermore, the culture conditions formaintaining the two cell types are quite distinct. ES cells self-renewin response to the cytokine leukaemia inhibitory factor (Lif) (Smithet al., 1988; Williams et al., 1988) and either serum, bonemorphogenetic protein, or the inhibition of Mek/Erk signalling(Burdon et al., 1999; Ying et al., 2003; Ying et al., 2008). They aredriven into differentiation by FGF/Erk signalling (Kunath et al.,2007; Stavridis et al., 2007). EpiSCs, by contrast, are maintained byFGF and activin (Brons et al., 2007).The molecular basis for restriction of egg cylinder epiblast andEpiSCs as compared with naïve epiblast and ES cells is presentlyunclear. This issue acquires added significance in light of evidencethat human embryo-derived stem cells are more akin to EpiSCs thanto ground state ES cells (Brons et al., 2007; Rossant, 2008; Tesar etal., 2007). Here, we report on requirements for the interconversionof mouse ES cells and EpiSCs.MATERIALS AND METHODSEpiSC derivation and cultureEpiSCs were derived from E5.75 mouse embryos using activin A (20 ng/ml)and Fgf2 (12 ng/ml) essentially as described (Brons et al., 2007), except thatwe employed N2B27 medium (Ying and Smith, 2003). OE cell lines werederived from F1 embryos carrying an Oct4GiP (eGFPiresPuro) transgene(Ying et al., 2002). EpiSCs were also derived from non-transgenic strain 129embryos. Cells were used between 10 and 25 passages. Differentiated cellscould be eliminated as required from OE cultures by puromycin (1 μg/ml)selection for expression of the Oct4GiP transgene.Embryonic stem cell and induced pluripotent stem (iPS) cell culture2i/Lif comprises the Mek inhibitor PD0325901 (1 μM), the Gsk3 inhibitorCHIR99021 (3 μM), and leukaemia inhibitory factor (Lif, 100 U/ml) inN2B27 medium (Ying et al., 2008). Cultured cells were expanded bydissociation with trypsin and replating every 3 days.Klf4 reverts developmentally programmed restriction ofground state pluripotencyGe Guo1,2, Jian Yang1,2, Jennifer Nichols1,3, John Simon Hall1,2,*, Isobel Eyres1, William Mansfield1and Austin Smith1,2,†Mouse embryonic stem (ES) cells derived from pluripotent early epiblast contribute functionally differentiated progeny to all foetallineages of chimaeras. By contrast, epistem cell (EpiSC) lines from post-implantation epithelialised epiblast are unable to colonisethe embryo even though they express the core pluripotency genes Oct4, Sox2 and Nanog. We examined interconversion betweenthese two cell types. ES cells can readily become EpiSCs in response to growth factor cues. By contrast, EpiSCs do not change into EScells. We exploited PiggyBac transposition to introduce a single reprogramming factor, Klf4, into EpiSCs. No effect was apparent inEpiSC culture conditions, but in ground state ES cell conditions a fraction of cells formed undifferentiated colonies. These EpiSC-derived induced pluripotent stem (Epi-iPS) cells activated expression of ES cell-specific transcripts including endogenous Klf4, anddownregulated Markers of lineage specification. X chromosome silencing in female cells, a feature of the EpiSC state, was erased inEpi-iPS cells. They produced high-contribution chimaeras that yielded germline transmission. These properties were maintainedafter Cre-mediated deletion of the Klf4 transgene, formally demonstrating complete and stable reprogramming of developmentalphenotype. Thus, re-expression of Klf4 in an appropriate environment can regenerate the naïve ground state from EpiSCs.Reprogramming is dependent on suppression of extrinsic growth factor stimuli and proceeds to completion in less than 1% of cells.This substantiates the argument that EpiSCs are developmentally, epigenetically and functionally differentiated from ES cells.However, because a single transgene is the minimum requirement to attain the ground state, EpiSCs offer an attractive opportunityfor screening for unknown components of the reprogramming process.KEY WORDS: Induced pluripotent stem (iPS) cell, Chimaera, Leukaemia inhibitory factor (Lif), Reprogramming, Mitogen-activated proteinkinase (Erk) kinase (Mek/Mkk), Embryonic stem (ES) cellDevelopment 136, 1063-1069 (2009) doi:10.1242/dev.0309571Wellcome Trust Centre for Stem Cell Research, 2Department of Biochemistry, and3Department of Physiology, Development and Neuroscience, University ofCambridge, Tennis Court Road, Cambridge CB2 1QR, UK.*Current address: The Paterson Institute, University of Manchester, Manchester,M20 4BX, UK†Author for correspondence (e-mail: austin.smith@cscr.cam.ac.uk)Accepted 30 January 2009DEVELOPMENT 1064ES cells overexpressing Klf4 were generated by electroporation ofOct4βgeo reporter cells (IOUD2) (Burdon et al., 1999) with apPyCAGKlf4iP construct followed by puromycin selection (1 μg/ml). ForES cell to EpiSC differentiation, cells were plated at a density of 1-2⫻104per cm2in fibronectin-coated plates. Twenty-four hours after plating, themedium was changed to N2B27 containing activin A and Fgf2. Thereafter,cells were maintained in EpiSC culture conditions and passaged every 2-3days. For colony formation, 1000 cells were plated per well in fibronectin-coated 6-well plates in activin A/Fgf2, or in laminin-coated plates in 2i/Lif.After 6 days, colonies were fixed and stained for alkaline phosphatase.Colonies were scored using ImageJ software.PiggyBac vector transfectionTo establish PiggyBac (PB) transgenic EpiSC lines, 1⫻106cells were co-transfected using Lipofectamine 2000 (Invitrogen) with 1 μg ofpGG137Klf4 or control pGG131 vector plus 2-3 μg of the PBase-expressingvector pCAGPBase (Wang et al., 2008). Transfection efficiency wasevaluated by flow cytometry for DsRed expression. To select for stabletransfectants, hygromycin (200 μg/ml) was applied for at least 5 days. Todelete transgenes, 1⫻105cells were transfected with 1 μg of Cre expressionplasmid using Lipofectamine 2000. Five days after transfection, DsRed-negative cells were purified and individually deposited into a 96-well plateusing a MoFlo high-performance cell sorter (DakoCytomation). Afterexpansion, genomic PCR was employed to identify revertants lacking theKlf4 transgene. RT-PCR was used to confirm the lack of Klf4 transgene andof DsRed expression. iPS cell induction and propagationEpiSCs, either stable transfectants isolated after hygromycin selection orcells immediately after transfection, were plated at a density of 1⫻104,5⫻104and 1⫻105cells per well of 6-well tissue culture plates in EpiSCculture condition. After 24 hours, medium was replaced with that containing2i/Lif and subsequently refreshed every other day. The number of Oct4-GFP-positive clones was manually counted using fluorescence microscopy.ES cell-like clones were picked after 14 days in 2i/Lif and subsequentlyexpanded by Accutase (PAA Laboratories) dissociation and replating every3-4 days.RT-PCRTotal RNA was prepared using the RNeasy Mini Kit (Qiagen) with DNaseItreatment. First-strand cDNA was synthesised using Superscript III reversetranscriptase (Invitrogen). Unless specified otherwise, real-time PCR wasperformed using Taqman Gene Expression Assays (Applied Biosystems).Gene expression was determined relative to Gapdh using the ΔCt method.Expression of the Klf4 transgene and of DsRed was determined by standardcurve calibration. All quantitative PCR (qPCR) reactions were performed ina 7900HT Fast Real-Time PCR System (Applied Biosystems).Taqman probesOct4, Mm00658129_gH; Klf4, Mm00516104_m1; Klf2, Mm01244979_g1;Klf5, Mm00456521_m1; Nanog, Mm02384862_g1; Rex1,Mm03053975_g; Fgf5, Mm00438615_m1; Lefty, Mm00438615_m1;brachyury (T), Mm01318252_m1; Nr0b1, Mm00431729_m1; Stella(Dppa3), Mm00836373_g1; Gapdh, 4352339E; β-actin (Actb), 4352341E.Chimaera productionTerm chimaeras were produced by microinjection into C57BL/6 blastocysts.Selected female chimaeras were mated with C57BL/6J black males.Germline transmission from cultured cell-derived oocytes manifests inagouti offspring.RESULTS AND DISCUSSIONWe derived EpiSCs from E5.75 mouse embryos carrying theOct4GiP transgene (Ying et al., 2002). Cell lines were establishedand maintained without feeders in serum-free N2B27 medium (Yingand Smith, 2003) supplemented with activin A and Fgf2 (bFGF)(Brons et al., 2007). They grew as monolayers of closely apposedcells on a fibronectin substrate (Fig. 1A). The majority of cellsexpressed the Oct4-GFP reporter (Fig. 1A). Consistent with theoriginal descriptions (Brons et al., 2007; Tesar et al., 2007), theEpiSCs we derived expressed the pluripotency markers Oct4 andNanog, but not the early epiblast marker Stella (Dppa3) (Fig. 1B).EpiSCs also differ from ES cells by upregulation of the post-implantation markers Fgf5, T (brachyury) and Lefty (see Fig. S1 inthe supplementary material). We established both male and femaleEpiSC lines. Immunofluorescence revealed a prominent body ofnuclear staining for the repressive histone modificationtrimethylated H3 lysine 27 (me3H3K27) in the female line (Fig.1C). This is diagnostic of a silent X chromosome (Silva et al., 2003).Thus, an emphatic epigenetic distinction between early and lateepiblast is conserved in ES cells and EpiSCs, respectively. This isreflected in a differential ability to colonise chimaeric embryos(Tesar et al., 2007). We found that after morula aggregation,Oct4GiP EpiSCs could mix with inner cell mass (ICM) cells inblastocysts, but that they quickly downregulated GFP. Consistentwith this, no contribution was detectable in egg cylinders afterembryo transfer (see Fig. S6 in the supplementary material).EpiSCs also lose expression of Oct4 and differentiate whentransferred to conventional mouse ES cell culture conditions (Bronset al., 2007). Recently, however, it has been established that smallmolecules that selectively inhibit the Mek/Erk MAP kinasesignalling cascade and glycogen synthase kinase 3 (Gsk3) provide,in combination with Lif, an optimal environment for derivation andpropagation of ES cells from different rodent backgrounds in serum-free medium (Buehr et al., 2008; Ying et al., 2008) (J.N.,unpublished). The combination of two inhibitors with Lif (2i/Lif)also promotes the generation of iPS cells (Silva et al., 2008). Wetherefore tested whether EpiSCs cultured in 2i/Lif might acquirefeatures of ground state pluripotency. However, after transfer into2i/Lif, EpiSCs underwent massive differentiation and death suchthat Oct4-GFP-expressing cells were entirely eliminated by 3 days(Fig. 1D). Some differentiated cells persisted, but in multipleplatings of 1⫻107EpiSCs not a single Oct4-GFP-expressing colonywas obtained. Since genetic background has a strong influence onthe derivation and propagation of ES cells and on iPS cell generation(Batlle-Morera et al., 2008; Silva et al., 2008), we also examinedEpiSCs from the permissive 129 strain. These EpiSCs also failed tosurvive in 2i/Lif (data not shown). We conclude that the EpiSCrepresents a stable cell state that does not naturally revert to naïvepluripotent status.The origin of ES cells and EpiSCs from early and late epiblast,respectively, suggests that ES cells might be capable of becomingEpiSCs. Indeed, ES cells transferred into EpiSC culture conditionscontinued to proliferate. After passaging, cultures became relativelyhomogenous and EpiSC-like. Thereafter, they displayed the markerprofile of EpiSCs rather than of ES cells, with maintained Oct4,reduced Nanog and downregulated Rex1 (Zfp42 – Mouse GenomeInformatics), Nr0b1 and Klf4 (Fig. 1E; see Fig. S2 in thesupplementary material). Furthermore, EpiSCs derived from femaleES cells showed a coincidence of Oct4 expression and Xchromosome inactivation (Fig. 1F). This signature distinguishesEpiSCs from ES cells and differentiated somatic cell types. Toconfirm that this ES cell-derived EpiSC state was trulydifferentiated, we transferred cells back into 2i/Lif. Occasional EScell-like colonies could initially be recovered, but not after four ormore passages in activin plus Fgf2 (data not shown). We concludethat ES cells differentiate into EpiSCs, although a minority ofundifferentiated cells persists for a while, as is commonly observedin other in vitro ES cell differentiation schema (Lowell et al., 2006;Smith, 2001).RESEARCH REPORT Development 136 (7)DEVELOPMENT One of the genes prominently downregulated duringdifferentiation of ES cells into EpiSCs is Klf4. Klf4 has beenimplicated in ES cell self-renewal (Jiang et al., 2008; Li et al., 2005).Klf4 is induced by Lif/Stat3 signalling in ES cells, but not in EpiSCs(Fig. 2A). To test whether Klf4 might regulate the ES cell to EpiSCtransition, we stably transfected ES cells with a Klf4 expressionplasmid. These cells show greatly reduced dependency on Lif forself-renewal, as previously reported (Li et al., 2005). On transfer toEpiSC culture conditions, however, they responded similarly toparental ES cells, growing as a monolayer and downregulating EScell-specific marker expression while maintaining Oct4 (Fig. 2B).This indicates that forced expression of Klf4 does not preventconversion into EpiSCs. However, even after ten passages in activinand Fgf2, ES cell colonies were obtained at low frequency upontransfer to 2i/Lif (Fig. 2C). Therefore, constitutive Klf4 either allowslong-term persistence of a small fraction of undifferentiated ES cells,or enables a fraction of EpiSCs to dedifferentiate and regain theground state.To distinguish between these possibilities, we investigatedwhether forced expression of Klf4 in embryo-derived EpiSCs couldinduce ground state pluripotency. We used PiggyBac (PB) vector-chromosome transposition to achieve high efficiency stabletransfection (Wang et al., 2008). The PB vector containsindependent CAG promoter units directing expression of the Klf41065RESEARCH REPORTReprogramming of epistem cells by Klf4Fig. 1. EpiSCs are distinct from, and do not spontaneously convert to, ES cells. (A) Phase contrast and fluorescence images of establishedEpiSC line. (B) qRT-PCR analysis of marker gene expression in ES cells and EpiSCs. ES, ES cells in 2i/Lif. Epi6 and Epi7 are two independent EpiSClines. y-axis, relative expression normalised to Gapdh. (C)Immunostaining of male and female EpiSCs for me3H3K27 and Oct4. White arrowindicates focus of staining diagnostic of an inactive X chromosome. (D) EpiSCs lose Oct4 expression and differentiate or die in 2i/Lif. AF, EpiSCcultured in activin A plus Fgf2. (E) qRT-PCR analysis of ES cell differentiation into EpiSCs upon culture in Fgf2 and activin. Epi3 and Epi10 indicatecells cultured in Fgf2 and activin for three and ten passages, respectively. y-axis, relative expression normalised to Gapdh. (F) Oct4 and me3H3K27immunostaining of female ES cell-derived EpiSCs. EpiSCs both express Oct4 and exhibit a nuclear body indicative of the inactive X (white arrow).Blue arrow indicates a dividing cell.DEVELOPMENT 1066open reading frame and of a DsRed reporter with a linkedhygromycin resistance gene (Fig. 2D). LoxP sites adjacent to the PBterminal repeats allow for excision of both expression units. FewerDsRed-expressing cells were obtained after Klf4 transfection thanfollowing control vector transfection (data not shown) and the levelof DsRed expression was reduced. Overexpression of Klf4 mighttherefore be toxic to EpiSCs. Nonetheless, Klf4/DsRed-expressingEpiSCs were isolated following hygromycin selection (Fig. 2E). Inactivin and Fgf2 they did not upregulate ES cell-specific genes (Fig.2F) and female cells maintained an inactive X chromosome asjudged by me3H3K27 staining (see Fig. S3 in the supplementarymaterial). We conclude that the expression of Klf4 at a similar RNAlevel to that present in ES cells is not alone sufficient to reset EpiSCsand instate full pluripotency in cells maintained in activin and Fgf2.We then examined the effect of Klf4 transfection in 2i/Lifconditions, which have been shown to promote the final stages ofiPS cell generation from neural stem cells and fibroblasts (Silva etal., 2008). Klf4-transfected EpiSCs were transferred into 2i/Lif 48or 72 hours after transfection. They exhibited a wave ofdifferentiation and cell death, similar to non-transfected EpiSCs.RESEARCH REPORT Development 136 (7)Fig. 2. Klf4 neither prevents ES cell differentiation into EpiSCs nor converts an EpiSC population into ES cells in the presence of activinand FGF. (A) qRT-PCR analysis of Lif induction of Klf4 in ES cells but not in EpiSCs. Cells were stimulated with Lif (+LIF) for 1 hour. (B)ES cellsconstitutively expressing Klf4 acquire an EpiSC marker profile in Fgf2 plus activin A. MT, empty vector transfectants. P0, P2 and P10 indicatepassage numbers in Fgf2/activin. (C) Constitutive Klf4 expression permits continued recovery of ES cell colonies after culture in activin and Fgf2. Onethousand cells were plated for each sample in triplicate at the indicated passage (P) number. MT, empty vector transfectants; K4, Klf4 transfectants.(D)PiggyBac vector for expression of Klf4 (pGG137Klf4), and control PiggyBac vector (pGG131). Arrows (P) indicate PCR primers used to amplifythe PB LTR fragment after Cre-mediated recombination. (E) Hygromycin-selected Klf4 and control vector-transfected EpiSCs. (F)qRT-PCR analysisshowing that forced Klf4 expression does not induce ES cell marker gene expression in EpiSC culture. ES, ES cells; Epi, EpiSCs; Vec, EpiSCtransfected with control vector pGG131; Klf4, EpiSCs transfected with pGG137Klf4. y-axis, relative expression normalised to Gapdh.DEVELOPMENT After 4 days in 2i/Lif, however, multiple Oct4-GFP-positivecolonies emerged (Fig. 3A). These colonies had the tightly packedthree-dimensional aggregate form typical of ES cells in 2i. Theyarose at a frequency of 0.1-0.2% of cells surviving transfection (21colonies from 1⫻104cells in one typical experiment). GFP-positivecolonies were never obtained in 2i/Lif from multiple control vectortransfections. We then tested whether stably transfected EpiSCspropagated in activin and Fgf2 could convert in 2i/Lif. Klf4transfectants generated colonies in 2i/Lif with similar kinetics tocells transferred directly after transfection (see Fig. S4 in thesupplementary material). The yield was higher, at ~1%. This couldpoint to some element of reprogramming proceeding in the stabletransfectants, but could also be explained by elimination of non-transfectants and cells with toxic overexpression of Klf4.Significantly, the frequency did not noticeably increase withpassaging of the stable transfectants, indicating that if there is anypartial reprogramming this does not accumulate in the cultures.Ten out of 12 colonies picked from a transfection with transfer to2i/Lif after 72 hours expanded with undifferentiated morphologyand stable expression of Oct4-GFP. qRT-PCR analysis showed themarker profile of ES cells, with upregulation of Stella and Klf2.Conversely, Fgf5 and brachyury mRNAs were lost (Fig. 3B; see Fig.S5 in the supplementary material). We examined me3H3K27immunostaining and found that the nuclear body corresponding tothe inactive X chromosome was lost in Oct4-GFP-positive cells aftertransfer to 2i/Lif (Fig. 3C). A potential disadvantage of using the PBvector and CAG promoter is that transgenes might not be silenced.However, in each of the GFP-positive clones we observed partial orcomplete loss of visible DsRed expression (Fig. 3D,E), althoughqRT-PCR analysis revealed that the transgenes were not completelysilenced (Fig. 3F).We injected cells without visible DsRed expression into C57BL/6blastocysts. Healthy chimaeras were obtained with extensive agouticoat colour contributions (Fig. 3G). Female chimaeras mated withC57BL/6 males produced agouti offspring, indicating transmissionof the cultured cell genome. This confirms that the developmentalcapacity has been fully derestricted and the authentic pluripotentstate established. These cells should therefore be considered asEpiSC-derived iPS cells, or Epi-iPS cells.We examined the copy number of PB integrations by genomicPCR analysis of ten Epi-iPS cell clones. Each Epi-iPS cell linecarried 1-3 PB insertions (Fig. 4A). To determine whether the low,but still detectable, Klf4 transgene expression might play a role inmaintaining the induced phenotype we excised the transgene1067RESEARCH REPORTReprogramming of epistem cells by Klf4Fig. 3. EpiSCs transfected with Klf4 can convert to ground state pluripotency. (A) Oct4-positive colonies obtained by transfection with Klf4and transfer to 2i/Lif after 72 hours. Images were taken after 9 days in 2i/Lif. (B) qRT-PCR analysis of marker gene expression in ES cells, EpiSCs andderivative Epi-iPS cells isolated in 2i/Lif. y-axis, relative expression normalised to Gapdh. (C) me3H3K27 staining of female EpiSCs and derivative Epi-iPS cells. (D) Images of Epi-iPS colonies after 10 days in 2i/Lif, showing mutually exclusive expression of DsRed and Oct4-GFP. (E) Flow cytometryanalysis of four expanded Epi-iPS clones. Two clones retain weak but detectable red fluorescence. (F)qRT-PCR analysis of Klf4 transgene and DsRedexpression in Epi-iPS cell clones and parental EpiSC line. y-axis, relative expression normalised to Gapdh. (G) Chimeric mouse produced from theK4C12 Epi-iPS clone and agouti germline offspring.DEVELOPMENT 1068copies. We chose two DsRed-positive clones and transfected eachwith a Cre expression plasmid. After 5 days, cells that no longerexpressed DsRed were isolated using flow cytometry with single-cell deposition into 96-well plates (Fig. 4B). Resulting cloneswere screened by genomic PCR for absence of the Klf4 transgeneand presence of a reverted PB fragment (Fig. 4C). Two thirds ofthe expanded clones retained only the PB terminal repeats. RT-PCR analysis failed to detect expression of the Klf4 transgene orDsRed from these revertants (Fig. 4D). They retained ES cellmorphology, Oct4-GFP expression and ES cell marker profile(Fig. 4E,F). The X chromosome silencing mark, me3H3K27, wasundetectable (Fig. 4G). These cells incorporated efficiently intothe ICM and subsequently the egg cylinder after morulaaggregation (see Fig. S6 in the supplementary material). Weinjected transgene-deleted cells into blastocysts and obtainedviable high-contribution chimaeras (see Table S1 in thesupplementary material). Female chimaeras from two out of threeclones produced agouti offspring in their first litter (Fig. 4H),indicative of transmission of iPS cell-derived oocytes. Therefore,complete removal of the Klf4 transgene does not destabilise theinduced ground state. This establishes that reprogramming hasbeen finalised and does not depend upon ongoing transgeneexpression or insertional mutagenesis.The restricted potency of EpiSCs as compared with ES cellsappears to mirror the developmental progression from naïve pre-implantation epiblast to epithelialised egg cylinder (Rossant,2008). ES cells in vitro recapitulate this conversion with stablealterations in gene expression, growth factor dependence andepigenetic status. These differentiation changes may becompletely reversed by re-expression of a single gene that isnormally downregulated in EpiSCs. Klf4 in combination withculture in 2i/Lif reverts EpiSCs to the naïve ground state. This ismediated by transcriptional resetting accompanied by activationof contrasting epigenetic processes that restrict expression ofexogenous DNA sequences and erase X chromosomeinactivation. Interestingly, although colonies with features of iPScells are detectable within 7 days of Klf4 transfection, all clonesrecovered showed stable integration of at least one copy of thetransgene, suggesting a requirement for sustained expression toeffect reprogramming. However, the combination of PB-mediatedRESEARCH REPORT Development 136 (7)Fig. 4. Retention of ground state pluripotency after transgene excision. (A) Splinkerette-PCR reveals 1-3 PB insertions in each iPS clone.(B) Flow cytometry showing the DsRed-negative population in the K4C3 line before and after Cre transfection. (C) Genomic PCR showing loss of theKlf4 transgene and gain of the PB-LTR fragment in two revertant clones. (D) RT-PCR analysis showing the lack of Klf4 transgene and DsRedexpression in expanded Cre-reverted cells. (E)Marker gene expression in Cre-reverted Epi-iPS cells (iPS-Cre) compared with ES cells and EpiSCs. Dataare for two independent Cre-revertant clones derived from each of Epi-iPS cell lines K4C3 and K4C5. y-axis, relative expression normalised toGapdh. (F) Maintained morphology and Oct4-GFP expression in a Cre-reverted Epi-iPS cell line. (G) me3H3K27 staining of Klf4 transgene-deleted iPScells as compared with parental EpiSCs. (H) Chimeric mouse made with revertant K4C3-A3 cells, and agouti offspring denoting germlinetransmission.DEVELOPMENT low copy number integration and Cre-mediated excisionfacilitated formal proof that once attained, the Epi-iPS cell statedoes not require the continuous presence of introduced DNAelements (Okita et al., 2008; Stadtfeld et al., 2008).It is apparent that the exogenous transcription factor requirementsfor inducing pluripotency vary with the starting cell type as do theefficiency and kinetics of molecular reprogramming (Aoi et al.,2008; Kim et al., 2008; Silva et al., 2008). It is striking, however,that even though other core components of the pluripotent networkare already present, only ~1% of Klf4-expressing EpiSCs becomeiPS cells. This emphasises that even though there are transcriptionalsimilarities, EpiSCs are truly differentiated from ground state EScells. Their reprogramming efficiency is limited in an analogousmanner to that of somatic cells by currently unknown parameters.However, because a single transgene is sufficient, we suggest thatEpiSCs might provide an attractive system in which to screen fornew components of the reprogramming process.Finally, continuous expression of Klf4 does not prevent ES celldifferentiation into EpiSCs when exposed to inductive extrinsicfactors. Nonetheless, downregulation of Klf4 may help to ensuredevelopmental restriction of epithelialised epiblast in the embryoand safeguard against dedifferentiation to a naïve and teratogeniccondition. We suggest that the creation of iPS cells might beintimately related, mechanistically, to the molecular transitionsthrough which ground state pluripotency is generated and thenrestricted in the early phases of mammalian embryogenesis.We thank Jose Silva for discussion and comments on the manuscript. MaryChol generated the morula aggregation chimaeras, Marko Hyvonen providedrecombinant activin A and Fgf2, and Rachel Walker provided flow cytometryassistance. We thank Adrian Woodhouse, Samuel Jameson and colleagues formouse husbandry. This research was supported by the Medical ResearchCouncil, The Wellcome Trust and the EC Project EuroSystem. G.G. is a MedicalResearch Council Stem Cell Career Development Fellow and A.S. is a MedicalResearch Council Professor. Deposited in PMC for release after 6 months.Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/cgi/content/full/136/7/1063/DC1ReferencesAoi, T., Yae, K., Nakagawa, M., Ichisaka, T., Okita, K., Takahashi, K., Chiba, T.and Yamanaka, S. (2008). Generation of pluripotent stem cells from adultmouse liver and stomach cells. Science 321, 699-702.Batlle-Morera, L., Smith, A. G. and Nichols, J. (2008). 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Recent studies showed that there are different pluripotent states that correspond to the sequential developmental stages in embryos (1,2). These states can be captured in vitro using defined culture conditions, and they are characterized by distinct developmental potential, gene expression profile, and epigenetic status (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Among them, the naïve pluripotent state is the earliest stage in pluripotency during embryonic development. ...... In order to study the naïve and primed pluripotent states, we cultured ESCs in the 2i/LIF (2iL) or FGF2/Activin-A/XAV-939 (FAX) medium as previously described (3,4,8,9,16). We were also able to induce the transition from the naïve to the primed state by switching the cells from the 2iL to FGF2/Activin-A (FA) medium (Figure 1A) (58). ...INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cellsArticleJun 2021NUCLEIC ACIDS RESHongyao YuJiajia WangBrad Lackford Guang HuThe INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. In the naïve state, INO80 pre-marked gene promoters that would adopt bivalent histone modifications by H3K4me3 and H3K27me3 upon transition into the primed state. In the primed state, in contrast to its known role in H2A.Z exchange, INO80 promoted H2A.Z occupancy at these bivalent promoters and facilitated H3K27me3 installation and maintenance as well as downstream gene repression. Together, our results identified an unexpected function of INO80 in H2A.Z deposition and gene regulation. We showed that INO80-dependent H2A.Z occupancy is a critical licensing step for the bivalent domains, and thereby uncovered an epigenetic mechanism by which chromatin remodeling, histone variant deposition and histone modification coordinately control cell fate.ViewShow abstract... In contrast, primed mEpiSCs fail in germline competence and even rarely produce chimeras [1, 2], suggesting their reduced developmental and differentiation capacity. Under defined in vitro culture conditions, the primed mouse epiblast-like cells (mEpiLCs) can be established from naïve mESCs [10][11][12]. Naïve and primed pluripotent states exhibit different molecular signatures in the epigenome and transcriptome profile [3,10,11,[13][14][15][16][17][18]. ...... Under defined in vitro culture conditions, the primed mouse epiblast-like cells (mEpiLCs) can be established from naïve mESCs [10][11][12]. Naïve and primed pluripotent states exhibit different molecular signatures in the epigenome and transcriptome profile [3,10,11,[13][14][15][16][17][18]. ...Elevated retrotransposon activity and genomic instability in primed pluripotent stem cellsArticleFull-text availableJul 2021GENOME BIOL Haifeng Fu Weiyu ZhangNiannian Li Lin LiuBackgroundNaïve and primed pluripotent stem cells (PSCs) represent two different pluripotent states. Primed PSCs following in vitro culture exhibit lower developmental potency as evidenced by failure in germline chimera assays, unlike mouse naïve PSCs. However, the molecular mechanisms underlying the lower developmental competency of primed PSCs remain elusive.ResultsWe examine the regulation of telomere maintenance, retrotransposon activity, and genomic stability of primed PSCs and compare them with naïve PSCs. Surprisingly, primed PSCs only minimally maintain telomeres and show fragile telomeres, associated with declined DNA recombination and repair activity, in contrast to naïve PSCs that robustly elongate telomeres. Also, we identify LINE1 family integrant L1Md_T as naïve-specific retrotransposon and ERVK family integrant IAPEz to define primed PSCs, and their transcription is differentially regulated by heterochromatic histones and Dnmt3b. Notably, genomic instability of primed PSCs is increased, in association with aberrant retrotransposon activity.ConclusionsOur data suggest that fragile telomere, retrotransposon-associated genomic instability, and declined DNA recombination repair, together with reduced function of cell cycle and mitochondria, increased apoptosis, and differentiation properties may link to compromised developmental potency of primed PSCs, noticeably distinguishable from naïve PSCs.ViewShow abstract... Extensive efforts have been made to identify approaches able to reverse the two states of pluripotent stem cells and mainly involve either specific culture conditions with different factors or forced naive gene expression (Bao et al., 2009;Guo et al., 2009;Guo and Smith, 2010;Okashita et al., 2016;Chen et al., 2018;Du et al., 2018;Pastor et al., 2018;Qiu et al., 2015;Rathjen et al., 1999;Zhou et al., 2010;Tai and Ying, 2013;Yu S. et al., 2020). D Aniello et al. (2017) proposed the pivotal roles of vitamin C and L-proline in controlling the pluripotency continuum from naive to primed states by affecting global DNA methylation, transcriptional profile, and energy metabolism. ...Establishment of Mouse Primed Stem Cells by Combination of Activin and LIF SignalingArticleFull-text availableAug 2021Mengyi WeiYanglin ChenChaoyue Zhao Siqin BaoIn mice, embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) are established from pre-and post-implantation embryos and represent the naive and primed state, respectively. Herein we used mouse leukemia inhibitory factor (LIF), which supports ESCs self-renewal and Activin A (Act A), which is the main factor in maintaining EpiSCs in post-implantation epiblast cultures, to derive a primed stem cell line named ALSCs. Like EpiSCs, ALSCs express key pluripotent genes Oct4, Sox2, and Nanog; one X chromosome was inactivated; and the cells failed to contribute to chimera formation in vivo. Notably, compared to EpiSCs, ALSCs efficiently reversed to ESCs (rESCs) on activation of Wnt signaling. Moreover, we also discovered that culturing EpiSCs in AL medium for several passages favored Wnt signaling-driven naive pluripotency. Our results show that ALSCs is a primed state stem cell and represents a simple model to study the control of pluripotency fate and conversion from the primed to the naive state.ViewShow abstract... This is particularly interesting, as KLF4 is one of the transcription factors involved in the regulation of naïve pluripotency, and clearly the limiting factor in transition from primed to naïve pluripotency. Indeed, KLF4 overexpression has been shown to reprogram EpiSc to a naïve state [8], and its forced expression with KLF2 has been tested as a strategy to boost pluripotency in primed rabbit ESC [9]. ...Introduction of Mouse Embryonic Fibroblasts into Early Embryos: From Confusion to Constructive Discussion. Comment on Savatier, P. Introduction of Mouse Embryonic Fibroblasts into Early Embryos Causes Reprogramming and (Con)fusion. Cells 2021, 10, 772ArticleFull-text availableJun 2021 Krystyna Żyżyńska-Galeńska Jolanta KarasiewiczAgnieszka BernatWe would like to address the issues raised by Pierre Savatier in \"Introduction of Mouse Embryonic Fibroblasts into Early Embryos Causes Reprogramming and (Con)Fusion” [...]ViewShow abstract... Among them, Klf4, like other Yamanaka factors, is highly expressed in ESCs, and its expression drops dramatically after induction of differentiation, suggesting that Klf4 is likely involved with the core transcription factor circuitry of ESCs (Bourillot et al., 2009;Jiang et al., 2008). In line with this, functional inactivation of Klf4 induces spontaneous differentiation of ESCs, whereas its overexpression leads to enhanced self-renewal and delayed differentiation (Hall et al., 2009;Li et al., 2005). In addition, expression of Klf4 is controlled by leukemia inhibitory factor (LIF)-signal transduction and activator of transcription 3 (STAT3) signaling (Bourillot et al., 2009). ...Chapter 9 - Bacteria to form induced pluripotent stem cells In Advances in Stem Cell Biology, Methods in iPSC TechnologyChapterJun 2021Arif Istiaq Shah Adil Ishtiyaq Ahmad Mohammad Badrul Anam Kunimasa OhtaIn 2006, Takahashi and Yamanaka reported the generation of mouse and human induced pluripotent stem cells (iPS cells) by retroviral gene transduction of four transcription factors, famously known as Yamanaka factors (OCT4, SOX2, Klf4, and c-Myc) (Takahashi et al., 2007; Takahashi and Yamanaka, 2006). iPS cells show stemness properties (self-renewal and differentiation into three germ layers derivatives) that are comparable to the embryonic stem cells (ES cells). However, conventional iPS cells technique is not entirely safe because of its procedural difficulties, lower efficiency, and the risk of cancer formation (Luo et al., 2013; Rais et al., 2013). Meanwhile, several bacteria-mediated strategies have been identified that can reprogram differentiated cells into stem cell–like state and dedifferentiate into three germ layer derived cells. In 2012, Fuji et al. reported Helicobacter pylori bacteria can reprogram gastric epithelial cells into intestinal stem/progenitor-like cells in vivo by inducing homeobox transcription factors (CDX) during intestinal metaplasia (Fujii et al., 2012). In the same year, our group discovered that lactic acid bacteria (LAB) can reprogram human dermal fibroblast (HDF) into stem cell–like cells in vitro that can differentiate into all three germ layer derivatives (Ohta et al., 2012). Later, Masaki et al. reported that leprosy-causing bacterium Mycobacterium leprae reprograms adult Schwann cells into stem cell–like cells in vivo that differentiate into mesenchymal, skeletal, and smooth muscle cells facilitating infection dissemination (Masaki et al., 2013). In 2017, Ikeda et al. reported that Wolbachia pipientis bacterial proteins can accelerate traditional retroviral-mediated iPS cells generation process (Ikeda et al., 2017). All these studies substantiate the epigenetic potentials of bacteria or its material to induce stemness in differentiated cells. Such microbial potentials might be connected with the endosymbiotic theory, which states that eukaryotic cells are generated through endosymbiotic relationship between prokaryotes (Woese et al., 1990).In this chapter, we briefly discussed about several evidences of bacterial impact on cellular homeostasis and plasticity. We largely focused on LAB-mediated cell reprogramming of HDF cells as a potential technique for future iPS cells generation.ViewShow abstractInducible CRISPRa Screen Identifies Putative EnhancersArticleJul 2021J GENET GENOMICSZhongye DaiRui LiYuying Hou Xudong WuEnhancers are critical cis-regulatory elements that regulate spatiotemporal gene expression and control cell fates. However, the identification of enhancers in native cellular contexts still remains a challenge. Here we develop an inducible CRISPR activation (CRISPRa) system by transgenic expression of doxycycline (Dox) inducible dCas9-VPR in mouse embryonic stem cells (iVPR ESC). With this line, a simple introduction of specific gRNAs targeting promoters or enhancers allows us to realize the effect of CRISPRa in an inducible, reversible and Dox concentration-dependent manner. Taking advantage of this system, we induce tiled CRISPRa across genomic regions (105 kilobases) surrounding T (Brachyury), one of the key mesodermal development regulator genes. Moreover, we identify several CRISPRa-responsive elements with chromatin features of putative enhancers, including a region the homologous sequence in which humans harbors a body height risk variant. Genetic deletion of this region in ESC does affect subsequent T gene activation and osteogenic differentiation. Therefore, our inducible CRISPRa ESC line provides a convenient platform for high-throughput screens of putative enhancers.ViewShow abstractDifferential localization patterns of pyruvate kinase isoforms in murine naïve, formative, and primed pluripotent statesArticleJun 2021Exp Cell Res Joshua DierolfAndrew J. Watson Dean H BettsMouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) represent opposite ends of the pluripotency continuum, referred to as naïve and primed pluripotent states, respectively. These divergent pluripotent states differ in several ways including growth factor requirements, transcription factor expression, DNA methylation patterns, and metabolic profiles. Naïve cells employ both glycolysis and oxidative phosphorylation (OXPHOS), whereas primed cells preferentially utilize aerobic glycolysis, a trait shared with cancer cells referred to as the Warburg Effect. Until recently, metabolism has been regarded as a by-product of cell fate; however, evidence now supports metabolism as being a driver of stem cell state and fate decisions. Pyruvate kinase muscle isoforms (PKM1 and PKM2) are important for generating and maintaining pluripotent stem cells (PSCs) and mediating the Warburg effect. Both isoforms catalyze the last step of glycolysis generating adenosine triphosphate and pyruvate, however, the precise role(s) of PKM1/2 in naïve and primed pluripotency is not well understood. The primary objective of this study was to characterize the cellular expression and localization patterns of PKM1 and PKM2 in mESCs, chemically transitioned epiblast-like cells (mEpiLCs) representing formative pluripotency, and mEpiSCs using immunoblotting and confocal microscopy. The results indicate that PKM1 and PKM2 are not only localized to the cytoplasm but also accumulate in differential subnuclear regions of mESC, mEpiLCs and mEpiSCs as determined by a quantitative confocal microscopy employing orthogonal projections and airyscan processing. Importantly, we discovered that the subnuclear localization of PKM1/2 shifts during the transition from mESCs, mEpiLCs and mEpiSCs. Finally, we have comprehensively validated the appropriateness and power of the Pearson s correlation coefficient and Manders overlap coefficient for assessing nuclear and cytoplasmic protein colocalization in PSCs by immunofluorescence confocal microscopy. We propose that nuclear PKM1/2 may assist with distinct pluripotency state maintenance and lineage priming by non-canonical mechanisms. These results advance our understanding of the overall mechanisms controlling naïve, formative, and primed pluripotency.ViewShow abstractAMPK activation reverts mouse epiblast stem cells to naïve stateArticleFull-text availableJun 2021Yajing LiuJunko YamaneAkito TanakaJun K. YamashitaDespite increasing knowledge on primed and naive pluripotency, the cell signaling that regulates the pluripotency type in stem cells remains not fully understood. Here we show that AMP kinase (AMPK) activators can induce the reversion of primed mouse epiblast stem cells (mEpiSCs) to the naive pluripotent state. The addition of AMPK activators alone or together with leukemia inhibitory factor to primed mEpiSCs induced the appearance of naive-like cells. After passaging in naive culture conditions, the colony morphology, protein expression, and global gene expression profiles indicated the naive state, as did germline transmission ability. Loss-of-function and gain-of-function studies suggested that p38 is a critical downstream target in AMPK activation. Finally, single-cell RNA sequencing analysis revealed that the reversion process through AMPK signaling passes an intermediate naive-like population. In conclusion, the AMPK pathway is a critical driving force in the reversion of primed to naive pluripotency.ViewShow abstractInhibition of N-myristoyltransferase Promotes Naive Pluripotency in Mouse and Human Pluripotent Stem CellsPreprintFull-text availableJun 2021Junko YoshidaHitomi WatanabeKaori YamauchiKyoji HorieNaive and primed states are distinct states of pluripotency during early embryonic development that can be captured and converted to each other in vitro. To elucidate the regulatory mechanism of pluripotency, we performed a recessive genetic screen of homozygous mutant mouse embryonic stem cells (mESCs) and found that suppression of N-myristoyltransferase (Nmt) promotes naive pluripotency. Disruption of Nmt1 in mESCs conferred resistance to differentiation. Suppression of Nmt in mouse epiblast stem cells (mEpiSCs) promoted the conversion from the primed to the naive state. This effect was independent of Src, which is a major substrate of Nmt and is known to promote differentiation of mESCs. Suppression of Nmt in naive-state human induced pluripotent stem cells (hiPSCs) increased the expression of the naive-state marker. These results indicate that Nmt is a novel target for the regulation of naive pluripotency conserved between mice and humans.ViewShow abstractResidual pluripotency is required for inductive germ cell segregationArticleJun 2021EMBO REPShinya AramakiSaya Kagiwada Guangming WuHans R SchölerFine-tuned dissolution of pluripotency is critical for proper cell differentiation. Here we show that the mesodermal transcription factor, T, globally affects the properties of pluripotency through binding to Oct4 and to the loci of other pluripotency regulators. Strikingly, lower T levels coordinately affect naïve pluripotency, thereby directly activating the germ cell differentiation program, in contrast to the induction of germ cell fate of primed models. Contrary to the effect of lower T levels, higher T levels more severely affect the pluripotency state, concomitantly enhancing the somatic differentiation program and repressing the germ cell differentiation program. Consistent with such in vitro findings, nascent germ cells in vivo are detected in the region of lower T levels at the posterior primitive streak. Furthermore, T and core pluripotency regulators co-localize at the loci of multiple germ cell determinants responsible for germ cell development. In conclusion, our findings indicate that residual pluripotency establishes the earliest and fundamental regulatory mechanism for inductive germline segregation from somatic lineages.ViewShow abstractShow morePromotion Of Reprogramming To Ground State Pluripotency By Signal InhibitionArticleFull-text availableNov 2008 Jose CR SilvaOrnella Barrandon Jennifer NicholsAustin SmithInduced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency--including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras--were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1-2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon.ViewShow abstractGeneration of Mouse Induced Pluripotent Stem Cells Without Viral VectorsArticleFull-text availableNov 2008Science Keisuke OkitaMasato Nakagawa Hyen Jong HongShinya YamanakaInduced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by introducing Oct3/4 and Sox2with either Klf4 and c-Myc or Nanog and Lin28 using retroviruses or lentiviruses. Patient-specific iPS cells could be usefulin drug discovery and regenerative medicine. However, viral integration into the host genome increases the risk of tumorigenicity.Here, we report the generation of mouse iPS cells without viral vectors. Repeated transfection of two expression plasmids,one containing the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other containing the c-Myc cDNA, into mouseembryonic fibroblasts resulted in iPS cells without evidence of plasmid integration, which produced teratomas when transplantedinto mice and contributed to adult chimeras. The production of virus-free iPS cells, albeit from embryonic fibroblasts, addressesa critical safety concern for potential use of iPS cells in regenerative medicine.ViewShow abstractInduced Pluripotent Stem Cells Generated Without Viral IntegrationArticleFull-text availableOct 2008Science Matthias StadtfeldMasaki Nagaya Jochen Utikal Konrad HochedlingerPluripotent stem cells have been generated from mouse and human somatic cells by viral expression of the transcription factorsOct4, Sox2, Klf4, and c-Myc. A major limitation of this technology is the use of potentially harmful genome-integrating viruses.We generated mouse induced pluripotent stem (iPS) cells from fibroblasts and liver cells by using nonintegrating adenovirusestransiently expressing Oct4, Sox2, Klf4, and c-Myc. These adenoviral iPS (adeno-iPS) cells show DNA demethylation characteristicof reprogrammed cells, express endogenous pluripotency genes, form teratomas, and contribute to multiple tissues, includingthe germ line, in chimeric mice. Our results provide strong evidence that insertional mutagenesis is not required for in vitroreprogramming. Adenoviral reprogramming may provide an improved method for generating and studying patient-specific stem cellsand for comparing embryonic stem cells and iPS cells.ViewShow abstractChanging po tency by spontaneous fusionArticleJan 2002NatureQ.L. YingJ. NicholsE.P. EvansA.G. SmithViewIsolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cellsArticleDec 1981P NATL ACAD SCI USAGR MartinThis report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.ViewShow abstractInhibition of pluripotent stem cell differentiation by purified polypeptidesArticleJan 1988NATUREA. Smith John K HeathDebra D. DonaldsonD. RogersViewThe origin and efficient derivation of embryonic stem (ES) cells in the mouseArticleFeb 1999Pathol Biol Richard L GardnerViewChanging potency by spon - taneous fusionArticleJan 2002NatureQl Ying Jennifer NicholsEp EvansAg SmithViewCapture of Authentic Embryonic Stem Cells from Rat BlastocystsArticleJan 2009Mia Buehr Stephen Meek Kathryn BlairAustin SmithEmbryonic stem (ES) cells have been available from inbred mice since 1981 but have not been validated for other rodents. Failure to establish ES cells from a range of mammals challenges the identity of cultivated stem cells and our understanding of the pluripotent state. Here we investigated derivation of ES cells from the rat. We applied molecularly defined conditions designed to shield the ground state of authentic pluripotency from inductive differentiation stimuli. Undifferentiated cell lines developed that exhibited diagnostic features of ES cells including colonization of multiple tissues in viable chimeras. Definitive ES cell status was established by transmission of the cell line genome to offspring. Derivation of germline-competent ES cells from the rat paves the way to targeted genetic manipulation in this valuable biomedical model species. Rat ES cells will also provide a refined test-bed for functional evaluation of pluripotent stem cell-derived tissue repair and regeneration.ViewShow abstractParameters Influencing Derivation of Embryonic Stem Cells From Murine EmbryosArticleDec 2008Genesis Laura Batlle Austin Smith Jennifer NicholsThe derivation of ES cells is poorly understood and varies in efficiency between different strains of mice. We have investigated potential differences between embryos of permissive and recalcitrant strains during diapause and ES cell derivation. We found that in diapause embryos of the recalcitrant C57BL/6 and CBA strains, the epiblast failed to expand during the primary explant phase of ES cell derivation, whereas in the permissive 129 strain, it expanded dramatically. Epiblasts from the recalcitrant strains could be expanded by reducing Erk activation. Isolation of 129 epiblasts facilitated very efficient derivation of ES cell lines in serum- and feeder-free conditions, but reduction of Erk activity was required for derivation of ES cells from isolated C57BL/6 or CBA epiblasts. The results suggest that the discrepancy in ES cell derivation efficiency is not attributable merely to variable prodifferentiative effects of the extra-embryonic lineages but also to an intrinsic variability within the epiblast to maintain pluripotency.ViewShow abstractShow moreAdvertisementRecommendationsDiscover more about: Kruppel-Like Transcription FactorsProjectSingle cell resoled transcriptomics during mouse gastrulation Wajid Jawaid Private Profile Carla Mulas[...]Fernando Calero-NietoView projectArticleFull-text availableE-Cadherin Promotes Incorporation of Mouse Epiblast Stem Cells into Normal DevelopmentSeptember 2012 · PLoS ONE Satoshi OhtsukaSatomi Nishikawa-TorikaiHitoshi NiwaMouse epiblast stem cells (mEpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos. Their pluripotency is distinct from that of mouse embryonic stem cells (mESCs) in several cell biological criteria. One of the distinctions is that mEpiSCs contribute either not at all or at much lower efficiency to chimeric embryos after blastocyst injection compared to mESCs. ... [Show full abstract] However, here we showed that mEpiSCs can be incorporated into normal development after blastocyst injection by forced expression of the E-cadherin transgene for 2 days in culture. Using this strategy, mEpiSCs gave rise to live-born chimeras from 5% of the manipulated blastocysts. There were no obvious signs of reprogramming of mEpiSCs toward the mESC-like state during the 2 days after induction of the E-cadherin transgene, suggesting that mEpiSCs possess latent ability to integrate into the normal developmental process as its origin, epiblasts.View full-textArticleNaive and Primed Pluripotent StatesJuly 2009 · Cell Stem Cell Jennifer Nichols Austin SmithAfter maternal predetermination gives way to zygotic regulation, a ground state is established within the mammalian embryo. This tabula rasa for embryogenesis is present only transiently in the preimplantation epiblast. Here, we consider how unrestricted cells are first generated and then prepared for lineage commitment. We propose that two phases of pluripotency can be defined: naive and primed. ... [Show full abstract] This distinction extends to pluripotent stem cells derived from embryos or by molecular reprogramming ex vivo.Read moreArticleOct4 and LIF/Stat3 Additively Induce Krüppel Factors to Sustain Embryonic Stem Cell Self-RenewalDecember 2009 · Cell Stem Cell John S Hall Ge Guo Austin Smith[...] Jason WrayEmbryonic stem cell (ESC) pluripotency is dependent on an intrinsic gene regulatory network centered on Oct4. Propagation of the pluripotent state is stimulated by the cytokine leukemia inhibitory factor (LIF) acting through the transcriptional regulator Stat3. Here, we show that this extrinsic stimulus converges with the intrinsic circuitry in Krüppel-factor activation. Oct4 primarily induces ... [Show full abstract] Klf2 while LIF/Stat3 selectively enhances Klf4 expression. Overexpression of either factor reduces LIF dependence, but with quantitative and qualitative differences. Unlike Klf4, Klf2 increases ESC clonogenicity, maintains undifferentiated ESCs in the genetic absence of Stat3, and confers resistance to BMP-induced differentiation. ESCs expanded with Klf2 remain capable of contributing to adult chimeras. Postimplantation-embryo-derived EpiSCs lack both Klf2 and Klf4 and expression of either can reinstate naive pluripotency. These findings indicate that Oct4 and Stat3 intersect in directing expression of Klf transcriptional regulators with overlapping properties that additively reinforce ground-state ESC pluripotency, identity, and self-renewal.Read moreArticleA genome-wide screen in EpiSCs identifies Nr5a nuclear receptors as potent inducers of ground state...October 2010 · Development Ge Guo Austin SmithIn rodents, the naïve early epiblast undergoes profound morphogenetic, transcriptional and epigenetic changes after implantation. These differences are maintained between blastocyst-derived embryonic stem (ES) cells and egg cylinder-derived epiblast stem cells (EpiSCs). Notably, ES cells robustly colonise chimaeras, whereas EpiSCs show little or no contribution. ES cells self-renew independently ... [Show full abstract] of mitogenic growth factors, whereas EpiSCs require fibroblast growth factor. However, EpiSCs retain the core pluripotency factors Oct4 and Sox2 and the developmental barrier dividing them from unrestricted pluripotency can be surmounted by a single reprogramming factor. This provides an opportunity to identify molecules that can reset the naïve state. We undertook a forward genetic screen for effectors of EpiSC reprogramming, employing piggyBac transposition to activate endogenous gene expression at random and selecting for undifferentiated colonies in the absence of growth factor signalling. Three recovered clones harboured integrations that activate the closely related orphan nuclear receptor genes Nr5a1 and Nr5a2. Activity of Nr5a1 and Nr5a2 was confirmed by direct transfection. Reprogrammed colonies were obtained without transgene integration and at 10-fold higher frequency than with other single factors. Converted cells exhibited the diagnostic self-renewal characteristics, gene expression profile and X chromosome activation signature of ground state pluripotency. They efficiently produced adult chimaeras and gave germline transmission. Nr5a receptors regulate Oct4 transcription but this is insufficient for reprogramming. Intriguingly, unlike previously identified reprogramming molecules, Nr5a receptors play no evident role in ES cell self-renewal. This implies a different foundation for their capacity to reset pluripotency and suggests that further factors remain to be identified.Read moreArticleThe origin identity of embryonic stem cellsJanuary 2011 · Development Jennifer Nichols Austin SmithEmbryonic stem (ES) cells are used extensively in biomedical research and as a model with which to study early mammalian development, but their exact origin has been subject to much debate. They are routinely derived from pre-implantation embryos, but it has been suggested that the cells that give rise to ES cells might arise from epiblast cells that are already predisposed to a primordial germ ... [Show full abstract] cell (PGC) fate, which then progress to ES cell status via the PGC lineage. Based on recent findings, we propose here that ES cells can be derived directly from early epiblast cells and that ES cells might arise via two different routes that are dictated by their culture conditions.Read moreArticleFull-text availablePromotion Of Reprogramming To Ground State Pluripotency By Signal InhibitionNovember 2008 · PLoS Biology Jose CR SilvaOrnella Barrandon Jennifer Nichols[...]Austin SmithInduced pluripotent stem (iPS) cells are generated from somatic cells by genetic manipulation. Reprogramming entails multiple transgene integrations and occurs apparently stochastically in rare cells over many days. Tissue stem cells may be subject to less-stringent epigenetic restrictions than other cells and might therefore be more amenable to deprogramming. We report that brain-derived neural ... [Show full abstract] stem (NS) cells acquire undifferentiated morphology rapidly and at high frequency after a single round of transduction with reprogramming factors. However, critical attributes of true pluripotency--including stable expression of endogenous Oct4 and Nanog, epigenetic erasure of X chromosome silencing in female cells, and ability to colonise chimaeras--were not attained. We therefore applied molecularly defined conditions for the derivation and propagation of authentic pluripotent stem cells from embryos. We combined dual inhibition (2i) of mitogen-activated protein kinase signalling and glycogen synthase kinase-3 (GSK3) with the self-renewal cytokine leukaemia inhibitory factor (LIF). The 2i/LIF condition induced stable up-regulation of Oct4 and Nanog, reactivation of the X chromosome, transgene silencing, and competence for somatic and germline chimaerism. Using 2i /LIF, NS cell reprogramming required only 1-2 integrations of each transgene. Furthermore, transduction with Sox2 and c-Myc is dispensable, and Oct4 and Klf4 are sufficient to convert NS cells into chimaera-forming iPS cells. These findings demonstrate that somatic cell state influences requirements for reprogramming and delineate two phases in the process. The ability to capture pre-pluripotent cells that can advance to ground state pluripotency simply and with high efficiency opens a door to molecular dissection of this remarkable phenomenon.View full-textLast Updated: 30 Apr 2021Interested in research on Kruppel-Like Transcription Factors?Join ResearchGate to discover and stay up-to-date with the latest research from leading experts in Kruppel-Like Transcription Factors and many other scientific topics.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? Keep me logged inLog inorContinue with GoogleWelcome back! Please log in.Email · HintTip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? 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