Effect of Tet1-knockdown on gene expression in mouse ES cells cultured in ES and TS cell culture conditions
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Kian Peng Koh
PROVIDER: E-GEOD-26900 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells.
Koh Kian Peng KP Yabuuchi Akiko A Rao Sridhar S Huang Yun Y Cunniff Kerrianne K Nardone Julie J Laiho Asta A Tahiliani Mamta M Sommer Cesar A CA Mostoslavsky Gustavo G Lahesmaa Riitta R Orkin Stuart H SH Rodig Scott J SJ Daley George Q GQ Rao Anjana A
Cell stem cell 20110201 2
TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Here, we show that Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem cells (ESCs) and are induced concomitantly with 5hmC during reprogramming of fibroblasts to induced pluripotent stem cells. ESCs depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1 and display hyperactive Nodal signaling and skewed differentiation into the endoderm- ...[more]
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Project description:TET-family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Tet1 and Tet2 are Oct4-regulated enzymes that together sustain 5hmC in mouse embryonic stem (ES) cells. ES cells depleted of Tet1 by RNAi show diminished expression of the Nodal antagonist Lefty1, and display hyperactive Nodal signalling and skewed differentiation into the endoderm-mesoderm lineage in embryoid bodies in vitro. In Fgf4- and heparin-supplemented culture conditions that favor derivation of trophoblast stem (TS) cells, Tet1-depleted ES cells activate the trophoblast stem cell lineage determinant Elf5 and can colonize the placenta in mid-gestation embryo chimeras. Consistent with these findings, Tet1-depleted ES cells form aggressive hemorrhagic teratomas with increased endoderm, reduced neuroectoderm and ectopic appearance of trophoblastic giant cells. Thus Tet1 functions to regulate the lineage differentiation potential of ES cells. Here, we performed whole-genome transcriptome profiling of ES cells stably depleted of Tet1 by shRNA knockdown (Tet1-kd) cultured in either standard ES cell or in TS cell culture conditions. Gene expression changes in Tet1-kd ES cells were fairly modest compared to control (GFP-kd) cells, although gene ontology (GO) analysis of differentially expressed genes yielded many terms related to embryonic development and cell cycle regulation. In TS cell culture conditions, a core set of genes defining trophectodermal cell differentiation, including Cdx2, Eomes and Tead4, was enriched in Tet1-kd compared to GFP-kd cells.
2011-02-07 | GSE26900 | GEO
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: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).
2014-01-28 | GSE47719 | GEO
Project description:To understand the mechanism underlying the versatility in transcriptional regulation by Sox2, we compared genome-wide binding sites of Sox2 in embryonic stem (ES) cells and trophoblast stem (TS) cells by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). A tetracycline-inducible Oct3/4 knockout ES cell line ZHBTc4 was treated with Tet for 4 days in the presence of FGF4 and mouse embryonic fibroblasts (MEFs).
2013-10-17 | E-GEOD-28453 | biostudies-arrayexpress
Project description:Effect of Tet1-knockdown on gene expression in mouse ES cells cultured in ES and TS cell culture conditions
| PRJNA136101 | ENA
Project description:Trophoblast stem cells (TS cells), derived from the trophectoderm (TE) of blastocysts, require transcription factors (TFs) and external signals (Fgf4, Activin/Nodal/Tgfb) for self-renewal. While many reports have focused on TF networks that regulate embryonic stem cell (ES cell) self-renewal and pluripotency, little is know about TF networks that regulate self-renewal in TS cells. To further understand transcriptional networks in TS cells we used chromatin immunopreciptiation and DNA microarray analysis (ChIP-chip) to investigate targets of TFs Ap-2g (Tcfap2c), Eomes, Ets2, and Gata3, and a chromatin remodeling factor, Brg1 (Smarca4). We then evaluated the transcriptional states of target genes using transcriptome analysis and genome-wide analysis of histone H3 acetylation (AcH3). Our results describe previously unknown transcriptional networks in TS cells, including TF occupancy of genes involved in ES cell self-renewal and pluripotency, co-occupancy of multiple TFs at target genes, and transcriptional regulatory circuitry within the 5 factors. Through genome-wide mapping and global expression analysis of 5 TF target genes, our data provide a comprehensive analysis of transcriptional networks that regulate TS cell self-renewal.
2010-02-09 | GSE18507 | GEO
Project description:Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. Recent demonstration that members of the Ten-eleven translocation (Tet) family proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell self-renewal and maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), here we show that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite a general increase in levels of DNA methylation at Tet1 binding-sites, Tet1 depletion does not lead to down-regulation of all the Tet1 targets. Interestingly, while Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also required for repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators. Mouse ES cells infected with control knockdown (KD) or Tet1 KD lentiviruses were FACS-sorted for RNA extraction and hybridization on Affymetrix microarrays. We also investigated the effect of Nanog overexpression (OE) in Tet1 KD mouse ES cells on dys-regulated Tet1 targets. We have collected four biologically independent replicates for each treatment.
2011-03-30 | E-GEOD-26830 | biostudies-arrayexpress
Project description:To characterize the transdifferentiation of embryonic stem (ES) cells into trophoblast stem (TS) cells triggered by forced repression of Oct3/4, we performed whole-genome expression analysis after tetracycline (Tet)-induced knockout of Oct3/4. A Tet-inducible Oct3/4 knockout ES cell line ZHBTc4 was treated with Tet for 4 days in the presence of FGF4 and mouse embryonic fibroblasts (MEFs). A total of 15 samples from Day 0 to Day 4 were analyzed in three biological replicates.
2013-10-17 | E-GEOD-28452 | biostudies-arrayexpress
Project description:Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. Recent demonstration that members of the Ten-eleven translocation (Tet) family proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell self-renewal and maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), here we show that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite a general increase in levels of DNA methylation at Tet1 binding-sites, Tet1 depletion does not lead to down-regulation of all the Tet1 targets. Interestingly, while Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also required for repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators. Chromatin or genomic DNA extracted from control (Con) or Tet1 knockdown (KD) mouse ES cells was immunoprecipitated with indicated antibodies and analyzed by NimbleGen 2.1M mouse whole genome tiling microarrays (a 4-array set covering the entired non-repetitive portion of mouse genome). Whole cell extract (WCE) was used as input controls in IP/input experiments. In IP/IP experiments, immunoprecipitated DNA from Con KD and Tet1 KD ES cells was directly compared on the same microarrays.
2011-03-30 | E-GEOD-26827 | biostudies-arrayexpress
Project description:A blastocyst consists of three distinctive cell types: epiblast (EPI), trophoblast (TB), and primitive endoderm (PrE). Stem cell lines representing EPI and TB (embryonic stem (ES) cells and trophoblast stem (TS) cells) have been derived and they contribute to epiblast derivatives and trophoblast derivatives of stem cell-blastocyst chimeras, respectively. Although derived from PrE, extraembryonic endoderm (XEN) cells contribute to only a limited part of parietal endoderm (PE) but rarely to other PrE derivatives. Here we describe the establishment of primitive endoderm stem (PrES) cell lines in mice. PrES cells were derived and maintained in a serum-free media containing CHIR99021, FGF4, heparin, and PDGF-AA. RNA-seq analysis revealed that the transcriptome of PrES cells is globaly different from XEN cells: PrES cells express not only endoderm markers (Dab2, Gata4, Gata6, Sox17, etc.), but also pluripotent markers (Pou5f1, Cdh1, Nanog, Zfp42, etc.), and resembles in vivo founder PrE. PrES cells were rapidly and efficiently incorporated into PrE after blastocyst injection and efficiently contributed to all PrE derivatives, including PE, VE (visceral endoderm), and MZE (marginal zone endoderm) in chimeras. Importantly, PrES cells rescued embryonic lethality of PrE-depleted blastocysts by complementing all PrE derivatives. PrES cells thus not only contribute to understanding of the mechanisms of PrE specification but also provide a critical resource for artificial embryo reconstitution by stem cells alone.
2020-05-30 | GSE130957 | GEO