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.
Project description:ROS are frequently associated with deleterious effects caused by oxidative stress. Despite the harmful effects of non-specific oxidation, ROS also function as signal transduction molecules that regulate various biological processes, including stem cell proliferation and differentiation. Here we show that elevated ROS level by myxothiazol exposure makes murine P19 pluripotent stem cells more potent to differentiate in trophoblast cells, but not in neurons. Overall design: For gene expression analysis samples were extracted from P19 cells grown under normal conditions (4 independent samples) or treated with 5 nM myxothiazol for 12 h (3 independent samples).
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
Project description:Inhibitors of MEK1/2 and Gsk3b, known as '2i' culture conditions, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency in rodents 1,2. Here we show that the derivation of female mouse ESCs in the presence of 2i (2i-ESCs) results in a widespread loss of DNA methylation including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, the early-passage 2i-ESCs efficiently differentiate into somatic cells and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in the 2i-ESC-derived differentiated cells. Consistently, 2i-ESCs exhibit impairment of autonomous embryonic and placental development by tetraploid embryo complementation and nuclear transplantation. We identified the derivation conditions of female ESCs that display 2i-ESC-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ESCs exhibit ICR demethylation, regardless of culture conditions. Our results provide insights into derivation of female ESCs reminiscent of ICM of preimplantation embryos. S-ESCs are ESCs established under serum-containing medium. 2i_S_ESCs are ESCs established in 2i-containing medium, followed by maintenance in serum-containing medium. Overall design: Analyzing DNA methylation profiles and transcriptional profiles in 2i-ESCs, 2i-S-ESCs, 2i-MEFs, S-ESCs, t2i-ESCs, a2i-ESCs and MEFs for molecular analysis and 2n/4n injection and nuclear transfer for developmental assay for ESCs.
Project description:Cardiomyocytes can be differentiated from human pluripotent stem cells (hPSCs) in defined conditions, but efficient and consistent cardiomyocyte differentiation often requires expensive reagents such as B27 supplement or recombinant albumin. Using a chemically defined albumin-free (E8 basal) medium, we identified heparin as a novel factor that significantly promotes cardiomyocyte differentiation efficiency, and developed an efficient method to differentiate hPSCs into cardiomyocytes. The treatment of heparin helped cardiomyocyte differentiation consistently reach at least 80% purity (up to 95%) from more than 10 different hPSC lines in chemically defined DMEM/F-12 based medium on either Matrigel or defined matrices like Vitronectin and Synthemax. One of heparin’s main functions was to act as a WNT modulator that helped promote robust and consistent cardiomyocyte production. Our study provides an efficient, reliable, and cost-effective method for cardiomyocyte derivation from hPSCs that can be used for potential large-scale drug screening, disease modeling, and future cellular therapies. 12 human pluripotent stem cells (hPSCs) at three different cardiac differentiation times (0 Days, 3 Days, 6 Days, 10 Days) under different culture conditions (+/- Heparin, +/- IWP2).
Project description:Human dental pulp cells (hDPCs) are one of the promising resources for regenerative medicine and tissue engineering, including derivation of induced pluripotent stem cells (iPSCs). However, our current protocol uses reagents of animal origin, mainly fetal bovine serum (FBS) with potential risk of infectious diseases and unwanted immunogenicity. This time, we designed a chemically defined protocol to isolate and maintain the growth and differentiation potentials of hDPCs. Overall design: To investigate the effect of chemically defined condition on global gene expression in DPCs, we cultured them in FBS and chemically defined conditions.
Project description:We characterized the trophoblast stem cell epigenome and gene expression profiles in rat and mouse. We profiled 5 histone modifications (+ chromatin input) using ChIP-Seq, and digital expression profiles (3' RNA-Seq) for trophoblast stem cells derived from rat and mouse. Furthermore, for mouse, we profiled key trophoblast stem cell factors Elf5, Cdx2, and Eomes. We found that enhancer regions (defined as distal regions of H3K27ac/H3K4me1 enrichment) were enriched for species-specific endogenous retroviral elements.