Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

NEUROMESO

ABSTRACT: Exposure of mouse ESCs to a sequence of extrinsic signals, which recapitulates in vivo development, generates a bipotential neuromesodermal precursor (NMP) that can be directed to differentiate into either spinal cord or paraxial mesodermal tissue. To induce differentiation,mouse ES cells were plated on CellBINDSurface dishes (Corning) precoated with 0.1% gelatin (Sigma) at a density of 5x10^3 cells cm-2 in N2B27 medium. Cells were grown in N2B27 supplemented with 10ng/ml bFGF (R&D) for 3 days (D1-D3) and then were transferred into serum free media without bFGF (D3-D5). To induce ventral hindbrain identity NPCs (NH) 100nM RA (Sigma) and 500nM SAG (Calbiochem) was added from D3-D5. Spinal cord identity (NP) was induced by the addition of 5nM CHIR 99021 (Axon) from D2 to D3 followed by 100nM RA, 500nM SAG from D3-D5. To induce mesodermal differentiation the cells were treated with CHIR99021 from D2-D5.

INSTRUMENT(S): Illumina HiSeq 2500

ORGANISM(S): Mus musculus

SUBMITTER: MINA GOUTI

PROVIDER: E-MTAB-2268 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity.

Gouti Mina M Tsakiridis Anestis A Wymeersch Filip J FJ Huang Yali Y Kleinjung Jens J Wilson Valerie V Briscoe James J

PLoS biology 20140826 8

Cells of the spinal cord and somites arise from shared, dual-fated precursors, located towards the posterior of the elongating embryo. Here we show that these neuromesodermal progenitors (NMPs) can readily be generated in vitro from mouse and human pluripotent stem cells by activating Wnt and Fgf signalling, timed to emulate in vivo development. Similar to NMPs in vivo, these cells co-express the neural factor Sox2 and the mesodermal factor Brachyury and differentiate into neural and paraxial me ...[more]

PMID: 25157815

Similar Datasets

Project description:Longevity mechanisms increase lifespan by counteracting the effects of aging. However, whether longevity mechanisms counteract the effects of aging continually throughout life, or whether they act during specific periods of life, preventing changes that precede mortality is unclear. Here, we uncover transcriptional drift, a phenomenon that describes how aging causes genes within functional groups to change expression in opposing directions. These changes cause a transcriptome-wide loss in mRNA stoichiometry and loss of co-expression patterns in aging animals, as compared to young adults. Using Caenorhabditis elegans as a model, we show that extending lifespan by inhibiting serotonergic signals by the antidepressant mianserin attenuates transcriptional drift, allowing the preservation of a younger transcriptome into an older age. Our data are consistent with a model in which inhibition of serotonergic signals slows age-dependent physiological decline and the associated rise in mortality levels exclusively in young adults, thereby postponing the onset of major mortality. In this study set out to measure aging in the transcriptome by determining drift-variance changes with age in C.elegans. We set up three different cohorts of water or mianserin treated animals. The title of each cohort indicates the treatment (e.g. h2o or mia), the concentration (mia2, mia10, mia50), the day when the treatment was started (e.g. d1= day 1 of adulthood) and the day when the sample was collected (e.g. d10= day 10 of adulthood). cohort #1: Celegans was treated with water or mianserin (50uM) on day 1 and RNA was harvested on day1 (water only), d3, d5 and day 10 (file titles: h2o d1/d1, h2o d1/d3, h2o d1/d5, h2o d1/d10, mia50 d1/d3, mia50 d1/d5, mia50 d1/d10) cohort #2: Celegans was treated with mianserin (50uM) starting on day 3, and day 5, RNA was harvested on day 5 or 10 (file titles: mia50 d3/d10, mia50 d5/d10, mia50 d3/d5) cohort #3: Celegans was treated with mianserin 2 uM and 10 uM Mianserin on day 1 and Rna harvested on day 5 (file titles: mia2 d1/d5, mia10 d1/d5)

Project description:Background: Studies suggested that mesenchymal stem cells (MSCs) have intrinsic neurogenic potential and can be differentiated into neural stem cell/ neural progenitor cells (NPCs) under specific microenvironment. Manipulation of growth factors is one of the popular method to achieve trans-lineage differentiation of MSCs. Synergistic effect of epidermal growth factor (EGF) and fibroblast growth factor 2 (bFGF) have been widely identified as basic requirement for neural differentiation to take place. Insulin-like growth factor 1 (IGF-1) also known as somatomedin C is an important growth promoting protein during embryonic development which control numerous cellular responses and biological systems. Our recent study has found that the combination of EGF, bFGF and IGF-1 could significantly improved the growth and survivability of MSCs-derived NPCs. Therefore, to understand the genomic mechanism underlying the differentiation in vitro, we have studied the miRNAs profile of MSCs-derived NPCs under IGF-1 influenced conditioned microenvironments. Objectives: To evaluate the effects of IGF-1 in trans-lineage differentiation of MSCs, we have induced MSCs into neural lineage in 3 groups; Group A (positive control) - EGF+bFGF, Group B (Treatment) - EGF+bFGF+IGF-1, and Group C (negative control/ untreated). To unravel the role of regulatory miRNAs involved in the early differentiation, we have performed detailed miRNA profiling for MSCs-derived NPCs at three time intervals (day 1, day 3 and day 5). The data has explored crucial miRNAs involved in early differentiation of MSCs into NPCs. Stage specific MSCs-derived NPCs at Passage 1 were collected for total RNA extraction at three time-points (D1, D3 and D5) and hybridization on Affymetrix miRNA geneChip 2.0 arrays. Each experiment were repeated three times independently (Exp 1, Exp 2 and Exp 3). Group A served as positive control (EGF+bFGF), Group B as treatment (EGF+bFGF+IGF-1) and Group C without growth factor as negative control.

Project description:Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. We have identified a CDX2+/p63+ cytotrophoblast (CTB) subpopulation in the early post-implantation human placenta, which is significantly reduced later in gestation. CTB differentiate into different trophoblast subtypes, which are responsible for gas/nutrient exchange (syncytiotrophoblast/STB) and invasion and maternal vascular remodeling (extravillous trophoblast/EVT). Study of early human placental development is severely hampered by lack of a representative trophoblast stem cell (TSC) model, with the capacity for self-renewal and the ability to differentiate into both STB and EVT. We describe a reproducible protocol, using defined media containing BMP4, by which human embryonic stem cells (hESC) can be differentiated into CDX2+/p63+ CTB-like cells. These cells can be replated to further differentiate into STB- and EVT-like cells, based on marker expression, hormone secretion and invasive ability. Differentiation of hPSC-derived CTB in hypoxia leads to reduced hCG secretion and STB-associated gene expression, instead inducing EVT differentiation in a hypoxia-inducible factor-dependent manner. Human embryonic stem cells (hESC) (WA09/H9) were maintained on Geltrex-coated plates (BD Biosciences ) in StemPro (Thermo Fisher) + bFGF (12 ng/ml). Undifferentiated hESC (D-2) were switched to minimal media (EMIM (Erb et al., 2011) containing KO DMEM/F12 (Gibco), 1% Insulin-Transferrin-Selenium Mix (Sigma-Aldrich), 1% NEAA (Gibco), 2mM L-Glutamine (Corning), 0.1mM 2-mercaptoethanol (Gibo), and 2% BSA (Gemini Bio Products)) for 2 days then treated with BMP4 (StemRD, 10ng/ml) for 3 days in minimal media+BMP4 (D3). At D3, cells were replated onto Geltrex in Feeder Conditioned Media +BMP4 and cultured in normoxia (20% O2) or hypoxia (2% O2) for 2 days (D3+2) to assess the effect of hypoxia. Cells were infected with lentiviral shRNA for shScramble (control) or shARNT, the beta subunit of the Hypoxia Inducible Factor (HIF) complex to assess the effect of ARNT knockdown. Each sample includes biological triplicates.

Project description:VEGF165 is one of the key regulators of tumor development. Using HCT116 cells transfected with full-length vegf mRNA, full-length vegf mRNA with mutations of H9D and L14E, mutated vegf mRNAs lacking untranslated regions (UTRs), and 5â??UTR mutated between nt 591 and nt 746, we found that vegf 5â??UTR resided an anti-apoptotic activity against chemotherapy independently of VEGF165. We next established HCT116 clones stably expressing vegf 5â??UTR or the mutated vegf 5â??UTR. The cells expressing 5â??UTR, but not the mutated UTR, showed the drug-resistant phenotype, anchorage-independent growth, and rapid tumor growth when implanted in athymic nude mice. Microarray and real-time PCR showed that the vegf 5â??UTR-expressing tumors up-regulated anti-apoptotic genes including mia and down-regulated pro-apoptotic genes including fas, pdcd1, nrg1, and bax. Microarray analysis also revealed specific down-regulation of IFN-inducible genes (43 genes) in the growing tumors. HCT116 cells stably transcribing vegf 5â??UTR decreased STAT1 expression and IFN alpha-STAT1 pathway. In addition to 5-fluorouracil treatment, the vegf 5â??UTR-expressing tumors did not respond to IFN alpha therapy at all. This novel tumor-promoting function in the vegf 5â??UTR may partly explain the insufficiency of the VEGF-VEGFR strategies and suggest a vegf-targeted silencing strategy as a more effective anti-tumor therapy. Experiment Overall Design: We established HCT116 clones stably expressing vegf 5â??UTR (vegf 5â??-G5) and vegf 5â??UTR mutated between nt 591 and nt 746 (vegf 5â?? mut-D5) and implanted into the left and the right flanks of the identical mouse, respectively. The generated tumors on day 14 after injection were removed and total RNA was extracted from each tumor using RNeasy mini (Qiagen). After removing contaminated DNA by DNaseI treatment, 10 micro grams each of RNA of vegf 5â??-G5-derived tumor from 3 individual mice was mixed and prepared for microarray analysis as sample RNA. The reference RNA of vegf 5â?? mut-D5-derived tumor was arranged in the same manner. The quality of both RNA samples was confirmed using an Agilent 2100 Bioanalyzer. Microarray analysis was then performed: briefly, 1 Âµg of sample RNA (vegf 5â??-G5) and reference RNA (vegf 5â?? mut-D5) were converted to cDNA with oligo dT conjugated T7 promoter primer and reverse transcriptase. These cDNAs were amplified using T7 RNA polymerase while labeling with either Cy5-CTP or Cy3-CTP (Low RNA Fluorescent Linear Amplification Kit, Agilent Technologies). 750 ng of Cy3 and Cy5-labeled cRNA were mixed and hybridized on an Agilent 22k human 1A array in a hybridization oven at 65oC for 17 h as rotating. Following hybridization and washing of a slide according to manufactureâ??s protocol, the arrays were scanned using the Agilent DNA Microarray Scanner (G2565BA) and microarray images were analyzed using Agilent Feature Extraction Software (v7.5). The final signal intensity was resulted from subtracting background signal from row spot signal. The gene expression level was presented as ratio of sample intensity against reference intensity.

Dataset Information

NEUROMESO

Publications

In vitro generation of neuromesodermal progenitors reveals distinct roles for wnt signalling in the specification of spinal cord and paraxial mesoderm identity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure