title: Gut Microbiota Orchestrates Energy Homeostasis during Cold [16S rRNA gene V4-region miSeq]

status: Public on Dec 03 2015

city: Genève

first: Ozren

last: Stojanovic

address: 1, rue Michèl-Servet

department: Faculté de Médecine, Dept. PHYM

institute: University of Geneva

summary: Microbial functions in the host physiology are a result of co-evolution between microbial communities and their hosts. Here we show that cold exposure leads to marked shift of the microbiota composition, referred to as cold microbiota. Transplantation of the cold microbiota to germ-free mice is sufficient to increase the insulin sensitivity of the host, and enable complete tolerance to cold partly by promoting the white fat browning, leading to increased energy expenditure and fat loss. During prolonged cold however, the body weight loss is attenuated, caused by adaptive mechanisms maximising caloric uptake and increasing intestinal, villi and microvilli lengths. This increased absorptive surface is promoted by the cold microbiota - effect that can be diminished by co-transplanting the most downregulated bacterial strain from the Verrucomicrobia phylum, Akkermansia muciniphila, during the cold microbiota transfer. Our results demonstrate the microbiota as a key factor orchestrating the overall energy homeostasis during increased demand.

overall_design: C57BL/6J mice were put for 31 day on cold (6C) or room temperature. Fresh feces and cecum samples were collected, immediately frozen and stored. Bacterial DNA content was extracted using QIAamp Fast DNA stool Mini Kit (Qiagen). Bacterial DNA was PCR amplified with barcoded universal bacterial primers targeting variable regionV4 of 16SrRNA gene. Samples were pooled and sequenced with Ilumina MiSeq platform. Using QIIME and custom scripts, sequences were quality filtered and demultiplexed using exact matches to the supplied DNA barcodes. Resulting sequences were then searched against the Greengenes reference database of 16S rRNA gene sequences, clustered at 97% by uclust. The longest sequence from each Operation Taxonomic Unit (OTU) thus formed was then considered as the OTU representative sequence, and assigned taxonomic classification via Mothur's Bayesian classifier, trained against the Greengenes database clustered at 98%. Each listed sample is indivudual biological replicate.

first: Claire

last: Chevalier

first: Mirko

last: Trajkovski


title: HOX13 activity reprograms cis-regulatory modules during digit development (ChIP-Seq)

status: Public on Dec 14 2016

city: montreal

first: Rushikesh

last: Sheth

address: 110 avenue des pins ouest

department: Genetics and Development

country: Canada

institute: IRCM

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The combinatorial expression of the Hox genes along the body axes, referred to as the HOX code, is a major determinant of cell fate and plays a prevailing role in generating the animal body plan. In developing limb buds, the paralogous group 13 genes of the HoxA and HoxD clusters are essential for patterning the distal-most limb structures, the digits. Inactivation of HOXA13 and HOXD13 transcription factors (HOX13) leads to complete digit agenesis in mice, but how HOX13 regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here we performed genome-wide profiling of HOX13 by chromatin immunoprecipitation and analyzed the transcriptome and chromatin state of wild type early and late-distal limb buds, as well as Hoxa13-/-;Hoxd13-/- compound mutant limb buds. Our results show that inactivation of HOX13 impairs the activation and repression of putative cis-regulatory modules specific to the late-distal limb cells. Loss of HOX13 also disrupts the specific, spatial patterning of gene expression along the proximal-distal axis of the developing limb buds. These results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.

overall_design: dissected distal parts of e11.5 forelimb, of wild-type as well as Hoxa13-/-;Hoxd13-/- mice

first: Iros

last: Barozzi

first: David

last: Langlais

first: Marco

last: Osterwalder

first: Stephen

last: Nemec

first: H

middle: S

last: Stadler

first: Axel

last: Visel

first: Jacques

last: Drouin

first: Marie

last: Kmita


title: A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus

status: Public on May 19 2016

city: Yokohama

first: Nayuta

last: Yakushiji-Kaminatsui

state: Kanagawa

address: 1-7-22 Suehiro-cho, Tsurumi-ku

country: Japan

institute: RIKEN Center for Integrative Medical Sciences

type: Expression profiling by high throughput sequencing

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Dexamethasone stimulated gene expression in peripheral blood

status: Public on Nov 03 2015

city: Munich

first: Janine

last: Arloth

address: Kraepelinstr. 2-10

country: Germany

institute: Max Planck Institute of Psychiatry

summary: Whole genome gene expression data (baseline and following GR-stimulation with 1.5 mg dexamethasone p.o.) were analysed to identify glucocorticoid receptor (GR)-mediated changes in gene expression.

overall_design: A Dexamethasone Suppression Test was performed in 297 subjects. Baseline and stimulated (3 hours after 1.5 mg dexamethasone p.o.) whole blood samples were analyzed using Illumina Human HT-12 v3 and v4 arrays.

first: Elisabeth

middle: B

last: Binder


title: Characterization of perivascular MSC from the adult human brain

city: Muenster

first: Marcos

middle: J.

last: Araúzo-Bravo

address: Rogentstrasse

department: Cell and Developmental Biology

institute: Max Planck Institute for Molecular Biomedicine

summary:
Brain perivascular cells have been recently identified as new mesodermal cell type of the human brain.
These cells reside in the perivascular niche and were shown to have mesodermal and – to a lesser extend –
tissue-specific differentiation potential.
Mesenchymal stem cells (MSCs) are widely discussed for the use in cell therapy in many neurological disorders.
Therefore it is of importance to better understand the “intrinsic” MSC population of the human brain.
Here we systematically characterized adult human brain-derived pericytes during in vitro expansion and differentiation
and compared these cells to fetal and adult human brain-derived NSCs and adult human bone marrow derived MSCs.
We found that adult human brain pericytes can be isolated from hippocampal as well as cortical white matter,
are – in contrast to adult human NSCs – easily expandable in monolayer cultures and show high similarities to human bone
marrow-derived MSCs both regarding surface marker expression and whole transcriptome analysis.
Human brain pericytes differentiated only in negligible amounts into neuroectodermal cell types using various differentiation
conditions but efficiently differentiated into mesodermal progenies.
Thus bone marrow-derived MSCs resemble human brain pericytes and might be therefore very interesting for possible
autologous NPC-based treatment strategies,
cell therapeutic approaches of neurological diseases.
For the gene expression microarray analysis we used the Affymetrix U133A chips
The whole procedure was performed following the manufacturer's standard protocol (Affymetrix, Santa Clara, CA).
For the data processing, normalization was calculated with the GCRMA (GC content corrected Robust Multi-array Analysis) algorithm.
Data post-processing and graphics was performed with in-house developed functions in Matlab.

overall_design:
17 samples were analyzed
fNSC, Neural Stem Cell, 2 replicates
ANPC-hip, adult Neuroprogenitor - Cell Hippocampus, 3 replicates
ANPC-wm, adult Neuroprogenitor Cell - White Matter, 3 replicates
ABPMC-hip, adult Brain Perivascular Mesodermal Cell - Hippocampus, 3 replicates
ABPMC-wm, adult Brain Perivascular Mesodermal Cell - White Matter, 3 replicates
MSC, Mesenchymal Stem Cell, 3 replicates

first: Xenia

last: Lojewski

first: Sumitra

last: Srimarson

first: Juliane

last: Rauh

first: Silvan

last: Franke

first: Manja

last: Wobus

first: Verdon

last: Taylor

middle: J

first: Susanne

last: Hallmeyer-Elgner

first: Matthias

last: Kirsch

first: Johannes

last: Schwarz

first: Alexander

last: Storch

first: Andreas

last: Hermann


title: A Comprehensive Atlas of Arabidopsis Regulatory DNA [DAP-seq]

status: Public on May 25 2016

city: La Jolla

first: Joseph

middle: R

last: Ecker

state: CA

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

country: USA

institute: HHMI-Salk-Institute

summary: We have developed a high throughput, next-generation DNA sequencing assay for rapid transcription factor binding site (TFBS) discovery in a genomic context. DNA affinity purification sequencing (DAP-seq), which uses affinity-purified transcription factors (TFs) to capture genomic DNA fragments, was applied to all 1,725 Arabidopsis thaliana TFs. High confidence TFBS motifs for 529 TFs and genome-wide enrichment maps for 349 factors were identified. In total,~ 2.7 million TFBS were identified which predict thousands of TF target genes enriched for known and novel functions.. Comparison of TF-binding using cytosine-methylated and -unmethylated genomic DNA revealed a 2-50 fold inhibition at methylated motifs for ~82% (264) of factors tested while 4.6% (15) showed stronger binding to methylated motifs. Finally, we describe how binding of Arabidopsis and maize Auxin Response Factors (ARFs) at phased motif repeats is highly enriched at ARF target gene promoters and how this architecture may allow for stabilization of dimers/multimers.

sample_organism:
Arabidopsis thaliana
Zea mays

platform_organism:
Arabidopsis thaliana
Zea mays

overall_design:
Identification of sequence motifs for 530 transcription factors (529 Arabidopsis thaliana, 1 Zea mays) and genome-wide binding sites for 350 transcription factors (349 Arabidopsis thaliana, 1 Zea mays) by direct sequencing of affinity purified genomic DNA fragments.  Idenfication of binding sites for 343 transcription factors (Arabidopsis thaliana) by direct sequencing of affinity purified, PCR amplified DNA libraries. Comparison to ChIP-Seq for ABI5 (AT2G36270).
The 530 transcription factors refer to the number of unique proteins  assayed on non-amplified DNA libraries (i.e. "characteristics: DNA  source" = "col" or "zm").  Additional 5 factors have only data on amplified libraries (i.e. "characteristics: DNA source = colamp"), which do not represent the natural DNA methylation state of   the organism (therefore not counted; more details in the "description" field). A subset of 350 of the 530 have both motif and TFBS identification (i.e. "characteristics: subset = TFBS+motif"), and the rest 180 have only motif identification (i.e. "characteristics: subset = motif").

first: Ronan

middle: C

last: O'Malley

first: Shao-shan

last: Huang

first: Liang

last: Song

first: Mathew

middle: G

last: Lewsey

first: Anna

last: Bartlett

first: Mary

last: Galli

first: Andrea

last: Gallavotti


title: Loss of immunosuppressive and anti-inflammatory properties in transformed human mesenchymal stem cells

status: Public on May 14 2015

first: Marcos

last: Araúzo-Bravo

summary: We used collection of sequentially mutated BM-hMSCs ranging from wild type (no oncogenic hits) to fully transformed hMSCs (targeted with up six oncogenic mutations)to address whether BM-hMSCs at different stages of a well-characterized oncogenic process (normal, immortalized, transformed) retain immunomodulatory properties in vitro and in vivo. We characterize, for the first time, an oncogenic transformation-associated loss of the immunesuppressive and anti-inflammatory properties by hMSCs and identify candidate immune effectors underlying the loss of immunomodulation in transformed hMSCs.

overall_design: Wild type BM-hMSCs (MSC-0H-GFP) did not develop tumors, meanwhile hMSCs transformed sequentially transformed with 5 oncogenic lesions (p53 inactivation; Rb inactivation; expression of hTERT; stabilization of c-myc; and expression of oncogenic H-RAS) (MSC-5H-GFP) cells gave rise to undifferentiated sarcomas when inoculated into immunedeficient mice. The tumoral transformation process causes the loss of the immunosuppressive and anti-inflamatory propierties of MSC-5H-GFP cells as compared to those of the wild type MSC-0H-GFP cells. To understand the basis of this loss of immunosuppression in transformed hMSCs, gene expression analysis was performed to create the list of differentially expressed genes between MSC-0H-GFP and MSC-5H-GFP cells. Further analysis of altered signaling pathways and upstream regulators were performed using the Ingenuity Pathway Analysis software.

first: Rene

last: Rodriguez

first: Pablo

last: Menendez

first: Mario

last: Delgado


title: Germ cell nuclear factor regulates gametogenesis in developing gonads

status: Public on May 08 2015

summary:
Expression of germ cell nuclear factor (GCNF, Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.
For RNA isolation, PGCs were sorted by FACS, collected by centrifugation, lysed in RLT buffer (QIAGEN), and processed using the RNeasy micro and mini kits with on-column DNase digestion as per the manufacturer’s instructions. Integrity of RNA samples was quality checked using a 2100 Bioanalyzer (Agilent). When 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 hr 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 hr 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 an iScan reader and accompanying software. 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.

overall_design:
8 samples were analyzed:
ESC++, ESC control Oct4-GFP, 1 replicate
ESC--, ESC Gcnf -/- Oct4-GFP, 1 replicate
D15++, Day 15 in vitro derived PGCs from GCNF +/+ Oct4-GFP ESC, 1 replicate
D15--, Day 15 in vitro derived PGC from GCNF -/- Oct4-GFP ESC, 1 replicate
D12++, Day 12 in vitro derived PGC from GCNF +/+ Oct4-GFP ESC, 1 replicate
D12--, Day 12 in vitro derived PGC from GCNF -/- Oct4-GFP ESC, 1 replicate
EpiSC, EpiESC, 2 replicates

first: Davood

last: Sabour

first: Xueping

last: Xu

first: Arthur

last: Chung

first: Damian

middle: L

last: Menuet

first: Kinarm

last: Ko

first: Natalia

last: Tapia

first: Luca

last: Gentile

first: Boris

last: Greber

first: Karin

last: Hübner

first: Vittorio

last: Sebastiano

first: Guangming

last: Wu

first: Hans

last: Schöler

first: Austin

last: Cooney


title: Direct Conversion of Fibroblasts into Oligodendrocyte Progenitor Cells

status: Public on Nov 02 2015

first: Marcos

last: Araúzo-Bravo

summary: Many studies have already shown the reprogramming of somatic cells into other cell types such as neural stem cells, blood progenitor cells, and hepatocytes by inducing combinations of transcription factors. One of the recent development in cellular reprogramming is the direct reprogramming, that can change cell fate towards different lineages. This strategy provides an alternative to the use of pluripotent stem cells ruling out the concerns of tumorigenicity caused by undifferentiated cell populations. Here, we generated induced oligodendrocyte progenitor cells (iOPCs) from mouse fibroblasts by direct reprogramming. The generated iOPCs are homogenous, self-renewing, and multipotent. Once differentiated, the somatic stem cells exhibit morphological and molecular characteristics of oligodendrocyte progenitor cells (OPCs). Thus, we demonstrated that terminally differentiated somatic cells can be converted into functional iOPCs by induction of transcription factors offering a new strategies to cure myelin disorders.

overall_design: To identify the global gene expression profiles of iOPCs, we analyzed total 6 samples.

first: Hyunah

last: Lee

first: Myung

last: Park

last: Arauzo-Bravo

first: Holm

last: Zaeres

first: Jeong

last: Kim


title: Human PGC commitment shares key gene dynamics to mouse, but owns a unique PRDM14 expression pattern

status: Public on Apr 06 2015

summary:
The  molecular mechanisms of human primordial germ cell (PGC) specification are poorly understood due
to the inaccessibility of cell materials and the lack of an alternative in vitro model that enables
tracking of the earliest stages of germ cell development.
Here, we introduce a defined and efficient differentiation system for the induction of pre-migratory PGC-like cells
from human embryonic stem cells (ESCs)
and induced pluripotent stem cells (iPSCs).
By step-wise differentiation, we generated an OCT4+/ T+/BLIMP1+ cell population that transitioned into
STELLA expressing PGC-like cells
that exhibited a similar key gene expression as mouse PGCs as well as global epigenetic reprogramming.
Even though, these PGC-like cells expressed PRDM14 at very low levels,
they underwent activation of pluripotency/PGC genes,
suppression of neural induction and suppression of de novo DNA methylation,
events that are regulated by Prdm14 during mouse PGC specification.
This study demonstrates that human PGC commitment shares many key features with mouse PGC specification,
but harbors unique and so far unknown mechanisms that,
point to a novel human transcriptional regulation.

overall_design:
7 samples were analyzed.
ESC: Human Embryonic Stem Cells, 1 biological rep
iPSC: Human induced Pluripotent Stem Cells, 1 biological rep
d2: Human induced Pluripotent Stem Cells, 2 days differentiation treatment , 2 biological rep
d4PGCLC: Human induced Pluripotent Stem Cells, 4 days differentiation treatment  towards Primordial Germ Cells Like Cells, 1 biological rep
d6PGCLC: Human induced Pluripotent Stem Cells, 6 days differentiation treatment  towards Primordial Germ Cells Like Cells, 2 biological rep

first: Fumihiro

last: Sugawa

first: Kee-Pyo

first: Martin

last: Stehling

last: Hubner

last: Scholer


title: Transcriptional Landscape of Psoriasis Identifies the Involvement of IL36 and IL36RN

status: Public on Apr 24 2015

city: Perth

first: Sulev

last: Koks

state: Western Australia

address: 90 South Street

department: Centre for Molecular Medicine and Innovative Therapeutics

country: Australia

institute: Murdoch University

summary:
Background: In present study we performed whole transcriptome analysis in plaque psoriasis patients and compared lesional skin with non-lesional skin and with the skin from healthy controls. We sequenced total RNA from 12 lesional (LP), 12 non-lesional (NLP) and from 12 normal (C) skin biopsies.
Results: Compared with previous gene expression profiling studies we had three groups under analysis - LP, NLP and C. Using NLP samples allows to see the transcriptome of visually normal skin from psoriasis patient. In LP skin S100A12, S100A7A, LCE3E, DEFB4A, IL19 were found up regulated. In addition to already well-described genes, we also found several other, not so widely recognized transcripts, related to psoriasis. Namely, KLK9, OAS2, OAS3, PLA2G, IL36G, IL36RN were found to be significantly and consistently related to the psoriatic lesions. Ingenuity pathway analysis was used to define functional networks significantly enriched in the studied samples. The genes up-regulated in the LP samples were related to the innate immunity, IL17 and IL10 networks. In NLP samples innate immunity and IL17 network were activated, but activation of IL10 network was not evident. The transcriptional changes characteristic in the NLP samples can be considered as a molecular signature of “dormant psoriasis”.
Conclusions: Taken together, our study described the transcriptome profile characteristic for LP and NLP psoriatic skin. RNA profile of the NLP skin is in between the lesional and healthy skin, with its own specific pattern. We found that both LP and NLP have up-regulated IL17 network, whereas LP skin has up regulated IL10 related cytokines (IL19, IL20, IL24). Moreover, IL36G and IL36RN were identified as strong regulators of skin pathology in both LP and NLP skin samples, with stronger influence in LP samples.

overall_design: 36 samples, 24 from psoriasis and 12 from controls

first: Maris

last: Keermann

last: Kõks

first: Ene

last: Reimann

first: Ele

last: Prans

first: Kristi

last: Aabram

first: Külli

last: Kingo


title: A mechanism for the segregation of age in mammalian neural stem cells

status: Public on Sep 22 2015

last: Araúzo-Bravo

summary: Across life neural stem cells (NSCs) generate new neurons in the mammalian brain through asymmetric neurogenic and self-renewing cell divisions. However, the cellular mechanisms underlying NSC asymmetry remain unknown. Using fluorescence loss in photobleaching (FLIP) we here show that NSCs in vitro and within the developing forebrain generate a lateral diffusion barrier during cell division resulting in asymmetric segregation of cellular components. The strength of the diffusion barrier is dynamically regulated with age and depends on the proper function of lamin-associated nuclear envelope constituents. Strikingly, age-associated or experimental impairment of the diffusion barrier disrupts asymmetric segregation of damaged proteins, a product of aging. Thus, the data presented here identify a mechanism how age is asymmetrically distributed during somatic stem cell division.

overall_design:
6 samples were analyzed
YoungNSC, 3 replicates
OldNSC, 3 replicates

first: Darcie

last: Moore

first: G

middle: A

last: Pilz

first: Y

last: Barral

first: Sebastian

last: Jessberger


title: Human adult white matter progenitor cells are multipotent neuroprogenitors identical  to adult hippocampal progenitors

status: Public on Jun 10 2016

first: Marcos

summary: Adult neural progenitor cells (aNPCs) are a potential autologous cell source for cell replacement in neurologic diseases such as Parkinson’s disease or stroke or for cell-based gene therapy for neurometabolic diseases.  Easy accessibility,  long-term expandability and detailed characterization of NPC properties are important requisites for their future  translational/clinical applications. aNPC can be isolated from different regions of the adult human brain including the accessible subcortical white matter (aNPCWM), but systematic studies comparing long-term expanded aNPCWM with aNPC from neurogenic brain regions to check for their  NPC characteristics and performance are not available.  Freshly isolated cells from subcortical white matter and hippocampus (aNPCHIP) expressed oligodendrocyte progenitor cell (OPC) markers such as A2B5,  NG2 and OLIG2 in ~20% of cells but no neural stem cell (NSC) markers such CD133 (Prominin1),  NESTIN, SOX2 or PAX6.  The EGF receptor (EGFR) protein was expressed in 18% of aNPCWM and 7% of aNPCHIP,  but only a small fraction of 1 cell out 694 cells from white matter and only 1 out of  1,331 hippocampal cells were able to generate neurospheres.  Studies comparing subcortical aNPCWM with their hippocampal counterparts showed that both  NPC types expressed mainly markers of glial origin such as NG2,  A2B5 and OLIG2,  and the NSC/NPC marker Nestin,  but no pericyte markers.  Both NPC types were able to produce fully mature neurons,  astrocytes and oligodendrocytes in comparable amounts to fetal NSC.  Whole transcriptome analyses finally confirmed the strong similarity  of aNPCWM with aNPCHIP.  Our data show that aNPCWM are multipotent NPC with long-term expandability capacity similar to  NPC from hippocampus making them an easily accessible source for possible autologous NPC-based treatment strategies. Isolation and propagation of multipotent NPCs. Adult human hippocampal (hip) and subcortical white matter (wm) tissue was obtained from routine epilepsy surgery procedures following informed consent of the subjects. All procedures were in accordance with the Helsinki convention and approved by the Ethical Committee of the University of Dresden (No. 47032006).  All subjects underwent high-resolution magnetic resonance imaging excluding tumors and were screened for the presence of infectious disease.  In all cases the neuropathological examinations did not reveal evidences for tumor formation. Gene expression Single-channel oligonucleotide microarray analysis. For the gene expression microarray analysis we used the Affimetrix U133A chips containing  22.215 probe sets representing at least 12.905 individual genes.  The whole procedure was performed following the manufacturer’s standard protocol (Affimetrix, Santa Clara, CA).  For the data processing,  normalization was calculated with the GCRMA (GC content corrected Robust Multi-array Analysis) algorithm.

overall_design:
fNSC:     Human fetal neural stem cells, 2 biological rep
aNPChip:  Human adult neural progenitor cells isolated from hippocampus, 3 biological rep
aNPCwm:   Human adult neural progenitor cells isolated from white matter, 2 biological rep

first: Xenia

last: Lojewski

first: Andreas

last: Hermann

first: Florian

last: Wegner

last: Arauzo-Bravo

first: Susanne

last: Hallmeyer-Elgner

first: Matthias

last: Kirsch

first: Johannes

last: Schwarz

first: Hans

last: Schoeler

first: Alexander

last: Storch


title: Breaking the first lineage barrier: Direct induction of trophoblast stem cells from murine fibroblasts

status: Public on Oct 01 2015

summary: Trophoblast stem cells represent the stem cell population of the extra-embryonic lineage and arise as a result of the first cell fate decision. From blastocyst stage onwards, a distinct epigenetic lineage barrier strictly separates mouse embryonic and extra-embryonic lineages. Recently, it has been shown that this epigenetic barrier cannot be fully overcome as the expression of TS-determining factors in embryonic stem cells lead to incomplete transdifferentiation. Here, we demonstrate that transient expression of Tfap2c, Gata3, Eomes and Ets2 in fibroblasts suffices to generate cells which are almost identical to trophoblast stem cells based on morphology, expression and methylation pattern. Further, these induced trophoblast stem cells display transgene independent self-renewal, differentiate along the extra-embryonic lineage and chimerize the placenta upon blastocyst injection. Our findings provide insights into the transcription factor networks governing trophoblast stem cell identity and offer a new tool for studying the hierarchy of those factors.

overall_design:
11 samples were analyzed:
MEF4F, mouse embyronic fibroblasts (MEFs) transduced with Tfap2c, Gata3, Eomes and Ets2, 3 replicates.
TS, mouse TS-EGFP trophoblast stem cells, 2 replicates.
iTSC4F5, mouse induced trophoblast stem (TS) cells clone A from 4 factor transduced MEFs, 3 replicates.
iTSC4F8, mouse induced trophoblast stem (TS) cells clone D from 4 factor transduced MEFs, 3 replicates.

first: Caroline

last: Kubaczka

first: Claire

middle: E

last: Senner

first: Monika

last: Cierlitza

first: Peter

last: Kuckenberg

first: Michael

last: Peitz

first: Myriam

last: Hemberger

first: Hubert

last: Schorle


title: Inherited and somatic defects in DDX41 in myeloid neoplasms

status: Public on Apr 01 2015

city: Cleveland

first: Bartlomiej

last: Przychodzen

state: Ohio

address: 9600 Euclid Avenue

department: THOR

institute: Cleveland Clinic

type:
Genome variation profiling by SNP array
SNP genotyping by SNP array

summary: Most cases of adult myeloid neoplasms are routinely assumed to be sporadic.  Here, we describe an adult familial acute myeloid leukemia (AML) syndrome caused by germline mutations in the DEAD/H-Box helicase gene DDX41. DDX41 was also found to be affected by somatic mutations in sporadic cases of myeloid neoplasms as well as in a biallelic fashion in 50% of patients with germline DDX41 mutations. Moreover, corresponding deletions on 5q35.3 present in 6% of cases lead to haploinsufficient DDX41 expression. DDX41 lesions caused altered pre-mRNA splicing and RNA processing.  DDX41 is exemplary of other RNA helicase genes also affected by somatic mutations, suggesting that they constitute a family of tumor suppressor genes.

overall_design: Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from cryopreserved diagnostic bone marrow or peripheral blood samples.

first: Chantana

last: Polprasert


title: Asthma II

status: Public on Nov 14 2014

city: Pittsburgh

first: Jadranka

last: Milosevic

state: PA - Pennsylvania

address: 2330 Tilbury Avenue

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 asmathic (notSA) and severe asthmatic (SA) patients.   For demographics data, contact Dr.Sally Wenzel (wenzelse@upmc.edu)

overall_design: Bronchoscopy with endobronchial epithelial brushing was performed as previously described (Chu et al., 2002; Zhao et al., 2011). The bronchial brushings generally comprised >90% epithelial cells and were placed into Trizol for mRNA analysis. Total RNA extracted using Trizol according to the manufacturer's instructions. Cy3-CTP  labeled RNA was prepared according to the standard Agilent protocol from 50ng total RNA. Labeled RNA was hybridized for 17 hr at 65 C on Agilent gene expression array. Arrays were washed according to the manufacturer's protocol.   Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4X44K array slides.  Agilent Feature Extraction v10.7.3.1 was used with default parameters. Normalized signal intensity data  are presented in the matrix.  The data set was normalized by cyclic-LOESS with use of Bioconductor package as described previously (Wu W et. al. 2005)

first: Sally

last: Wenzel

first: Naftali

last: Kaminski


title: m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation

status: Public on Nov 02 2014

city: Rehovot

first: Noa

last: Novershtern

address: Weizmann Institute

department: Molecular Genetics

country: Israel

institute: Weizmann Institute of Science


title: Global transcript structure resolution of high gene density genomes through multi-platform data integration

status: Public on Jul 08 2016

city: New Orleans

first: Erik

middle: K

last: Flemington

state: LA

address: 1430 Tulane Ave., SL79

department: Pathology

institute: Tulane Health Sciences Center


title: H3K27 trimethylation in mouse peripheral nerve

city: Madison

first: John

last: Svaren

state: WI

address: 1500 Highland Ave.

department: Comparative Biosciences

institute: University of Wisconsin

summary: ChIP-seq of H3K27 trimethylation in sciatic nerve

overall_design: H3K27me3 ChIP-Seq with corresponding input

first: Holly

last: Hung


title: Cell Type Specific Enrichment Of Risk Associated Regulatory Elements At Ovarian Cancer Susceptibility Loci

status: Public on Apr 22 2015

city: Los Angeles

first: Simon

last: Gayther

address: 1441 Eastlake Ave.

institute: University of Southern California

summary: Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single nucleotide polymorphisms (SNPs) associated with cancer risk lie in non protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs), and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared to normal human mammary epithelial cells (HMECs) and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissues specific regulatory signatures to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P=3.8x10-30) OSECs (P=2.4x10-23) and HMECs (P=6.7x10-15) but not for EECs (P=0.45) or LNCaP cells (P=0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) indicating both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets germline genetic susceptibility variants for ovarian cancer

overall_design: FAIRE-Seq and ChIP-Seq of 2 different histone modifications in 5 cell types.

last: Coetzee

first: Howard

last: Shen

first: Dennis

last: Hazelett

first: Kate

last: Lawrenson

first: Karoline

last: Kucheunbaecker

first: Jonathan

last: Tyrer

first: Suhn

last: Rhie

first: Keren

last: Levanon

first: Alison

last: Karst

first: Ronny

last: Drapkin

first: Susan

last: Ramus

first: Fergus

last: Couch

first: Kenneth

last: Offit

first: Georgia

last: Chenevix-Trench

first: Alvaro

middle: N

last: Monteiro

first: Antonis

last: Antoniou

first: Matthew

last: Freedman

first: Gerhard

first: Paul

middle: D

last: Pharoah

first: Houtan

last: Noushmehr


title: Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

status: Public on Feb 20 2015

first: Marcos

last: Araúzo-Bravo

summary: Epigenetic memory in induced pluripotent stem cells (iPSCs), with regards to their somatic cell type of origin, might lead to variations in their differentiation capacities. In this context, iPSCs from human CD34+ hematopoietic stem cells (HSCs) might be more suitable for hematopoietic differentiation than commonly used fibroblast-derived iPSCs. To investigate the influence of an epigenetic memory on the ex vivo expansion of iPSCs into erythroid cells, we compared iPSCs from human neural stem cells (NSCs) and human cord blood-derived CD34+ HSCs and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells (RBCs). Although genome-wide DNA methylation profiling at all promoter regions demonstrates an epigenetic memory of iPSCs with regards to their somatic cell type of origin, we found a similar hematopoietic induction potential and erythroid differentiation pattern. All human iPSC lines showed terminal maturation into normoblasts and enucleated RBCs, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the CD34+ HSC-derived iPSCs. More detailed methylation analysis of the hematopoietic and erythrocyte promoters identified similar CpG methylation levels in the CD34+ iPSCs and NSC iPSCs, which confirms their comparable erythroid differentiation potential.

overall_design:
To investigate the influence of an epigenetic memory on the ex vivo expansion of iPSCs into erythroid cells, we compared iPSCs from human neural stem cells (NSCs) and human cord blood-derived CD34+ HSCs and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells (RBCs).
RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300ng of total RNA per sample was used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating the biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates.
8 samples were analyzed:
CD34 1, Human CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) population 1, 1 replicate
CD34 2, Human CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) population 2, 1 replicate
CD34 OSiPS 1, Human Human two factors (POU5F1, SOX2) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 1, 1 replicate
CD34 OSKMiPS 1, Human Human four factors (POU5F1, SOX2, KLF4, CMYC) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 1, 1 replicate
CD34 OSiPS 2, Human Human two factors (POU5F1, SOX2) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 2, 1 replicate
CD34 OSKMiPS 2, Human Human four factors (POU5F1, SOX2, KLF4, CMYC) induced Pluripotent Cell (iPSC) reprogrammed from CD34+ Cord blood CD34+ Hematopoyetic Stem Cell(HSC) induced Pluripotent Cell (iPSC) population 2, 1 replicate
H1, Human H1 embryonic stem cell (ESC), 1 replicate
H9, Human H9 embryonic stem cell (ESC), 1 replicate

first: Isabel

last: Dorn

first: Katharina

last: Klich

first: Martina

last: Radstaak

first: Simon

last: Santourlidis

first: Foued

last: Ghanjati

first: Teja

last: Radke

first: Katherina

last: Psathaki

first: Gunnar

last: Hargus

first: Jan

last: Kramer

first: Jeong

last: Kim

first: Gesine

last: Kögler

first: Peter

last: Wernet

first: Hans

last: Schöler

last: Schlenke

first: Holm

last: Zaehres


title: m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation (RNA-Seq)

first: Noa

last: Novershtern

summary: Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N6-methyladenosine (m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout pre-implantation epiblasts and naïve embryonic stem cells (ESCs) are depleted for m6A in mRNAs and yet, are viable. However, they fail to adequately terminate their naïve state, and subsequently undergo aberrant and restricted lineage priming at the post-implantation stage, leading to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo, and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

overall_design: polyA RNA-seq was measured in mouse embryonic stem cells (ESCs) and embroid bodies (EBs), each in WT and in Mettl3-KO cell lines. RNA-seq was measured also from WT mouse embronic fibroblasts (MEF). 3 biological replicates are available from ESCs and 2 from EBs. Replicate C in ESCs was measured alongside protein levels (SILAC) and was used for the analysis of that assay.

first: Sharon

last: Moshitch-Moshkovitz

first: Shay

last: Geula

first: Nitzan

last: Kol

first: Gidi

last: Rechavi

first: Jacob

last: Hanna


title: In vitro, genomic context identification of transcription factor binding sites

first: Joseph

last: Ecker

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Nanog-induced intestinal and colonic hyperplasia

status: Public on Aug 11 2014

first: Marcos

last: Araúzo-Bravo

summary:
Though expression of the homeobox transcription factor Nanog is generally restricted to pluripotent cells and early germ cells,  recent reports have found that Nanog is re-expressed in somatic and germ cell tumors associated with poor differentiation and aggressive disease progression.
To elucidate its oncogenic properties, a modified Tet-On system was utilised to generate Nanog-inducible mice.
By dexamethasone and doxycycline injection, similar Nanog expression levels as in wild-type embryonic stem cells were obtained in all major organs except for brain.
Ectopic Nanog expression resulted in intestinal and colonic epithelium hyperplasia-intestinal villi had doubled in length and hyperplastic epithelium outgrowths were seen after 7 days.
The tumor suppressor gene Cdx2 was downregulated at onset of Nanog expression, suggesting that crosstalk between Nanog and Cdx2 is conserved from early embryo development to adulthood.
Although the mice died before they could form tumors, Cdx2 repression and intestinal hyperplasia links Nanog to tumor initiation.

overall_design:
8 samples were analyzed.
Intestine-: Mouse intestine cells without Nanog overexpression, 2 biological rep
Intestine+: Mouse intestine cells with Nanog overexpression, 2 biological rep
Colon-: Mouse colon cells without Nanog overexpression, 2 biological rep
Colon+: Mouse colon cells with Nanog overexpression, 2 biological rep

first: Gerrit

last: Fishedick

first: Guandming

last: Wu

first: Boris

last: Greber

first: Gabriele

last: Köhler

first: Natalia

last: Tapia

first: Roberto

last: Iacone

first: Konstantinos

last: Anastassiadis


title: Transcriptome profiling of purified mouse mammary stem, progenitor and mature cell populations

status: Public on Mar 15 2015

city: Parkville

first: Matthew

last: Ritchie

state: Victoria

address: 1G Royal Parade

department: Epigenetics and Development Division

institute: The Walter and Eliza Hall Institute of Medical Research

summary:
Purpose: The aim of this study is to determine the absolute and relative expression levels of mRNA transcripts across multiple flow cytometrically sorted epithelial cell types including freshly isolated CD24+CD29hi mammary stem cell-enriched basal cells (MaSC/basal), CD24+CD29loCD61+ luminal progenitor-enriched (LP) and the CD24+CD29loCD61- mature luminal-enriched (ML) cell populations. Additionally, a comparison between these primary cell types and cultured MaSC/Basal-derived mammosphere cells (mammosphere) and the CommaD-βGeo (CommaDβ) cell line was performed.

Methods: Total RNA was extracted and purified from sorted luminal or basal populations from the mammary glands of female virgin 8- to 10-week-old FVB/N mice (3 independent samples per population), MaSC/Basal cells cultured for 1 week under mammosphere conditions and CommaDβ cells grown under maintenance conditions (Deugnier et al. 2006). Total RNA (100 ng) was used to generate sequencing libraries for whole transcriptome analysis following Illumina’s TruSeq RNA v2 sample preparation protocol. Completed libraries were sequenced on HiSeq 2000 with TruSeq SBS Kit v3- HS reagents (Illumina) as 100 bp single-end reads at the Australian Genome Research Facility (AGRF), Melbourne. Approximately 30 million 100 bp single-end reads were obtained for each sample. Reads were aligned to the mouse reference genome mm10 and mapped to known genomic features at the gene level using the Rsubread package (version 1.14.1) (Liao et al. 2013). Single reads were then summarized into gene-level counts using FeatureCounts (Liao et al. 2014).

overall_design: Total RNA was extracted and purified from sorted luminal or basal populations from the mammary glands of female virgin 8- to 10-week-old FVB/N mice (3 independent samples for population), MaSC/Basal cells cultured for 1 week under mammsophere conditions and CommaDβ cells grown under maintenance conditions (Deugnier et al. 2006) and their transcriptomes analysed by RNA-Seq.


title: Interactions of ARF6 with PIF4, BZR1, and RGA integrate auxin signaling with environmental and other hormonal signals in Arabidopsis

status: Public on Jun 03 2014

city: Stanford

first: Eunkyoo

last: Oh

address: 260 Panama Street

department: Plant Biology

institute: Carnegie Institution


title: m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation (3p-Seq)

first: Noa

last: Novershtern

summary: Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here we identify Mettl3, an N6-Methyladenosine (m6A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout pre-implantation epiblasts and naïve embryonic stem cells (ESCs) are depleted for m6A in mRNAs and yet, are viable. However, they fail to adequately terminate their naïve state, and subsequently undergo aberrant and restricted lineage priming at the post-implantation stage, leading to early embryonic lethality. m6A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo, and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.

overall_design: 3' polyA RNA-sequencing (equivalent to Digital Gene Expression) measured in mouse Embryonic Stem Cells (ESCs) and mouse Embriod bodies (EBs) 0,4 & 8 hours after treatment with Actinomycin which halts transcription. Measured in both WT and Mettl3-KO cells.


title: Dissecting the specific role of Oct4 during the early stage of reprogramming

status: Public on May 13 2014

summary:
Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs)  following the forced overexpression of a specific combination of transcription factors, of which Oct4 is essential. To better understand the mechanism underlying iPSC reprogramming, we investigated the immediate transcriptional response to ectopic Oct4 expression in somatic cells. To this end, we derived different somatic cell types from Oct4-inducible transgenic mice and induced Oct4 expression for different time periods. Using microarray analysis, we have identified early transcriptional Oct4 targets, including genes not previously known to interact with Oct4. Comparison of the sets of Oct4-regulated genes revealed pronounced differences among the individual cell types. Furthermore, a notable up-regulation of pluripotent markers could not be detected. In contrast, we observed a down-regulation of somatic markers specific to each cellular population. This finding suggests that Oct4 interferes with the somatic transcriptional program in a cell type-specific manner, thereby promoting an unstable epigenetic state that facilitates reprogramming rather than the direct initiation of a pluripotent gene expression network. Finally, we show that Mgarp, a gene expressed in pluripotent cells, is commonly up-regulated in all cell types after Oct4 induction. Unexpectedly, Mgarp expression decreases during the first steps of reprogramming due to a Klf4-dependent inhibition. Our data show that Oct4 counteracts Klf4 repressive activity and enhances Mgarp expression at the late stages of reprogramming. This temporal pattern of Mgarp expression is crucial for the efficient generation of iPSCs. In summary, our study provides new insights into the specific role that Oct4 plays during the early stages of reprogramming.
Different somatic cell types were derived from transgenic mice containing both a tetracycline-inducible transactivator (rtTA-M2) and a tetracycline operon-controlled Oct4 expression cassette (TO) (Hochedlinger 2005, Cell 121:465-477) that were mated with GOF18-GFP mice in which the green fluorescent protein (GFP) is driven by 18 kb of the Oct4 regulatory region (Oct4-GFP) (Yoshimizu 1999, Dev Growth Differ 41:675-684), thus indicating endogenous Oct4 expression. In addition, a control neural stem cell (CtrlNSC) line was generated from transgenic mice that entail a tetracycline-inducible transactivator (irtTA-VP16-GBD) (Anastassiadis 2002, Gene 298:159-172) plus an OG2 Oct4-GFP reporter transgene (Yoshimizu 1999),but lack a TO cassette. A second control consisted of wild type (C57BL/6) mouse embryonic fibroblasts (CtrlMEF). Oct4-inducible mouse embryonic fibroblasts (TO-MEFs) and neural stem cell (TO-NSCs) were derived from 14.5 days post coitum (d.p.c.) embryos and cultured as previously described (Conti 2005, PLoS Biol 3:e283). Primary bone marrow cells (TO-BMCs) were isolated from 6-week old mice after flushing femora and tibiae and plated onto dishes with a combined coating of gelatin (PAA, Pasching, Austria), laminin (Santa Cruz Biotechnology, Santa Cruz, CA), and poly-L-lysine (Sigma, St. Louis, MO). Adherent cells were then cultivated in DMEM Low Glucose (Gibco, Carlsbad, CA) with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM/L glutamine (all PAA). The transgenic Oct4 expression was induced by either 6 µg/ml doxycycline (Sigma) or by 2 µg/ml doxycycline (Dox) plus 10–7 M dexamethasone (Sigma), depending on the respectively contained tetracycline transactivator. 5-azacytidine (10 nM, Sigma) and cycloheximide (30 µg/ml, Sigma) were applied as indicated. Generation, culture, and differentiation of ESCs and iPSCs were performed as previously described (Tiemann 2011, Cytometry A 79:426-435).
Microarray Gene Expression Analysis
Samples from Oct4-inducible cell types and controls were collected at different time points (0, 6, 12, and 24 hours after induction). Biotin-labeled cRNA was prepared with the linear TotalPrep RNA Amplification Kit (Ambion, Austin, TX) from 500 ng original RNA and hybridized onto MouseRef 8 v2.0 Expression BeadChips (Illumina, San Diego, CA). The chips were stained with streptavidin-Cy3 (GE Healthcare, Little Chalfont, UK) and scanned using the iScan reader (Illumina). Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix robust multiarray analysis model. Variance stabilization was performed by log2 scaling and the R-Bioconductor lumi package was used for normalization.

overall_design:
17 samples were analyzed.
ESC: Mouse Embryonic Stem Cell (ESC)
TO_MEF_0: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 0h
TO_MEF-6: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 6h
TO_MEF-12: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 12h
TO_MEF-24: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 24h
TO_BMC-0: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 0h
TO_BMC-6: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 6h
TO_BMC-12: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 12h
TO_BMC-24: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 24h
TO_NSC-0: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 0h
TO_NSC-6: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 6h
TO_NSC-12: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 12h
TO_NSC-24: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 24h
Ctrl_MEF_0: Control wild type Mouse Embryonic Fibroblast (MEF) untreated
Ctrl_MEF_24: Control wild type Mouse Embryonic Fibroblast (MEF) 24h treated
Ctrl_NSC_0: Control wild type Mouse Neural Stem Cell (NSC) untreated
Ctrl_NSC_24: Control wild type Mouse Neural Stem Cell (NSC) 24h treated

first: Ulf

last: Tiemann

first: Adele

last: Marthaler

middle: U

last: Fischedick

first: Hans

last: Schöler


title: Reprogramming to pluripotency through a somatic stem cell intermediate

status: Public on Mar 28 2014

summary:
In the past years, transdifferentiation from one lineage to another has become an important research field. In that context, we recently reported the direct reprogramming of mouse embryonic fibroblasts (MEFs) into induced neural stem cells (iNSCs). In this study, we, for the first time, successfully reprogrammed these iNSCs into induced pluripotent stem cells (iPSCs), which we termed iNSC-derived iPSCs (iNdiPSCs). iNdiPSCs proved to be truly pluripotent, as shown by a pluripotent gene expression profile and the ability to differentiate into all three germ layers both, in vitro and in vivo. We could further show that iNdiPSCs do not retain an epigenetic memory neither from iNSCs nor MEFs. In conclusion, our data validate the stable somatic stem cell status of iNSCs and demonstrate their potential to give rise to bona fide iPSCs that are indistinguishable from other iPSCs or embryonic stem cells.
Cell culture conditions:
iNdiPSCs were grown on feeder cells in 2i medium
(Knockout DMEM supplemented with 20% Knockout serum replacement, 0.2x -Mercaptoethanol (all GIBCO),
1x nonessential amino acids and 1x penicillin/streptomycin/glutamine (PSG) (both PAA),
1nM PD0325901, 3nM CHIR99021 (both Axon Medchem), and 1000 U/ml leukemia inhibitory factor [in house preparation]).
Virus production and infection:
Commercially available pMX retroviruses expressing mouse Oct4 (Addgene # 13366) and Klf4 (# 13370) were transfected in 293T cells in iNSC medium:
DMEM/F-12 supplemented with B27 (both GIBCO),
10ng/ml EGF, 10ng/ml bFGF (both Peprotech) and 1x PSG.
Supernatant was collected after 48 h, filtered and used for infection of 5 x 104 iNSCs. After 48 h cells were cultured in 2i medium.
Alkaline phosphatase staining
Cells were fixed for 5 min in 4% paraformaldehyde (PFA) at room temperature (RT),
washed twice with phosphate buffered saline (PBS) (PAA) and stained with the SIGMA FAST Fast Red TR/Napthol AS-MX kit
(product no. F4648) for 20 min in the dark at RT.
Chromosome spread:
Cells were subjected to 100ng/ml KaryoMax Colcemid (GIBCO) for 3 h at 37C. Cells were harvested and resuspended by slowly adding 4 ml 0.56% prewarmed KCl,
incubated for 15 min at 37C and spun down at 900 rpm for 5 min. Fresh fixative
(3 parts methanol plus 1 part acetic acid) was very slowly added to the pellet to a final volume of 3 ml and incubated for 30 min at RT.
Cells were subsequently washed 3 times in fixative and resuspened in fixative. Approximately 20 microl were dropped from a height of about 2 m on a dry slide.
Drops were air dried for 30 min and mounted with DAPI containing mounting solution.
RNA extraction, cDNA synthesis and quantitative real time PCR (qRTPCR)
RNA was extracted using the QIAGEN RNeas Mini Kit (cat no. 74106) and cDNA synthesis performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).
qRTPCR was carried out using iTaq SYBR Green Supermix With ROX (Bio-Rad).
Oct4 promoter methylation analysis:
Bisulfite conversion was performed using the QIAGEN EpiTectﾮ Bisulfite Kit (Cat no. 59104).
Followed by nested PCR using a set of target region specific primers.
The first PCR was performed using 1micro-l of bisulfite converted and purified DNA,
SuperTaq polymerase (Promega) according to the manufacturers protocol and a total of 35 cycles with denaturation at 94C for 30 s,
followed by 30 s of annealing at 50C and 72C elongation for 1 min with an initial denaturation at 94C and a final elongation step at 72C for 3 min each.
For the second PCR, we used Taq polymerase according to the manufacturers protocol (New England Biolabs) and 1 microl of the first PCR product with denaturation at 95C for 30 s,
followed by 40 cycles of denaturation for 30 s at 95C, annealing for 30 s at 55C and elongation for 1 min at 68C,
and finally an additional elongation step for 5 min at 68C.
The amplified fragments were resolved on a 1.5% agarose gel,
purified using the QIAGEN QIAquick Gel Extraction Kit (Cat no. 28706) and subsequently cloned into the pCRII-TOPO-vector using the TOPO TA Cloning Kit (Invitrogen).
The cloned products were transformed into One Shot TOP10 competent bacteria (Invitrogen) and plated on agar plates.
The picked colonies were sent to GATC for sequencing.
The results were then analyzed with the QUMA software (online available at www.quma.cdb.riken.jp).
Immunocytochemistry:
Cells were fixed for 5 min in 4% PFA at RT, washed twice in PBS, permeabilized with 0.1% Triton X-100 for 10 min,
and washed again twice in PBS.
Followed by blocking with 5% bovine serum albumin (BSA) (GIBCO) for 1 h shaking at RT. Subsequently the first antibody was added in 1% BSA and incubated for 1 h shaking at RT.
The cells were again washed twice in PBS and subjected to the second antibody in 1% BSA for 1 h shaking at RT and then washed three times with PBS, including DAPI in the second wash.
The following primary antibodies were used: anti-Nanog (REC-RCAB0002P-F, Cosmo Bio Co.) 1:1000
anti SSEA1, (MC-480, Millipore), 1:160,  anti-3-Tubulin (T8660, Sigma),
1:2000,  anti-Smooth muscle actin (M0851, DAKO), 1:200,  anti-Sox17 (AF1924, R&D Systems),
1:100. The following secondary antibodies were used: Alexa Fluor 568 goat anti-rabbit, rabbit anti-mouse, and rabbit anti-goat (Invitrogen), all 1:2000.
DNA extraction and PCR:
DNA extraction was performed using the QIAGEN QIAampﾮ DNA Mini Kit (cat no. 51306).
PCR was carried out using Taq polymerase including buffers from New England Biolabs,
following their recommended protocol. The following primers were used for genotyping:
pMX-Oct4, -Klf4, -Sox2, -c-Myc and -Brn4.
In vitro differentiation:
First, hanging drops were formed placing 20ﾵl drops containing 600 cells each on the lid of a 10cm cell culture dish, which was then inverted.
After 3 days, hanging drops were transferred to gelatin-coated dishes and cultured in standard MEF medium (DMEM containing 20% fetal calf serum (both GIBCO) and 1x PSG),
which was also used for hanging drop formation. After approximately 10-14 days the cells were fixed and stained as described above.
Teratoma formation:
1 x 106 iNdiPSCs were injected subcutaneously into SCID mice. Tumors were resected after 4 weeks and fixed in 4% PFA.
Paraffin sections were cut, mounted on glass slides and stained with haematoxylin and eosin.
Animal handling was performed in accordance with the German animal protection law and the guidelines of the Max Planck Institute.
Microarray analysis:
Purified cRNA samples were prepared with the linear TotalPrep RNA Amplification Kit (Ambion) from 500 ng original RNA,
involving synthesis of double-stranded cDNA by T7-promoter-linked oligo(dT) primers and 14 h of in vitro transcription incorporating biotin-labeled nucleotides.
Hybridization onto MouseRef 8 v2.0 Expression BeadChips (Illumina) was performed according to the manufacturerﾒs recommendations.
Chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using an iScan reader (Illumina).
The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina).
Background correction was performed using the Affymetrix robust multiarray analysis (RMA) background correction model.
Variance stabilization was performed by log2 scaling,
and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor.

overall_design:
8 samples were analyzed.
MEF: Regular mouse embryonic fibroblasts with Tet-On background, 1 replicate
NSC: Regular mouse neural stems cells with Tet-On background, 1 replicate
iNSC: mouse induced neural stems cells, 1 replicate
iNdiPSC-1: mouse induced pluripotent cells (IPSs) derived from iNSC (clon 1), 1 replicate
iNdiPSC-4: mouse induced pluripotent cells (IPSs) derived from iNSC (clon 4), 1 replicate
NSC2FiPSC: mouse  induced pluripotent cells (IPSs) derived from regular NSC (using reprogramming 2 factors), 1 replicate
MEF4FiPSC: mouse  induced pluripotent cells (IPSs) derived from regular MEF (using reprogramming 4 factors), 1 replicate
ESC:  regular mouse embryonic stem cells with Tet-On background, 1 replicate

last: Arauzo-Bravo

first: Guangming

first: Holm

last: Zaeres

first: Dong

middle: W

last: Han

last: Schoeler


title: m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation (m6a-seq)

overall_design: m6A-seq was measured from total RNA in mouse embryonic stem cells (ESCs), embroid bodies (EBs) and embronic fibroblasts (MEF). 3 biological replicates are available from BVSC ESC line and EBs, and two biological replicates are available for MEFs. m6A-seq was also measured from total RNA in Mettl3 knockout ESCs. Each sample consist of IP to m6A and control input.

first: Sharon

last: Moshitch-Moshkovitz

first: Shay

last: Geula

first: Nitzan

last: Kol

first: Gidi

last: Rechavi

first: Jacob

last: Hanna


title: Reprogramming of two somatic nuclei in the same ooplasm leads to pluripotent embryonic stem cells

status: Public on Jul 11 2013

first: Marcos

last: Araúzo-Bravo

summary:
We provide proof that the oocyte is able to reprogram two somatic nuclei at once and therefore is not limited in its reprogramming capacity to a single nucleus. Mouse ooplasts transplanted with 2 somatic cell nuclei simultaneously (double nuclear transfer) support preimplantation development and the derivation of tetraploid NT embryonic stem cells (tNT-ES cells). These cells are unique in that they embody the first case of cloned ES cells containing two reprogrammed somatic genomes at the same time and exhibit characteristics of true pluripotency.
By assessing allelic expression of Oct4 in normal and double nucleus transplanted embryos throughout development we demonstrate that oocyte mediated reprogramming is embarked with a probabilistic component and that one reprogrammed genome can dominate the other during preimplantation development  even if merged into the same nucleus. Animal handling and cell recovery: Oocytes from mice of the B6C3F1 strain were used for SCNT and ICSI. Cumulus cells (for SCNT) and sperm (for ICSI) were either from a B6C3F1 or OG2 donor the latter expressing an Oct4 promoter-driven GFP transgene. Additionally cells from pure C56Bl/6 or C3H mice were retrieved for SCNT. Female mice were sacrificed by cervical dislocation 14 h after hCG injection  and oocytes retrieved from oviduct ampullae. Oocytes and cumulus cells were handled in Hepes-buffered CZB (HCZB) medium. Double nuclear transfer  ICSI and embryo culture: Micromanipulations and embryo culture were performed at 30°C (room temperature). For SCNT oocytes were first enucleated (spindle-chromosome complex removed) before transfer of one  two or three cell nuclei with a micropipette driven by a piezo actuator under Nomarski optics. The reconstructed oocytes were activated for 6 h in Calcium-free alpha-MEM supplemented with 10 mM SrCl2 and 5 micro-g mL-1 cytochalasin B. Intracytoplasmic sperm injection (ICSI) of one  two or four into intact oocytes was used as the fertilization control condition  using sperm from 8-week-old mice of B6C3F1 or OG2 strains. Embryos were placed in embryo culture medium and cultured at 37°C under 5% CO2. The activated oocytes were cultured in alpha-MEM at 37°C and under 5% CO2 until use. On day 5 after activation SCNT and ICSI blastocysts were processed for ESC derivation. Analysis of allelic expression of Oct4: RNA from single embryos was extracted using the ZR-RNA MicroPrep kit (Zymo Research). Complementary DNA synthesis was performed using the High Capacity cDNA Reverse Transcription Kit (Invitrogen) according to manufacturers instruction. A nested PCR was performed to amplify part of the Oct4 cDNA containing a BamH1 sensitive SNP. Primer sequences and PCR conditions are given in the supplementary methods. The PCR product was digested for 8h using 100 units of BamH1-HF (NEB) subsequently subjected to agarose gel electrophoreses and the results grouped into 3 categories (mainly C57Bl/6  balanced mainly C3H) according to the observed band patterns. Global transcriptome analysis by microarray: RNA samples to be analyzed by 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 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto a MouseRef-8 v2 expression BeadChip (Illumina) following the manufacturer 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 exclusively hybridized as biological replicates. Microarray data processing: 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 Background correction performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics was performed with in-house developed functions in Matlab.

overall_design:
8 samples were analyzed.
tNT#1: tetraploid nuclear transfer, p17, double sedimentation;
tNT#2: tetraploid nuclear transfer, p19, double sedimentation;
tNT#3: tetraploid nuclear transfer, p25, double sedimentation;
tNT#4: tetraploid nuclear transfer, p29, double sedimentation;
tNT#5: tetraploid nuclear transfer, p18, double sedimentation;
tNT#6: tetraploid nuclear transfer, p25, double sedimentation;
NT#1: nuclear transfer, double sedimentation;
NT#2: nuclear transfer, double sedimentation;

first: Matrin

last: Pfeiffer

first: Telma

last: Esteves

first: Sebastian

middle: T

last: Balbach

first: Martin

last: Stehling

first: Anna

last: Jauch

first: Francesca

last: Houghton

first: Michele

last: Boiani


title: FLT3 activation cooperates with MLL-AF4 fusion gene to abrogate the hematopoietic specification of human ESCs

status: Public on Jun 04 2013

summary: MLL-AF4 is a hallmark genomic aberration which arises prenatally in high-risk infant acute lymphoblastic leukemia (ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hemato-endothelial specification but was not sufficient for transformation. Additional cooperating genetic insults seem required for MLL-AF4-mediated leukemogenesis. FLT3 is highly expressed in MLL-AF4+ ALL through activating mutations (FLT3-TKD or FLT3-ITD) or increased transcriptional expression, being therefore considered a potential cooperating event in MLL-AF4+ ALL. Here, we explored the developmental impact of FLT3 activation on its own or in cooperation with MLL-AF4 in the hematopoietic fate of hESCs. FLT3 activation did not impact specification of CD45-CD31+ hemogenic precursors but significantly enhanced the formation of CD45+CD34+ and CD45+ blood cells and blood progenitors with clonogenic potential. Importantly, FLT3 activation through FLT3 mutations or FLT3-WT overexpression completely abrogated hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly impacts hESC specification rather than selective proliferation/survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling supported the limited impact of FLT3 activation on hESC specification towards CD45-hemogenic precursors and the enhanced hematopoiesis upon FLT3 activation, and inhibited hematopoiesis upon MLL-AF4 expression in FLT3-activated hESCs which was associated to large transcriptional changes and regulation of master early hematopoietic genes. Also, although FLT3 activation and MLL-AF4 cooperate to inhibit embryonic hematopoiesis the underlying molecular/genetic mechanisms differ depending on how FLT3 activation is achieved. Finally, FLT3 activation did not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells.

overall_design:
18 samples were analyzed.
CD45- hemogenic precursors EV, 2 biological rep
CD45- hemogenic precursors FLT3-TKD, 2 biological rep
CD45- hemogenic precursors FLT3-WT, 2 biological rep
CD45- hemogenic precursors FLT3-TKD/MLLAF4, 2 biological rep
CD45- hemogenic precursors FLT3-WT/MLLAF4, 2 biological rep
CD45+ blood cells EV, 1 biological rep
CD45+ blood cells FLT3-TKD, 2 biological rep
CD45+ blood cells FLT3-WT, 2 biological rep
CD45+ blood cells FLT3-TKD/MLLAF4, 2 biological rep
CD45+ blood cells FLT3-WT/MLLAF4, 1 biological rep

first: Clara

last: Bueno

first: Rosa

last: Montes

first: Oscar

last: Navarro-Montero

first: Verónica

last: Ayllón

last: Ramos-Mejia

first: Pedro

last: Real

first: Damià

last: Romero-Moya

first: Pablo

last: Menendez


title: The Effect of the Leptin Receptor Q223R Polymorphism on the Host Transcriptome Following Infection with E. histolytica.

status: Public on Jun 05 2013

city: Hamilton

first: Dan

last: Sturdevant

state: MT

address: 903 S 4th street

department: NIAID

institute: NIH

summary: Resistance to amebiasis is associated with a polymorphism in the leptin receptor. Previous studies demonstrated that humans with the ancestral Q223 leptin receptor allele were nearly four times less likely to be infected with Entamoeba histolytica than those carrying the mutant R223 allele. We hypothesized that the Q223 allele protected against E. histolytica via STAT3-mediated transcription of genes required for mucosal immunity. To test this, mice containing the humanized LEPR Q or R allele at codon 223 were intracecally infected with E. histolytica. Susceptibility to amebiasis was assessed, and cecal tissues analyzed for changes in gene expression. By 72 h post-challenge all Q223 mice had cleared E. histolytica, whereas 39% of 223R mice were infected. 37 genes were differentially expressed in response to infection at 72 h, including pro-inflammatory genes (CXCL2, calprotectin (S100A8/9), Pla2g7, Itbg2, and MMP9) and functions pertaining to the movement and activity of immune cells. A comparison at 12 h post-challenge of infected Q223 vs. R223 mice identified a subset of differentially-expressed genes, many of which were closely linked to leptin signaling. Further analyses indicated that the Q223 gene expression pattern was consistent with a suppressed apoptotic response to infection, while 223R showed increased cellular proliferation and recruitment. These studies are the first to illuminate the downstream effects of leptin receptor polymorphisms on intestinal infection by E. histolytica. As such, they are important for the insight that they provide to this previously uncharacterized mechanism of mucosal immunity. Resistance to amebiasis is associated with a polymorphism in the leptin receptor. Previous studies demonstrated that humans with the ancestral Q223 leptin receptor allele were nearly four times less likely to be infected with Entamoeba histolytica than those carrying the mutant R223 allele. We hypothesized that the Q223 allele protected against E. histolytica via STAT3-mediated transcription of genes required for mucosal immunity. To test this, mice containing the humanized LEPR Q or R allele at codon 223 were intracecally infected with E. histolytica. Susceptibility to amebiasis was assessed, and cecal tissues analyzed for changes in gene expression. By 72 h post-challenge all Q223 mice had cleared E. histolytica, whereas 39% of 223R mice were infected. 37 genes were differentially expressed in response to infection at 72 h, including pro-inflammatory genes (CXCL2, calprotectin (S100A8/9), Pla2g7, Itbg2, and MMP9) and functions pertaining to the movement and activity of immune cells. A comparison at 12 h post-challenge of infected Q223 vs. R223 mice identified a subset of differentially-expressed genes, many of which were closely linked to leptin signaling. Further analyses indicated that the Q223 gene expression pattern was consistent with a suppressed apoptotic response to infection, while 223R showed increased cellular proliferation and recruitment. These studies are the first to illuminate the downstream effects of leptin receptor polymorphisms on intestinal infection by E. histolytica. As such, they are important for the insight that they provide to this previously uncharacterized mechanism of mucosal immunity.

overall_design: Control (non-infected QQ or RR) vs. infected with E. histolytica at two time points (12hour and 72 hour)

first: Nicole

middle: M

last: Mackey-Lawrence

first: Xiaoti

last: Guo

first: Daniel

first: Kimmo

last: Virtaneva

first: Matthew

last: Hernandez

first: Eric

last: Houpt

first: Alan

last: Sher

first: Stephen

middle: F

last: Porcella

first: William

last: Petri Jr


title: Gut Microbiota Orchestrates Energy Homeostasis during Cold

status: Public on Dec 03 2015

first: Ozren

last: Stojanovic

country: Switzerland

sample_organism:
Mus musculus
mouse gut metagenome

platform_organism:
Mus musculus
mouse gut metagenome


title: Hypoxia induces pluripotency in primordial germ cells by HIF1 alpha stabilization and Oct4 deregulation

status: Public on Sep 18 2014

first: Marcos

last: Araúzo-Bravo

summary:
To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). We found 11 genes  ≥1.5-fold overexpressed in EGCs. None of the genes identified were Yamanaka's genes, but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGL), which differentiate into cells of the three germ layers in vitro, and contribute to the ICM in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1? stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signalling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. The data suggests that the properties of stemness, pluripotency and selfrenewal, are differentially regulated in primordial germ cell reprogramming induced by hypoxia.
8.5 dpc Oct4-GFP PGCs cultured in hypoxia conditions for 6 days were isolated by FACS. To eliminate the possibility of differences due solely to anaerobic respiration, TGC-10 EGC line was exposed to the hypoxia-mimetic agent Cl2Co at 150 microM for four hours prior to RNA isolation. RNA samples to be analyzed by 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 (Tetu-Bio), 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 MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer's instructions. After washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. 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 the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of RBioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clustering of genes and samples was performed with one minus correlation metric and the unweighted average distance (UPGMA) (also known as group average) linkage method.

overall_design:
14 samples were analyzed
EG: pluripotent Embryonic Germ Cells (EGCs), 2 replicates
hiEGL1: hypoxia-induced EGC-like cells (hiEGL), 2 replicates
hiEGL2: hypoxia-induced EGC-like cells (hiEGL), 2 replicates
ESCm: Embryonic Stem Cells (ESCs) male, 2 replicates
ESCf: Embryonic Stem Cells (ESCs) female, 2 replicates
EpiT9: Epiblast Stem Cells T9, 1 replicate
EpiE3: Epiblast Stem Cells E3, 1 replicate
PGC11.5: unipotent Primordial Germ Cells (PGCs) from dpc 11.5 , 2 replicates

first: Pilar

last: Lopez-Iglesias

first: Yago

last: Alcaina

first: Natalia

last: Tapia

first: Sabour

last: Davood

first: Edurne

last: Berra

first: Nunez-OMara

last: Analia

first: Manuel

last: Nistal

first: Sagrario

last: Ortega

first: Peter

last: Donovan

first: Hans

last: Schoeler

first: Maria

last: De Miguel


title: m6A mRNA Methylation Facilitates Resolution of Naïve Pluripotency Towards Differentiation (Ribo-seq)

first: Noa

last: Novershtern

overall_design: Ribosome footprint (Ribo-Seq) was measured from mouse embryonic stem cells and mouse embriod bodies, in WT and Mettl3-KO cell lines.

first: Sharon

last: Moshitch-Moshkovitz

first: Shay

last: Geula

first: Nitzan

last: Kol

first: Gidi

last: Rechavi

first: Jacob

last: Hanna


title: Efficient hematopoietic redifferentiation of induced pluripotent stem cells derived from primitive murine bone marrow cells

status: Public on Aug 10 2012

first: Marcos

last: Araúzo-Bravo

summary:
Heterogeneity among iPSC lines with regard to their gene expression profile and differentiation potential has been described and has been at least partly linked to the tissue of origin.  We generated iPSCs from primitive (linneg) and non-adherent differentiated (linpos) bone marrow cells (BM-iPSC), and compared their differentiation potential to that of fibroblast-derived iPSCs (Fib-iPSC) and ESCs.  In the undifferentiated state, individual iPSC clones but also ESCs proved remarkably similar when analyzed for alkaline phosphatase and SSEA-1 staining, endogenous expression of the pluripotency genes Nanog, Oct4, and Sox2, or global gene expression profiles.  However, substantial differences between iPSC clones were observed after induction of differentiation, which became most obvious upon cytokine-mediated instruction towards the hematopoietic lineage.  All three BM-iPSC lines derived from undifferentiated cells yielded high proportions of cells expressing the  hematopoietic differentiation marker CD41, and in two of these lines, high proportions of CD41+/CD45+ cells were detected. In contrast, little hematopoiesis-specific surface marker expression was detected in linpos BM-iPSC and FIB-iPSC lines.  These results were corroborated by functional studies demonstrating robust colony outgrowth from hematopoietic progenitors in two of the linneg BM-iPSCs only.  Thus, in summary our data demonstrate efficient generation of iPSCs from primitive hematopoietic tissue as well as efficient hematopoietic  redifferentiation for linneg BM-iPSC lines, thereby further supporting the notion of an epigenetic memory in iPSCs.

Murine embryonic fibroblasts (MEFs) from C3H mice were cultured in low-glucose DMEM supplemented with 10% heat-inactivated fetal calf serum gold (PAA, Pasching, Austria), penicillin-streptomycin, 1 mM L-glutamine and 0.05 mM beta-mercaptoethanol on gelatine-coated dishes.  C3H MEFs were grown to confluence, inactivated with 10 ug/ml Mitomycin C (Sigma) and used as feeder layers. Virus production was performed in a four plasmid-manner. Briefly, 3.5x10^6 293T cells were seeded 24h prior to transfection in 10 cm dishes.  293T cells were cultivated in high-glucose DMEM (Gibco) supplemented with 10% heat-inactivated FCS, penicillin-streptomycin and 1 mM L-glutamine.  Cells were transfected with 5 ug lentiviral vector, 8 ug pcDNA3.GP.4xCTE (expressing HIV-1 gag/pol), 5 ug pRSV-Rev and 2 ug pMD.G (encoding the VSV glycoprotein) using the calcium phosphate method in the presence of HEPES and chloroquine.  Supernatants were harvested 48h and 72h after transfection, filtered and subsequently 50x concentrated by ultracentrifugation.  Titers determined based on real-time PCR, were in the range of 1-5x10^7/ml.

For iPSC generation, bone marrow cells were isolated from femurs and tibias of Oct4-GFP transgenic mice (OG2) and immunomagnetically separated into lineage negative (Lin-) and lineage positive (Lin+) populations using the mouse lineage depletion kit (Miltenyi Biotec).  Lin- cells were cultivated in serum-free StemSpan medium (Stem Cell Technology) supplemented with 2 mM L-glutamine, penicillin-streptomycin, 10 ng/ml mSCF, 20 ng/ml mTPO, 20 ng/ml, 20 ng/ml IGF-2 and 10 ng/ml FGF-1 (all Peprotech).  Lin+ cells were cultivated in Iscove's modified eagle medium (IMDM), supplemented with 15% heat-inactivated FCS, 1 mM L-glutamine, penicillin-streptomycin, 100 ng/ml mSCF, 100 ng/ml mFLT3-L, 10 ng/ml hIL-3 and 100 ng/ml hIL-11.  Both Lin- and Lin+ cells were pre-stimulated in the aforementioned media for 48 h.  Thereafter, 2x10^5 Lin- and and Lin+ bone marrow cells were transduced on Retronection-coated plates (Takara) with lentiviral vectors encoding for human Oct4, Sox2, Klf4 and c-Myc using a multiplicity of infection (MOI) of 50 per virus.  Twenty-four hours after transduction, media were supplemented with 2 mM valproic acid.  Transduced bone marrow cells were kept in hematopoietic medium until 5 or 7 days post transduction (p.t.)  and then transferred onto Mitomycin C-treated MEF feeders on gelatine-coated dishes.  Henceforward, cells were cultivated in ES cell medium (knockout DMEM (Gibco), 15% ES-tested FCS, 1 mM L-glutamine, 0.1 mM non-essential amino acids (Gibco), 100 uM beta-mercaptoethanol (Sigma), penicillin-streptomycin and 103 units/ml leukemia inhibitory factor (LIF, provided by the Max-Planck-Institute, Munster, Germany).  Upon appearance of GFP-positive ESC-like colonies, single colonies were picked based on morphology and GFP expression.  Murine ESCs and iPSCs were cultured on Mitomycin C-treated MEF feeders in the aforementioned ES medium.  Murine ESCs and iPSCs were passaged every 2-3 days.  The murine embryonic fibroblast-derived iPSC lines (MEF-iPS, 3FLV2, 4FLV1) were generated by transduction of OG2-MEFs with the same lentiviral vector constructs using standard technology.  For iPSC lines 3FLV2 and 4FLV1, complete reprogramming was demonstrated by alkaline phosphatase and SSEA1-staining, pluripotency factor expression and teratoma formation.

overall_design:
7 samples were analyzed:

Lin-OG2-BM: Lineage-negative bone marrow cells isolated from OG2 mice, 1 biological rep.
OG2-ESC: OG2 control ES cells, 1 biological rep.
Lin-iPSCs112: iPSCs generated from OG2 lineage-negative bone marrow cells, line 1.1.2, 1 biological rep.
Lin-iPSCs7: iPSCs generated from OG2 lineage-negative bone marrow cells, line 7, 1 biological rep.
Lin-iPSCs9: iPSCs generated from OG2 lineage-negative bone marrow cells, line 9, 1 biological rep.
Lin+iPSCs11: iPSCs generated from OG2 lineage-positive bone marrow cells, line 1.1, 1 biological rep.
MEF-iPSC:iPSCs generated from OG2 MEFs, 1 biological rep.

first: Nils

last: Pfaff

first: Nico

last: Lachmann

first: Malte

last: Sgodda

last: Arauzo-Bravo

first: Boris

last: Greber

first: Wilfried

last: Kues

first: Silke

last: Glage

first: Christopher

last: Baum

first: Heiner

last: Niemann

first: Axel

last: Schambach

first: Tobias

last: Cantz

first: Thomas

last: Moritz


title: Origin-dependent neural cell identities in differentiated human iPS cells in vitro and after transplantation into the rodent brain

status: Public on Sep 12 2014

summary: The differentiation capability of induced pluripotent stem cells (iPSCs) towards certain cell types for disease modelling and drug screening assays might be influenced by their somatic cell of origin. Here, we have compared the neural induction of human iPSCs generated from either fetal neural stem cells (fNSCs), dermal fibroblasts or CD34+ hematopoietic stem cells. Neural progenitor cells (NSCs) and neurons could be generated at similar efficiencies from all iPSCs. Whole genome transcriptome analysis and an expression analysis of neural genes in iPSC-derived NSCs revealed a separation of neuroectoderm- from mesoderm-derived cells. Furthermore, we found genes, which were similarly expressed between fNSCs and neuroectoderm- but not mesoderm-derived NSCs. Notably, these neural signatures were retained after transplantation into the cortex of mice and paralleled with increased survival and integration of neuroectoderm-derived cells in vivo. These results indicated distinct origin- dependent neural cell identities in differentiated human iPSCs both in vitro and in vivo.

overall_design:
RNA samples for microarray analysis were prepared using RNeasy columns (Qiagen, Germany) with on-column DNA digestion. 300 ng of total RNA per sample were used as the input in the linear amplification protocol (Ambion), which involved the synthesis of T7-linked double-stranded cDNAs and 12hrs of in vitro transcription incorporating biotin-labeled nucleotides. Purified and labeled cRNA was then hybridized for 18 hrs onto HumanHT-12 v4 expression BeadChips (Illumina, USA) following the manufacturer's instructions. After the recommended washing, the chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and the accompanying software. The samples were exclusively hybridized as biological replicates. The RNA samples from in vivo transplanted and FACS sorted cells were amplified using the TargetAmp 2-Round Biotin-aRNA Amplification Kit 3.0 (Epicentre, USA) from 100 pg to 50 microg.
43 samples were analyzed
Fib, Human fibroblast F134, 2 replicates
CD34+, Human Cord Blood CD34+ Hematopoyetic Stem Cell (HSC), 1 replicate
fNSC, Human fetal Neural Stem Cells (NSC), 1 replicate
fNSC-1FiPSCa-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates
fNSC-1FiPSCb-NSC, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates
fNSC-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates
CD34-2FiPSCa-NSC, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates
CD34-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, in vitro differentiated to NSC, 2 replicates
CD34-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, in vitro differentiated to NSC, 2 replicates
Fib-4FiPSCa-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vitro differentiated to NSC, 2 replicates
Fib-4FiPSCb-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, in vitro differentiated to NSC, 2 replicates
Fib-4FiPSCc-NSC, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone c, in vitro differentiated to NSC, 1 replicate
H1-ESC-NSC, Human H1 embryonic stem cell (ESC), in vitro differentiated to NSC, 2 replicates
HUES6-ESC-NSC, Human HUES6 embryonic stem cell (ESC), in vitro differentiated to NSC, 1 replicate
fNSC-1FiPSCa, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate
fNSC-1FiPSCb, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone b, 1 replicate
fNSC-2FiPSCa, Human two factors (POU5F1, KLF4) fetal Neural Stems Cell (NSC) induced reprogrammed cell (iPSC) clone a, 1 replicate
CD34-2FiPSCa, Human two factors (POU5F1, KLF4) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate
CD34-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone a, 1 replicate
CD34-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) cord blood CD34+ Hematopoyetic Stem Cell (HSC) induced reprogrammed cell (iPSC) clone b, 1 replicate
Fib-4FiPSCa, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, 1 replicate
Fib-4FiPSCb, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone b, 1 replicate
H1-ESC, Human H1 embryonic stem cell (ESC), 3 replicates
HUESC6-ESC, Human HUESC6 embryonic stem cell (ESC) , 2 replicates
Fib-4FiPSCa-NSC-In Vivo, Human four factors (POU5F1, KLF4, SOX2, CMYC) fibroblast induced reprogrammed cell (iPSCs) clone a, in vivo differentiated to NSC, 2 replicates
fNSC-1FiPSCa-NSC-In Vivo, Human one factor (POU5F1) fetal Neural Stem Cell (NSC) induced reprogrammed cell (iPSC) clone a, in vivo differentiated to NSC, 4 replicates

first: Gunnar

last: Hargus

first: Marc

last: Ehrlich

first: Anna-Lena

last: Hallmann

first: Kathrin

last: Hemmer

first: Peter

last: Reinhardt

first: Kee-Pyo

last: Kim

first: Kenjiro

last: Adachi

first: Simon

last: Santourlidis

first: Foued

last: Ghanjati

first: Mareike

last: Fauser

first: Christiana

last: Ossig

first: Alexander

last: Storch

first: Jeong

middle: B

first: Jens

last: Schwamborn

first: Jared

last: Sterneckert

first: Hans

last: Schöler

first: Tanja

last: Kuhlmann

first: Holm

last: Zaehres


title: Interactions of ARF6 with PIF4, BZR1, and RGA integrate auxin signaling with environmental and other hormonal signals in Arabidopsis [ChIP-Seq]

first: Eunkyoo

last: Oh

summary: Auxin is a major plant hormone for both development and environmental adaptation. Auxin responses are context dependent and highly modulated by light, temperature, the circadian clock, brassinosteroid, and gibberellin, but the underlying mechanisms remain unclear. Here, we show that auxin signaling integrates with other signals through direct interactions of AUXIN RESPONSE FACTOR6 (ARF6) with PHYTOCHROME INTERACTING FACTOR4 (PIF4), the brassinosteroid-signaling transcription factor BZR1, and the gibberellin-signaling repressor RGA.  ChIP-Seq and RNA-Seq experiments show that ARF6, PIF4, and BZR1 bind to largely overlapping targets in the genome and synergistically activate gene expression. In vitro and in vivo assays show that ARF6-promoter binding is enhanced by PIF4 and BZR1 but blocked by RGA. Furthermore, a tripartite HLH/bHLH module feedback regulates PIF activity and thus modulates auxin sensitivity according to additional developmental and environmental cues. Our results demonstrate a central growth-regulation transcriptional network that coordinates hormonal, environmental, and developmental control of cell elongation and plant growth.

overall_design: Genome-wide identification of ARF6 DNA-binding sites in etiolated Arabidopsis seedlings.

first: Zhiyong

last: Wang


title: Reprogramming to pluripotency is an ancient trait of vertebrate Oct4 and Pou2 proteins

status: Public on Mar 23 2014

first: Marcos

middle: J.

last: Araúzo-Bravo

summary: The evolutionary origins of the gene network underlying cellular pluripotency, a central theme in developmental biology, have yet to be elucidated. In mammals, Oct4 is a factor crucial in the reprogramming of differentiated cells into induced pluripotent stem cells. The Oct4 and Pou2 genes evolved from a POU class V gene ancestor, but it is unknown whether pluripotency induced by Oct4 gene activity is a feature specific to mammals or was already present in ancestral vertebrates. Here we report that different vertebrate Pou2 and Oct4 homologues can induce pluripotency in mouse and human fibroblasts and that the inability of zebrafish Pou2 to establish pluripotency is not representative of all Pou2 genes, as medaka Pou2 and axolotl Pou2 are able to reprogram somatic cells into pluripotent cells. Therefore, our results indicate that induction of pluripotency is not a feature specific to mammals, but existed in the Oct4/Pou2 common ancestral vertebrate.

overall_design:
16 samples were analyzed
Notation:
O: stands for OCT4 reprogramming factor from human; o: stands for Oct4 reprogramming factor from Axolotl
S: stands for SOX2 reprogramming factor from human; s: stands for SOX2 reprogramming factor from Axolotl
K: stands for KLF4 reprogramming factor from human


title: Gut Microbiota Orchestrates Energy Homeostasis during Cold [RNA-Seq]

first: Ozren

last: Stojanovic

summary: Microbial functions in the host physiology are a result of co-evolution between microbial communities and their hosts. Here we show that cold exposure leads to marked shift of the microbiota composition, referred to as cold microbiota. Transplantation of the cold microbiota to germ-free mice is sufficient to increase the insulin sensitivity of the host, and enable complete tolerance to cold partly by promoting the white fat browning, leading to increased energy expenditure and fat loss. During prolonged cold however, the body weight loss is attenuated, caused by adaptive mechanisms maximising caloric uptake and increasing intestinal, villi and microvilli lengths. This increased absorptive surface is promoted by the cold microbiota - effect that can be diminished by co-transplanting the most downregulated bacterial strain from the Verrucomicrobia phylum, Akkermansia muciniphila, during the cold microbiota transfer. Our results demonstrate the microbiota as a key factor orchestrating the overall energy homeostasis during increased demand.

overall_design: Mice were kept 30 days at room temperature or at 6C, 2 per cage, under SPF conditions, with or without administration of antibiotic coctail in drinking water (whole microbiota depletion). Fasted 5h before sacrifice. Segments of proximal jejunum were isoated, flushed gently with PBS and frozen. Each of 12 samples is a pool of two biological replicates (2 biological replicates of the same condition combined into one sample)

first: Claire

last: Chevalier

first: Mirko

last: Trajkovski