Human Hypoblast Formation Is Not Dependent on FGF Signalling

Developmental Biology 361 (2012) 358–363

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Developmental Biology

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Human hypoblast formation is not dependent on FGF signalling

Mila Roode a,b, Kathryn Blair a,c, Philip Snell d, Kay Elder d, Sally Marchant e, Austin Smith a,c, Jennifer Nichols a,b,⁎

a Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK b Department of Physiology, Development and Neuroscience, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK c Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK d Bourn Hall Clinic, Bourn, Cambridge CB23 2TN, UK e Centre for Reproductive Medicine, Barts and The London, West Smithﬁeld, London EC1A 7BE, UK

article info abstract

Article history: Mouse embryos segregate three different lineages during preimplantation development: trophoblast, epi- Received for publication 24 August 2011 blast and hypoblast. These differentiation processes are associated with restricted expression of key tran- Revised 24 October 2011 scription factors (Cdx2, Oct4, Nanog and Gata6). The mechanisms of segregation have been extensively Accepted 26 October 2011 studied in the mouse, but are not as well characterised in other species. In the human embryo, hypoblast dif- Available online 31 October 2011 ferentiation has not previously been characterised. Here we demonstrate co-exclusive immunolocalisation of Nanog and Gata4 in human blastocysts, implying segregation of epiblast and hypoblast, as in rodent embryos. Keywords: Pluripotency However, the formation of hypoblast in the human is apparently not dependent upon FGF signalling, in con- Epiblast trast to rodent embryos. Nonetheless, the persistence of Nanog-positive cells in embryos following treatment Hypoblast with FGF inhibitors is suggestive of a transient naïve pluripotent population in the human blastocyst, which Fibroblast growth factor may be similar to rodent epiblast and ES cells but is not sustained during conventional human ES cell deriva- Human ES cell derivation tion protocols. © 2011 Elsevier Inc.Open access under CC BY license.

Introduction and hypoblast lineages are morphologically distinct within the murine ICM by embryonic day (E) 4.5, just before implantation. The mouse embryo undergoes two crucial lineage segregation Studying the mechanisms of early lineage segregation has provided events before implantation in the uterus. The first is the separation insight into how the pluripotent epiblast is specified, and this has en- of trophectoderm from the inner cell mass (ICM); the second is the abled development of efficient strategies to isolate ES cells from murine segregation of the epiblast and hypoblast in the ICM. The trophecto- embryos, either by physical separation (Brook and Gardner, 1997)or derm and hypoblast lineages give rise to extra-embryonic tissues application of specific inhibitors to block differentiation (Buehr et al., that facilitate the implantation of the embryo into the uterus and for- 2008; Li et al., 2008; Nichols et al., 2009; Ying et al., 2008). mation of the placenta. The epiblast forms a pool of pluripotent cells Pluripotency is defined as the ability of a cell to give rise to differ- that will give rise to the foetus in collaboration with patterning sig- ent tissues representative of all three of the embryonic germ layers: nals from the extra-embryonic tissues (Beddington and Robertson, ectoderm, mesoderm and endoderm. It can be considered as two CORE Metadata, citation and similar papers at core.ac.uk 1999; Gardner and Beddington, 1988). The pre-implantation epiblast states (Nichols and Smith, 2009). Naïve (or ‘ground state’) pluripo- Provided by Elsevier - Publisher Connector is also the source of embryonic stem (ES) cells (Brook and Gardner, tency is represented by the newly segregated pre-implantation epi- 1997). blast and rodent ES cells. Following implantation, the epiblast The separation of the trophectoderm and ICM in mouse embryos responds to signals from the extra-embryonic tissues and becomes is marked by mutually exclusive expression of the two transcription primed for differentiation. Primed pluripotency is also exhibited by factors Oct4 and Cdx2 (Niwa et al., 2005). The segregation of epiblast the epiblast stem cell lines (EpiSCs) derived from post-implantation and hypoblast from the cells of the ICM is suggested to involve Nanog epiblasts in culture (Brons et al., 2007; Tesar et al., 2007). In addition and Gata6. These two factors are initially expressed in an overlapping to their ability to give rise to multiple tissue types, naïve and primed manner in the ICM but then become mutually exclusive in the late pluripotent cells are both characterised by expression of the core mouse blastocyst (Chazaud et al., 2006; Plusa et al., 2008). Epiblast pluripotency factors Oct4, Sox2 and Nanog, but differ in their expression spectrum of other genes, their culture requirements and their ability to resume normal development when placed in the embryonic environment. Interestingly, although pluripotent cell lines have been ⁎ Corresponding author at: Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK. Fax: +44 1223 760241. derived from primate blastocysts (Suemori et al., 2001; Thomson E-mail address: [email protected] (J. Nichols). et al., 1995; Thomson et al., 1998), these cells more closely resemble

EpiSCs than murine ES cells (Brons et al., 2007; Tesar et al., 2007). Sig- was Sprague–Dawley (SD). Animals were maintained by in-house nificantly, both primate ES cells and EpiSCs require exogenous FGF in breeding on a lighting regime of 14 hours light and 10 hours darkness order to self-renew, in contrast to rodent ES cells. with food and water supplied ad libitum. Prior to caging with stud Naïve pluripotency can readily be captured from embryos of mice males, female mice were oestrous selected by visual inspection of and rats when fibroblast growth factor (FGF)/Erk signalling are inhib- the vagina (Champlin et al., 1973). Female rats were selected for oes- ited in combination with glycogen synthase kinase 3 (GSK3) inhibi- trus using a vaginal impedance monitor (Muromachi Kikai, Tokyo, tion, a system known as ‘2i’, short for ‘two inhibitors’ (Buehr et al., Japan, MK-11)(Koto et al., 1987). Detection of a copulation plug the 2008; Li et al., 2008; Nichols et al., 2009; Ying et al., 2008). Analysis following morning was used to confirm successful mating; the result- of murine embryos cultured from the 8-cell-stage in 2i revealed that ing embryos were then considered to be E0.5. For all experiments, the the resulting ICM consisted almost entirely of cells expressing high embryos from at least two females were pooled and randomly levels of Nanog and no Gata4, indicating that putative hypoblast assigned to experimental groups. Mouse and rat embryos were cells had become epiblast (Nichols et al., 2009). The efficient deriva- flushed from oviducts using M2 medium at E2.5 and E3.5 respective- tion of murine ES cells in 2i may therefore be attributable in part to ly. Mouse embryos were cultured in BlastAssist® for 2 days before blockade of hypoblast. Further evidence that formation of hypoblast being moved to N2B27 as expanded blastocysts. Rat embryos were in the mouse is dependent upon FGF signalling is provided by the cultured in BlastAssist® for 1 day, after which they were switched demonstration that supplementation of the embryo culture medium to N2B27. Embryos were cultured at 5% oxygen, 7% carbon dioxide with high levels of recombinant FGF4 diverts ICM cells to hypoblast and 37 °C. (Yamanaka et al., 2010). The 2i culture regime provides a highly selective environment in Immunostaining which cells not exhibiting properties of naive pluripotency fail to thrive, and are thus eliminated. EpiSCs and human ES cells do not survive in 2i Embryos were processed for immunohistochemistry as described (Guo et al., 2009; Nichols and Smith, 2009). So far there have been no previously (Nichols et al., 2009). Briefly, they were fixed in 4% para- reports of the derivation of naïve pluripotent cells from non-rodent em- formaldehyde in PBS for 15 minutes, then rinsed in PBS containing bryos. For practical purposes, naïve human ES cells would be highly de- 3 mg/ml polyvinylpyrolidone (PBS/PVP; P0930, Sigma), permeabi- sirable, but attempts to generate human ES cells by culturing embryos lised in PBS/PVP containing 0.25% Triton X-100 (23,472-9, Sigma) in 2i have failed (JN, MR, unpublished). We have attempted to find an for 30 minutes and blocked in blocking buffer, which comprised PBS explanation for this failure by first investigating lineage segregation in containing 0.1% BSA, 0.01% Tween 20 (P1379, Sigma) and 2% donkey human embryos, then analysing their responses to the application of in- serum. Primary antibodies were Oct4 (C-10 sc-5279, Santa Cruz Bio- hibitors, specifically in the maintenance of Nanog expression and the tech, Santa Cruz, CA, USA), Nanog (ab21624 or ab21603, Abcam, Cam- emergence of Gata4-expressing cells. bridge, UK), Gata4 (C-20 sc-1237, Santa Cruz), Gata6 (R&D Systems, AF1700) and Sox17 (R&D Systems, AF1924). Antibodies were diluted Materials and methods 1:100 (Oct-4 was used at 1:200) in blocking buffer and embryos were incubated in the appropriate antibody solution at 4 °C overnight. They Human embryos were rinsed three times in blocking buffer for 15 minutes each, and incubated in secondary antibody solution for 1 hour at room temper- Human embryos surplus to IVF requirements were donated to re- ature. Secondary antibodies raised in Donkey labelled with Alexa search after informed patient consent, with approval of local ethics fluorophores (Invitrogen, Paisley, UK) were diluted 1:500 in blocking committees and the UK Human Fertilisation and Embryology Author- buffer (anti-mouse IgG2b for Oct4:647; anti-rabbit IgG for Nanog:488; ity (Research licence R0178). Embryos were obtained from Bourn Hall anti-goat IgG for Gata4, Gata6 and Sox17:555). Embryos were then Clinic, Cambridgeshire. Embryos were thawed using EmbroThawTM rinsed three times in blocking buffer, incubated briefly in increasing Kit (EMF40_T, Fertipro) according to the manufacturer's instructions. concentrations of Vectashield (H-1200, Vector Labs, Peterborough, Embryos were cultured to day 3 in EmbryoAssist™ (12140010, Ori- UK) before mounting on glass slides in small drops of concentrated gio), after which they were changed to BlastAssist® (12160010, Ori- Vectashield with DAPI, and subsequently sealed with nail varnish. gio) until the appearance of a cavity, upon which they were transferred to N2B27 medium (SCS-SF-NB-02, Stem Cell Sciences) Image collection and analysis (Nichols and Ying, 2006; Ying and Smith, 2003) for further culture. Embryos were cultured in 50 μl drops of medium under mineral oil Embryos were imaged on either a Leica TCS SP5 confocal microscope (MINOIL500, Fertipro) and changed to fresh medium every 2 days. or Revolution XD Confocal System (Andor). Reconstructions of three- Embryos were cultured in a humidified atmosphere at 5% oxygen, dimensional images from sections and cell counts were performed 7% carbon dioxide and 37 °C. Embryos were exposed to inhibitors using Leica or Imaris software. from day 3 of development. Culture medium was supplemented with inhibitors in the combina- Statistical analysis tions specified in the text: PD0325901, a Mek inhibitor with an IC50 value in the 20–50 nM range in human cells (Ciuffreda et al., 2009), Probability (P) values were established using Student's t-test for

0.5 μMor1μM; Chir99021, a GSK3β inhibitor with an IC50 value of comparison between two samples. 10 nM in human cells (Ring et al., 2003), 3 μM (both synthesised in the Division of Signal Transduction Therapy, University of Dundee, Dundee, Results

UK) and PD173074 (Sigma), a pan-FGF receptor inhibitor with an IC50 value in the range of 5–22 nM for human FGFR1–5(Byron et al., 2008; Human embryo culture to the late blastocyst stage Grand et al., 2004; Mohammadi et al., 1998; Skaper et al., 2000; St Bernard et al., 2005; Stavridis et al., 2007), 100 nM. Human embryos were previously thawed and cultured in standard IVF medium to the blastocyst stage (day 6–7) at 20% oxygen. Blastocyst Rodent embryos formation rates were low (17.5%, 25/143) and many blastocysts did not progress beyond the initial cavitation stages. The use of 5% oxygen has The strains of mice used in this study were MF1 outbred, and F1 been reported to be beneficial for blastocyst formation rates hybrids between C57BL/6/Ola and CBA/Ca. The strain of rat used (Dumoulin et al., 1999; Waldenstrom et al., 2009). By reducing the 360 M. Roode et al. / Developmental Biology 361 (2012) 358–363 concentration of oxygen in the culture regime to 5%, our blastocyst for- are the components of 2i that effectively maintain naïve pluripotency mation rates increased significantly (35.1%, 67/191), with many embryos in cultures of murine ES cells (Ying et al., 2008). Gata4-positive cells progressing to day 7 and older. Therefore, for all subsequent experiments were still observed in embryos developed under these conditions ahumidified atmosphere of 5% oxygen, 7% carbon dioxide and 37 °C was (Fig. 2B, D). In all three conditions that target FGF/Erk signalling, adopted. human embryos possess Gata4 and Nanog-positive cells that are exclusive to one another. Thus, the segregation of epiblast and hypo- Putative hypoblast is segregated by day 7 of human development blast within the human embryo appears not to be dependent on FGF signalling. The interplay between Nanog and Gata6 is suggested to be crucial To eliminate the human-specific culture regime as a potential var- for the epiblast/hypoblast fate decision in mouse embryos (Plusa iable in the response of embryos to FGF signalling inhibition, we cul- et al., 2008). Although Nanog protein has been shown to localise to tured murine embryos from the 8-cell-stage under identical a sub-population of cells in the ICM of human embryos at the mid/ conditions. Mouse embryos responded as previously reported to late blastocyst stage (Cauffman et al., 2009; Hyslop et al., 2005; 1 μM PD0325901 and 2i (Nichols et al., 2009)(Fig. 2E, G). We also Kimber et al., 2008), the identity of the Nanog-negative ICM cells used the combination of PD0325901 and PD173074 that has been has not yet been investigated. We therefore investigated the expres- shown to block hypoblast formation (Yamanaka et al., 2010). This sion of the hypoblast markers Gata6, Gata4 and Sox17 in human also prevented formation of Gata4 positive cells. Finally, we cultured blastocysts. rat embryos under these conditions and found very similar responses We first examined human embryos at 6 days post-fertilisation. (Fig. 2F, G). This indicates that the effect of FGF inhibition is not spe- Nanog is confined to a few inner cells within the embryo (Fig. 1A). cific to the mouse. Gata6 and Oct4 are widely expressed throughout the embryo, with In both mouse and rat embryos, 1 μM PD0325901 reproducibly levels varying between individual cells, consistent with previous blocks the formation of the hypoblast. The addition of Chir99021 to data (Cauffman et al., 2005; Cauffman et al., 2006; Chen et al., 2009; PD0325901 (2i) seems to facilitate some hypoblast cells escaping Kimber et al., 2008). Some inner cells exhibit high levels of Nanog this block, especially in the rat embryo (Fig. 2G). The combination and low levels of Gata6 (Fig. 1B, arrows), whilst in other cells both of 0.5 μM PD0325901 and PD173074 reduces the emergence of hypo- markers are expressed at about the same level (Fig. 1B, arrowheads). blast compared to control embryos, but is not as effective in blocking This pattern is similar to that observed in the early mouse blastocyst hypoblast as 1 μM PD0325901. Despite the consistency of 1 μM (Plusa et al., 2008). PD0325901 in its effects on mouse and rat embryos, it did not elimi- At day 7 of development, Gata4 and Sox17, both markers of differ- nate the formation of Gata4-positive cells in the human embryo. entiated hypoblast, are restricted to a narrow subset of cells within However, it should be noted that culturing human embryos for ex- the embryo (Fig. 1C). Significantly, at this stage of development, this tended periods in FGF signalling inhibitors resulted in no detectable pattern is mutually exclusive with Nanog expression. Some embryos detrimental effect on the Nanog positive population of cells. show Gata4 or Sox17-positive cells in what appears to be an epithelial layer overlying the Nanog-positive epiblast on the blastocoelic sur- Discussion face (Figs. 1C and 2C). This mirrors delamination of the hypoblast seen in rodent blastocysts. Oct4 protein is much more restricted to The first two lineage decisions that mouse embryos undertake cells of the ICM than in earlier blastocysts, with staining in both the during preimplantation development have been extensively studied. epiblast and hypoblast (Fig. 1C), as is the case with early murine hy- Detailed examination of these decisions has not been performed in poblast (Silva et al., 2009). This may be due to slight differences in the human embryos due to the scarcity of available material and the developmental age of the embryos, most likely reflecting variability in sub-optimal nature of culture regimes. Whilst the differentiation of their development in vitro. These embryos tended to exhibit reduced ICM and trophectoderm has been observed in human embryos total cell number, consistent with this (Fig. 1C, D). These observations (Cauffman et al., 2009; Chen et al., 2009), there has been little evi- suggest that the human embryo at day 7 resembles the mouse em- dence for the segregation of the ICM into epiblast and hypoblast. bryo at E4.5 when all three embryonic lineages can be distinguished. Nanog has previously been shown to be restricted to a subset of ICM cells within the embryo at the blastocyst stage (Cauffman et al., Hypoblast segregation is not dependent upon FGF/Erk signalling 2009), but it was not known if the Nanog-negative cells within the ICM represented hypoblast. Fig. 1C demonstrates Gata4 expression FGF/Mek inhibition in mouse preimplantation embryos has a in cells negative for Nanog, indicating that the hypoblast is segregated striking effect on lineage segregation (Chazaud et al., 2006; Nichols from the epiblast within the ICM by day 7 of human development. A et al., 2009; Yamanaka et al., 2010). Inhibition of FGF signalling di- population of cells negative for Nanog is also immunoreactive to verts all cells of the ICM to the epiblast fate, bypassing the hypoblast. Sox17 (Fig. 1D), an additional marker of hypoblast in the mouse, pro- We were interested in investigating whether the formation of human viding further evidence that hypoblast is formed in human embryos. hypoblast is also dependent on FGF signalling. If so, this would indi- We have previously reported that the segregation of hypoblast in cate that the segregation of these lineages may be based on a con- mouse embryos is dependent upon FGF signalling (Nichols et al., served mechanism. We performed experiments with human 2009). We sought to examine if this was also the case in human em- embryos, adding inhibitors from the 6–8 cell stage. 1 μM PD0325901 bryos. Mouse embryos behaved as we have previously reported when is effective to block the formation of hypoblast in murine embryos if exposed to small molecule inhibitors targeting the FGF pathway applied before its segregation (Nichols et al., 2009). However, all when we cultured them in the human embryo culturing regime human embryos treated with 1 μM PD0325901 that developed to (Fig. 2E, G). The effect of inhibiting FGF signalling on lineage alloca- late blastocysts showed Gata4 expression in a subset of cells, separate tion in mouse embryos is not specific to this species: rat embryos be- from the Nanog-expressing population (Fig. 2A, D). To test whether haved in a similar way (Fig. 2F, G). This observation indicates that the an alternative downstream pathway for FGF signalling is adopted role of FGF/Erk signalling is conserved amongst the early embryos of during human hypoblast induction, we introduced an inhibitor of these two species. the FGF receptor, PD173074, in combination with PD0325901. Using The effect of FGF inhibition on human embryos shows a number of this combination, Gata4-positive cells were still identified in all the differences from the effects observed in rodents, even though the human embryos investigated (Fig. 2C, D). We examined whether concentrations used were well above their respective IC50 values. PD0325901 could work synergistically with Chir99021, since these Gata4-positive hypoblast still forms under conditions where it is M. Roode et al. / Developmental Biology 361 (2012) 358–363 361

Fig. 1. Human embryos segregate putative hypoblast by day 7 of development. Human embryos were thawed and cultured in standard IVF medium until they formed cavitated blastocysts, upon which they were moved to N2B27 medium. Embryos were ﬁxed and immunostained for Oct4 (white), Nanog (green) and Gata6 (red). At day 6 of in vitro development (A) Nanog is restricted to a few cells within the embryo, whilst Gata6 and Oct4 are broadly expressed. Confocal images of two representative embryos with a maximum projection of the 3D reconstruction of the blastocyst are shown. (B) A single slice in a z stack of each of the two embryos shown in (A), indicating that Nanog and Gata6 can both be expressed highly in the same cell (arrowheads) or that Gata6 can be low as Nanog is high (arrows). (C and D) Embryos were developed to day 7 in vitro and immunostained for Nanog (green), Oct4 (white) and Gata4 (red) (C) or Sox17 (red) (D). In contrast to the stainings observed at day 6, Oct4 is restricted to the cells of the ICM. Gata4 and Sox17 are restricted to a subset of cells within the embryo, distinct from the Nanog positive cells: the putative hypoblast. In all embryos nuclei were counterstained with DAPI (blue). The total number of cells in each embryo is written in the top right hand corner of the panel. blocked in rodent embryos (Fig. 2A–D), indicating that the formation this family. Furthermore, TGFβ has been shown to maintain self- of human hypoblast is not dependent on FGF signalling. The molecu- renewal of trophectoderm stem cells (Erlebacher et al., 2004). It lar mechanism of hypoblast segregation in the human embryo is still may therefore be anticipated that blocking this pathway could shield unknown. Possible candidates include the transforming growth factor pluripotent cells from diverting to extra-embryonic lineages. The (TGF) β family proteins that operate via SMAD transcription factors. mitogen-activated protein kinase P38α has been suggested to play a Human ES cell lines can be propagated in a state of primed pluripo- role in branching morphogenesis of embryonic lung epithelium (Liu tency in the presence of Activin (Vallier et al., 2005), a member of et al., 2008), a derivative of the deﬁnitive endoderm that shares an 362 M. Roode et al. / Developmental Biology 361 (2012) 358–363

Fig. 2. Effect of FGF/Erk signalling inhibition on human epiblast and hypoblast compared with mouse and rat. Human embryos were thawed and cultured in standard IVF medium until they formed cavitated blastocysts, upon which they were moved to N2B27 medium. Embryos were exposed to inhibitors from the 6–8 cell stage and developed until day 7 in vitro. Embryos were immunostained for Oct4 (white), Nanog (green) and Gata4 (red). Confocal images were taken and 3D reconstructions of the embryos created. The addition of 1 μM PD0325901 (A), 2i (B) or 0.5 μM PD0325901 and 100 nM PD173074 (C) did not eliminate the segregation of the putative hypoblast as indicated by the expression of Gata4. (D) Blastocysts were variable in their number of Nanog and Gata4-expressing cells within each experimental group and across experimental groups. This may be due to the inher- ent variation between human embryos in vitro. The number of cells per embryo is written above each bar in the graph. Mouse (E) and rat (F) embryos were cultured from the 8-cell- stage under the same culture regime as human embryos. The addition of small molecules that inhibit the FGF/Erk pathway result in the loss of hypoblast in these embryos, indicating that hypoblast formation is dependent on FGF signalling in both the mouse and rat. The Nanog antibody utilised has a lower affinity for the rat protein (F) than the mouse (E). (G) Cells of the epiblast and hypoblast were counted in 3D reconstruction of embryos. * indicates a Pb0.05 indicating a statistically significant difference between two data sets. The statistical differences of the no factors group and inhibitor conditions were not plotted for clarity. In all embryos nuclei were counterstained with DAPI (blue). overlapping molecular profile with the hypoblast. Although we have cannot survive exposure to 2i (Nichols and Smith, 2009). If the failed to detect any effect of suppression of hypoblast formation by Nanog-positive cells in the human embryo were the in vivo counter- applying inhibitors of either of these pathways to early mouse or rat part of human ES cells, they may be expected to deteriorate in the embryos (data not shown), they have not yet been applied to presence of small molecules targeting FGF/Erk signalling. The sur- human embryos. vival of Nanog-positive cells within the embryo suggests that it The Nanog-positive epiblast compartment is not reduced in may ultimately be possible to isolate cells that are independent of human embryos when they are cultured in the presence of inhibi- FGF/Erk signalling from these embryos (Hanna et al., 2010; Smith, tors for FGF/Erk and Gsk3 signalling (Fig. 2D). Human ES cells 2010). M. Roode et al. / Developmental Biology 361 (2012) 358–363 363

Acknowledgements differentiation of human embryonic stem cells to extraembryonic lineages. Stem Cells 23, 1035–1043. Kimber, S.J., Sneddon, S.F., Bloor, D.J., El-Bareg, A.M., Hawkhead, J.A., Metcalfe, A.D., We would like to thank all of the patients who kindly donated Houghton, F.D., Leese, H.J., Rutherford, A., Lieberman, B.A., et al., 2008. Expression their embryos to research, and all staff at Bourn Hall and Barts for pro- of genes involved in early cell fate decisions in human embryos and their regulation by growth factors. Reproduction 135, 635–647. viding embryos. We also thank Samuel Jameson and staff for animal Koto, M., Miwa, M., Togashi, M., Tsuji, K., Okamoto, M., Adachi, J., 1987. A method for husbandry and Peter Humphreys for assistance with imaging. This detecting the optimum day for mating during the 4-day estrous cycle in the rat; work was conducted in compliance with HFEA regulation and ethical measuring the value of electrical impedance of the vagina. Jikken Dobutsu 36, – guidelines under the authority of licence R0178. Research was funded 195 198. Li, P., Tong, C., Mehrian-Shai, R., Jia, L., Wu, N., Yan, Y., Maxson, R.E., Schulze, E.N., Song, by the Wellcome Trust, Medical Research Council and the Biotechnology H., Hsieh, C.L., et al., 2008. Germline competent embryonic stem cells derived from and Biological Sciences Research Council. rat blastocysts. Cell 135, 1299–1310. Liu, Y., Martinez, L., Ebine, K., Abe, M.K., 2008. Role for mitogen-activated protein kinase p38 alpha in lung epithelial branching morphogenesis. Dev. Biol. 314, 224–235. References Mohammadi, M., Froum, S., Hamby, J.M., Schroeder, M.C., Panek, R.L., Lu, G.H., Eliseenkova, A.V., Green, D., Schlessinger, J., Hubbard, S.R., 1998. Crystal structure of an angiogenesis Beddington, R.S., Robertson, E.J., 1999. Axis development and early asymmetry in inhibitor bound to the FGF receptor tyrosine kinase domain. EMBO J. 17, 5896–5904. mammals. Cell 96, 195–209. Nichols, J., Smith, A., 2009. Naive and primed pluripotent states. Cell Stem Cell 4, Brons, I.G., Smithers, L.E., Trotter, M.W., Rugg-Gunn, P., Sun, B., de Sousa, Chuva, Lopes, 487–492. S.M., Howlett, S.K., Clarkson, A., Ahrlund-Richter, L., Pedersen, R.A., et al., 2007. Nichols, J., Ying, Q.L., 2006. Derivation and propagation of embryonic stem cells in Derivation of pluripotent epiblast stem cells from mammalian embryos. Nature serum- and feeder-free culture. Meth. Mol. Biol. 329, 91–98. 448, 191–195. Nichols, J., Silva, J., Roode, M., Smith, A., 2009. Suppression of Erk signalling promotes Brook, F.A., Gardner, R.L., 1997. The origin and efficient derivation of embryonic stem ground state pluripotency in the mouse embryo. Development 136, 3215–3222. cells in the mouse. Proc. Natl. Acad. Sci. U. S. A. 94, 5709–5712. Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi, K., Yagi, R., Rossant, J., 2005. Buehr, M., Meek, S., Blair, K., Yang, J., Ure, J., Silva, J., McLay, R., Hall, J., Ying, Q.L., Smith, Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. A., 2008. Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, Cell 123, 917–929. 1287–1298. Plusa, B., Piliszek, A., Frankenberg, S., Artus, J., Hadjantonakis, A.K., 2008. Distinct sequential Byron, S.A., Gartside, M.G., Wellens, C.L., Mallon, M.A., Keenan, J.B., Powell, M.A., Goodfellow, cell behaviours direct primitive endoderm formation in the mouse blastocyst. Develop- P.J., Pollock, P.M., 2008. Inhibition of activated fibroblast growth factor receptor 2 in ment 135, 3081–3091. endometrial cancer cells induces cell death despite PTEN abrogation. Cancer Res. 68, Ring, D.B., Johnson, K.W., Henriksen, E.J., Nuss, J.M., Goff, D., Kinnick, T.R., Ma, S.T., 6902–6907. Reeder, J.W., Samuels, I., Slabiak, T., et al., 2003. Selective glycogen synthase kinase Cauffman, G., Van de Velde, H., Liebaers, I., Van Steirteghem, A., 2005. Oct-4 mRNA and 3 inhibitors potentiate insulin activation of glucose transport and utilization in protein expression during human preimplantation development. Mol. Hum. vitro and in vivo. Diabetes 52, 588–595. Reprod. 11, 173–181. Silva, J., Nichols, J., Theunissen, T.W., Guo, G., van Oosten, A.L., Barrandon, O., Wray, J., Cauffman, G., Liebaers, I., Van Steirteghem, A., Van de Velde, H., 2006. POU5F1 isoforms Yamanaka, S., Chambers, I., Smith, A., 2009. Nanog is the gateway to the pluripotent show different expression patterns in human embryonic stem cells and preimplan- ground state. Cell 138, 722–737. tation embryos. Stem Cells 24, 2685–2691. Skaper, S.D., Kee, W.J., Facci, L., Macdonald, G., Doherty, P., Walsh, F.S., 2000. The FGFR1 Cauffman, G., De Rycke, M., Sermon, K., Liebaers, I., Van de Velde, H., 2009. Markers that inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and define stemness in ESC are unable to identify the totipotent cells in human preimplan- neurotropic effects. J. Neurochem. 75, 1520–1527. tation embryos. Hum. Reprod. 24, 63–70. Smith, A., 2010. Pluripotent stem cells: private obsession and public expectation. EMBO Champlin, A.K., Dorr, D.L., Gates, A.H., 1973. Determining the stage of the estrous cycle Mol. Med. 2, 113–116. in the mouse by the appearance of the vagina. Biol. Reprod. 8, 491–494. St Bernard, R., Zheng, L., Liu, W., Winer, D., Asa, S.L., Ezzat, S., 2005. Fibroblast growth Chazaud, C., Yamanaka, Y., Pawson, T., Rossant, J., 2006. Early lineage segregation between factor receptors as molecular targets in thyroid carcinoma. Endocrinology 146, epiblast and primitive endoderm in mouse blastocysts through the Grb2-MAPK path- 1145–1153. way. Dev. Cell 10, 615–624. Stavridis, M.P., Lunn, J.S., Collins, B.J., Storey, K.G., 2007. A discrete period of FGF-induced Chen, A.E., Egli, D., Niakan, K., Deng, J., Akutsu, H., Yamaki, M., Cowan, C., Fitz-Gerald, C., Erk1/2 signalling is required for vertebrate neural specification. Development 134, Zhang, K., Melton, D.A., et al., 2009. Optimal timing of inner cell mass isolation increases 2889–2894. the efficiency of human embryonic stem cell derivation and allows generation of sibling Suemori, H., Tada, T., Torii, R., Hosoi, Y., Kobayashi, K., Imahie, H., Kondo, Y., Iritani, A., cell lines. Cell Stem Cell 4, 103–106. Nakatsuji, N., 2001. Establishment of embryonic stem cell lines from cynomolgus Ciuffreda, P., Casati, S., De Mieri, M., Ferraboschi, P., 2009. Corticosteroids 21-glucuronides: monkey blastocysts produced by IVF or ICSI. Dev. Dyn. 222, 273–279. synthesis and complete characterization by (1)H and (13)C NMR. Steroids 74, Tesar, P.J., Chenoweth, J.G., Brook, F.A., Davies, T.J., Evans, E.P., Mack, D.L., Gardner, R.L., 870–875. McKay, R.D., 2007. New cell lines from mouse epiblast share defining features with Dumoulin, J.C., Meijers, C.J., Bras, M., Coonen, E., Geraedts, J.P., Evers, J.L., 1999. Effect of human embryonic stem cells. Nature 448, 196–199. oxygen concentration on human in-vitro fertilization and embryo culture. Hum. Thomson, J.A., Kalishman, J., Golos, T.G., Durning, M., Harris, C.P., Becker, R.A., Hearn, Reprod. 14, 465–469. J.P., 1995. Isolation of a primate embryonic stem cell line. Proc. Natl. Acad. Sci. Erlebacher, A., Price, K.A., Glimcher, L.H., 2004. Maintenance of mouse trophoblast stem U. S. A. 92, 7844–7848. cell proliferation by TGF-beta/activin. Dev. Biol. 275, 158–169. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, Gardner, R.L., Beddington, R.S., 1988. Multi-lineage 'stem' cells in the mammalian embryo. V.S., Jones, J.M., 1998. Embryonic stem cell lines derived from human blastocysts. J. Cell Sci. Suppl. 10, 11–27. Science 282, 1145–1147. Grand, E.K., Chase, A.J., Heath, C., Rahemtulla, A., Cross, N.C., 2004. Targeting FGFR3 in Vallier, L., Alexander, M., Pedersen, R.A., 2005. Activin/Nodal and FGF pathways cooperate multiple myeloma: inhibition of t(4;14)-positive cells by SU5402 and PD173074. to maintain pluripotency of human embryonic stem cells. J. Cell Sci. 118, 4495–4509. Leukemia 18, 962–966. Waldenstrom, U., Engstrom, A.B., Hellberg, D., Nilsson, S., 2009. Low-oxygen compared Guo, G., Yang, J., Nichols, J., Hall, J.S., Eyres, I., Mansfield, W., Smith, A., 2009. Klf4 reverts with high-oxygen atmosphere in blastocyst culture, a prospective randomized developmentally programmed restriction of ground state pluripotency. Development study. Fertil. Steril. 91, 2461–2465. 136, 1063–1069. Yamanaka, Y., Lanner, F., Rossant, J., 2010. FGF signal-dependent segregation of primitive Hanna, J., Cheng, A.W., Saha, K., Kim, J., Lengner, C.J., Soldner, F., Cassady, J.P., Muffat, J., endoderm and epiblast in the mouse blastocyst. Development 137, 715–724. Carey, B.W., Jaenisch, R., 2010. Human embryonic stem cells with biological and Ying, Q.L., Smith, A.G., 2003. Defined conditions for neural commitment and differentiation. epigenetic characteristics similar to those of mouse ESCs. Proc. Natl. Acad. Sci. Meth. Enzymol. 365, 327–341. U. S. A. 107, 9222–9227. Ying, Q.L., Wray, J., Nichols, J., Batlle-Morera, L., Doble, B., Woodgett, J., Cohen, P., Smith, Hyslop, L., Stojkovic, M., Armstrong, L., Walter, T., Stojkovic, P., Przyborski, S., Herbert, A., 2008. The ground state of embryonic stem cell self-renewal. Nature 453, M., Murdoch, A., Strachan, T., Lako, M., 2005. Downregulation of NANOG induces 519–523.