Establishment of trophoblast stem cells under defined culture conditions in mice
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE59107 | GEO | 2014/07/08
SECONDARY ACCESSION(S): PRJNA254454
REPOSITORIES: GEO
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Project description:The inner cell mass (ICM) and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE) exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES) cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS) cells have not been established. Here, we report that addition of FGF2, activin A, XAV939, and Y27632 are necessary and sufficient for derivation of TS cells from both of E3.5 blastocysts and E6.5 early postimplantation extraembryonic ectoderm. Moreover, the undifferentiated TS cell state can be stably maintained in chemically defined culture conditions. Cells derived in this manner expressed TS cell marker genes, including Eomes, Elf5, Cdx2, Klf5, Cdh1, Esrrb, Sox2, and Tcfap2c; differentiated into all trophoblast subtypes (trophoblast giant cells, spongiotrophoblast, and labyrinthine trophoblast) in vitro; and exclusively contributed to trophoblast lineages in chimeric animals. This delineation of minimal requirements for derivation and self-renewal provides a defined platform for precise description and dissection of the molecular state of TS cells. Total RNA was purified by using RNeasy Mini kit (QIAGEN). Microarray targets from 200 ng total RNA were synthesized and labelled using the Low RNA Input Linear Amp Kit (Agilent) and hybridized to Mouse 4x44K Ver.2.0 arrays. Arrays were scanned on an Agilent Technologies Microarray scanner and signal intensities were calculated in Agilent Feature Extraction 10.7.3.1 software.
2014-07-08 | E-GEOD-59107 | biostudies-arrayexpress
Project description:During mammalian embryonic development, the first lineage commitment event gives rise to two distinct cell populations: the trophectoderm (TE) and the inner cell mass (ICM). The TE consists of outer cells of the blastocyst and ultimately forms the placenta while the ICM gives rise to all the embryonic tissues. Numerous transcription factors (TFs) guiding ICM differentiation into different embryonic tissues have been characterized. However, only a few TFs that are required for TE specification and differentiation have been identified, and much less is understood as to how these TFs interact with other TFs or with their chromosomal targets in order to drive cell fate towards TE lineage. Understanding trophectoderm development is crucial because cells in this lineage are required for proper embryo implantation in the uterus. Defects in this lineage can cause early failure of pregnancy as well as other pregnancy related disorders such as preeclampsia and intrauterine growth restriction (IUGR). Here, we characterize the function of one of TE-specific TF, Fosl1, which was previously suggested as having some role in placental development. We utilized mouse embryonic stem (ES) cells (derived from ICM) and showed that ectopic expression of Fosl1 can transdifferentiate ES cells to differentiated TS cells (trophoblast giant-like cells). We show that Fosl1 does so by directly binding and activating TE specific genes and genes associated with epithelial-mesenchymal transition (EMT). Using mouse trophoblast stem (TS) cells, we also establish that Fosl1 is required for specification of TS cells to trophoblast giant cells (TGCs) subtype. Therefore, we postulate that Fosl1 is a key regulator of TS cell differentiation.
2018-04-25 | GSE99984 | GEO
Project description:During mammalian embryonic development, the first lineage commitment event gives rise to two distinct cell populations: the trophectoderm (TE) and the inner cell mass (ICM). The TE consists of outer cells of the blastocyst and ultimately forms the placenta while the ICM gives rise to all the embryonic tissues. Numerous transcription factors (TFs) guiding ICM differentiation into different embryonic tissues have been characterized. However, only a few TFs that are required for TE specification and differentiation have been identified, and much less is understood as to how these TFs interact with other TFs or with their chromosomal targets in order to drive cell fate towards TE lineage. Understanding trophectoderm development is crucial because cells in this lineage are required for proper embryo implantation in the uterus. Defects in this lineage can cause early failure of pregnancy as well as other pregnancy related disorders such as preeclampsia and intrauterine growth restriction (IUGR). Here, we characterize the function of one of TE-specific TF, Fosl1, which was previously suggested as having some role in placental development. We utilized mouse embryonic stem (ES) cells (derived from ICM) and showed that ectopic expression of Fosl1 can transdifferentiate ES cells to differentiated TS cells (trophoblast giant-like cells). We show that Fosl1 does so by directly binding and activating TE specific genes and genes associated with epithelial-mesenchymal transition (EMT). Using mouse trophoblast stem (TS) cells, we also establish that Fosl1 is required for specification of TS cells to trophoblast giant cells (TGCs) subtype. Therefore, we postulate that Fosl1 is a key regulator of TS cell differentiation.
2018-04-25 | GSE99983 | GEO
Project description:We attempted to identify candidate genes that are expressed more highly in the ICM than in TE cells. Mouse ES cells are cultured from the ICM, whereas mouse TS cells are cultured from the TE. Although these cells have been cultured in vitro, they represent the in vitro equivalents of the ICM and TE. Therefore, genes which are expressed more highly in ES than TS in microarray studies were good candidates for genes predominantly expressed in the ICM in blastocysts. We attempted to identify candidate genes that are expressed more highly in the ICM than in TE cells. Mouse ES cells are cultured from the ICM, whereas mouse TS cells are cultured from the TE. Although these cells have been cultured in vitro, they represent the in vitro equivalents of the ICM and TE. Therefore, genes which are expressed more highly in ES than TS in microarray studies were good candidates for genes predominantly expressed in the ICM in blastocysts.
2010-06-09 | E-GEOD-3214 | biostudies-arrayexpress
Project description:Trophoblast stem (TS) cells are in vitro equivalents to the precursor cells of the placenta. In mice, TS cells can be derived either from polar trophectoderm (TE) of blastocyst outgrowths or from postimplantation extraembryonic ectoderm (ExE), originating from polar TE. Since their first successful derivation in 1998 TS cells have been cultured in serum-rich medium in the presence of fibroblast growth factor 4 (FGF4), the cofactor heparin and embryonic fibroblast conditioned medium. Here, we developed and tested a simple medium consisting of ten chemically defined ingredients for routine culture of TS cells. The defined medium (TX medium) supports growth and self-renewal of TS cells grown on Matrigel-coated dishes over multiple (>20) passages. TS cells cultured under these conditions retain expression of key trophoblast stem cell-associated markers. Global gene expression profiling of cells cultured in standard and TX media conditions revealed that 99.6 % of genes are similarly expressed. DNA methylation profiling confirmed the faithful propagation of epigenomic characteristics of TS cells cultured in TX compared to standard media. Importantly, TX media also supported the derivation of new TS cell lines directly from post implantation embryos. In addition, TS cells cultured in TX retain their ability to differentiate into all derivatives of the trophectodermal lineage in vitro and give rise to haemorrhagic lesions in nude mice, indistinguishable from cells grown in standard conditions. Further, injection into blastocysts proofs chimerization potential after TX culture. The fact that TX media formulation no longer requires fetal bovine serum and conditioned medium facilitates and standardizes the culture of this extraembryonic lineage in vitro. Cell culture. TS cell lines TS-LacZ and TS-L5 have been derived from blastocysts and post implantation embryos us according to standard protocols. Cells were grown on tissue culture plastic (TPP) at 37°C in humidified incubators containing 5% CO2 (Heraeus). For conventional stem cell culture cells were cultured in serum containing medium supplemented with 70% CM, 25 ng/ml human recombinant FGF4 (Reliatech) and 1 µg/ml Heparin (Sigma-Aldrich), hereafter named TS medium. For differentiation experiments TS medium without CM, FGF4 and heparin (TS -CMF4H) was used. For stem cell culture in defined medium cells were cultured on Matrigel-coated dishes in TX medium. TX medium formulation: DMEM/F12 (Life Technologies), 64 mg/l l-ascorbic acid-2-phosphate magnesium (Sigma), 14 microg/l sodium selenite (Sigma-Aldrich), 25 ng/ml human recombinant FGF4 (Reliatech), 19.4 mg/l insulin (Sigma-Aldrich), 543 mg/l NaHCO3 (Sigma-Aldrich), 10.7 mg/l holo-transferrin (Sigma-Aldrich), 2 ng/ml human recombinant TGF-beta1 (Peprotech), 1 microg/ml Heparin (Sigma-Aldrich), 2 mM L-glutamine (PAN-biotech), 1% penicillin and streptomycin (PAN-biotech). Medium was prepared without growth factors, stored at 4°C and used within two weeks. FGF4, heparin and TGFß were added to the medium prior to use. Medium was changed every other day. Cells were passaged when sub confluent, by incubation in trypsin-EDTA (PAN-biotech) for 4 min at 37°C. The enzyme was inactivated by addition of trypsin inhibitor (Life Technologies) in defined culture and by serum containing medium in case of conventional culture. Cells were dissociated and plated in fresh medium. Passage numbers are represented as x + y, where x represents the passage number at which cells were transferred to TX medium, and y being the passage number in TX. For cryopreservation, cells were harvested and pellets were re-suspended either in FBS/10%DMSO (standard conditions) or in KnockOut Serum Replacement/10%DMSO (Life Technologies). Plate coating. Tissue culture dishes for culture of TS cells in TX medium were coated with growth factor reduced Matrigel (BD Biosciences), which was diluted 1:30 in ice cold DMEM/F12 and incubated over night at 4°C. Coating solution was aspirated immediately before plating cells. For initial coating tests, plates were incubated either with Matrigel, poly-L-lysine, gelatine or fibronectin, respectively. Poly-L-lysine was used at 0.01 % (w/v) in PBS (PAN-biotech) for 30 min at 37°C and washed once with PBS prior to use. Gelatine coating was performed with 0.1% (w/v) gelatine solution in PBS for 30 min at 37°C. Dishes were incubated with fibronectin solution (16.7 µg/ml) for 30 min in the incubator, washed once with PBS and used thereafter. Isolation of TS cells from post-implantation (E6.5) embryos Embryos were dissected in PBS on day E6.5 and transferred in PBS/10% (v/v) FBS and kept on ice. Before dissociation, embryos were washed in PBS twice. Whole conceptuses were transferred into an U-bottom 96-well plate and incubated in 40 µl trypsin-EDTA (PAN-biotech) for 15 min in the incubator. The content of the well was pipetted up and down several times and transferred to a 24 well plate on pre-plated irradiated MEFS (gammaMEFs) in either TS medium with 1.5x FGF4 and heparin concentration (TS+1.5xF4H) or TX medium with 1.5x FGF4 and heparin (TX+1.5xF4H). TS cell colonies appeared after 2-4 days. Immunofluorescent staining, western blotting. Before staining, cells were washed with PBS once and fixed for 10 min with 4% paraformaldehyde (Sigma-Aldrich). Permeabilization of the cells was performed with 0.5 % (v/v) Triton X-100 (Sigma-Aldrich) in PBS for 10 min and subsequent incubation for 30 min in blocking buffer (2% bovine serum albumin [Sigma-Aldrich], 0.1% (v/v) Triton X-100 in PBS). Samples were incubated overnight at 4°C with the following primary antibodies: Cdx2 (1:200, Cdx2-88, BioGenex Inc), Eomes (1:500,ab23345, Abcam), Tfap2c (1:300, sc-8977, Santa Cruz). After three washing steps, detection was performed with appropriate Alexa Fluor-conjugated antibodies (Invitrogen) at a 1:500 dilution in blocking buffer for 1 h at room temperature in the dark. After secondary antibody incubation, nuclei were stained with Hoechst (Sigma-Aldrich). Cells stained without primary antibodies served as controls. Western blotting was performed with 23 µg of cell lysates from TS cells cultured in TS and TX media by SDS-polycrylamide gel electrophoresis and blotted onto PVDF membranes. Membranes were blocked with 5% (v/v) BSA or 5% (v/v) non-fat milk powder in TBST (TBS with 0.1% Tween). Membranes were incubated with primary antibodies at room temperature for 2 h, washed three times with TBST and labeled with HRP conjugated antibodies (Dako) for 1 h at room temperature. After three washing steps with TBST, membranes were incubated in ECL substrate (Thermo Scientific). RNA isolation and analysis. RNA was isolated from cells using the RNeasy Minikit (Qiagen) according to the manufacturer's protocol. 500 ng total RNA was used for cDNA synthesis by RevertAidPremium (Thermo Scientific). For gene expression microarray analysis, RNA quality was tested using the Agilent 2100 Bioanalyzer (Agilent Technologies). 100 ng total RNA were used for cDNA synthesis. Biotin labeling and fragmentation were performed according to the Affymetrix user manual. cRNA was hybridized for 16h to Mouse Genome 430 2.0 GeneChip expression arrays (Affymetrix). After hybridization, arrays were washed and stained automatically on a GeneChip Fluidics station 450 (Affymetrix) and scanned with a GeneChip scanner 3000 7G (Affymetrix). 6 samples were analyzed. TSGFPTS: Mouse trophoblast stem cell EGFP cell line cultered in standard trophoblast stem cell medium, 1 biological rep TSGFPTX: Mouse trophoblast stem cell EGFP cell line cultered in defined TX medium, 1 biological rep TSL5TS: Mouse trophoblast stem cell L5 cell line cultered in standard trophoblast stem cell medium, 1 biological rep TSL5TX: Mouse trophoblast stem cell L5 cell line cultered in defined TX medium, 1 biological rep TSLacZTS: Mouse trophoblast stem cell LacZ cell line cultered in standard trophoblast stem cell medium, 1 biological rep TSLacZTX: Mouse trophoblast stem cell LacZ cell line cultered in defined TX medium, 1 biological rep
2014-01-28 | E-GEOD-47719 | biostudies-arrayexpress
Project description:We attempted to identify candidate genes that are expressed more highly in the ICM than in TE cells. Mouse ES cells are cultured from the ICM, whereas mouse TS cells are cultured from the TE. Although these cells have been cultured in vitro, they represent the in vitro equivalents of the ICM and TE. Therefore, genes which are expressed more highly in ES than TS in microarray studies were good candidates for genes predominantly expressed in the ICM in blastocysts.
2005-01-01 | E-MNIA-73 | biostudies-arrayexpress
Project description:The histone H3 lysine 9 (H3K9) methyltransferase Eset is an epigenetic regulator critical for the development of the inner cell mass (ICM). Although ICM-derived embryonic stem (ES) cells are normally unable to contribute to the trophectoderm (TE) in blastocysts, we find that depletion of Eset by shRNAs leads to differentiation with the formation of trophoblast-like cells and induction of trophoblast-associated gene expression. Using ChIP-seq analyses, we identified Eset target genes with Eset-dependent H3K9 trimethylation. We confirmed that genes that are preferentially expressed in the TE (Tcfap2a and Cdx2) are bound and repressed by Eset. Single cell PCR analysis shows that the expression of Cdx2 and Tcfap2a is also induced in Eset-depleted morula cells. Importantly, Eset-depleted cells can incorporate into the TE of a blastocyst and subsequently placental tissues. Co-immunoprecipitation and ChIP assays further demonstrates that Eset interacts with Oct4, which in turn recruits Eset to silence these trophoblast-associated genes. Our result suggests that Eset restricts the extraembryonic trophoblast lineage potential of pluripotent cells and links an epigenetic regulator to key cell fate decision through a pluripotency factor. Keywords: Epigenetics Examine Eset-binding sites and compare the H3K9me3 state between Eset knockdown mouse ES cells and control knockdown mouse ES cells.
2009-11-04 | E-GEOD-17642 | biostudies-arrayexpress
Project description:We attempted to identify candidate genes that are expressed more highly in the ICM than in TE cells. Mouse ES cells are cultured from the ICM, whereas mouse TS cells are cultured from the TE. Although these cells have been cultured in vitro, they represent the in vitro equivalents of the ICM and TE. Therefore, genes which are expressed more highly in ES than TS in microarray studies were good candidates for genes predominantly expressed in the ICM in blastocysts. Keywords: strain or line design
2005-08-30 | GSE3214 | GEO
Project description:Expression profiling of wild-type and Prdm1 null mouse trophoblast giant cell cultures using Illumina whole genome mouse V2 arrays. The hypothesis tested was that Prdm1/Blimp1 regulates expression of genes required for spiral artery trophoblast giant cell function. Prdm1 null and littermate control wild-type trophoblast stem cell clones were generated from blastocyst outgrowths. Total RNA was obtained from multiple replicates of four wild-type TS cell clones and four Prdm1 null TS cell clones differenitated for zero, two, four and six days by growth factor withdrawal and hybridized to Illumina WG6_V2 arrays
2016-03-14 | E-GEOD-74409 | biostudies-arrayexpress
Project description:To identify splicing regulators with unique regulation in placenta and the trophectoderm lineage, we sequenced trophoblast stem (TS) and Extraembryonic endoderm stem (XEN) cells.
2022-07-01 | GSE181334 | GEO