Transcriptional profiling of human embryonic stem cell lines induced with BMP4 or Wnt3a or Wnt3a/BMP4.
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ABSTRACT: MIXL1-GFP reporter lines were differentiated as Spin EBs in APEL medium supplemented with Wnt3a alone, BMP4 alone or Wnt3a/BMP4 in combination. EBs induced in the absence of growth factors were used as control. EBs induced with BMP4 or Wnt3a/BMP4 were FACS sorted based on E-CADHERIN and GFP expression. Unsorted EBs and sorted fractions were subjected to Illumina microarray processing.
Project description:MIXL1-GFP reporter lines (HESC3MIXL1-GFP/w and MEL1MIXL1-GFP/w, referred to as HES3 and MEL1 in this submission) were differentiated as spin embryonic bodies (Ebs) supplemented with mesodermal inducing growth factors (BMP4,SCF, VEGF based). Methylcellulose hematopoietic colony forming assays were performed by culturing dissociated day 4 EBs in the presence of hematopoietic growth factors (VEGF, SCF, IL3, IL6, TPO, EPO and FLT3L) for 7-9 days. Addition of WNT3a to the methylcellulose lead to the formation of compact, mesodermal colonies, which we term 'mesosphere' (Meso-balls). We demonstrated that the inclusion of WNT3a in the methylcellulose could be replaced with BIO, a GSK3 inhibitor, acting as a canonical WNT signaling agonist. Mesospheres formed in the culture supplemented with BIO molecules were termed 'BIO-balls'. Expression profiling between 'meso-balls' and 'BIO-balls' were compared and analyzed for markers assisiting in defining their phenotypes.
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:Differentiation of INSGFP/w hESCs using published protocols demonstrated that all GFP+ cells co-expressed insulin, confirming the fidelity of the reporter gene. INS-GFP+ cells also co-expressed glucagon and somatostatin, confirming prior studies regarding the polyhormonal nature of early hESC derived insulin-expressing cells. INSGFP/w hESCs were employed to develop a 96 well format spin Embryoid Body (EB) differentiation protocol that utilized the recombinant protein based fully defined medium, APEL. Like INS-GFP+ cells generated with other methods, those derived using the spin EB protocol expressed a collection of pancreatic related transcription factors including ISL1, PAX6 and NKX2.2. However, in contrast to previous methods, the spin EB protocol yielded INS-GFP+ cells that also co-expressed the beta-cell transcription factor, NKX6.1 and comprised a substantial proportion of monohormonal insulin+ cells.
Project description:Analysis of genes enriched in MIXL1+ sorted cells during primitive streak induction identified Apelin receptor (APLNR), a highly conserved member of the G-protein coupled receptor family. Depending upon the growth factor conditions used for differentiation, APLNR+ cells identified both posterior mesodermal and anterior mesendodermal components of the primitive streak. APLNR+ cells isolated from mesodermal differentiated cultures were enriched in hematopoietic blast colony forming cells (Bl-CFCs) and the addition of Apelin peptide enhanced the frequency and growth of both hematopoietic colonies and hESC-derived endothelial cells. These studies identified APLNR as a marker of primitive streak-like cells differentiated from hESCs and defined a novel role for Apelin as a regulator of early human hematopoiesis.
Project description:Human cardiomyocytes can be generated from human embryonic stem cells (hESCs) in vitro by a variety of methods, including co-culture with visceral endoderm-like cell lines and growth factor directed differentiation as monolayers or three-dimensional embryonic bodies. To enable the identification, purification and characterisation of human embryonic stem cell derived cardiomyocytes (CMs) and cardiac progenitor cells (CPCs), we introduced sequences encoding GFP into the NKX2-5 locus by homologous recombination. We found that NKX2-5GFP hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells and cardiomyocytes and the standardization of differentiation protocols.
Project description:Transcriptional profiling of HeLa cells treated with Wnt3a at 0, 2 and 6 hours to identify transcriptional regulators controlling lipid homeostasis. Cells were treated with control or Wnt3a conditioned media for the indicated times before RNA extraction and sequencing.
Project description:Wnt signaling is upregulated frequently in several cancers, including sarcomas. Since, there is cell-context dependent variation in the target gene expression, to identify canonical Wnt targets in sarcomas, we used human mesenchymal stem cells. Human mesenchymal stem cells were treated with 100ng/ml recombinant Wnt3a either 6 hrs or 24 hrs. Total RNA was extracted from untreated and Wnt3a treated samples using Qiagen's RNA extraction kit.
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.
Project description:The goal of this project was to elucidate the target genes and transcriptional networks activated by Wnt3a during gastrulation, a complex morphogenetic process in which the embryonic germ layers are formed and the vertebrate body plan is established. To identify downstream targets of Wnt3a, transcriptional profiling was performed using Affymetrix GeneChips on both wildtype and Wnt3a null embryos at embryonic days (E) 7.75-8. In order to maximize the likelihood of identifying direct target genes of Wnt3a, RNA was isolated from the node and primitive streak (NPS) region, an area where Wnt3a is highly expressed and at a time 12-18 hours before the morpholical phenotype of Wnt3a mutants becomes apparent.
Project description:The gene targeted NKX2.1GFP/w hESC line was differentiated as spin EBs in BPEL medium supplemented with FGF2 (50-100 ng/ml). At day 7 of the differentiation, the spin EBs were pulsed with all-trans retinoic acid (10-5 M) for 72 hours. At day 10, spin EBs were removed from retinoic acid containing media and transferred from suspension to adherent culture in BEL medium containing no growth factors. Day 23 EBs were FACS sorted based on E-CADHERIN and NKX2.1-GFP expression. Day 0 hESCs, day 7 EBs, day 10 EBs with or without RA treatment and day 23 unsorted EBs and sorted fractions were subjected to Illumina microarray processing.