Gene Expression of ESC and iPSC lines after specific differentiation
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Sergio Ruiz
PROVIDER: E-GEOD-39210 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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.
2012-10-17 | E-GEOD-30125 | biostudies-arrayexpress
Project description:To induce the differentiation, undifferentiated human pluripotent stem cells (both hESC andh iPSC; T0 time point) were transferred into 20% O2 atmosphere environment and treated with mTESR1 basal media supplemented with 1 μM ATRA (Sigma-Aldrich) and 25 ng/ml BMP4 (R&D) for 7 days (Induction). To select for the cells that acquired early ectodermal fate, cells were harvested and re-plated onto freshly prepared 3D human dermal fibroblast ECM at a density of 5-10x10^3 cells per cm2 and grown in DMEM:Ham F12 (3:1) (Life Technologies) supplemented with 1 μM ATRA and 25 ng/ml BMP4 for a further 7 days (Selection). To enrich for putative epidermal progenitors, rapid-adhesion to type IV collagen-coated dishes was used, and the rapidly-adhering cells were cultured in DK SFM supplemented with 1 μM ATRA for 7 days (Enrichment). After that, the cells were cultured in EpiLife medium (Life Technologies) for a further 7 days (Expansion) before final harvest (T3 time point) and analysis. We analyzed here gene expression profiles of undifferentiated hESC/hiPSC (T0), hESC/hIPC-derived keratinocytes (T3) and primary normal human keratinocytes from skin biopsy (NHK). We found that hESC/hIPC-derived keratinocytes are similar to NHK. Biological triplicates of undifferentiated (T0) hESC (KCL034) and hiPSC lines (iKCL004, iKCL011) were compared with hESC/hiPSC-derived keratinocytes (T3), primary human keratinocyes (NHK) and fibroblasts (BJ).
2015-04-01 | E-GEOD-55898 | biostudies-arrayexpress
Project description:Analyzed differentially expressed genes among FOP- or resFOP-iMSCs treated by several ligands: Activin-A, 100 ng/mL; BMP-7, 100 ng/mL; TGF-B3, 10 ng/mL
2015-12-09 | GSE62783 | GEO
Project description:Human embryonic stem (hES) cells have the capacities to propagate for extended periods and to differentiate into cell types from all three germ layers both in vitro and in vivo. These characteristics of self-renewal and pluripotency enable hES cells having the potential to provide an unlimited supply of different cell types for tissue replacement, drug screening, and functional genomics studies. The hES-T3 cells with normal female karyotype cultured on either mouse embryonic fibroblasts (MEF) in hES medium (containing 4 ng/ml bFGF) (T3MF) or feeder-free Matrigel in MEF-conditioned medium (supplemented with additional 4 ng/ml bFGF) (T3CM) were found to express very similar profiles of mRNAs and microRNAs, indicating that the unlimited self-renewal and pluripotency of hES cells can be maintained by continuing culture on these two conditions. However, the expression profiles, especially microRNAs, of the hES-T3 cells cultured on Matrigel in hES medium supplemented with 4 ng/ml bFGF and 5 ng/ml activin A (T3BA) were found to be different from those of T3MF and T3CM cells. In T3BA cells, four hES cell-specific microRNAs miR-372, miR-302d, miR-367 and miR-200c, as well as three other microRNAs miR-199a, miR-19a and miR-217, were found to be up-regulated, whereas five miRNAs miR-19b, miR-221, miR-222, let-7b and let-7c were down-regulated by activin A. Thirteen abundantly differentially expressed mRNAs, including NR4A2, ERBB4, CXCR4, PCDH9, TMEFF2, CD24 and COX6A1 genes, targeted by seven over-expressed miRNAs were identified by inverse expression levels of these seven microRNAs to their target mRNAs in T3BA and T3CM cells. The NR4A2, ERBB4 and CXCR4 target genes were further found to be regulated by EGF and/or TNF. The 50 abundantly differentially expressed genes targeted by five under-expressed miRNAs were also identified. The abundantly expressed mRNAs in T3BA and T3CM cells were also analyzed for the network and signaling pathways, and roles of activin A in cell proliferation and differentiation were found. These findings will help elucidate the complex signaling network which maintains the self-renewal and pluripotency of hES cells. In this investigation, both miRNA and mRNA expression profiles from human embryonic stem cells grown on MEF feeder (T3MF), feeder-free Matrigel in MEF-conditioned medium (T3CM) and in hES medium (containing 4 ng/ml bFGF) supplemented with 5 ng/ml activin A (T3BA) were quantitatively determined. Several target genes of T3BA and T3CM cells-specific miRNAs were identified. ***This submission represents the mRNA expression component of the study only***
2010-09-10 | E-GEOD-16910 | biostudies-arrayexpress
Project description:We used RNA-Seq to analyse the interactions between Bmp4 and Wnt at a genome-wide level in EpiSCs treated for 48 hrs with Bmp4 and/or Wnt3a in the presence of Activin and bFGF. Control EpiSC were cultured in the presence of IWP2 for 48h. Cells were cultured with BMP4 with or without IWP2; Wnt3a and Wnt3a with BMP4 for 48h.
2014-12-24 | E-GEOD-62205 | biostudies-arrayexpress
Project description:We cultured hESC and hiPSC lines and compared the transcriptome of untreated cells with cells treated with Activin or BMP4 during 5 days
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Project description:The Wnt3a/?-catenin and Activin/Smad2,3 signaling pathways synergize to induce endodermal differentiation of human embryonic stem cells, however the mechanism is not well-understood. Using ChIP-seq and GRO-seq analyses, we report here that hESC enhancers, including Wnt3a/LEF-1 sites, hold enhancer RNAPII complexes (eRNAPII) containing high levels of Ser5P and low Ser7P. In Wnt3a signaling, ?-catenin recruits cohesin to the LEF-1:eRNAPII sites to induce enhancer-promoter looping and activate transcription of mesoendodermal (ME) genes. However, paused Ser5P-RNAPII complexes accumulate at these genes, indicating that elongation remains limiting. Subsequent Activin/Smad2,3 signaling increases P-TEFb occupancy, CTD-Ser7P, and productive elongation at ME genes. Additionally, ME genes, including EOMES and MIXL1, are repressed by the Hippo regulator, Yap1, an essential pluripotency factor. GRO-seq experiments indicate that Yap1 blocks nascent transcription and controls NELF occupancy on ME genes. Thus, Wnt3a/?-catenin and Activin/Smad2,3 pathways up-regulate transcription initiation and elongation, respectively, to overcome Yap1 repression during early hESC differentiation ChIP-seq and GROseq experiments in H1 hESCs. Cells were treated with Wnt3a (200ng/ml), Activin A (100ng/ml) or Wnt3a+Activin A (W200ng/ml+A100ng/ml) for 4h (ChIP-seq) or 6h (GRO-seq). GRO-seq in YAP depleted cells were carried out following transfection with control or YAP siRNAs . After 48h transfection, cells were left untreated or treated with Wnt3a+Activin (W200ng/ml+A100ng/ml) for additional 6h.
2015-05-04 | E-GEOD-64758 | biostudies-arrayexpress
Project description:Members of the transforming growth factor (TGF)-β superfamily play essential roles in the pluripotency, self-renewal, and differentiation of embryonic stem cells. While bone morphogenic proteins maintain pluripotency of undifferentiated mouse ES cells, the role of Activin/Nodal signaling is less clear. To determine the target genes of Activin/Nodal-Smad2 signaling in undifferentiated embryonic stem cells, changes in gene expression were examined following stimulation with recombinant Activin (2 hours) or after inhibition of Activin/Nodal with SB431542 (24 hours) using defined media culture conditions with LIF and 20 ng/mL BMP4. SB431542 is a specific inhibitor of ALK4/5/7 receptors and antagonizes both Activin and Nodal signaling. Via western analysis, Activin stimulation increased pSmad2 in ES cells after 2 hours, and treatment with SB431542 for 24 hours virtually eliminated pSmad2. Total Smad2 expression remained unchanged through these manipulations. RNA from cells treated with Activin or SB431542 was extracted by standard methods with Qiagen RNeasy columns. The RNA was analyzed with the Mouse Genome 430A Array from Affymetrix. Samples were performed in duplicate, and RNA from cells treated with Activin or SB431542 was compared to untreated embryonic stem cells.
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Project description:For definitive endoderm differentiation, iPS cells (day0) were treated with 100 ng ml-1 activin A and 3μM CHIR99021 for 1 day and 100 ng ml-1 activin A for the following two days. Definitive endoderm was subsequently treated with 3uM CHIR99021 and 500ng ml-1 FGF4 for mid/hindgut differentiation. Mid/hindgut floating spheroids (fl; t-Spheroids) were collected from culture medium from day6 to day 8. On day6, mid/hindgut cells were dissociated into single cells and seeded onto EZSPHERE plate to generate suspension spheroids(EZ; s-Spheroids). RNA of fl spheroids and EZ spheroids were extracted using QIAGEN RNeasy kit.
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Project description:Activin/Nodal/TGF-β signaling pathway plays a major role in maintaining mouse epiblast stem cells (mEpiSCs). The mEpiSC medium which contains Activin A and bFGF induces differentiation of mouse embryonic stem cells (mESCs) to mEpiSC. Here we show that Activin A also has an ability to efficiently propagate mESCs without differentiation to mEpiSCs when combined with a MEK inhibitor PD0325901. mESCs cultured in Activin+PD retained high-level expression of naive pluripotency-related transcription factors. Genome-wide analysis revealed that the gene expression profile of mESCs cultured in Activin+PD resembles that of mESCs cultured in 2i. mESCs cultured in Activin+PD also showed features which are related to naive pluripotency of mESCs, including the preferential usage of the Oct4 distal enhancer and the self-renewal response to Wnt pathway activation. Our finding reveals a role of Activin/Nodal/TGF-β signaling in stabilizing self-renewal gene regulatory networks in mESCs. To compare the gene expression patterns of mESCs cultured in Activin+PD, 2i and LIF+BMP4 and mEpiSCs, we performed genome-wide gene expression analysis by using Affymetrix GeneChip oligonucleotide microarrays
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