Project description:This experiment is part of the FunGenES project (FunGenES - Functional Genomics in Embryonic Stem Cells partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org). The experiment was conducted at the University Nice Sophia-Antipolis, Nice, France. The aim of this project is to identify new regulators, and new markers of the early steps of adipocyte commitment.Addition of RA on early-differentiating mES cells (Hanging drops, Day 3) orientates their commitment to the adipocyte lineage, while inhibiting myogenesis. The effect of RA on adipogenesis is mediated by retinoic acid receptor beta (RARb), since it can be mimiked by the addition of CD2314 compound, a specific agonist of this receptor isotype. In contrast, mES cell adipogenesis can be bloked by artificial activation of the Wnt pathway using GSK3 inhibitors such as lithium, or Bio.To decipher the genetic pathways that control the early steps of adipogenesis, we have compared the transcriptome of mES cells stimulated to differentiate using adipogenesis-enhancing (CD2314) or -inhibitory (BIO, or CD2314+BIO) treatments (treatment between day 3 and day 6). We have analysed the transcriptome of these cells at day 3 (before treatment), at day 6 (just after stimulation or inhibition of the early steps of adipocyte commitment) and at day 11 (to investigate later regulators of adipogenesis). Materials and methods: CGR8 at different time point during adipogenic differentiation. - Induction of differentiation by removal of LIF at day 0, hanging drops from day 0 to 3. - At day 3, harvesting of the hanging drops, and start adipogenesis-enhancing (CD2314) or -inhibitory (BIO, or CD2314+BIO) treatments (treatment from day 3 to 6, EBs in suspension on petri dishes).- At day 6, harvesting of the EBs and plating on gelatin-treated TC plates in normal culture medium ( GMEM+10%serum).- At Day 7, treatment with final inducers of adipogenic differentiation (Insuline, T3, BRL). Same medium until end point of differentiation (Day 21). Relationships between samples: day03= CGR8, day 3 of differentiation, hanging drops from day 0 (removal of LIF) and 3. day06_0= day 6 of differentiation, control condition from day 3 to 6 (GMEM+serum)day06_bio= day 6 of differentiation, treatment with Bio from day 3 to day 6. day06_cd= day 6 of differentiation,treatment with CD2314 from day 3 to day 6.day06_cd_bio= day 6 of differentiation,treatment with CD2314 plus Bio from day 3 to day 6.Day11_0= day 11 of differentiation, cells in control condition from day 3 to 6 (GMEM+serum).Day11_bio= day 11 of differentiation, cells treated with Bio from day 3 to day 6.Day11_cd= day 11 of differentiation,cells treated with CD2314 from day 3 to day 6.Day11_cd_bio= day 11 of differentiation,cells treated with CD2314 plus Bio from day 3 to day 6.Treatments: - At day 3, addition of adipogenesis-enhancing (CD2314) or -inhibitory (BIO, or CD2314+BIO) treatments (treatment from day 3 to 6).- At Day 7, treatment with final inducers of adipogenic differentiation (Insuline, T3, BRL). O= No treatment CD= treatment w Culture conditions; - Hanging drops from day 0 to Day 3.- Suspension culture between day 3 to 6, Petri dishes.- Plating on gelatine from day 6 to end point.

Project description:This experiment is part of the FunGenES project (FunGenES - Functional Genomics in Embryonic Stem Cells partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org).The experiment was conducted at the Unyversity of Bath, Bath, UK. Aim is to identify genes regulated by PI3K-dependent signaling in undifferentiated ES cells. Materials and methods: E14tg2a cell line was used. Cells were cultured in Knock-out DMEM supplemented with 15% (v/v) Knock-out serum replacement, 1000U/ml LIF, 2mM glutamine, 50 ?M 2-mercaptothanol and 1x NEAA (as described in Handbook of work methods, 4.3.1.1). Cells were plated at 5x105 per 10cm gelatin-coated TC dish for 24h. ES cells were then starved for 24h in media lacking LIF and pre-incubated for 30 minutes with 5 <B5>M LY294002 (the concentration of this PI3K inhibitor that we have shown to perturb self-renewal in the presence of LIF and which knocks down PI3K signals) or DMSO alone. Cells were then stimulated with 103U/ml mLIF (Chemicon) for 2h (which gave maximal activation of STAT3), 24h, 48h or 72h.</p>\n<h3>Relationships between samples</h3>\n <p ALIGN="JUSTIFY">Samples were either maintained in LIF or LIF plus LY294002 (PI3K inhibitor) for the required treatment times.</p>\n<h3>Treatments</h3>\n <p ALIGN="JUSTIFY">Treatment times with PI3K inhibitor LY294002 at 5 <B5>M <96> 2h (n=5), 24h (n=3), 48h (n=3), 72h (n=3). DMSO was used as vehicle alone treatment control.</p>\n<h3>Culture conditions</h3>\n <p ALIGN="JUSTIFY">Gelatin-coated TC dishes (Nunc)</p>\n<h3>RNA isolation method</h3>\n <p ALIGN="JUSTIFY">RNA Easy with on-column DNase treatment</p>\n<h3>Experimental description</h3>\n <p ALIGN="JUSTIFY">This information is included above. Briefly, E14tg2a ES cells were either maintained in LIF or LIF plus LY294002 (5 <B5>M) for 2, 24, 48 or 72 hours, after which RNA was extracted from each sample and sent for expression profiling. During this time-course, only 72h time points plus LY294002 showed phenotypic evidence of differentiation. </p>\n

Dataset Information

Transcription profiling of mouse embryonic stem cell line CGR8 grown in presence of Leukemia Inhibitory Factor and treated with trichostatin A

Publications

Histone deacetylase inhibition accelerates the early events of stem cell differentiation: transcriptomic and epigenetic analysis.

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