title: Derivation and maintenance of murine trophoblast stem cells under defined conditions

status: Public on Jan 28 2014

summary: 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).

overall_design:
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

first: Caroline

last: Kubaczka

last: Senner

middle: J

last: Arauzo-Bravo

first: Neha

last: Sharma

first: Peter

last: Kuckenberg

first: Astrid

last: Becker

first: Andreas

last: Zimmer

first: Oliver

last: Brüstle

first: Michael

last: Peitz

first: Myriam

last: Hemberger

first: Hubert

last: Schorle


title: miRNA profiling of serum, liver and white adipose tissues.

status: Public on Oct 02 2014

city: Okayama

first: Jun

last: Wada

address: 2-5-1 Shikata-cho, Kita-ku

department: Department of Nephrology, Rheumatology, Endocrinology and Metabolism

country: Japan

institute: Okayama University

type: Non-coding RNA profiling by high throughput sequencing

summary: Male C57BL/6J mice were housed in cages and maintained on a 12-hour light-dark cycle. STD (NMF; Oriental Yeast) and HFHS chow (D12331; Research Diet, New Brunswick, NJ) were used, the mice were sacrificed at 20 weeks of age.

overall_design: Total RNAs from serum, liver and epididymal fat were subjected to Illumina TruSeq Small RNA preparation and sequencing.


title: High-Resolution Transcriptome Maps Reveal Strain-Specific Regulatory Features of Multiple Campylobacter jejuni Isolates

status: Public on May 27 2013

city: Cologne

first: Konrad

middle: U.

last: Förstner

state: North Rhine-Westphalia

address: Gleueler Str. 60

department: Information Services

country: Germany

institute: ZB MED - Information Centre for Life Sciences

type: Expression profiling by high throughput sequencing

summary: Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA–seq (dRNA–seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA–seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA–seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA–seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level.

sample_organism: Campylobacter jejuni subsp. jejuni NCTC 11168 = ATCC 700819

sample_organism: Campylobacter jejuni subsp. jejuni 81-176

sample_organism: Campylobacter jejuni subsp. jejuni 81116

overall_design: Our dRNA-seq study of multiple C. jejuni strains represents the first comparative analysis of the primary transcriptomes of multiple strains and provides new insights into riboregulation in this bacterial pathogen.

first: Gaurav

last: Dugar

first: Alexander

last: Herbig

middle: U

first: Nadja

last: Heidrich

first: Richard

last: Reinhardt

first: Kay

last: Nieselt

first: Cynthia

middle: M


title: Asthma

status: Public on Jul 01 2014

city: Pittsburgh

first: Jadranka

last: Milosevic

state: PA - Pennsylvania

address: 2330 Tilbury Avenue

country: USA

institute: University of Pittsburgh

summary: Bronchial Epithelial Cells were isolated processed as described (Chu et al., 2002 and Zhao et al., 2011). The objective of the study was to identify differentially expressed genes between normal control (NC), mild-moderate asthmatic (MMA) and severe asthmatic (SA) patients.

overall_design: Isolated fresh bronchial epithelial cells were placed into trizol for further analysis.

first: Sally

middle: E

last: Wenzel

first: Naftali

last: Kaminski


title: Distinct developmental ground states of different epiblast stem cell lines determine pluripotency features

status: Public on Aug 10 2012

city: Muenster

first: Marcos

middle: J.

last: Araúzo-Bravo

address: Rogentstrasse

department: Cell and Developmental Biology

institute: Max Planck Institute for Molecular Biomedicine

summary:
Epiblast stem cells (EpiSC) are pluripotent stem cells derived from mouse postimplantation embryos between E5.5 to E7.5, a time window in which gastrulation commences. Therefore, EpiSC represent a valuable tool for studying mammalian postimplantation development in vitro. Beyond their pluripotent features, EpiSC can also be reprogrammed into a mouse embryonic stem cell-like (mESC) state. Published reports showed that EpiSC reversion requires transcription factor overexpression, while others suggest that applying stringent mESC culture conditions alone was sufficient to revert EpiSC to mESC-like cells. In an effort to clarify these discrepancies, we systematically compared a panel of independent EpiSC lines. Indeed, the different lines shared a number of defining EpiSC characteristics such as teratoma formation ability, regardless of the day of isolation from the embryo. However, despite being grown in identical, self-renewal-promoting conditions, some lines displayed strong expression of genes associated with mesendodermal differentiation. Strikingly, these same lines were not revertable to a mESC-like state by applying stringent mESC culture conditions. Moreover, expression of mesendodermal marker genes correlated in a negative manner with the ability to efficiently undergo neural induction. Overall, our data suggest that different EpiSC lines may self-renew in distinct developmental ground states, which has important consequences for functional readouts such as revert ability to a mESC-like state and directed differentiation.
For EpiSC lines derivation E5.5 embryos were plated onto irradiated mouse embryonic feeder cells (MEF cells) seeded at low density. Outgrowths were picked and expanded manually. Basal medium consisted of Knock-Out-DMEM, 20% Knock-Out Serum Replacement, Non Essential Amino Acids, L-Glutamine and beta-Mercaptoethanol (all Gibco). For cultivation on feeders 5ng/ml bFGF (Peprotech) was added to the medium to yield EpiSC medium. For preparation of mouse embryonic fibroblast-conditioned medium, EpiSC medium incubated on inactivated MEFs, followed by addition of another 5ng/ml bFGF (CM+). For feeder free culture, EpiSCs were seeded onto FCS-coated dishes. EpiSC were routinely passaged using 1mg/ml Collagenase IV (Invitrogen). Mouse ESC were cultured in standard conditions - Knock-Out-DMEM, 15% Knock-Out Serum Replacement (both Gibco), 5% fetal calf serum (Biowest), Non Essential Amino Acids, L-Glutamine and beta-Mercaptoethanol (all Gibco), supplemented with either homemade or commercially available LIF (ESGRO) at 1000U/ml. Some experiments were carried out in chemically defined N2B27 medium 11 in the presence of 10ng/ml bFGF. For reversion experiments, EpiSC were harvested using Accutase (Invitrogen) and seeded as single cells at approximately 16.000 cells / well of a 6-well plate (Sarstedt) in CM+. After 24h medium was changed to PCL medium (basal medium, LIF (1000U/ml), PD0325901 (Axon Medchem 1µM), and CHIR99021 (Axon Medchem 3µM). After 48h treatment, reversion medium was further supplemented with 10ÂµM SB431542 (Sigma) to eliminate remaining EpiSC.

overall_design:
7 samples were analyzed:
OG2.1: OG2.1 EpiSC, 1 biological rep
OG2.2: OG2.2 EpiSC, 1 biological rep
E3: E3 EpiSC, 1 biological rep
E5: E5 EpiSC, 1 biological rep
T9: T9 EpiSC, 1 biological rep
C1a1: C1a1 EpiSC, 1 biological rep
mESC: R1 ESC, 1 biological rep

first: Christof

last: Bernemann

first: Boris

last: Greber

first: Kinarm

last: Ko

first: Jared

last: Sterneckert

first: Dong

middle: W

last: Han

first: Hans

middle: R

last: Schöler


title: Isolation of Novel Multipotent Neural Crest-Derived Stem Cells from Adult Human Inferior Turbinate

status: Public on Mar 19 2012

summary: Adult human neural crest-derived stem cells are of extraordinary high plasticity and promising candidates for the use in regenerative medicine. Here we describe for the first time a novel neural crest-derived stem cell population within the respiratory epithelium of adult human inferior turbinate. In contrast to superior and middle turbinates, high amounts of source material could be isolated from human inferior turbinates using minimally-invasive surgery methods isolation is efficient even from older patients. Within their endogenous niche, inferior turbinate stem cells (ITSCs) expressed high levels of nestin, p75NTR and S100. Immuno-electron microscopy using anti-p75 antibodies displayed that ITSCs are of glial origin and closely related to non-myelinating Schwann cells. Cultivated ITSCs were positive for nestin and S100 as well as the neural crest markers Slug and Sox10. Whole genome microarray analysis showed pronounced differences to human ES cells in respect to pluripotency markers OCT4, SOX2, LIN28 and NANOG, whereas expression of WDR5, KLF4 and c-MYC was nearly similar. ITSCs were able to differentiate into ectodermal, mesodermal and endodermal cell types. Additionally ITSCs are able to survive and perform neural crest typical chain migration in vivo when transplanted into chicken embryos. However ITSCs do not form teratomas in immunodeficient mice. Finally, we developed a separation strategy based on magnetic cell sorting of p75 positive ITSCs, which formed larger neurospheres and proliferated faster than p75 negative ITSCs. Taken together our study describes a novel, readily accessible source of multipotent human neural crest-derived stem cells for potential cell-replacement therapy. RNA samples to be analyzed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion) which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto HumanHT-12 v4 expression BeadChips (Illumina) following the manufacturers instructions. After washing as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software.

overall_design:
12 samples were analyzed.
Donor I: Donor I sorted p75 positive Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor II: Donor II sorted p75 positive Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor III: Donor III sorted p75 positive Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor IV: Donor IV sorted p75 positive Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor V: Donor V sorted p75 positive Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor I-: Donor I sorted p75 negative Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor II-: Donor II sorted p75 negative Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor III-: Donor III sorted p75 negative Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor IV-: Donor IV sorted p75 negative Inferior Turbinate stem cells (ITSC), 1 biological rep
Donor V-: Donor V sorted p75 negative Inferior Turbinate stem cells (ITSC), 1 biological rep
HUESC6: Human Embryonic Stem Cells HUESC6, 2 biological rep

first: Stefan

last: Hauser

first: Darius

last: Widera

first: Firas

last: Qunneis

first: Janine

last: Müller

first: Christin

last: Zander

first: Johannes

last: Greiner

first: Christina

last: Strauss

first: Patrick

last: Lüningschrör

first: Peter

last: Heimann

first: Hartmut

last: Schwarze

first: Holger

last: Sudhoff

first: Holm

last: Zaehres

last: Schröler

first: Chistian

last: Kaltschmidt

first: Barbara


title: Generation of Induced Pluripotent Stem Cells from Human Testis Biopsy Derived Fibroblastic Cells

status: Public on Apr 02 2011

first: Marcos

last: Araúzo-Bravo

summary:
Although reported more than once, the generation of pluripotent stem cell populations from human testis remains questionable. We recently showed that the so - called human adult germline stem cells (haGSCs), which were claimed to be pluripotent, showed a gene expression profile comparable to human fibroblasts, suggesting that these cells are originated from a fibroblastic cells. We could derive human testicular fibroblast cells (hTFCs) from several routine testicular biopsies, naming them human testicular fibroblastic cells (hTFCs). The true conversion of unipotent mouse GSCs, also known as spermatogonial stem cells into pluripotent stem cells was recently shown. However, this conversion was not observed for human cells so far.
We suggest that hTFCs isolated from routine biopsies can help in the understanding, modeling and also regenerative medicine for various diseases after reprogramming them to human induced pluripotent stem cells (iPS). We successfully reprogrammed hTFCs into human iPS cells by transducing them retrovirally with reprogramming factors as already demonstrated for human dermal fibroblasts. These hTFCs derived iPS cells are comparable with human embryonic stem cells from global gene expression, promoter methylation, and they show pluripotency judged by in vitro and in vivo differentiation.

overall_design:
For transcriptome profiling, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit - Ambion) following the manufacturer's instructions (IVT: 12h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto human-8 v3 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials/instrumentation supplied/suggested by the manufacturer.
8 samples were analyzed:
NCL: NCL3 hESCs grown on feeders, bulk harvest at semi-sub confluence (1 replicate)
HuES6: HuES6 hESCs grown on feeders, bulk harvest at semi-sub confluence (1 replicate)
HT3: Human fibroblast-like testis cells, line 3 (1 replicate)
HT16: Human fibroblast-like testis cells, line 16 (1 replicate)
FS: FS human foreskin fibroblasts (1 replicate)
BJ: BJ human fibroblasts (1 replicate)
iPS3: hiPSC line 2 derived from HT3 (1 replicate)
iPS16: hiPSC line 2 derived from HT16 (1 replicate)

first: Kinarm

last: Ko

last: Reinhardt

first: Rebekka

middle: K

last: Schneider

first: Natalia

last: Tapia

first: Julee

last: Kim

first: Boris

last: Greber

first: Luca

last: Gentile

first: Sabine

last: Kliesch

first: Martin

last: Zenke


title: Metabolic control of adult neural stem cell activity by FASN-dependent lipogenesis

status: Public on Nov 02 2012

summary: Mechanisms controlling the proliferative activity of neural stem/progenitor cells (NSPCs) play a pivotal role to ensure life-long neurogenesis in the mammalian brain. How metabolic programs are coupled with NSPC activity remains unknown. Here we show that fatty acid synthase (FASN), the key enzyme of de novo lipogenesis, is highly active in adult NSPCs and that conditional deletion of FASN in NSPCs impairs adult neurogenesis. The rate of de novo lipid synthesis and subsequent proliferation of NSPCs is regulated by Spot14, a gene we found to be selectively expressed in low proliferating adult NSPCs. Spot14 reduces the availability of malonyl-CoA, which is an essential substrate for FASN to fuel lipogenesis. Thus, we here identified a functional coupling between the regulation of lipid metabolism and adult NSPC proliferation.

overall_design:
6 samples were analyzed.
Spot14+: Spot14 GFP positive mouse neural stem cell, 3 biological rep
Spot14-: Spot14 GFP negative mouse neural stem cell, 3 biological rep

first: Marlen

last: Knobloch

first: Simon

last: Braun

first: Luis

last: Zurkirchen

first: Carolin

last: von Schoultz

first: Nicola

last: Zamboni

first: Werner

last: Kovacs

first: Ozlem

last: Karalay

first: Ueli

last: Suter

first: Raquel

middle: A

last: Machado

first: Marta

last: Roccio

first: Matthias

middle: P

last: Lutolf

first: Clay

middle: F

last: Semenkovich

first: Sebastian

last: Jessberger


title: Mutating Zta (182N) changes sequence specific DNA binding  (5mCG)

status: Public on Jun 04 2019

city: Bethesda

first: Desiree

last: Tillo

state: MD

address: 41 Center Dr, Room D310

institute: NIH/NCI

summary: The Epstein-Barr virus (EBV) bZIP transcription factor (TF) Zta is a sequence-specific DNA binding protein that recognizes both unmethylated and methylated DNA.  To study the contribution of the conserved N182 amino acid to sequence specific Zta DNA binding, we replaced it with five other amino acids:  serine (S), glutamine (Q), threonine (T), isoleucine (I), or valine (V).  We used protein binding microarrays (PBMs) to evaluate sequence-specific DNA binding to four types of double-stranded DNA: 1) DNA with cytosine in both strands (DNA(C|C), 2) DNA with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (DNA(5mC|C)), 3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (DNA(5hmC|C)), and 4) DNA with methylated cytosine in both strands in all CG dinucleotides (DNA(5mCG)).  With unmethylated DNA, Zta(N182S) binds variants of the consensus TRE (TGA-G/C-TCA) motif, such as TGA-G/C-TGA and TCA-G/C-TGA where C at position 3 is replaced with G in one or both half sites of the motif.  Zta(N182S) also binds stronger to DNAs containing modified cytosines compared to wildtype. Zta(N182Q) binds new sequences containing GTAA with DNA(C|C), DNA(5mC|C) and DNA(5hmC|C) where C at position 3 is replaced with A.  Zta(N182I) and Zta(N182V) bind sequence specifically to DNA(5mC|C), and weakly with all other types of DNA examined.  Zta(N182T) DNA binding is weaker to all types of DNA examined.  Our data highlight that mutation of ZtaN182 with the hydrophilic amino acids serine and glutamine alters Zta sequence specific DNA binding, while mutation with hydrophobic amino acids (I and V) increases binding to DNA(5mC|C).

overall_design: Protein binding microarray (PBM) experiments were performed for the DNA binding domain of the Epstein Barr Virus Zta protein and for 5 mutant constructs of Zta in which the asparagine (N) at position 182 of wild type Zta was changed to serine (N182S), glutamine (N182Q), threonine (N182T), isoleucine (N182I), or valine (N182V).   Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to double-stranded and methylated or unmethylated 44K Agilent microarrays, containing a DeBruijn sequence design, in order to determine their sequence preferences. Details of the methylated PBM protocol are described in Mann et al., Genome Research, 2013


title: Transcription factor TFAP2C regulates major programs required for murine fetal germ cell maintenance and haploinsufficiency predisposes to teratomas in male mice

status: Public on Jan 30 2014

first: Marcos

last: Araúzo-Bravo

summary: Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor Tcfap2c / TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of Tcfap2c in primordial germ cell-like cells. We show that loss of Tcfap2c affects many aspects of the genetic network regulating germ cell biology, such as downregulation maturation markers and induction of markers indicative of somatic differentiation, cell cycle, epigenetic remodeling, and pluripotency associated genes. Chromatin-immunoprecipitation analyses demonstrated binding of Tcfap2c to regulatory regions of deregulated genes (Sfrp1, Dmrt1, Nanos3, c-Kit, Cdk6, Cdkn1a, Fgf4, Klf4, Dnmt3b and Dnmt3l) suggesting that these genes are direct transcriptional targets of Tcfap2c in primordial germ cells. Since Tcfap2c deficient primordial germ cell like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the Tcfap2c-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for Tcfap2c develop germ cell cancer with high incidence. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate, that mice with a heterozygous deletion of the Tcfap2c target gene Nanos3 are also prone to develop teratoma. These data highlight Tcfap2c as a critical and dose-sensitive regulator of germ cell fate.

overall_design:
8 samples were analyzed.
Ctrl ESC:     Control mouse embryonic stem cells (ESCs), 2 biological rep
KO ESC:       Tcfap2c knock-out mouse embryonic stem cells (ESCs), 2 biological rep
Ctrl PGC:     Control mouse primordial germ cells (PGCs), 2 biological rep
KO PGC:       Tcfap2c knock-out  mouse primordial germ cells (PGCs), 2 biological rep

first: Jana

last: Schemmer

last: Arauzo-Bravo

first: Natalie

last: Haas

last: Schaefer

first: Susanne

middle: N

last: Weber

first: Astrid

last: Becker

first: Dawid

last: Eckert

first: Andreas

last: Zimmer

first: Daniel

last: Nettersheim

first: Hubert

last: Schorle


title: Identification of genes specific to mouse primordial germ cells through dynamic global gene expression

status: Public on Nov 16 2010

summary: The molecular mechanisms underlying the commitment of cells to the germ cell lineage and the subsequent formation of germ cells during mammalian development remain poorly understood due to an inability to obtain sufficient amounts of cellular materials to conduct thorough in-vitro analyses. Although primordial germ cells (PGCs) have been generated and isolated from embryonic stem cells (ESCs) in vitro, concurrent expression of several cell surface receptors and transcription factors, at various levels, confounds the identification of these cells. To date, only a few genes that mark the onset of germ cell commitment to the epiblast, including tissue nonspecific alkaline phosphatase (TNAP), Blimp1, Stella, and Fragilis, have been used with some degree of success to detect PGC formation in in-vitro model systems. In light of the current literature, the generation of an unlimited supply of germ cells and gametes from pluripotent stem cells would greatly facilitate studies into reproductive development. To accomplish this, we would need to discover novel markers that are specifically expressed in germ cells. Here we identified four genes that are specifically expressed in male and female germ cells, both in vivo and in vitro, but are not expressed in somatic tissues or ESCs. Expression of these genes therefore allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro.

overall_design:
For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using RNeasy Micro kits with on-column DNase digestion as per the manufacturer's instructions. Integrity of RNA samples was quality-checked using a 2100 Bioanalyzer (Agilent). If possible, 300 ng of total RNA per sample was used as starting material for linear amplification (Illumina TotalPrep RNA amplification kit, Ambion), which involved synthesis of T7-linked double-stranded cDNA and 14 hrs of in-vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then quality-checked on a 2100 Bioanalyser, and hybridized as biological or technical duplicates for 18 hrs onto MouseRef-8 v2 gene expression BeadChips (Illumina), following the manufacturer's instructions. After being washed, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader and accompanying software.

44 samples were analyzed.
ESCm: OG2 male Embryonic Stem Cell, +/+ Oct4-GFP ES cells, 1 biological rep
11.5: 11.5 embryonic day PGC (Primordial Germ Cell), 2 biological reps
12.5F: 12.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
13.5F: 13.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
14.5F: 14.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
15.5F: 15.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
16.5F: 16.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
17.5F: 17.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
18.5F: 18.5 embryonic day female PGC (Primordial Germ Cell), 2 biological reps
12.5M: 12.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
13.5M: 13.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
14.5M: 14.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
15.5M: 15.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
16.5M: 16.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
17.5M: 17.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
18.5M: 18.5 embryonic day male PGC (Primordial Germ Cell), 2 biological reps
8M: 8 day after birth male PGC (Primordial Germ Cell), 2 biological reps
10M: 10 day after birth male PGC (Primordial Germ Cell), 2 biological reps
12M:  12 day after birth male PGC (Primordial Germ Cell), 2 biological reps
14M: 14 day after birth male PGC (Primordial Germ Cell), 1 biological rep
16M: 16 day after birth male PGC (Primordial Germ Cell), 2 biological reps
18M: 18 day after birth male PGC (Primordial Germ Cell), 2 biological reps
20M: 20 day after birth male PGC (Primordial Germ Cell), 2 biological reps

first: Davood

last: Sabour

first: Karin

last: Hübner

first: Kinarm

last: Ko

first: Boris

last: Greber

first: Luca

last: Gentile

first: Martin

last: Stehling

first: Hans

last: Schöler


title: Direct Reprogramming of Fibroblasts into Neural Stem Cells by Defined Factors

status: Public on May 03 2012

last: Araúzo-Bravo

summary:
Recent advances in the stem cell biology have revealed that cell type-specific transcription factors could reset the somatic memory and induce direct reprogramming into specific cellular identities. The induction of pluripotency in terminally differentiated cells has been a major achievement in the field of direct reprogramming. Recent studies have shown that fibroblasts could be directly converted into specific cell types, such as neurons, cardiomyocytes, blood progenitor cells, and epiblast stem cells, without first passing through an induced pluripotent stem cell state3-7. However, direct reprogramming of differentiated cells into somatic stem cell types has not been described yet4. Here we show that a combination of neural-specific transcription factors (Sox2, Klf4, c-Myc, Brn4/Pou3f4 or Sox2, Klf4, c-Myc, Brn4, E47/Tcf3) can induce a neural stem cell (NSC) fate on the fibroblasts. Induced neural stem cells (iNSCs) showed morphology, gene expression, epigenetic features, differentiation potential, and functionality similar to wild-type NSCs. Therefore, our data suggest that cell type-specific defined factors can induce specific stem cell identities on somatic cells.
Fibroblasts (5 x 104 cells) were infected with retroviruses for two days, and cells were maintained in NSC media:  DMEM/F-12 supplemented with N2 or B27 supplements (Gibco-BRL), 10 ng/ml EGF, 10 ng/ml bFGF (both from Invitrogen), 50 ug/ml BSA (Fraction V Gibco-BRL), and 1x penicillin/streptomycin/glutamine (Gibco-BRL). In order to establish stable iNSC lines, were either manually picked mature iNSC clumps or passaged and seeded the whole dishes of cells onto either gelatin- or laminin-coated dishes. RNA samples to be analysed on microarrays were prepared using QIAGEN RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input RNA into a linear amplification protocol (Ambion) involving synthesis of T7-linked double-stranded cDNA and 12 h of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was hybridised onto MouseRef-8 v2 expression BeadChips (Illumina) for 18 h according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using iScan reader (Illumina) and accompanying software.
Samples were hybridised as biological replicates.

overall_design:
9 samples were analyzed.
Fibroblast: CF1 Mouse embryonic fibroblasts, 1 biological rep
Control NSC: OG2-Rosa Neural Stem Cells, 2 biological rep
4F iNSC (late): 4 factors induced Neural Stem Cells (late passage), 2 biological rep
4F iNSC (early): 4 factors induced Neural Stem Cells (early passage), 2 biological rep
5F iNSC: 5 factors induced Neural Stem Cells, 2 biological rep

first: DongWook

last: Han

first: Holm

last: Zaehres

last: Hermann

first: Natalia

last: Tapia

first: JiHun

last: Yang

first: Sören

last: Moritz


title: ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses

first: Marcos

summary:
With the increasing use of Assisted Reproductive Technologies (ART) for treatment of human infertility,  there is an increasing requirement for embryo culture conditions that perform as similar to nature as possible.
How good the match, however, cannot be tested experimentally in human.
We solved the central question of how well ART culture protocols prepare embryos for postimplantation development, under the provisions of the 'mouse embryo assay' (MEA).
Our side-by-side comparison of 8 conditions [i.e., 3 culture conditions (KSOM, HTF  and ISDM1) plus the in vivo system in two different mouse strains (B6 and CD1)] shows that mouse embryos cultured under ART conditions are differentially primed for postimplantation development,  and that certain ART protocols outperform the oviduct.
The distinct performances of blastocysts formed in ART vs. oviduct do not correlate with any significant transcriptome changes, whereas protein analysis by immunoconfocal microscopy reveals differences in the allocation of embryonic cells to the three germ layers of blastocysts.
We conclude that in vitro technology is not always a defective copy of nature, and that the choice of ART protocol primes the embryos for subsequent  development.

overall_design:
22 samples were analyzed.
B6KSOM: Mouse B6  background, E3.5 blastocysts in KSOM medium, 3 biological rep
B6HTF: Mouse B6  background, E3.5 blastocysts in HTF medium, 3 biological rep
B6ISM1: Mouse B6  background, E3.5 blastocysts in ISM1 medium, 3 biological rep
B6vivo: Mouse B6  background, E3.5 blastocysts in vivo, 3 biological rep
CD1KSOM: Mouse CD1  background, E3.5 blastocysts in KSOM medium, 1 biological rep
CD1HTF: Mouse CD1  background, E3.5 blastocysts in HTF medium, 3 biological rep
CD1ISM1: Mouse CD1  background, E3.5 blastocysts in ISM1 medium, 3 biological rep
CD1vivo: Mouse CD1  background, E3.5 blastocysts in vivo, 3 biological rep

first: Michele

last: Boiani

first: Telma

middle: C

last: Esteves

first: Séverine

last: Le Gac

first: Verena

last: Nordhoff

first: Stefan

last: Schlatt

first: Caroline

last: Schwarzer


title: Generation of Autologous Pluripotent Stem Cells from Adult Testis Biopsy

status: Public on Jan 23 2011

summary:
In regenerative medicine, histocompatibility of pluripotent stem cells is required to solve the problem of immunorejection after therapeutic transplantation. In this study, we show that autologous germline stem cells (GSCs), often called spermatogonial stem cells, could be derived by testis biopsy from individual mice and that GSCs subsequently could be dedifferentiated into autologous germline-derived pluripotent stem (gPS) cells.
The establishment of GSCs by testicular biopsy in the mouse model can prove the principle of human clinical application to derive autologous GSCs to generate patient-specific pluripotent cells for regenerative medicine.

overall_design:
For transcriptome profiling, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit - Ambion) following the manufacturer's instructions (IVT: 12h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto mouse-8 v2 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials / instrumentation supplied / suggested by the manufacturer
13 samples were analyzed, GSC1: Mouse Germ Stem Cells OG2, line 1 (1 replicate); GSC2: Mouse Germ Stem Cells OG2, line 2 (1 replicate); GSC3: Mouse Germ Stem Cells OG2, line 3 (1 replicate); GSCr1: Mouse Germ Stem Cells OG2-ROSA, line 1 (1 replicate); GSCr2: Mouse Germ Stem Cells OG2-ROSA, line 2 (2 replicate); GSCr3: Mouse Germ Stem Cells OG2-ROSA, line 3 (1 replicate); ESC:  Mouse Embryonic Stem Cells (duplicate) gPS1: Mouse clonal germ Pluripotent Stem cells from OG2, line 1, (1 replicate); gPS2: Mouse clonal germ Pluripotent Stem cells from OG2, line 2, (1 replicate); gPS3: Mouse clonal germ Pluripotent Stem cells from OG2, line 3, (1 replicate); gPSr1: Mouse clonal germ Pluripotent Stem cells from OG2-ROSA, line 1, (1 replicate); gPSr2: Mouse clonal germ Pluripotent Stem cells from OG2-ROSA, line 2, (1 replicate); gPSr3: Mouse clonal germ Pluripotent Stem cells from OG2-ROSA, line 3, (1 replicate).

first: Kinarm

last: Ko

first: Guangming

last: Wu

first: Julee

last: Kim

last: Greber

first: Jury

last: Francine

first: Jörg

last: Mühlisch

first: Jin

middle: Y

last: Joo

first: Michel

last: Frühwald


title: An analysis of global gene expression reveals molecular and signalling pathways hallmarks of neural stem cell survival and expansion in response to FGF-2 and EGF

status: Public on Apr 27 2012

last: Araúzo-Bravo

summary:
The culture of neural stem cells (NSCs) as floating neurospheres has become widely used as an experimental model to analyse the properties of NSCs. Although the neurosphere model has existed for two decades, there is still no standard protocol to grow NSCs in this way. Thus, we have analysed the consequences of the frequency of growth factor (FGF-2 and EGF) addition to embryonic and adult olfactory bulb stem cells (eOBSCs and aOBSCs) cultures, specifically in terms of proliferation, cell cycle progression, death and differentiation, as well as on global changes in gene expression and signaling pathways. We found that addition of FGF-2 and EGF every two or four days rather than daily significantly reduces the volume of the neurospheres and the total number of cells, changes that were more evident in aOBSC than in eOBSC cultures. The reduction in neurosphere size was mainly due to an increase in cell death and occurs without major changes in the cell cycle parameters tested. Moreover, partial deprivation of FGF-2 and EGF produces a mild increase in aOBSC differentiation during the proliferative phase. Remarkably, these effects were accompanied by a significant upregulation in the expression of genes involved in cell death regulation (Cryab), lipid catabolic processes (Pla2g7), cell adhesion (Dscaml1), cell differentiation (Dscaml1, Gpr17, S100b) and signal transduction (Gpr17, Ndrg2), among others. These findings support that continuous supply of FGF-2 and EGF is critical to maintain the viability/survival of NSCs in culture and reveals novel molecular hallmarks of NSC maintenance/survival and expansion in response to these growth factors.
Total RNA was extracted from aOBSC cultures using the Trizol reagent (Invitrogen) and purified with Qiagen RNeasy Mini Kit separation columns (Qiagen). The RNA was sent to the Genomic Unit of CNB (Centro Nacional de Biotecnologia, Madrid, Spain). RNA integrity was corroborated by using Bioanalyzer. Then, cDNA was synthesized and hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA, http://www.affymetrix.com) which contain a total of 45101 transcripts to assess and compare the overall gene expression profiles.

overall_design:
9 samples were analyzed.
Ctr:     Control mouse adult olfatory bulb stem cells (aOBSC) cultured with daily added Fgf2 and Egf, 3 biological rep
C2:      Mouse adult olfatory bulb stem cells (aOBSC) cultured with Fgf2 and Egf added every two days, 3 biological rep
C4:      Mouse adult olfatory bulb stem cells (aOBSC) cultured with Fgf2 and Egf added every four days, 3 biological rep

first: Vanesa

last: Nieto-Estavez

first: Jaime

last: Pignatelli

last: Arazo-Bravo

first: Anaha

last: Hurtado-Chong

first: Carlos

last: Vicario-Abejan


title: Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages

status: Public on Feb 24 2011

first: Marcos

summary: Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.

overall_design:
RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates.

6 samples were analyzed, five of them in duplicate and one of them (T9-EpiSC) a single time (11 total samples).  ESC: Mouse ESC male;  EpiSC: Mouse EpiSC male GOF18;  Epi-Sox2: Mouse EpiSC Sox2 male GOF18 (Overexpressing WT Sox2) cultured in condition EpiSC medium (CM);  EpiSC-GFP-: Mouse E3 EpiSC grown in CM and FACS-sorted for GFP-;  EpiSC-GFP+: Mouse E3 EpiSC grown in CM and FACS-sorted for GFP+;  T9-EpiSC: Mouse T9 EpiSC grown on medium-density CF1 MEFs in UM, 2d -Fgf2, harvested without MEFs.

The supplementary file 'GSE17984_non-normalized_data.txt' contains non-normalized data for Samples GSM450294-GSM450304.

first: Dong

last: Han

first: Natalia

last: Tapia

first: Boris

first: Christof

last: Bernemann

first: Martin

last: Stehling

first: Jeong

middle: T

last: Do

first: Hans

last: Schöler


title: Induction of pluripotency in human cord blood unrestricted somatic stem cells

status: Public on Jun 22 2010

summary: Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of the transcription factors OCT4, SOX2, KLF4, c-MYC as well as NANOG and LIN28. Here we report generation of induced pluripotent stem cells from human umbilical cord blood derived unrestricted somatic stem cells (USSC) using retroviral expression of the transcription factors OCT4, SOX2, KLF4 and C-MYC and evaluation of their molecular signature and differentiation potential in comparison to human embryonic stem cells. The reprogrammed cells (HUiPS) were analysed morphologically, by qRT-PCR, global miRNA and epigenetic profiling and gene expression microarrays, as well as in their in vitro and in vivo differentiation potential by embryoid body formation and teratoma assay. The cord blood iPS cells are highly similar to human embryonic stem cells morphologically, at their molecular signature as well as in their in vitro and in vivo differentiation potential. Human cord blood derived unrestricted somatic stem cells offer an attractive source of cells for the generation of induced pluripotent stem cells. Our findings open novel perspectives to generate HLA matched pluripotent stem cell banks based on existing cord blood banks. Besides its obvious relevance of such a second generation cord blood iPS bank for pharmacological and toxicological testing, its application for autologous or allogenic regenerative cell transplantation appears feasible.

overall_design:
For transcriptome profiling, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit - Ambion) following the manufacturer's instructions (IVT: 10h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto HumanRef-8 v3 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials / instrumentation supplied / suggested by the manufacturer.
Six sample types were analyzed, each one of them in duplicate.
USSC: human umbilical cord blood unrestricted somatic stem cells (duplicates)
HUiPS: human iPS cells from human umbilical cord blood USSC, hand-picked cols (duplicates)
H9 hESC: H9 human ESCs grown on low-density CF1 MEFs (duplicates)
H1 hESC: H1 human ESCs grown on low-density CF1 MEFs (duplicates)
1F hNiPS: One factor (Oct4) human iPS cells from hNSCs, hand-picked cols (duplicates)
2F hNiPS: Two factors (Oct4, Klf4) human iPS cells from hNSCs, hand-picked cols (duplicates)

first: H

last: Zaehres

first: G

last: Kögler

first: M

last: Bleidissel

first: S

last: Santourlidis

last: Weinhold

first: B

last: Greber

first: J

middle: B

last: Kim

first: A

last: Buchheiser

last: Liedtke

first: N

last: Graffmann

first: X

last: Zhao

last: Meyer

first: P

last: Reinhardt

last: Burr

last: Waclawczyk

first: C

last: Ortmeier

last: Uhrberg

first: T

last: Cantz

last: Wernet


title: Sox2 Levels Define Pluripotent Ground States and Reprogramming Potential

status: Public on Dec 31 2010

first: Marcos

last: Araúzo-Bravo

summary: Pluripotent stem cell lines derived from embryos of different stages have distinct pluripotent ground states, but similar levels of the transcription factor Oct4. Epiblast-derived pluripotent stem cells (EpiSCs), in contrast to embryonic stem (ES) cells, cannot form chimeras. We show that EpiSCs express lower levels of the transcription factors Sox2 and Klf4 than ES cells and have limited reprogramming potential, as shown by cell fusion. Sox2 overexpression dramatically increases the reprogramming potential, chimera formation, and germline contribution of EpiSCs. Therefore, although Oct4 is essential for reprogramming, the level of Sox2 defines both the reprogramming capability and the pluripotent ground states.

overall_design:
RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates. 12 sample types were analyzed, each of them in duplicate.
ESCm: Mouse ESC male; ESCf: Mouse ESC OG2 female; F9 EC: F9 EC (mouse embryonic carcinoma cell); F9-Sox2: F9 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCf: Mouse EpiSC OG2 female; Epi-Sox2f: Mouse EpiSC Sox2 (OG2 female) overexpressing wild type Sox2; P19 EC: P19 EC (mouse embryonic carcinoma cell); P19-Sox2: P19 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCm: Mouse EpiSC (GOF18 male) (duplicates); EpiSox2mL2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in condition EpiSC medium (CM); EpiSox2mE1: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like1); EpiSox2mE2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like2).

first: Dong

last: Han

last: Do

first: Natalia

last: Tapia

first: Jin

last: Joo

middle: J

last: Bernemann

first: Boris

last: Stehling

first: Jared

last: Sterneckert

first: Holm

last: Zaehres

last: Schöler


title: Direct Reprogramming of Fibroblasts into Epiblast Stem Cells

status: Public on Dec 08 2010

summary: Embryonic stem cells (ESCs), which are derived from the primitive ectoderm of pre-implantation blastocysts, are pluripotent cells and can thus contribute to the formation of all somatic cell lineages in chimeric animals.  Similarly, epiblast stem cells (EpiSCs), which are derived from epiblast tissue of post-implantation embryos,  are also pluripotent and can give rise to derivatives of all three germ layers in teratoma assays.  Introduction of the four transcription factors Oct4/Sox2/Klf4/c-Myc into somatic cells has been shown  to generate induced pluripotent stem cells (iPSCs).  iPSCs exhibit ESC-like properties and are virtually identical to ESCs with respect to a number of charactertistics.  However, generation of EpiSC-like cells by direct reprogramming of somatic cells using these  transcription factors has not been shown to date.  Here we show that Yamanaka’s four transcription factors can be used to directly generate EpiSC-like iPS cells (ePSCs) under EpiSC culture conditions.  These ePSCs are identical to EpiSCs with respect to morphology, gene expression pattern, epigenetic status, and chimera formation.  This is the first study to demonstrate that the culture environment in transcription factor-mediated reprogramming  determines the cell fate of the reprogrammed cell.  We could therefore envision that we eventually are able to shape the identity of a directly reprogrammed cell at will simply by modulating the culture conditions.

overall_design:
RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion.  500 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides.  Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina)  according to the manufacturer's instructions.  After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software.  Samples were hybridized as biological replicates.

24 samples were analyzed:
MEF: Female mouse embryonic fibroblast, 2 biological rep
ESC OG2: OG2 female embryonic stem cell, 2 biological rep
EpiSC OG2: OG2 female EpiSC grown as single cells with conditioned medium, 2 biological rep
EpiSC GOF18: GOF18 EpiSC grown as single cells with conditioned medium, 2 biological rep
EpiSC T9b: T9 EpiSC on MEFs in activin-containing CDM, harvested w/o feeders, subconfluent, 2 biological rep
ePSC L4: EpiSC-like iPS Cells L4, MEF reprogrammed to EpiSCs with 4F, line L4, grown with LIF and activin, 2 biological rep
ePSC L7: EpiSC-like iPS cells L7, MEF reprogrammed to EpiSCs with 4F, line L7, grown with LIF and activin, 2 biological rep
ePSC C1: EpiSC-like iPS Cells C1, MEF reprogrammed to EpiSCs with 4F, line C1, grown with LIF and activin, 2 biological rep
ePSC C3: EpiSC-like iPS Cells C3, MEF reprogrammed to EpiSCs with 4F, line C3, grown with LIF and activin, 2 biological rep
ePSC-R: EpiSC-like iPS Cells reverted, L4 ePSC cells reverted to an ESC-like state using Klf4 virus, 2 biological rep
ePSC-RC1: EpiSC-like iPS Cells reverted, L4 ePSR Cre1, Cre-treated L4 ePSR cells, line 1, 2 biological rep
ePSC-RC2: EpiSC-like iPS Cells reverted, L4 ePSR Cre2, Cre-treated L4 ePSR cells, line 2, 2 biological rep

last: Greber

first: Guangming

last: Wu

first: Kinarm

last: Ko

first: Martin

first: Hans


title: Chromatin-Remodeling Components of the BAF Complex Facilitate Reprogramming

status: Public on Jun 14 2010

summary:
Human and mouse somatic cells can be reprogrammed by the combination of Oct4, Sox2, Klf4, and c-Myc, but the efficiency of reprogramming is low. To better understand the process of reprogramming we sought to identify factors that mediate reprogramming at higher efficiency. For this we established an assay to screen nuclear fractions from the extracts of pluripotent cells based on Oct4 reactivation. We identified components of the ATP-dependent SWI/SNF chromatin-remodeling complex, which when used along with the above four factors increase reprogramming efficiency by five-fold and improve the quality of the reprogrammed cells. These cells were found to be capable of germline transmission and exhibited pluripotency according to gene expression and in vivo and in vitro assays. SWI/SNF was found to replace c-Myc and mediate its effects by facilitating recruitment of Oct4 on target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells.

DNA-free RNA samples to be hybridized on Illumina expression BeadChips were processed using a linear amplification kit (Ambion) (generating IVT duration: 12h). cRNA samples were quality-checked on a 2100 Bioanalyzer (Agilent) and hybridized as biological triplicates onto MouseWG-6 V2 chips as recommended and using materials / reagents provided by the manufacturer (hybridization time: 18h). The Myc probe on the V2 arrays was found to be defective. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina), background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model, variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clusters of genes and samples were performed with a one minus correlation metric and the average (unweighted pair group) linkage method.

overall_design:
4 samples were analyzed, each one them by triplicate.

ESC: Mouse Embryonic Stem Cells (OG2 mix female and male)
MEF: Mouse Embryonic Fibroblasts (OG2 male)
iPS4F: Mouse induced Pluripotent Stem cells with 4 factors (Oct4, Sox2, Klf4, c-myc)
iPS6F: Mouse induced Pluripotent Stem cells with 6 factors (Oct4, Sox2, Klf4, c-myc, Brg1, Baf155)

first: Nishant

last: Singhal

first: Johannes

last: Graumann

first: Luca

last: Gentile

first: Mathias

last: Mann


title: Generation of parthenogenetic iPS cells from parthenogenetic neural stem cell

status: Public on Jan 23 2010

last: Araúzo-Bravo

summary: In pluripotential reprogramming, a pluripotent state is established within somatic cells. In this study, we have generated induced pluripotent stem (iPS) cells from bi-maternal (uniparental) parthenogenetic neural stem cells (pNSCs) by transduction with four (Oct4, Klf4, Sox2, and c-Myc) or two (Oct4 and Klf4) transcription factors. The parthenogenetic iPS (piPS) cells directly reprogrammed from pNSCs were able to generate germline-competent himeras, and hierarchical clustering analysis showed that piPS cells were clustered more closer to parthenogenetic ES cells than normal female ES cells. Interestingly, piPS cells showed loss of parthenogenetic-specific imprinting patterns of donor cells. Microarray data also showed that the maternally imprinted genes, which were not expressed in pNSCs, were upregulated in piPS cells, indicating that pluripotential reprogramming lead to induce loss of imprinting as well as re-establishment of various features of pluripotent cells in parthenogenetic somatic cells.

overall_design:
5 samples were analyzed by microarray, each one them in duplicate.
fNSC: Mouse female NSC (Neural Stem Cell)
pNSC: Mouse parthenogenetic NSC (Neural Stem Cell)
piPS-2F: Mouse parthenogenetic induced pluripotent cells derived from NSC overexpressing Oct4 and Klf4
pESC-B: Mouse parthenogenetic ESC (Embryonic Stem Cell) SSEA-1 sorted
fESC: Mouse female ESC (Embryonic Stem Cell) OG2

first: Jeong

first: Min

last: Kim

first: Ingeborg

last: Sobek-Klocke

first: Hyung

last: Chung


title: Direct Reprogramming of Human Neural Stem Cells by OCT4

status: Public on Sep 23 2009

first: Marcos

summary: Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by ectopic expression of the four transcription factors (OCT4, SOX2, c-MYC, KLF4). We previously reported that Oct4 alone is sufficient to directly reprogram adult mouse neural stem cells (NSCs) to iPS cells. Here, we report the generation of one-factor (1F) human iPS from human NSCs (1F hNiPS) by ectopic expression of Oct4 alone. 1F hNiPS cells resemble human embryonic stem cells (hESCs) in global gene expression profiles, epigenetic status and pluripotency in vitro and in vivo. These findings demonstrate that the transcription factor OCT4 is sufficient to reprogram human NSCs to pluripotency. 1F iPS cell generation will accelerate this field further towards understanding reprogramming and generating patient-specific pluripotent stem cells.

overall_design:
For transcriptome profiling, 400 ng of total DNA-free RNA was used as input for labelled cRNA synthesis (Illumina TotalPrep RNA Amplification Kit - Ambion) following the manufacturer's instructions (IVT: 10h). Quality-checked cRNA samples were hybridized as biological or technical duplicates for 18 h onto HumanRef-8 v3 expression BeadChips (Illumina), washed, stained, and scanned following guidelines and using materials / instrumentation supplied / suggested by the manufacturer. Five sample types were analyzed, each one of them in duplicate.
hNSC: human fetal neural stem cells (duplicates); 1F hNiPS: One factor (Oct4) human iPS cells from hNSCs, hand-picked cols (duplicates); 2F hNiPS: Two factors (Oct4,Klf4) human iPS cells from hNSCs, hand-picked cols (duplicates); H9 hESC: H9 human ESCs grown on low-density CF1 MEFs (duplicates); H1 hESC: H1 human ESCs grown on low-density CF1 MEFs (duplicates).

first: Johann

last: Meyer

first: Kook

middle: I

last: Park

first: Nae

last: Yoo


title: Induction of Pluripotency in Adult Unipotent Germline Stem Cells

status: Public on Jul 29 2009

summary: Mouse and human stem cells with features similar to those of embryonic stem cells have been derived from testicular cells. Although pluripotent stem cells have been obtained from defined germline stem cells (GSCs) of mouse neonatal testis, only multipotent stem cells have been obtained so far from defined cells of mouse adult testis. In this study we describe a robust and reproducible protocol for obtaining germline-derived pluripotent stem (gPS) cells from adult unipotent GSCs. Pluripotency of gPS cells was confirmed by in vitro and in vivo differentiation, including germ cell contribution and transmission. As determined by clonal analyses gPS cells indeed originate from unipotent GSCs. We propose that the conversion process requires a GSC culture microenvironment that depends on the initial number of plated GSCs and the length of culture time.

overall_design:
Nine samples were analyzed.
GSC1: Mouse Germ Stem Cells, line 1 (1 replicate), GSC2: Mouse Germ Stem Cells, line 2 (1 replicate), GSC3: Mouse Germ Stem Cells, line 3 (1 replicate), GSC4: Mouse Germ Stem Cells, line 4 (1 replicate)
gPS1: Mouse clonal germ Pluripotent Stem cells, line 1,  GFP-sorted (1 replicate), gPS2: Mouse clonal germ Pluripotent Stem cells, line 2,  GFP-sorted (1 replicate), gPS3: Mouse clonal germ Pluripotent Stem cells, line 3,  GFP-sorted (1 replicate)
ESC:  Mouse Embryonic Stem Cells (duplicate)

first: Natalia

last: Tapia

first: Jeong-Beom

first: Philipp

last: Sasse

first: Tamara

last: Glaser

first: David

last: Ruau

first: Dong-Wook

last: Han

first: Boris

first: Kirsten

last: Hausdörfer

first: Vittorio

last: Sebastiano

last: Stehling

first: Bernd

last: Fleischmann

first: Oliver

last: Brüstle

last: Zenke


title: Human tumor cell lines treated with interferon-alpha-2a

status: Public on May 16 2010

city: Bern

first: Fredy

last: Siegrist

address: Freiburgstrasse 15

department: Department of Nephrology

country: Switzerland

institute: University Hospital Bern

summary: A hallmark of resistance to type I interferons (IFNs) is the lack of antiproliferative responses. Foser et al. 2006 have shown that costimulation with IFN-alpa and transforming growth factor beta-1 (TGF-beta) potentiates antiproliferative activity in a sensitive (ME15) and resistant (D10) human melanoma cell line. Gene induction by small concentration of interferon-alpha-2a (100 U/ml) was analyzed in human cancer cell lines with differences in sensitivity to IFN-alpha and TGF-beta. Among other antiproliferative genes we found upregulated IFITM3 gene expression levels and inducibility of this antiproliferative protein in interferon sensitive cell lines.

overall_design: A panel of 10 human cancer cell lines (ME-15, D10, LS174T, AsPC1, A549, MIAPACA, CaLu6, HCT116, PANC1, JUSO) were treated with 100 U/ml IFN-alpha. In addition to 24 hours treatment with (D) or without IFN-alpha (C) as control, gene expression levels of IFN-alpha treated cells at 4 hours (H) were analyzed. mRNA abundance was measured in triplicates using commercial Illumina microarrays.

first: Rachel

last: Scott

first: Ulrich

last: Certa


title: virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages

status: Public on May 05 2009

city: Hamilton

first: Dan

last: Sturdevant

state: MT

address: 903 S 4th street

department: NIAID

institute: NIH

summary:
The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Although some Francisella determinants of intracellular growth have been identified, much remains to be understood about the pathogenesis of this organism. In particular, how Francisella responds to its intracellular environment could provide clues about its intracellular biology and reveal pathogenic determinants based on their intracellular expression profiles. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages. Phagocytosed bacteria rapidly responded to their intracellular environment and progressively altered their transcriptional profile over time. Differential gene expression profiles were revealed that correlated with specific intracellular locations of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of a subset of transport and metabolic genes characterized the cytosolic replication stages. Expression of the Francisella Pathogenicity Island (FPI) genes, the functions of which are associated with intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci putatively encoding hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated during the intracellular cycle. In-frame deletion of FTT0383, the Schu S4 ortholog of fevR, of FTT0369c or FTT1676 abolished the ability of Schu S4 to either survive or proliferate intracellularly, demonstrating that bacterial factors of intracellular pathogenesis can be identified based on their intracellular expression profile. In conclusion, establishing the intracellular transcriptome of Francisella has revealed important aspects of its intracellular biology and identified novel virulence determinants of this pathogen.

Keywords: Time series

sample_organism: Francisella tularensis subsp. tularensis SCHU S4

platform_organism:
Borrelia hermsii
Francisella tularensis
Coxiella burnetii
Rickettsia prowazekii
Burkholderia mallei
Brucella suis

overall_design: Intracellular cycle within primary murine macrophages using the time series of zero, one, two, four, eight, twelve, sixteen and twenty-four hours

first: Tara

middle: D

last: Wehrly

first: Audrey

last: Chong

first: Kimmo

last: Virtaneva

first: Robert

last: Child

first: Jessica

last: Edwards

first: Vinod

last: Nair

first: Elizabeth

last: Fischer

first: Luke

last: Wicke

first: Alissa

last: Curda

first: John

last: Kupko

first: Craig

last: Martens

first: Stephen

last: Porcella

first: Jean

last: Celli


title: Melanoma SK-Mel-103 cell line : control vs. treated with pIC/pIC+PEI

status: Public on Jan 01 2010

city: Madrid

first: Maria

middle: S

last: Soengas

address: Melchor Fernandez Almagro, 3

department: Molecular Pathology

country: Spain

institute: Spanish National Cancer Research Institute

summary:
Transcriptional profiling of the melanoma SK-mel-103 comparing control untreated cells with cells treated with 1ug/ml of polyinosine-polycytidylic acid (pIC) or 1ug/ml of pIC with polyethyleneimine (PEI) for 4 or 10 hours.

Keywords: Treatment

overall_design: Four-condition experiment, SK-Mel-103 untreated vs. SK-Mel-103 treated with pIC/pIC+PEI at 4 and 10 hours. One replicate per array.

first: Damia

last: Tormo


title: Embryonic stem cells derived from developmentally incompetent embryos deficient in maternal and zygotic Cdx2

status: Public on Dec 30 2008

first: Marcos

last: Araúzo-Bravo

summary: Separation of cell lineages during early mammalian development is required to establish the pluripotent founder cell population that will give rise to the embryo proper and a functional trophoblast to support its development. We systemically assessed the role of the homeobox gene Cdx2 in vivo and in vitro development with an RNAi approach. Effective elimination of both maternal and zygotic Cdx2 resulted in typical phenotypes of Cdx2-mutant embryos, such as failure of hatching and implantation. However, the blastulation and expression of TE specific markers in these Cdx2-deficient embryos excluded the possibility of Cdx2 to act as a TE determinant, although compromised structure and functioning of TE was observed and the resulted embryos were not viable. Strikingly, the efficiency of stem cell derivation was significantly higher than control when embryos were put on MEF at the 8-cell stage and the derived stem cells were fully pluripotent as shown by chimera and tetraploid complementation experiments. Comparative genomic hybridization of wild type and Cdx2 mutant at 8-cell and blastocyst mouse embryos were performed.

overall_design: 8-cell biological duplicates and blastocyst stage biological triplicates embryos were used.The hybridization experiments were duplicated in a reciprocal labeling manner to reduce dye integration bias (dye-swaps).

first: Guangming

last: Wu

first: Luca

last: Gentile

first: Jeong

last: Tae Do

first: Tobias

last: Cantz

first: Julien

last: Sutter

first: Katherina

last: Psathaki

first: Telma

last: Esteves

last: Arauzo-Bravo

first: Claudia

last: Ortmeier

first: Takuya

last: Fuchikami

first: Kuniya

last: Abe

first: Hans

last: Scholer


title: Expression data from E11.5 mouse embryonic forelimbs - various mutant conditions or dissected limb domains

status: Public on Sep 15 2008

city: Austin

first: Steven

middle: A.

last: Vokes

state: Texas

address: 2500 Speedway, STOP A5000

department: Molecular Biosciences

institute: University of Texas at Austin

summary:
Sonic hedgehog (Shh) signals via Gli transcription factors to direct digit number and identity in the vertebrate limb. We have characterized the Gli-dependent cis-regulatory network through a combination of whole genome ChIP-on-chip and transcriptional profiling of the developing mouse limb.
In this dataset, we include the expression data obtained from dissected mouse forelimbs using a variety of gain- and loss-of-function hedgehog pathway mutants, as well as limbs dissected into responsive (posterior 2/3ds) and non-responsive (anterior 1/3d) Hh tissues. These data are used to obtain 753 genes that are differentially expressed in response to Shh signaling.
Keywords: Comparison of genetic samples

overall_design: 28 Total samples were analyzed. We generated the following pairwise comparisons using PowerExpress: Shh<WT; SmoM2>WT; Gli3<WT; Gli3>WT; Ant<Post; Ant>Post. Genes with an FDR≤10% and a fold-change ≥2 were selected. We did not generate pairwise comparisons for a certain combinations with SmoGli3 and Gli3 mutants because data from these arrays contained significant variability.  To identify additional genes that were Shh-responsive, we performed the following multiple sample comparisons using an FDR≤10% and a posterior probability cutoff of ≤25%: 1.) Ant<Post and Shh<WT<SmoM2, 2.) Ant<Post and Shh<WT<SmoM2 and Gli3>SmoGli3, 3.) Ant<Post and Shh<WT<SmoM2 and WT>SmoGli3

first: Hongkai

last: Ji


title: A Genome-Scale Analysis of the Cis-Regulatory Circuitry Underlying Hedgehog Mediated Patterning of the Mammalian Limb

summary:
Sonic hedgehog (Shh) signals via Gli transcription factors to direct digit number and identity in the vertebrate limb. We have characterized the Gli-dependent cis-regulatory network through a combination of whole genome ChIP-on-chip and transcriptional profiling of the developing mouse limb. These analyses identified approximately 5,000 high quality Gli3 binding sites, including all known Gli-dependent enhancers. Discrete binding regions exhibit a higher-order clustering, highlighting the complexity of cis-regulatory interactions. Further, Gli3 binds inertly to previously identified neural-specific Gli enhancers, demonstrating the accessibility of their cis-regulatory elements. Intersection of DNA binding data with gene expression profiles predicted 205 putative limb target genes. A subset of putative cis-regulatory regions were analyzed in transgenic embryos, establishing Blimp1 as a direct Gli target and identifying Gli-activator signaling in a direct, long-range regulation of the BMP antagonist Gremlin. In contrast, a long range silencer cassette downstream of Hand2 likely mediates Gli3 repression in the anterior limb. These studies provide the first comprehensive characterization of the transcriptional output of a Shh-patterning process in the mammalian embryo and a framework for elaborating regulatory networks in the developing limb.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Identification of Gli-Act targets in the mouse ventral neural tube by ChIP

status: Public on Apr 20 2007

type: Genome binding/occupancy profiling by genome tiling array

summary:
In studies investigating Sonic hedgehog (Shh) mediated patterning of the ventral neural tube, a process where Shh acts as a morphogen, we have investigated the transcriptional network underlying neural tube specification. Adopting an ES-cell based Shh neuronal specification assay (embryoid body; EBs) and a FLAG-tagged Gli protein (a transcriptional effector of the Shh pathway), we identified a number of direct targets of Gli action using Chromatin Immunoprecipitation (ChIP). These results will provide a first survey of the genome level response to this critical signaling input in vertebrate patterning
Keywords: ChIP-chip, Gli transcription factors, Gli1, neural specification, mouse

overall_design: Custom genomic tiling arrays based on expression data from a variety of Hh mutant backgrounds were constructed. These include 132 genes tiled with 60-mer oligo probes at a density of one probe per 125 bases spanning between 50-125kb of DNA. This region spans the TSS. Therefore there is very substantial upstream coverage and more variable downstream coverage (depending on gene size). There are three biological replicates, each with 4 technical replicates. The 4 technical replicates contain dye-swaps such that 1 and 2 are identical and reps 3 and 4 are identical. Within each hybridization, the Input sample is a small fraction of sheared chromatin that was not precipitated with the M2 Flag antibody as was the experimental ChIP sample, but was otherwise treated equivalently.

first: Wing

middle: H

last: Wong

first: Andrew

last: McMahon

first: Scott

last: McCuine

first: Shane

last: Giles


title: Genome-Wide Gli3 Binding Sites in the E11.5 Mouse Limb Bud

summary:
Sonic hedgehog (Shh) signals via Gli transcription factors to direct digit number and identity in the vertebrate limb. We have characterized the Gli-dependent cis-regulatory network through a combination of whole genome ChIP-on-chip and transcriptional profiling of the developing mouse limb. These analyses identified approximately 5,000 high quality Gli3 binding sites, including all known Gli-dependent enhancers. Discrete binding regions exhibit a higher-order clustering, highlighting the complexity of cis-regulatory interactions. Further, Gli3 binds inertly to previously identified neural-specific Gli enhancers, demonstrating the accessibility of their cis-regulatory elements. Intersection of DNA binding data with gene expression profiles predicted 205 putative limb target genes.

The supplementary bed file contains all 5,274 high quality binding Gli3 binding sites reported in the paper.

overall_design: RosaGli3T-FLAG limb buds ChIPed with FLAG (Gli3-repressor binding sites) vs. Input in E11.5 mouse limb buds.


title: Transcriptional Profiling of Hh-Treated Embryoid Bodies

summary:
This experiment was specifically designed to measure neural targets of Shh signaling, we sought to profile the genes upregulated by Hh signaling in the ventral neural tube to obtain a valid dataset.  To obtain ventral-specific markers, we generated retinoic acid-treated EBs grown in the presence or absence of HH-Ag.  We did not observe induction of ventral Hh markers in RA-treated constitutive Gli1FLAG EBs and used these for the control, baseline set.  The presence of FoxA2, Nkx2.9 and Nkx6.1 amongst the top 10 genes based on expression levels suggests that profiling significantly enriches for Hh-dependent cell types.   As expected, the benchmark standard Gli1 was not up-regulated in our array, since it is constitutively expressed in the control as well.
Keywords: neural progenitors, embryoid bodies, differentiation, Hedgehog, retinoic acid

overall_design: There are a total of 8 samples. Four biological replicates of Retinoic-acid treated EBs (Baseline) and 4 additional biological samples of retinoic acid + Hh-Ag treated EBs (induced sample).


title: Somatic cell nuclear reprogramming by mouse oocytes endures beyond reproductive decline

status: Public on Aug 16 2011

first: Marcos

middle: J.

last: Araúzo-Bravo

summary:
The mammalian oocyte has the unique feature of supporting fertilization and normal development while being able of reprogramming the nuclei of somatic cells towards pluripotency and occasionally even totipotency. Whilst oocyte quality is known to decay with somatic aging it is not a given that different biological functions decay concurrently. In this study we tested whether oocyte reprogramming ability decreases with somatic aging. We show that oocytes isolated from mice aged beyond the usual reproductive age (climacteric) yield ooplasts that retain reprogramming capacity after somatic nuclear transfer (SCNT) at levels similar to ooplasts of young donors. Despite differences in transcriptome between ooplasts of old and young mice gene expression profiles of the resultant SCNT blastocysts were very similar. Furthermore albeit results showing global down-regulation of apoptosis-related genes in climacteric ooplasts and the reported beneficial effect of inhibiting p53 on transcription factor-induced (iPS) pluripotency reprogramming capacity of young ooplasts was not affected by specifically blocking p53. Our observations strongly suggest that the outcome of oocyte-induced reprogramming is determined by the availability of intrinsic reprogramming factors tightly regulated throughout aging as well as intracellular and environmental checkpoints during pre-implantation development towards selection of the successfully reprogrammed embryos. While oocytes are not regenerated but rather last for life we further propose that these cells can still be a resource for somatic reprogramming when they cease to be considered safe for sexual reproduction.
Total mRNA was isolated for samples from young and climacteric mice (pools of 20 ooplasts and 20 SCNT blastocysts in duplicate) using a RNeasy Mini Kit (Qiagen Valencia CA USA) as described by the manufacturer (no DNase treatment). RNA concentrations as well as purity and integrity check were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit (Agilent Technologies Palo Alto CA USA). RiboAmp HS Plus Amplification Kit (MDS Analytical Technologies Ismaning Germany) was used to amplify the total mRNA with two rounds of amplification according to the manufacturerâ??s instructions. Amplified RNA was eluted with 15 ÂµL RE Buffer included in the RiboAmp HS Plus Amplification Kit. RNA concentrations were determined with Agilent Bioanalyzer 2100 and RNA Pico 6000 Lab-Chip Kit. Turbo Labeling CY3 Kit (MDS Analytical Technologies) was used to label 3 Âµg of amplified RNA with Cy3. Concentration and frequency of incorporation were measured with NanoPhotometer (Implen). Microarray wash and detection of the labeled RNA on GeneChips were carried out according to the manufacturerâ??s instructions (Agilent Technologies). Gene expression profiling was performed using Agilent Whole Mouse Genome Oligo Microarrays (4 x 44k each array containing 41174 features). Array image acquisition and feature extraction was performed using Agilent G2505B Microarray Scanner and Feature Extraction software v.9.5 (Agilent Technologies).

overall_design: 6 samples were analyzed. YoungOoplasts: young ooplasts 1 biological rep ClimactericOoplasts: climacteric ooplasts 1 biological rep YoungBlastocysts: young blastocysts 2 biological rep ClimactericBlastocysts: climacteric blastocysts 2 biological rep

first: Sebastian

last: Balbach

first: Martina

last: Sinn

first: Martin

last: Pfeiffer

first: Michele

last: Boiani