Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 1. Genomic organization of the human ELF5 locus and transcript isoform expression in placenta and trophoblast cell lines. (A) Diagram of the exon–intron structure of the human ELF5 locus and annotated splice var- iants. Position of primers used is indicated. Filled boxes represent open- reading frames and open boxes represent untranslated regions. (B) RT–PCR analysis with isoform-specific and common primers reveals that ELF5-2b is the expressed splice variant in placenta and the trophoblast-like cell line TCL-1, but that it is absent from the first trimester mesenchymal-like cell line TCL-2. (C) RT–PCR with primers spanning exons 3 and 4 demonstrates that the annotated ELF5-2bDex3/4 variant is not present in placenta and chor- iocarcinoma and trophoblast-like cell lines JEG-3 and TCL-1.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Variant Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 2. Expression of trophoblast stem cell genes and epigenetic regulation of ELF5 in placenta throughout gestation. (A) RT–PCR analysis of ELF5, CDX2 and EOMES (i.e. genes important for trophoblast stem cell self-renewal and proliferation in the mouse) on human placental villous samples ranging from 7 weeks of gestation to term. Four independent term placental samples were investigated. The choriocarcinoma cell line JEG-3 was included as control. Colour-inverted photographs of ethidium bromide stained gels are shown. All three genes are expressed in placenta, but CDX2 is not detected from the second trimester onwards even when the PCRs are over-cycled. (B) Quantitative RT–PCR (qPCR) analysis of ELF5, CDX2 and EOMES on the same samples used in (A). ELF5 is down- regulated in second and third trimesters, whereas no overall regulation with gestational age was observed for EOMES. (C) Comparison of expression levels between first trimester and term. ELF5 expression is significantly reduced at term when compared with first trimester, CDX2 is absent from term placentas. (D) Bisulphite sequencing analysis of the ELF5 promoter region. Filled circles indicate methylated cytosine residues. ELF5 is extremely hypomethylated in the first trimester and acquires higher DNA methylation levels in second and third trimester, correlating with transcriptional down-regulation at these stages. (E) DNA methylation analysis of an extended region between 2400 bp and +400 bp around the transcriptional start site of ELF5. Hypomethylation correlates with ELF5 expression in JEG-3 cells and, conversely, ELF5 is hypermethylated and not expressed in TCL-2 cells. The methylation pattern in TCL-1 cells reveals a critical stretch of five CpG residues (grey box) at the immediate transcriptional start site that needs to be unmethylated for ELF5 to be expressed.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Staining, Quantitative RT-PCR, Comparison, Bisulfite Sequencing, Methylation, DNA Methylation Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 3. Immunofluorescence localization of ELF5 to cytotrophoblasts in the human placenta. (A) Overview of 11 week placental villous cross-section shows ELF5 localization to nuclei of villous cytotrophoblasts, but absence from nuclei of the overlying syncytiotrophoblast layer. Cytotrophoblasts are a proliferative cell population that continuously divide to replenish the overlying syncytium. (B) Co-localization with cytokeratin 7 (CK7) confirms the trophoblast identity of ELF5-positive cells. (C) Confocal image of a double staining of ELF5 and the villous cytotrophoblast marker SPINT1 (also known as HAI-1) shows that every ELF5-positive nucleus resides within the cytotrophoblast layer. Top row 6 week, bottom row 11 week placenta. (D) Confocal image analysis of an 11 week villous section stained for ELF5 and the extravillous cytotrophoblast (EVT) marker integrin alpha-5 (ITGA5). ELF5 is detected only in nuclei at the proliferative base, but not further distal along the EVT column where cells adopt an invasive phenotype and lose proliferative potential. (E) ELF5 is also absent from post- mitotic interstitial and endovascular EVTs within the decidual bed.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Double Staining, Marker, Staining

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 4. CDX2 identifies a subset of ELF5-positive cytotrophoblasts as a TS-like compartment that is regulated by FGFR2. (A) ELF5 co-localizes with FGFR2 in villous cytotrophoblasts as identified by confocal image analysis of double immunofluorescence stainings of 11 week placental sections. Since FGF signalling has been implicated in TS cell proliferation in mice and humans and can activate ELF5 expression in other tissues, FGF/FGFR2 may induce ELF5 expression within a putative TS cell niche in the human placenta. (B) Double staining of a 6 week placental section for ELF5 and CDX2. Larger groups of CDX2-positive cells are detected only in early gestation up to 8.5–9 weeks. CDX2 is mostly co-expressed with ELF5 (arrowheads). (C) Dual labelling of 6 week placental section for CDX2 and the proliferation marker Ki67. CDX2-expressing cytotrophoblasts preferentially stain positive for Ki67, indicating their high proliferation rate. CDX2 and Ki67 are restricted to the proximal end of cytotrophoblast cell columns (highlighted by the boxed area). The white arrows indicate the direction of progressive extravillous trophoblast (EVT) differentiation and migration.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Double Staining, Marker, Staining, Migration

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 5. Inter-regulatory network of trophoblast transcription factors CDX2, EOMES and ELF5. (A) Chromatin immunoprecipitation assays show that CDX2 binds to the ELF5 promoter region in JEG-3 and TCL-1 cells where ELF5 is hypomethylated and expressed, but not in TCL-2 cells where ELF5 is hypermethylated and repressed. (B) In turn, ELF5 binds to the CDX2 and EOMES promoter regions in JEG-3 and TCL-1 cells where it is expressed, but not in TCL-2 cells from which it is absent, thereby establishing a transcrip- tional feedback loop between all three transcription factors. Binding to the EOMES promoter region was more consistent and is indicative of a more effi- cient, stronger interaction than with the CDX2 upstream region, consistent with results observed in mouse trophoblast (9).

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Chromatin Immunoprecipitation, Binding Assay

Journal: Human molecular genetics

Article Title: ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta.

doi: 10.1093/hmg/ddq128

Figure Lengend Snippet: Figure 6. Trophoblast transcription factor expression and epigenetic regulation of ELF5 in human ES cells and derived trophoblast cell lines. (A) Initial bisul- phite sequencing analysis of two pooled hES cell lines and derived trophoblast cells indicates a high degree of DNA methylation at the ELF5 promoter despite the limited trophoblast differentiation potential. (B) RT–PCR and (C) qPCR analysis for trophoblast transcription factors ELF5, CDX2 and EOMES on six differ- ent hES cells lines (Shef1, Shef4–7, H7), including one subclone with an abnormal karyotype (Shef5a), two derived cytotrophoblast cell lines (TrophH7 and TrophShef4), the JEG-3, TCL-1 and TCL-2 cell lines, an 8+4 week placenta for relative comparison of expression levels and a colorectal cancer cell line (DKO4) as positive control for CDX2 expression (27). Colour-inverted photographs of ethidium bromide stained gels are shown. ELF5 is detectable in some hES cell lines, albeit at very low levels. Higher expression levels of CDX2 and EOMES may relate to their function within the embryonic lineage and is not directly indicative of trophoblast differentiation potential. Strikingly, in contrast to their expression in placenta, all three genes are absent from the hES-derived tropho- blast cell lines. (D) Normalization of qPCR data to Shef6, one of the most highly ELF5 expressing hES cell lines, in comparison with JEG-3, TCL-1 and TCL-2 cell lines as well as a first trimester placenta sample demonstrates the comparatively negligible amount of ELF5 expression in hES cells that is approximately 300-fold less than in normal trophoblast in vivo. (E) Bisulphite sequencing analysis of the ELF5 promoter in three different hES cell lines and two derived trophoblast cell lines shows relatively little epigenetic variability between different hES cell lines. Hypermethylation correlates with extremely low ELF5 expression levels. (F) Elf5 is also highly methylated in three independent mouse epiblast stem cell lines and (G) in two human-induced pluripotent stem cell lines derived from kereatinocytes and fibroblasts.

Article Snippet: For each immunoprecipitation reaction, 50 mg of chromatin was pre-cleared and incubated overnight at 48C with 5 mg of anti-ELF5 antibody (Santa Cruz Biotechnology), anti-CDX2 antibody (BioGenex) or control antibody bound to Protein G sepharose beads (Amersham).

Techniques: Expressing, Derivative Assay, Sequencing, DNA Methylation Assay, Reverse Transcription Polymerase Chain Reaction, Comparison, Positive Control, Staining, In Vivo, Bisulfite Sequencing, Methylation

Journal: Developmental Cell

Article Title: Maternal DNA Methylation Regulates Early Trophoblast Development

doi: 10.1016/j.devcel.2015.12.027

Figure Lengend Snippet: Scml2 Is Controlled by DNA Methylation and Affects SynT Formation and Cell Adhesion (A) RT-qPCR of Dnmt3a mKO EPCs confirms that Scml2 is controlled by oocyte methylation. (B) Methylation analysis by Sequenom MassARRAY in E7.5 male EPCs, confirming the DMR at an intragenic TSS of Scml2 . Each data point may include more than one CpG from the amplicon, as indicated on the x axis. (C) RT-qPCR analysis of TSCs grown in FGF+ (TSC conditions) or FGF− (differentiation conditions) medium for 6 days, with or without Scml2 overexpression. (D) Expression of Syna is reduced in Dnmt3a mKO EPCs, whereas markers of SynT-II Synb and Cebpa are unaffected. (E) E-cadherin staining of two independent Scml2 knockout clones from TKO TSCs shows a rescue of the morphological alterations seen in TKO TSCs. (F) Scml2 KO on TKO TSCs also rescues the defect in cell adhesion to cell culture wells in the absence of laminin. (G) RT-qPCR analysis of TKO Scml2 KO clones shows maintained expression of the TSC marker Cdx2 ; the expression of genes involved in cell adhesion is not rescued upon Scml2 deletion. Error bars represent SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001; t test comparing WT and Dnmt3a mKO EPCs (A and D) or Scml2 -expressing TSCs versus vector control (C), or ANOVA with post hoc tests comparing Ctrl with DHet/DKO (B) or TKO TSC lines with WT TSCs. See also Figure S6 .

Article Snippet: Paraffin sections were deparaffinized with Histo-Clear and dehydrated through an ethanol series, followed either by standard H&E staining or antigen retrieval by boiling slides for 30 min (in 1 mM EDTA, 0.05% Tween 20, pH 8) and cooling at room temperature for 20 min. After blocking with 1% BSA overnight, sections were incubated with a rabbit monoclonal anti-CDX2 antibody (EPR2764Y, Novus Biologicals, 1:250 dilution) for 2 hr.

Techniques: DNA Methylation Assay, Quantitative RT-PCR, Methylation, Amplification, Over Expression, Expressing, Staining, Knock-Out, Clone Assay, Cell Culture, Marker, Plasmid Preparation, Control

Journal: Tissue Engineering Part C: Methods

Article Title: Cell Adhesion Minimization by a Novel Mesh Culture Method Mechanically Directs Trophoblast Differentiation and Self-Assembly Organization of Human Pluripotent Stem Cells

doi: 10.1089/ten.tec.2015.0038

Figure Lengend Snippet: FIG. 4. Results of immunofluorescence microscopy performed to characterize cysts formed as a result of hiPSC differentiation on mesh. (A) Bright field (first image of row) and immunofluorescence images of a cyst stained for hCG-b subunit of hCG hormone (third image). Hoechst-labeled nuclei is shown in the second image, and merged image in the last image of the row. (B) Immunofluorescence images of a cyst showing the expression of CDX2 (third image). (C) Comparative immunofluorescence images of an EB stained for CDX2. Scale bar represent 100 mm. EB, embryonic body; hCG, human chorionic gonadotropin.

Article Snippet: Primary antibodies used in this study were mouse anti human OCT4 (Santa Cruz) at 1:50 dilution, goat anti human CDX2 (R&D Systems) at 1:200 dilution, rabbit anti human NANOG (Cell Signaling Technology) at 1:100 dilution, polyclonal rabbit anti human CG (Abcam) at 1:200 dilution, monoclonal Anti-vinculin antibody produced in mouse (clone hVIN-1; Sigma Aldrich), and mouse monoclonal anti-human E-cadherin (Takara Bio) at a dilution of 1:200 dilution.

Techniques: Microscopy, Staining, Labeling, Expressing