Gene expression profiling of Human ES cell line (hESC) derived embryoid body (EB) treated with H3R17methylation inhibitor TBBD
ABSTRACT: H3R17 methylation inhibitor TBBD associated differential gene expression in embryoid bodies was assessed by microarray analysis treated with 10uM TBBD for 48 hours compared to DMSO control treated Ebs Affymetrix one-color experiment,Organism: Homo sapiens[HuGene-1_0-st] Affymetrix Human Gene 1.0 ST Array
Project description:Many large noncoding RNAs (lncRNAs) regulate chromatin, but the mechanisms by which they localize to genomic targets remain unexplored. Here we investigate the localization mechanisms of Xist during X-chromosome inactivation (XCI), a paradigm of lncRNA-mediated chromatin regulation. During the maintenance of XCI, Xist binds broadly across the X-chromosome. During initiation of XCI, Xist initially transfers to distal regions across the X-chromosome that are not defined by specific sequences. Instead, Xist identifies these regions by exploiting the three-dimensional conformation of the X-chromosome. Xist initially accumulates on the periphery of actively transcribed regions and requires its silencing domain to spread across active regions. This suggests a model where Xist coats the entire X-chromosome by searching in three dimensions, modifying chromosome structure, and spreading to newly accessible locations. We examined the genomic localization of the Xist lncRNA using RNA Antisense Purification (RAP) in multiple cell contexts: 1) differentiated female cells (MLFs); 2) a time-course of Xist localization in male embryonic stem (ES) cells where the endogenous Xist promoter is replaced by a tet-inducible one (pSM33); 3) a time-course of Xist localization in differentiating female ES cells (F1 2-1); and 4) wild-type (delXF6) and A-repeat deletion (delSXC9) Xist transgenes incorporated into the Hprt locus under the control of a tet-inducible promoter.
Project description:The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Towards this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines. We used microarrays to compare the gene expression profiles between human ES cell lines, iPS cell lines, fibroblasts and embryoid bodies, and to identify cell-line specific outlier genes. 20 human ES cell lines (HUES1, HUES3, HUES6, HUES8, HUES9, HUES13, HUES28, HUES44, HUES45, HUES48, HUES49, HUES53, HUES62, HUES63, HUES64, HUES65, HUES66, H1, H7, H9), 12 human iPS cell lines (hiPS 11a, hiPS 11b, hiPS 11c, hiPS 15b, hiPS 17a, hiPS 17b, hiPS 18a, hiPS 18b, hiPS 18c, hiPS 20b, hiPS 27b, hiPS 27e), 6 fibroblast cell lines (hFib 11, hFib 15, hFib 17, hFib 18, hFib 20, hFib 27), 5 embryoid bodies (hEB16d HUES1, hEB16d HUES3, hEB16d HUES6, hEB16d HUES45, hEB16d H1)
Project description:Eed (embryonic ectoderm development) is a core component of the Polycomb Repressive Complex 2 (PRC2) which catalyzes the methylation of histone H3 lysine 27 (H3K27). Trimethylated H3K27 (H3K27me3) can act as a signal for PRC1 recruitment in the process of gene silencing and chromatin condensation. Previous studies with Eed KO ESCs revealed a failure to down-regulate a limited list of pluripotency factors in differentiating ESCs. Our aim was to analyze the consequences of Eed KO for ESC differentiation. To this end we first analyzed ESC differentiation in the absence of Eed and employed in silico data to assess pluripotency gene expression and H3K27me3 patterns. We linked these data to expression analyses of wildtype and Eed KO ESCs. We observed that in wildtype ESCs a subset of pluripotency genes including Oct4, Nanog, Sox2 and Oct4 target genes progressively gain H3K27me3 during differentiation. These genes remain expressed in differentiating Eed KO ESCs. This suggests that the deregulation of a limited set of pluripotency factors impedes ESC differentiation. Global analyses of H3K27me3 and Oct4 ChIP-seq data indicate that in ESCs the binding of Oct4 to promoter regions is not a general predictor for PRC2-mediated silencing during differentiation. However, motif analyses suggest a binding of Oct4 together with Sox2 and Nanog at promoters of genes that are PRC2-dependently silenced during differentiation. In summary, our data further characterize Eed function in ESCs by showing that Eed/PRC2 is essential for the onset of ESC differentiation. RNAs obtained from undifferentiated (d0) wild type and Eed KO ESCs and from day 3 (d3) and day 7 (d7) respective Ebs were subjected to Affymetrix Mouse Gene 1.0 ST Array. 24 samples in total.
Project description:Snai1 is a master factor of epithelial to mesenchymal transitioin (EMT), however, its role in embryonic stem cell (ESC) differentiation and lineage commitment remains undefined. We used microarrays to compare the global programme of gene expression between control and Snai1 knockout ESCs-derived EB and teratoma. For EBs, control and Snai1 knockout ESCs were cultured as embryoid bodies in spotaneous differentiation media, RNA of 5 days EBs were collected for Affymetrix microarrays. For teratomas, control and Snai1 knockout ESCs were injected into nude mice to form teratomas. RNA of 6 weeks were collected for Affymetrix microarrays.
Project description:This experiment was specifically designed to measure neural targets of Shh signaling, we sought to profile the genes upregulated by Hh signaling in the ventral neural tube to obtain a valid dataset. To obtain ventral-specific markers, we generated retinoic acid-treated EBs grown in the presence or absence of HH-Ag. We did not observe induction of ventral Hh markers in RA-treated constitutive Gli1FLAG EBs and used these for the control, baseline set. The presence of FoxA2, Nkx2.9 and Nkx6.1 amongst the top 10 genes based on expression levels suggests that profiling significantly enriches for Hh-dependent cell types. As expected, the benchmark standard Gli1 was not up-regulated in our array, since it is constitutively expressed in the control as well. Experiment Overall Design: There are a total of 8 samples. Four biological replicates of Retinoic-acid treated EBs (Baseline) and 4 additional biological samples of retinoic acid + Hh-Ag treated EBs (induced sample).
Project description:In this study we have compared functional and molecular properties of highly purified murine induced pluripotent stem (iPS) cell- and embryonic stem (ES) cell-derived cardiomyocytes (CM). In order to obtain large amounts of purified CM, we have generated a transgenic murine iPS cell line, which expresses puromycin resistance protein N-acetyltransferase and EGFP under the control of the cardiomyocyte-specific α-myosin heavy chain promoter (alphaMHC-Puro-IRES-GFP, aPiG). We demonstrate that murine aPIG-iPS and aPIG-ES cells differentiate into spontaneously beating CM at comparable efficiencies. When selected with puromycin both cell types yielded more than 97% pure population of CMs. Both aPIG-iPS and aPIG-ES cell-derived CM express typical cardiac transcripts and structural proteins and possess similar sarcomeric organization. Action potential recordings revealed that iPS- and ES cell-derived CM respond to beta-adrenergic and muscarinic receptor modulation, express functional voltage-gated sodium, calcium and potassium channels and possess comparable current densities. Comparison of global gene expression profiles of CM generated from iPS and ES cells revealed that both cell types cluster close to each other but are highly distant to undifferentiated ES or iPS cells as well as unpurified iPS and ES cell-derived embryoid bodies (EB). Both iPS and ES cell-derived CMs express genes and functional categories typical for CM. They are enriched in genes involved in transcription and genes coding for structural proteins involved in cardiac muscle contraction and relaxation. They also express genes involved in heart and muscle developmental processes, ion export and ion binding processes and various metabolic processes for ATP synthesis. These CMs downregulate genes involved in immune response, cell cycle and cell division, thus demonstrating the CMs population is mitotically inactive. Most surface signaling pathways are also downregulated. Thus, a transgenic aPiG-iPS cell line can provide a robust supply of highly purified and functional CMs for future in vitro and in vivo studies. Seven different experimental groups were included into analysis: undifferentiated murine ES cells (1) and undifferentiated murine iPS cells (2), murine ES cell-derived embyroid bodies (3) and murine iPS cell-derived embryoid bodies at day 16 of differentiation (4), murine ES cell-derived cardiomyocytes (5) and murine iPS cell-derived cardiomyocytes (6) at day 16 of differentiation (they were generated by puromycin selection for 7 days prior to RNA isolation). Adult mouse tail tip fibroblasts (7) were used as a control for iPS cells. Total RNA samples were prepared from three independent biological replicates in groups 1-6. In group 7, single RNA probes were analyzed as three technical replicates.
Project description:Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMPK, a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK-/- EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs over-expressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. 2 WT and 2 AMPK DKO ESC lines were differentiated into embryoid bodies (EBs) for various lengths of time (2, 4, 8, and 12 days) in high and low glucose conditions. Both ESC and EB samples were profiled by mRNA-seq to examine how global gene expression changes associated with ESC differentiation are affected by AMPK deletion.
Project description:Goal was to identify yeast genes whose expression changed as a function of the shift from growth in bulk culture to growth in an air-liquid interfacial biofilm. Experiment Overall Design: Cells were grown 24 h at 30 C in YEPD to a density of about 500,000,000 cells/ml. At harvest, sugar was found to be depleted as measured by an enzymatic dip stick (Diastic, Bayer). Cells were pelleted and washed twice in sterile distilled water by centrifugation and then diluted 10-fold into 100 ml of Flor medium (YNB + 4% ethanol + leucine + histidine + uracil) in triplicate 250 ml beakers. Cultures were then grown statically in Flor medium at 27 C. Within a few hours following inoculation, the bulk liquid appeared to be clear, no biofilm was evident by visual observation, and a layer of settled cells was evident at the bottom of the beakers. After 48 h, a visible air-liquid interfacial biofilm covered the entire surface while the thickness of the layer of settled cells appeared unchanged. After 48 h, biofilm cells (floaters) were collected by aseptic aspiration. Once the biofilm cells were removed, cells at the bottom of the beaker (sinkers) were collected similarly. Cells from both populations were washed once in sterile distilled water by centrifugation prior to RNA isolation. Significant cell clumping was evident in both populations of cells by microscopic observation. While cell viability was estimated by plating on YEPD, the resultant cfu/ml values could not be directly correlated with cell counts in a hemacytometer because clumps of cells containing at least one viable cell presumably produced only a single colony. Further, counting individual cells accurately in the numerous clumps containing large numbers of cells was not possible. Nonetheless, when cells were counted (clumps were counted as single cells) and compared to cfu/ml for the same suspension, the cfu/ml values were about 2-fold higher than the corresponding values for cell counts using the hemacytometer for both biofilm and bottom layer cells.
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 University of Cologne, Cologne, Germany. Goal of the experiment is the identification of genes involved in endothelial differentiation. This is of great interest for the understanding of the cellular and molecular mechanisms involved in the development of new blood vessels. Mouse embryonic stem (mES) cells serve as a potential source of endothelial cells for transcriptomic analysis. We isolated endothelial cells from 8-days old embryoid bodies by immuno-magnetic separation using platelet endothelial cell adhesion molecule-1 (also known as CD31) expressed on both early and mature endothelial cells.