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1.  Global Mapping of DNA Methylation in Mouse Promoters Reveals Epigenetic Reprogramming of Pluripotency Genes 
PLoS Genetics  2008;4(6):e1000116.
DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency.
Author Summary
Large scale epigenetic reprogramming occurs in mammalian germ cells and the early embryo. The biological purpose of this reprogramming is largely unknown, although it has been suggested that it may be required for the embryonic genome to return to a pluripotent state. We carried out a genome-wide screen of promoter methylation in the mouse, comparing germ cells with pluripotent cells, multipotent cells, and more differentiated cell types. We find that promoter methylation is an epigenetic signature of developmental potency. Genes linked to pluripotency are generally hypomethylated in stem cells and hypermethylated (and silenced) in more differentiated cell types, and our genome-wide screen provides new candidates for the regulation of pluripotency. Importantly, germ cells resemble pluripotent cell types in that most promoters have been reprogrammed. However, a small group of key pluripotency regulators (including Nanog), are methylated in mature germ cells, presumably in order to suppress pluripotency at critical stages of germ cell differentiation. Indeed, methylation in these genes becomes reprogrammed after fertilisation so that the embryo can regain totipotency. This work, therefore, shows for the first time that epigenetic reprogramming is crucial for maintaining the pluripotency of germ and embryonic stem cells.
doi:10.1371/journal.pgen.1000116
PMCID: PMC2432031  PMID: 18584034
2.  Cross-Species Genome Wide Expression Analysis during Pluripotent Cell Determination in Mouse and Rat Preimplantation Embryos 
PLoS ONE  2012;7(10):e47107.
The transition between morula and blastocyst stage during preimplantation development represents the first differentiation event of embryogenesis. Morula cells undergo the first cellular specialization and produce two well-defined populations of cells, the trophoblast and the inner cell mass (ICM). Embryonic stem cells (ESCs) with unlimited self-renewal capacity are believed to represent the in vitro counterpart of the ICM. Both mouse and rat ESCs can be derived from the ICM cells, but their in vitro stability differs. In this study we performed a microarray analysis in which we compared the transcriptome of mouse and rat morula, blastocyst, and ICM. This cross-species comparison represents a good model for understanding the differences in derivation and cultivation of ESCs observed in the two species. In order to identify alternative regulation of important molecular mechanisms the investigation of differential gene expression between the two species was extended at the level of signaling pathways, gene families, and single selected genes of interest. Some of the genes differentially expressed between the two species are already known to be important factors in the maintenance of pluripotency in ESCs, like for example Sox2 or Stat3, or play a role in reprogramming somatic cells to pluripotency like c-Myc, Klf4 and p53 and therefore represent interesting candidates to further analyze in vitro in the rat ESCs. This is the first study investigating the gene expression changes during the transition from morula to blastocyst in the rat preimplantation development. Our data show that in the pluripotent pool of cells of the rat and mouse preimplantation embryo substantial differential regulation of genes is present, which might explain the difficulties observed for the derivation and culture of rat ESCs using mouse conditions.
doi:10.1371/journal.pone.0047107
PMCID: PMC3471948  PMID: 23077551
3.  HIPPO Pathway Members Restrict SOX2 to the Inner Cell Mass Where It Promotes ICM Fates in the Mouse Blastocyst 
PLoS Genetics  2014;10(10):e1004618.
Pluripotent epiblast (EPI) cells, present in the inner cell mass (ICM) of the mouse blastocyst, are progenitors of both embryonic stem (ES) cells and the fetus. Discovering how pluripotency genes regulate cell fate decisions in the blastocyst provides a valuable way to understand how pluripotency is normally established. EPI cells are specified by two consecutive cell fate decisions. The first decision segregates ICM from trophectoderm (TE), an extraembryonic cell type. The second decision subdivides ICM into EPI and primitive endoderm (PE), another extraembryonic cell type. Here, we investigate the roles and regulation of the pluripotency gene Sox2 during blastocyst formation. First, we investigate the regulation of Sox2 patterning and show that SOX2 is restricted to ICM progenitors prior to blastocyst formation by members of the HIPPO pathway, independent of CDX2, the TE transcription factor that restricts Oct4 and Nanog to the ICM. Second, we investigate the requirement for Sox2 in cell fate specification during blastocyst formation. We show that neither maternal (M) nor zygotic (Z) Sox2 is required for blastocyst formation, nor for initial expression of the pluripotency genes Oct4 or Nanog in the ICM. Rather, Z Sox2 initially promotes development of the primitive endoderm (PE) non cell-autonomously via FGF4, and then later maintains expression of pluripotency genes in the ICM. The significance of these observations is that 1) ICM and TE genes are spatially patterned in parallel prior to blastocyst formation and 2) both the roles and regulation of Sox2 in the blastocyst are unique compared to other pluripotency factors such as Oct4 or Nanog.
Author Summary
Pluripotent stem cells can give rise to any cell type in the body, making them an attractive tool for regenerative medicine. Pluripotent stem cells can be derived from the mammalian embryo at the blastocyst stage or they can be created from mature adult cells by reprogramming. During reprogramming, SOX2 helps establish pluripotency, but it is not clear how SOX2 establishes pluripotency in the blastocyst. We evaluated where SOX2 is present, how SOX2 is regulated, and where SOX2 is active during blastocyst formation. Our data show that the roles and the regulation of SOX2 are unique compared to other pluripotency/reprogramming factors, such as OCT4 and NANOG. SOX2 marks pluripotent cells earlier than do other factors, but does not regulate pluripotency until several days later. Rather, the earlier role of SOX2 is to help establish the yolk sac lineage, which is essential for gestation.
doi:10.1371/journal.pgen.1004618
PMCID: PMC4207610  PMID: 25340657
4.  Global gene expression of the inner cell mass and trophectoderm of the bovine blastocyst 
Background
The first distinct differentiation event in mammals occurs at the blastocyst stage when totipotent blastomeres differentiate into either pluripotent inner cell mass (ICM) or multipotent trophectoderm (TE). Here we determined, for the first time, global gene expression patterns in the ICM and TE isolated from bovine blastocysts. The ICM and TE were isolated from blastocysts harvested at day 8 after insemination by magnetic activated cell sorting, and cDNA sequenced using the SOLiD 4.0 system.
Results
A total of 870 genes were differentially expressed between ICM and TE. Several genes characteristic of ICM (for example, NANOG, SOX2, and STAT3) and TE (ELF5, GATA3, and KRT18) in mouse and human showed similar patterns in bovine. Other genes, however, showed differences in expression between ICM and TE that deviates from the expected based on mouse and human.
Conclusion
Analysis of gene expression indicated that differentiation of blastomeres of the morula-stage embryo into the ICM and TE of the blastocyst is accompanied by differences between the two cell lineages in expression of genes controlling metabolic processes, endocytosis, hatching from the zona pellucida, paracrine and endocrine signaling with the mother, and genes supporting the changes in cellular architecture, stemness, and hematopoiesis necessary for development of the trophoblast.
doi:10.1186/1471-213X-12-33
PMCID: PMC3514149  PMID: 23126590
Blastocyst; Trophectoderm; Inner cell mass; Development
5.  Differentiation of nonhuman primate embryonic stem cells along neural lineages 
The differentiation of embryonic stem cells (ESCs) into neurons and glial cells represents a promising cell-based therapy for neurodegenerative diseases. Because the rhesus macaque is physiologically and phylogenetically similar to humans, it is a clinically relevant animal model for ESC research. In this study, the pluripotency and neural differentiation potential of a rhesus monkey ESC line (ORMES6) was investigated. ORMES6 was derived from an in vitro produced blastocyst, which is the same way human ESCs have been derived. ORMES6 stably expressed the embryonic transcription factors POU5F1 (Oct4), Sox2 and NANOG. Stage-specific embryonic antigen 4 (SSEA 4) and the glycoproteins TRA-1-60 and TRA-1-81 were also expressed. The embryoid bodies (EBs) formed from ORMES6 ESCs spontaneously gave rise to cells of three germ layers. After exposure to basic fibroblast growth factor (bFGF) for 14–16 days, columnar rosette cells formed in the EB outgrowths. Sox2, microtubule-associated protein (MAP2), β-tublinIII and glial fibrillary acidic protein (GFAP) genes and Nestin, FoxD3, Pax6 and β-tublinIII antigens were expressed in the rosette cells. Oct4 and NANOG expression were remarkably down-regulated in these cells. After removal of bFGF from the medium, the rosette cells differentiated along neural lineages. The differentiated cells expressed MAP2, β-tublinIII, Neuro D and GFAP genes. Most differentiated cells expressed early neuron-specific antigen β-tublinIII (73±4.7%) and some expressed intermediate neuron antigen MAP2 (18±7.2%). However, some differentiated cells expressed the glial cell antigens A2B5 (7.17%±1.2%), GFAP (4.93±1.9%), S100 (7±3.5%) and O4 (0.2±70.2%). The rosette cells were transplanted into the striatum of immune-deficient NIHIII mice. The cells persisted for approximately 2 weeks and expressed Ki67, NeuN, MAP2 and GFAP. These results demonstrate that the rhesus monkey ESC line ORMES6 retains the pluripotent characteristics of ESCs and can be efficiently induced to differentiate along neural lineages.
doi:10.1016/j.diff.2008.10.014
PMCID: PMC2749555  PMID: 19272521
Embryonic stem cell; Rhesus monkey; Neurogenesis; Neural lineage differentiation
6.  X-chromosome inactivation in monkey embryos and pluripotent stem cells 
Developmental biology  2012;371(2):146-155.
Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI. We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo primate pluripotent cells.
doi:10.1016/j.ydbio.2012.08.009
PMCID: PMC3466365  PMID: 22935618
X-inactivation; Embryonic Stem Cells; Blastocyst; Inner Cell Mass; Primates
7.  Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos 
PLoS ONE  2013;8(4):e61900.
Background
The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells.
Methodology/Significant Principal Findings
In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively.
Conclusion/Significance
Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras.
doi:10.1371/journal.pone.0061900
PMCID: PMC3633951  PMID: 23626746
8.  Ethanol, Acetaldehyde and Estradiol Affect Growth and Differentiation of Rhesus Monkey Embryonic Stem Cells 
Background
The timing of the origins of fetal alcohol syndrome have been difficult to determine, in part because of the challenge associated with in vivo studies of the peri-implantation stage of embryonic development. Because embryonic stem cells (ESCs) are derived from blastocyst stage embryos, they are used as a model for early embryo development.
Methods
Rhesus monkey ESC lines (ORMES-6 and -7) were treated with 0, 0.01%, 0.1%, or 1.0% ethanol, 1.0% ethanol with estradiol or 0.00025% acetaldehyde with or without estradiol for 4 weeks.
Results
Although control ESCs remained unchanged, abnormal morphology of ESCs in the ethanol and acetaldehyde treatment groups was observed before 2 weeks of treatment. Immunofluorescence staining of key pluripotency markers ( TRA-1- 81 and alkaline phosphatase) indicated a loss of ESC pluripotency in the 1.0% ethanol group. ORMES-7 was more sensitive to effects of ethanol than ORMES-6.
Conclusions
Estradiol appeared to increase sensitivity to ethanol in the ORMES-6 and -7 cell line. The morphological changes and labeling for pluripotency, proliferation and apoptosis demonstrated that ethanol affects how these early cells develop in culture, their differentiation state in particular. The effects of ethanol may be mediated in part through metabolic pathways regulating acetaldehyde formation, and while potentially accentuated by estradiol in some individuals, how remains to be determined.
doi:10.1111/j.1530-0277.2011.01490.x
PMCID: PMC3139714  PMID: 21438889
Primate; fetal alcohol syndrome; embryo development
9.  SOX2 Modulates Reprogramming of Gene Expression in Two-Cell Mouse Embryos1  
Biology of Reproduction  2011;85(2):409-416.
Sox2 is a key gene that controls transcriptional networks required for pluripotency. The role of Sox2 in the developmental transition of a highly differentiated oocyte to totipotent blastomeres of the early preimplantation embryo, however, is not known. We report that Sox2, which is localized in the nucleus, is first zygotically expressed during the 2-cell stage and that its expression dramatically increases between the morula and blastocyst stages. Injecting a cRNA encoding Sox2 into 1-cell embryos resulted in overexpression of SOX2 by approximately 70% and developmental arrest at the 2-cell stage, whereas injecting cRNAs encoding Pou5f1, Myc (also known as c-Myc), or Klf4 has little effect on the ability of 2-cell embryos to cleave to the 4-cell stage. Global transcription assessed by bromo uridine triphosphate incorporation is reduced by approximately 15%, and transcript profiling revealed that approximately 15% of zygotically expressed genes are dramatically repressed in 2-cell embryos overexpressing SOX2. Furthermore, overexpressing a dominant-negative SOX2 perturbs reprogramming of gene expression in 2-cell embryos, though to a much lesser extent than that observed following overexpression of SOX2, and leads to developmental failure after the 2-cell stage but before the 8-cell stage. Results of these experiments implicate Sox2 as a critical transcriptional regulator in the oocyte-to-embryo transition that entails formation of totipotent blastomeres and indicate that the amount of Sox2 is critical for successful execution of this transition.
Overexpression of SOX2 or a dominant-negative form inhibits embryo development beyond the 2-cell stage by perturbing the reprogramming of gene expression during genome activation that is necessary for continued development.
doi:10.1095/biolreprod.111.090886
PMCID: PMC3142263  PMID: 21543769
chromatin; early development; embryo; gene expression; transcriptional regulation
10.  Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo 
PLoS ONE  2010;5(11):e13952.
Background
In preimplantation mammalian development the transcription factor Sox2 (SRY-related HMG-box gene 2) forms a complex with Oct4 and functions in maintenance of self-renewal of the pluripotent inner cell mass (ICM). Previously it was shown that Sox2−/− embryos die soon after implantation. However, maternal Sox2 transcripts may mask an earlier phenotype. We investigated whether Sox2 is involved in controlling cell fate decisions at an earlier stage.
Methods and Findings
We addressed the question of an earlier role for Sox2 using RNAi, which removes both maternal and embryonic Sox2 mRNA present during the preimplantation period. By depleting both maternal and embryonic Sox2 mRNA at the 2-cell stage and monitoring embryo development in vitro we show that, in the absence of Sox2, embryos arrest at the morula stage and fail to form trophectoderm (TE) or cavitate. Following knock-down of Sox2 via three different short interfering RNA (siRNA) constructs in 2-cell stage mouse embryos, we have shown that the majority of embryos (76%) arrest at the morula stage or slightly earlier and only 18.7–21% form blastocysts compared to 76.2–83% in control groups. In Sox2 siRNA-treated embryos expression of pluripotency associated markers Oct4 and Nanog remained unaffected, whereas TE associated markers Tead4, Yap, Cdx2, Eomes, Fgfr2, as well as Fgf4, were downregulated in the absence of Sox2. Apoptosis was also increased in Sox2 knock-down embryos. Rescue experiments using cell-permeant Sox2 protein resulted in increased blastocyst formation from 18.7% to 62.6% and restoration of Sox2, Oct4, Cdx2 and Yap protein levels in the rescued Sox2-siRNA blastocysts.
Conclusion and Significance
We conclude that the first essential function of Sox2 in the preimplantation mouse embryo is to facilitate establishment of the trophectoderm lineage. Our findings provide a novel insight into the first differentiation event within the preimplantation embryo, namely the segregation of the ICM and TE lineages.
doi:10.1371/journal.pone.0013952
PMCID: PMC2980489  PMID: 21103067
11.  Promoter analysis of the rabbit POU5F1 gene and its expression in preimplantation stage embryos 
BMC Molecular Biology  2009;10:88.
Background
The POU5F1 gene encodes the octamer-binding transcription factor-4 (Oct4). It is crucial in the regulation of pluripotency during embryonic development and widely used as molecular marker of embryonic stem cells (ESCs). The objective of this study was to identify and to analyse the promoter region of rabbit POU5F1 gene; furthermore to examine its expression pattern in preimplantation stage rabbit embryos.
Results
The upstream region of rabbit POU5F1 was subcloned sequenced and four highly conserved promoter regions (CR1-4) were identified. The highest degree of similarity on sequence level was found among the conserved domains between rabbit and human. Among the enhancers the proximal enhancer region (PE-1A) exhibited the highest degree of homology (96.4%). Furthermore, the CR4 regulator domain containing the distal enhancer (DE-2A) was responsible for stem cell-specific expression. Also, BAC library screen revealed the existence of a processed pseudogene of rabbit POU5F1. The results of quantitative real-time PCR experiments showed that POU5F1 mRNA was abundantly present in oocytes and zygotes, but it was gradually reduced until the activation of the embryonic genome, thereafter a continuous increase in POU5F1 mRNA level was observed until blastocyst stage. By using the XYClone laser system the inner cell mass (ICM) and trophoblast portions of embryos were microdissected and examined separately and POU5F1 mRNA was detected in both cell types.
Conclusion
In this study we provide a comparative sequence analysis of the regulatory region of rabbit POU5F1 gene. Our data suggest that the POU5F1 gene is strictly regulated during early mammalian development. We proposed that the well conserved CR4 region containing the DE-2A enhancer is responsible for the highly conserved ESC specific gene expression. Notably, we are the first to report that the rabbit POU5F1 is not restricted to ICM cells only, but it is expressed in trophoblast cells as well. This information may be well applicable to investigate further the possible phylogenetic role and the regulation of POU5F1 gene.
doi:10.1186/1471-2199-10-88
PMCID: PMC2751759  PMID: 19732419
12.  Interspecies Somatic Cell Nuclear Transfer Is Dependent on Compatible Mitochondrial DNA and Reprogramming Factors 
PLoS ONE  2011;6(4):e14805.
Interspecies somatic cell nuclear transfer (iSCNT) involves the transfer of a nucleus or cell from one species into the cytoplasm of an enucleated oocyte from another. Once activated, reconstructed oocytes can be cultured in vitro to blastocyst, the final stage of preimplantation development. However, they often arrest during the early stages of preimplantation development; fail to reprogramme the somatic nucleus; and eliminate the accompanying donor cell's mitochondrial DNA (mtDNA) in favour of the recipient oocyte's genetically more divergent population. This last point has consequences for the production of ATP by the electron transfer chain, which is encoded by nuclear and mtDNA. Using a murine-porcine interspecies model, we investigated the importance of nuclear-cytoplasmic compatibility on successful development. Initially, we transferred murine fetal fibroblasts into enucleated porcine oocytes, which resulted in extremely low blastocyst rates (0.48%); and failure to replicate nuclear DNA and express Oct-4, the key marker of reprogramming. Using allele specific-PCR, we detected peak levels of murine mtDNA at 0.14±0.055% of total mtDNA at the 2-cell embryo stage and then at ever-decreasing levels to the blastocyst stage (<0.001%). Furthermore, these embryos had an overall mtDNA profile similar to porcine embryos. We then depleted porcine oocytes of their mtDNA using 10 µM 2′,3′-dideoxycytidine and transferred murine somatic cells along with murine embryonic stem cell extract, which expressed key pluripotent genes associated with reprogramming and contained mitochondria, into these oocytes. Blastocyst rates increased significantly (3.38%) compared to embryos generated from non-supplemented oocytes (P<0.01). They also had significantly more murine mtDNA at the 2-cell stage than the non-supplemented embryos, which was maintained throughout early preimplantation development. At later stages, these embryos possessed 49.99±2.97% murine mtDNA. They also exhibited an mtDNA profile similar to murine preimplantation embryos. Overall, these data demonstrate that the addition of species compatible mtDNA and reprogramming factors improves developmental outcomes for iSCNT embryos.
doi:10.1371/journal.pone.0014805
PMCID: PMC3083390  PMID: 21556135
13.  The chromatin remodeling factor Chd1l is required in the preimplantation embryo 
Biology Open  2012;2(2):121-131.
Summary
During preimplantation development, the embryo must establish totipotency and enact the earliest differentiation choices, processes that involve extensive chromatin modification. To identify novel developmental regulators, we screened for genes that are preferentially transcribed in the pluripotent inner cell mass (ICM) of the mouse blastocyst. Genes that encode chromatin remodeling factors were prominently represented in the ICM, including Chd1l, a member of the Snf2 gene family. Chd1l is developmentally regulated and expressed in embryonic stem (ES) cells, but its role in development has not been investigated. Here we show that inhibiting Chd1l protein production by microinjection of antisense morpholinos causes arrest prior to the blastocyst stage. Despite this important function in vivo, Chd1l is non-essential for cultured ES cell survival, pluripotency, or differentiation, suggesting that Chd1l is vital for events in embryos that are distinct from events in ES cells. Our data reveal a novel role for the chromatin remodeling factor Chd1l in the earliest cell divisions of mammalian development.
doi:10.1242/bio.20122949
PMCID: PMC3575647  PMID: 23429299
Chd1l; ALC1; Preimplantation development; ICM; ES cells; Chromatin remodeling
14.  Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos 
Human Reproduction (Oxford, England)  2009;24(11):2709-2717.
BACKGROUND
Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres.
METHODS
Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs).
RESULTS
Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype.
CONCLUSION
We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.
doi:10.1093/humrep/dep262
PMCID: PMC2763129  PMID: 19633307
human embryonic stem cell line; single blastomere; morula; 4-cell stage human embryo
15.  Defining the Genomic Signature of Totipotency and Pluripotency during Early Human Development 
PLoS ONE  2013;8(4):e62135.
The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs), still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs) from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS) and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes), in vivo pluripotency (20 genes), and in vitro pluripotency (107 genes), and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions.
doi:10.1371/journal.pone.0062135
PMCID: PMC3629124  PMID: 23614026
16.  Transcriptome Analysis of Mouse Stem Cells and Early Embryos 
PLoS Biology  2003;1(3):e74.
Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.
250,000 EST sequences from oocytes, blastocysts, and embryonic and adult stem cells contribute to the annotation of the mouse genome and suggest genes that contribute to the unique features of these developmental stages and cell types
doi:10.1371/journal.pbio.0000074
PMCID: PMC300684  PMID: 14691545
17.  Stress Induces AMP-Dependent Loss of Potency Factors Id2 and Cdx2 in Early Embryos and Stem Cells 
Stem Cells and Development  2013;22(10):1564-1575.
The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5 h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo.
doi:10.1089/scd.2012.0352
PMCID: PMC3653377  PMID: 23316940
18.  Functional Heterogeneity of Embryonic Stem Cells Revealed through Translational Amplification of an Early Endodermal Transcript 
PLoS Biology  2010;8(5):e1000379.
Detection of low-level, lineage-specific transcription aids in the identification of lineage-primed populations of ES cells provides a new framework for pluripotency.
ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically “undifferentiated” cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V+S+), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly when introduced back into morulae or blastocysts, the V+S+ population is not effective at contributing to the epiblast and can contribute to the extra-embryonic visceral and parietal endoderm, while the V−S+ population generates high contribution chimeras. Taken together our data support a model in which ES cell culture has trapped a set of interconvertible cell states reminiscent of the early stages in blastocyst differentiation that may exist only transiently in the early embryo.
Author Summary
Embryonic stem (ES) cells are karyotypically normal, embryo-derived cell lines that are pluripotent, i.e. capable of generating all the cell types of the future organism, but not the extra-embryonic lineages. What gives ES cells this unique capacity? Here, we use a fluorescent reporter cell line that employs translational amplification to visualize single ES cells expressing low levels of lineage-specific genes. With this reporter we split ES cell cultures into two fractions that both express certain stem cell markers but only one of which expresses low levels of an endodermal marker gene. Following purification, single cells from either fraction are equally competent to re-establish a heterogeneous culture. However, when challenged to differentiate immediately after purification, each exhibits strong lineage bias, with the endoderm marker-expressing fraction unexpectedly able to contribute to the extra-embryonic endoderm in chimeric embryos. These data suggest that ES cells expand under steady-state conditions as a heterogeneous mix of lineage-biased—but not lineage-committed—cell types. We propose that these observed uncommitted substates exist temporarily in vivo, but are perpetuated in vitro under the selectively self-renewing conditions of ES cell culture. Our findings suggest that pluripotency is determined by the capacity of a mixed population of lineage-biased intermediates to commit to different cell fates in specific contexts.
doi:10.1371/journal.pbio.1000379
PMCID: PMC2876051  PMID: 20520791
19.  The primate preimplantation embryo is a target for relaxin during early pregnancy 
Fertility and sterility  2011;96(1):203-207.
OBJECTIVE
To determine if preimplantation embryos are targets for relaxin secreted from the corpus luteum of the menstrual cycle.
DESIGN
Rhesus monkey oocytes obtained from females undergoing controlled ovarian hyperstimulation were inseminated and the resulting embryos were cultured in medium with or without recombinant human relaxin (20 ng/ml) for 8 days.
SETTING
Research laboratory.
ANIMALS
Rhesus monkey.
INTERVENTIONS
Controlled ovarian stimulation to obtain oocytes for in vitro produced embryos that were cultured with or without human recombinant relaxin.
MAIN OUTCOME MEASURES
The rate of blastocyst development and the percentage of blastocysts and ICM/TE ratio were measured on Day 8 of culture. The presence of relaxin receptor (RXFP1) mRNA in 8 cell embryos was observed by array hybridization.
RESULTS
RXFP1 receptor expression was localized to the inner cell mass of blastocysts as shown by immunohistochemistry. The percentage of embryos that developed to blastocyst and the inner cell mass/ trophectoderm cell ratio was unchanged with relaxin supplementation, however the relaxin-treated embryos developed into blastocysts significantly sooner than untreated embryos.
CONCLUSIONS
These results are the first evidence that the preimplantation primate embryo is a target for relaxin and that the addition of relaxin to in vitro culture medium enhances rhesus monkey embryo development.
doi:10.1016/j.fertnstert.2011.05.016
PMCID: PMC3129389  PMID: 21645893
gene expression; granulosa cells; blastocyst; rhesus macaque
20.  High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization 
Gene expression patterns : GEP  2005;6(2):213-224.
Mammalian preimplantation embryos provide an excellent opportunity to study temporal and spatial gene expression in whole mount in situ hybridization (WISH). However, large-scale studies are made difficult by the size of the embryos (∼60 μm diameter) and their fragility. We have developed a chamber system that allows parallel processing of embryos without the aid of a microscope. We first selected 91 candidate genes that were transcription factors highly expressed in blastocysts, and more highly expressed in embryonic (ES) than in trophoblast (TS) stem cells. We then used the WISH to identify 48 genes expressed predominantly in the ICM and to follow several of these genes in all seven preimplantation stages. The ICM-predominant expressions of these genes suggest their involvement in the pluripotency of embryonic cells. This system provides a useful tool to a systematic genome-scale analysis of preimplantation embryos.
doi:10.1016/j.modgep.2005.06.003
PMCID: PMC1850761  PMID: 16325481
preimplantation embryo; whole mount in situ hybridization; High-throughput screen; hybridization chamber; ICM; TE
21.  The Roles of Parathyroid Hormone-Like Hormone during Mouse Preimplantation Embryonic Development 
PLoS ONE  2012;7(7):e40528.
Parathyroid hormone-like hormone (PTHLH) was first identified as a parathyroid hormone (PTH)-like factor responsible for humoral hypercalcemia in malignancies in the 1980s. Previous studies demonstrated that PTHLH is expressed in multiple tissues and is an important regulator of cellular and organ growth, development, migration, differentiation, and survival. However, there is a lack of data on the expression and function of PTHLH during preimplantation embryonic development. In this study, we investigated the expression characteristics and functions of PTHLH during mouse preimplantation embryonic development. The results show that Pthlh is expressed in mouse oocytes and preimplantation embryos at all developmental stages, with the highest expression at the MII stage of the oocytes and the lowest expression at the blastocyst stage of the preimplantation embryos. The siRNA-mediated depletion of Pthlh at the MII stage oocytes or the 1-cell stage embryos significantly decreased the blastocyst formation rate, while this effect could be corrected by culturing the Pthlh depleted embryos in the medium containing PTHLH protein. Moreover, expression of the pluripotency-related genes Nanog and Pou5f1 was significantly reduced in Pthlh-depleted embryos at the morula stage. Additionally, histone acetylation patterns were altered by Pthlh depletion. These results suggest that PTHLH plays important roles during mouse preimplantation embryonic development.
doi:10.1371/journal.pone.0040528
PMCID: PMC3396650  PMID: 22808183
22.  Characterization of Trophoblast and Extraembryonic Endoderm Cell Lineages Derived from Rat Preimplantation Embryos 
PLoS ONE  2010;5(3):e9794.
Background
Previous attempts to isolate pluripotent cell lines from rat preimplantation embryo in mouse embryonic stem (ES) cell culture conditions (serum and LIF) were unsuccessful, however the resulting cells exhibited the expression of such traditional pluripotency markers as SSEA-1 and alkaline phosphatase. We addressed the question, which kind of cell lineages are produced from rat preimplantation embryo under “classical” mouse ES conditions.
Results
We characterized two cell lines (C5 and B10) which were obtained from rat blastocysts in medium with serum and LIF. In the B10 cell line we found the expression of genes known to be expressed in trophoblast, Cdx-2, cytokeratin-7, and Hand-1. Also, B10 cells invaded the trophectodermal layer upon injection into rat blastocysts. In contrast to mouse Trophoblast Stem (TS) cells proliferation of B10 cells occurred independently of FGF4. Cells of the C5 line expressed traditional markers of extraembryonic-endoderm (XEN) cells, in particular, GATA-4, but also the pluripotency markers SSEA-1 and Oct-4. C5 cell proliferation exhibited dependence on LIF, which is not known to be required by mouse XEN cells.
Conclusions
Our results confirm and extend previous findings about differences between blastocyst-derived cell lines of rat and mice. Our data show, that the B10 cell line represents a population of FGF4-independent rat TS-like cells. C5 cells show features that have recently become known as characteristic of rat XEN cells. Early passages of C5 and B10 cells contained both, TS and XEN cells. We speculate, that mechanisms maintaining self-renewal of cell lineages in rat preimplantation embryo and their in vitro counterparts, including ES, TS and XEN cells are different than in respective mouse lineages.
doi:10.1371/journal.pone.0009794
PMCID: PMC2848026  PMID: 20369002
23.  CDX2 in the Formation of the Trophectoderm Lineage in Primate Embryos 
Developmental biology  2009;335(1):179-187.
The first lineage decision during mammalian development is the establishment of the trophectoderm (TE) and the inner cell mass (ICM). The caudal-type homeodomain protein Cdx2 is implicated in the formation and maintenance of the TE in the mouse. However, the role of CDX2 during early embryonic development in primates is unknown. Here, we demonstrated that CDX2 mRNA levels were detectable in rhesus monkey oocytes, significantly upregulated in pronuclear stage zygotes, diminished in early cleaving embryos but restored again in compact morula and blastocyst stages. CDX2 protein was localized to the nucleus of TE cells but absent altogether in the ICM. Knockdown of CDX2 in monkey oocytes resulted in formation of early blastocyst-like embryos that failed to expand and ceased development. However, the ICM lineage of CDX2-deficient embryos supported the isolation of functional embryonic stem cells. These results provide evidence that CDX2 plays an essential role in functional TE formation during primate embryonic development.
doi:10.1016/j.ydbio.2009.08.025
PMCID: PMC2764001  PMID: 19733166
Embryo; CDX2; Trophectoderm; Primates; Embryonic stem cells
24.  Co-localization of NANOG and OCT4 in human pre-implantation embryos and in human embryonic stem cells 
Purpose
NANOG and OCT4 are required for the maintenance of pluripotency in embryonic stem cells (ESCs). These proteins are also expressed in the inner cell mass (ICM) of the mouse pre-implantation embryo.
Methods
Immunohistochemistry was used to show the presence of NANOG and OCT4 protein, and in situ hybridization was used to localize NANOG mRNA in human embryos from two-cell to blastocyst stage, and in human ESCs (hESCs).
Results
Nanog and Oct4 were co-localized in human embryos from morula and blastocyst stages. NANOG mRNA was detected in a group of cells in the morula, in cells of the ICM of blastocysts, and evenly in hESCs. All non-differentiated hESCs expressed NANOG and OCT4 protein. Pluripotent cells expressing NANOG and Oct4 were eccentrically localized, probably in polarized cells in a human compacted morula, which appears to be different from expression in murine embryos.
Conclusion
In this study, we demonstrate that whole mount in situ hybridization is amenable to localization of mRNAs in human development, as in other species.
doi:10.1007/s10815-012-9824-9
PMCID: PMC3492569  PMID: 22743827
Pluripotency; Embryo; Embryonic stem cells; NANOG; In situ hybridization
25.  The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways 
eLife  2014;3:e02224.
The neural fate commitment of pluripotent stem cells requires the repression of extrinsic inhibitory signals and the activation of intrinsic positive transcription factors. However, how these two events are integrated to ensure appropriate neural conversion remains unclear. In this study, we showed that Pou3f1 is essential for the neural differentiation of mouse embryonic stem cells (ESCs), specifically during the transition from epiblast stem cells (EpiSCs) to neural progenitor cells (NPCs). Chimeric analysis showed that Pou3f1 knockdown leads to a markedly decreased incorporation of ESCs in the neuroectoderm. By contrast, Pou3f1-overexpressing ESC derivatives preferentially contribute to the neuroectoderm. Genome-wide ChIP-seq and RNA-seq analyses indicated that Pou3f1 is an upstream activator of neural lineage genes, and also is a repressor of BMP and Wnt signaling. Our results established that Pou3f1 promotes the neural fate commitment of pluripotent stem cells through a dual role, activating internal neural induction programs and antagonizing extrinsic neural inhibitory signals.
DOI: http://dx.doi.org/10.7554/eLife.02224.001
eLife digest
After an egg has been fertilized, it undergoes a series of divisions to produce a ball of cells known as a blastocyst. The cells within the blastocyst are pluripotent stem cells, which have the potential to become many different types of cell. After a few days, the stem cells organize into three layers—an innermost layer called the endoderm, a middle layer of mesoderm, and an outer layer of ectoderm—that ultimately give rise to different types of tissues.
The brain and nervous system are formed from cells in the neuroectoderm, which is part of the ectoderm. Now, Zhu et al. have shown that a transcription factor called Pou3f1 triggers stem cells within a region of the ectoderm to turn into neural progenitor cells, thereby generating the neuroectoderm. These neural progenitor cells then go on to become neurons and glial cells that make up the brain and nervous system.
Using a virus to reduce levels of Pou3f1 in embryonic stem cells grown in a dish led to a drop in the number of stem cells that committed to neural progenitor cells. Overexpressing Pou3f1 in the stem cells restored the number of neural progenitor cells. Together these results showed that Pou3f1 is both necessary and sufficient for the conversion of embryonic stem cells into future neurons and glia.
The same result was seen when embryonic stem cells containing either reduced or elevated levels of Pou3f1 were injected into 2.5-day-old mouse blastocysts, which were then implanted into surrogate females. The resulting embryos comprised some cells with normal levels of Pou3f1, and others with either too little or too much. Cells with elevated Pou3f1 mostly became neural progenitors, whereas those with reduced levels rarely did so. Gene expression studies revealed that Pou3f1 promoted the formation of neural progenitor cells by activating the expression of pro-neuronal genes inside the stem cells, and by blocking anti-neuronal pathways called Wnt/BMP signaling cascades initiated outside the cells.
By revealing the two roles of Pou3f1, Zhu et al. have increased our understanding of one of the earliest stages of nervous system development. Further work is required to determine exactly how Pou3f1 exerts its effects and, in particular, whether it performs its two roles simultaneously or in sequence.
DOI: http://dx.doi.org/10.7554/eLife.02224.002
doi:10.7554/eLife.02224
PMCID: PMC4095939  PMID: 24929964
Pou3f1; neural fate commitment; pluripotent stem cell; intrinsic factor; extrinsic signal; BMP/Wnt pathways; chicken; mouse

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