Transcription profiling by array of mouse embryonic stem cells during differentiation
ABSTRACT: ES cells are differentiated into mesenderm cell linage by Activin A treatment. During this differentaition, gene expression profile of ES cells, day 4 mesendoderm cell, day 5 mesendoderm cells and mesoderm cells are examined.
Project description:ES cells are differentiated into mesenderm cell linage by Activin A treatment. During this differentaition, gene expression profile of ES cells, day 4 mesendoderm cell, day 5 mesendoderm cells and mesoderm cells are examined.
Project description:The mechanisms by which human embryonic stem cells (hESC) differentiate to endodermal lineage have not been extensively studied. Mathematical models can aid in the identification of mechanistic information. In this work we use a population-based modeling approach to understand the mechanism of endoderm induction in hESC, performed experimentally with exposure to Activin A and Activin A supplemented with growth factors (basic fibroblast growth factor (FGF2) and bone morphogenetic protein 4 (BMP4)). The differentiating cell population is analyzed daily for cellular growth, cell death, and expression of the endoderm proteins Sox17 and CXCR4. The stochastic model starts with a population of undifferentiated cells, wherefrom it evolves in time by assigning each cell a propensity to proliferate, die and differentiate using certain user defined rules. Twelve alternate mechanisms which might describe the observed dynamics were simulated, and an ensemble parameter estimation was performed on each mechanism. A comparison of the quality of agreement of experimental data with simulations for several competing mechanisms led to the identification of one which adequately describes the observed dynamics under both induction conditions. The results indicate that hESC commitment to endoderm occurs through an intermediate mesendoderm germ layer which further differentiates into mesoderm and endoderm, and that during induction proliferation of the endoderm germ layer is promoted. Furthermore, our model suggests that CXCR4 is expressed in mesendoderm and endoderm, but is not expressed in mesoderm. Comparison between the two induction conditions indicates that supplementing FGF2 and BMP4 to Activin A enhances the kinetics of differentiation than Activin A alone. This mechanistic information can aid in the derivation of functional, mature cells from their progenitors. While applied to initial endoderm commitment of hESC, the model is general enough to be applicable either to a system of adult stem cells or later stages of ESC differentiation.
Project description:Bone morphogenetic protein (BMP) signaling is known to support differentiation of human embryonic stem cells (hESCs) into mesoderm and extraembryonic lineages, whereas other signaling pathways can largely influence this lineage specification. Here, we set out to reinvestigate the influence of ACTIVIN/NODAL and fibroblast growth factor (FGF) pathways on the lineage choices made by hESCs during BMP4-driven differentiation. We show that BMP activation, coupled with inhibition of both ACTIVIN/NODAL and FGF signaling, induces differentiation of hESCs, specifically to ?hCG hormone-secreting multinucleated syncytiotrophoblast and does not support induction of embryonic and extraembryonic lineages, extravillous trophoblast, and primitive endoderm. It has been previously reported that FGF2 can switch BMP4-induced hESC differentiation outcome to mesendoderm. Here, we show that FGF inhibition alone, or in combination with either ACTIVIN/NODAL inhibition or BMP activation, supports hESC differentiation to hCG-secreting syncytiotrophoblast. We show that the inhibition of the FGF pathway acts as a key in directing BMP4-mediated hESC differentiation to syncytiotrophoblast.
Project description:Wnt/beta-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.We tested the hypothesis that Wnt/beta-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/beta-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for beta-myosin heavy chain (beta-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/beta-catenin signaling is required for Smad1 activation by BMP4.Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/beta-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.
Project description:Bone morphogenetic protein (BMP) signaling is known to support differentiation of human embryonic stem cells (hESCs) into mesoderm and extraembryonic lineages, whereas other signaling pathways can largely influence this lineage specification. Here, we set out to reinvestigate the influence of ACTIVIN/NODAL and fibroblast growth factor (FGF) pathways on the lineage choices made by hESCs during BMP4-driven differentiation. We show that BMP activation, coupled with inhibition of both ACTIVIN/NODAL and FGF signaling, induces differentiation of hESCs, specifically to M-NM-2hCG hormone-secreting multinucleated syncytiotrophoblast and does not support induction of embryonic and extraembryonic lineages, extravillous trophoblast, and primitive endoderm. It has been previously reported that FGF2 can switch BMP4-induced hESC differentiation outcome to mesendoderm. Here, we show that FGF inhibition alone, or in combination with either ACTIVIN/NODAL inhibition or BMP activation, supports hESC differentiation to hCG-secreting syncytiotrophoblast. We show that the inhibition of the FGF pathway acts as a key in directing BMP4-mediated hESC differentiation to syncytiotrophoblast. Human embryonic Stem Cells (hESCs) were treated under defined conditions (N2B27) with BMP4 (B), SB431542 (SB) (ACTIVIN/NODAL inhibitor), SU5402 (SU) (FGFR1 inhibitor), FGF2 (F) either alone or in various combinations as mentioned, followed by isolation of total RNA.
Project description:Embryonic stem (ES) cells have been shown to recapitulate normal developmental stages. They are therefore a highly useful tool in the study of developmental biology. Profiling of ES cell-derived cells has yielded important information about the characteristics of differentiated cells, and allowed the identification of novel marker genes and pathways of differentiation. In this review, we focus on recent results from profiling studies of mouse embryos, human islets, and human ES cell-derived differentiated cells from several research groups. Global gene expression data from mouse embryos have been used to identify novel genes or pathways involved in the developmental process, and to search for transcription factors that regulate direct reprogramming. We introduce gene expression databases of human pancreas cells (Beta Cell Gene Atlas, EuroDia database), and summarize profiling studies of islet- or human ES cell-derived pancreatic cells, with a focus on gene expression, microRNAs, epigenetics, and protein expression. Then, we describe our gene expression profile analyses and our search for novel endoderm, or pancreatic, progenitor marker genes. We differentiated mouse ES cells into mesendoderm, definitive endoderm (DE), mesoderm, ectoderm, and Pdx1-expressing pancreatic lineages, and performed DNA microarray analyses. Genes specifically expressed in DE, and/or in Pdx1-expressing cells, were extracted and their expression patterns in normal embryonic development were studied by in situ hybridization. Out of 54 genes examined, 27 were expressed in the DE of E8.5 mouse embryos, and 15 genes were expressed in distinct domains in the pancreatic buds of E14.5 mouse embryos. Akr1c19, Aebp2, Pbxip1, and Creb3l1 were all novel, and none has been described as being expressed, either in the DE, or in the pancreas. By introducing the profiling results of ES cell-derived cells, the benefits of using ES cells to study early embryonic development will be discussed.
Project description:To investigate the role of histone H3K27 demethylase UTX in embryonic stem (ES) cell differentiation, we have generated UTX knockout (KO) and enzyme-dead knock-in male ES cells. Deletion of the X-chromosome-encoded UTX gene in male ES cells markedly decreases expression of the paralogous UTY gene encoded by Y chromosome, but has no effect on global H3K27me3 level, Hox gene expression, or ES cell self-renewal. However, UTX KO cells show severe defects in mesoderm differentiation and induction of Brachyury, a transcription factor essential for mesoderm development. Surprisingly, UTX regulates mesoderm differentiation and Brachyury expression independent of its enzymatic activity. UTY, which lacks detectable demethylase activity, compensates for the loss of UTX in regulating Brachyury expression. UTX and UTY bind directly to Brachyury promoter and are required for Wnt/?-catenin signaling-induced Brachyury expression in ES cells. Interestingly, male UTX KO embryos express normal levels of UTY and survive until birth. In contrast, female UTX KO mice, which lack the UTY gene, show embryonic lethality before embryonic day 11.5. Female UTX KO embryos show severe defects in both Brachyury expression and embryonic development of mesoderm-derived posterior notochord, cardiac, and hematopoietic tissues. These results indicate that UTX controls mesoderm differentiation and Brachyury expression independent of H3K27 demethylase activity, and suggest that UTX and UTY are functionally redundant in ES cell differentiation and early embryonic development.
Project description:Nodal/Activin signaling directs mesendoderm specification in early vertebrate embryogenesis. We have characterized transcriptional profiling of human embryonic stem cells and Activin-treated cells at different timepoints. In this dataset, we include the timecourse gene expression data obtained from hESC differentiation post Activin treatment, examining at day 0, 1, 3 and 5. Four biological replicates were used for day 0, three for day 1, three for day 3, and four for day 5. Cells were harvested and used for total RNA preparation using Trizol (Invitrogen). Gene expression was analyzed using GeneChip Human Exon 1.0 ST Array (Affymetrix).
Project description:This study aimed to examine gene expression in human ES cells (the RUNX1C GFP reporter line) differentiated towrads hameatopoietic mesoderm in a defined serum free medium. At day 7 of differentiation, the cells were sorted into fractions based on CD34 and CD41 expression and the four fractions analysed by microarray. The total number of samples analysed was 13. Undifferentiated hESC (RUNX1C GFP/w, based on the HES3 cell line) plus samples from d1 to d8 of differentiation comprised one experiment (9 samples) and four flow sorted fractions from d7 differentiated cells (CD34-CD41-, CD34lo CD41-, CD34hi CD41- and CD34lo CD41lo) comprised the second experiment. The parent cell line was maintained on mouse feeder cells in KOSR containing medium supplemented with 10 ng/ml FGF2. Differentiation was performed as spin EBs in APEL medium (Ng et al Nature Protocols 2008). For the first 4 days, medium was supplemented with BMP4, VEGF, SCF and Activin. Medium was changed at d4 to fresh APEL medium supplemented with BMP4, VEGF, SCF, FGF2 and IGF2.
Project description:<h4>Background</h4>Mesendoderm induction during embryonic stem cell (ESC) differentiation in vitro is stimulated by the Transforming Growth Factor and Wingless (Wnt) families of growth factors.<h4>Principal findings</h4>We identified the periods during which Bone Morphogenetic Protein (BMP) 4, Wnt3a or Activin A were able to induce expression of the mesendoderm marker, Mixl1, in differentiating mouse ESCs. BMP4 and Wnt3a were required between differentiation day (d) 1.5 and 3 to most effectively induce Mixl1, whilst Activin A induced Mixl1 expression in ESC when added between d2 and d4, indicating a subtle difference in the requirement for Activin receptor signalling in this process. Stimulation of ESCs with these factors at earlier or later times resulted in little Mixl1 induction, suggesting that the differentiating ESCs passed through 'temporal windows' in which they sequentially gained and lost competence to respond to each growth factor. Inhibition of either Activin or Wnt signalling blocked Mixl1 induction by any of the three mesendoderm-inducing factors. Mixing experiments in which chimeric EBs were formed between growth factor-treated and untreated ESCs revealed that BMP, Activin and Wnt signalling all contributed to the propagation of paracrine mesendoderm inducing signals between adjacent cells. Finally, we demonstrated that the differentiating cells passed through 'exit gates' after which point they were no longer dependent on signalling from inducing molecules for Mixl1 expression.<h4>Conclusions</h4>These studies suggest that differentiating ESCs are directed by an interconnected network of growth factors similar to those present in early embryos and that the timing of growth factor activity is critical for mesendoderm induction.