Transcription profiling of human cord blood derived CD34+ hematopoietic stem/progenitor cells to identifyTGF-beta1 target genes in human dendritic cells (DC).
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ABSTRACT: CD34+ hematopoietic stem/progenitor cells were isolated from human cord blood and amplified in vitro for 10-14 days in serum-free medium with specific cytokines (Ju et al., Eur. J. Cell Biol. 82, 75-86, 2003; Hacker et al., Nat. Immunol. 4, 380-386, 2003). Cultured progenitor cells were induced to differentiate into DC in RPMI medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 0.1 microM Beta-mercaptoethanol, 100 U/ml penicillin and streptomycin (GIBCO-BRL) and 500 U/ml GM-CSF, 500 U/ml IL-4 for 6 days with or without 10 ng/ml TGF-beta1 as indicated (0.5x10E6 cells/ml). Every 2 days growth factors were added and cells were maintained at 0.5x10E6 cells/ml cell density. RNA was prepared and subjected to microarray analysis. Experiment Overall Design: Dendritic cells (DC) were treated for various periods of time (4, 16 and 36 hours) with TGF-beta1 (10 ng/ml) or left untreated. Experiment Overall Design: DC untreated Experiment Overall Design: DC + TGF-beta1 for 4 hours Experiment Overall Design: DC + TGF-beta1 for 16 hours Experiment Overall Design: DC + TGF-beta1 for 36 hours
Project description:This SuperSeries is composed of the following subset Series:; GSE5150: TGF-beta1 target genes in human hematopoietic stem/progenitor cells. GSE5151: TGF-beta1 target genes in human dendritic cells (DC). Experiment Overall Design: Refer to individual Series
Project description:CD34+ hematopoietic stem/progenitor cells were isolated from human cord blood and amplified in vitro for 10-14 days in serum-free medium with specific cytokines (Ju et al., Eur. J. Cell Biol. 82, 75-86, 2003; Hacker et al., Nat. Immunol. 4, 380-386, 2003). Cells were then treated with TGF-beta1 for various periods of time (2, 4, 16 hours) and RNA was prepared and subjected to microarray analysis. Experiment Overall Design: CD34+ hematopoietic stem/progenitor cells (HPC) were amplified in vitro and treated with TGF-beta1 (10 ng/ml) for 2, 4 and 16 hours. Experiment Overall Design: HPC untreated Experiment Overall Design: HPC + TGF-beta1 for 2 hours Experiment Overall Design: HPC + TGF-beta1 for 4 hours Experiment Overall Design: HPC + TGF-beta1 for 16 hours
Project description:Dendritic cells (DC) develop from hematopoietic stem cells, which is guided by instructive signals through cytokines. DC development progresses from multipotent progenitors (MPP) via common DC progenitors (CDP) into DC. Flt3 ligand (Flt3L) signaling via the Flt3/Stat3 pathway is of pivotal importance for DC development under steady state conditions. Additional factors produced during steady state or inflammation, such as TGF-beta1 or GM-CSF, also influence the differentiation potential of MPP and CDP. Here, we studied how gp130, GM-CSF and TGF-beta1 signaling influence DC lineage commitment from MPP to CDP and further into DC. We observed that activation of gp130 signaling promotes expansion of MPP. Additionally, gp130 signaling inhibited Flt3L-driven DC differentiation, but had little effect on GM-CSF-driven DC development. The inflammatory cytokine GM-CSF induces differentiation of MPP into inflammatory DC and blocks steady state DC development. Global transcriptome analysis revealed a GM-CSF-driven gene expression repertoire that primes MPP for differentiation into inflammatory DC. Finally, TGF-beta1 induces expression of DC-lineage affiliated genes in MPP, including Flt3, Irf-4 and Irf-8. Under inflammatory conditions, however, the effect of TGF- beta1 is altered: Flt3 is not upregulated, indicating that an inflammatory environment inhibits steady state DC development. Altogether, our data indicate that distinct cytokine signals produced during steady state or inflammation have a different outcome on DC lineage commitment and differentiation.
Project description:Background: The composition of Emdogain® (EMD) is unclear, but it has been postulated to contain transforming growth factor-beta-1 (TGF-beta1) as the main functioning cytokine. The purpose of this study was to compare target genes of EMD and TGF-beta1 on invasive oral carcinoma HSC-3 cells. Particular focus was on matrix metalloproteinase (MMP) family genes. <br>Methodology/Principal Findings: Affymetrix microarrays were conducted to study differentially (P<0.05) expressed genes in HSC-3 cells after 6h and 24h of EMD (200 ?g/ml) or TGF-beta1 (10 ng/ml) incubations. Gene Ontology (GO) enrichment analyses from the regulated genes were also conducted. After 6h of EMD or TGF-?1 treatment, 48 and 393 genes, respectively, were regulated. After 24h incubations, only 12 genes by EMD but 346 genes by TGF-beta1 were regulated. Among the most regulated genes by both of the study reagents were several genes commonly regulated in carcinomas, including MMP-9 and -10. The expression of MMP-10 by EMD treated carcinoma cells was also verified in protein level. <br>Conclusions/Significance: Our results show that EMD and TGF-beta1 can regulate genes related to carcinogenesis, but TGF-beta1 regulates significantly greater number of genes. This suggests that TGF-beta1 cannot be present in EMD at the studied concentration, if present at all.<br>
Project description:Dendritic cells (DC) develop from hematopoietic stem cells, which is guided by instructive signals through cytokines. DC development progresses from multipotent progenitors (MPP) via common DC progenitors (CDP) into DC. Flt3 ligand (Flt3L) signaling via the Flt3/Stat3 pathway is of pivotal importance for DC development under steady state conditions. Additional factors produced during steady state or inflammation, such as TGF-beta1 or GM-CSF, also influence the differentiation potential of MPP and CDP. Here, we studied how gp130, GM-CSF and TGF-beta1 signaling influence DC lineage commitment from MPP to CDP and further into DC. We observed that activation of gp130 signaling promotes expansion of MPP. Additionally, gp130 signaling inhibited Flt3L-driven DC differentiation, but had little effect on GM-CSF-driven DC development. The inflammatory cytokine GM-CSF induces differentiation of MPP into inflammatory DC and blocks steady state DC development. Global transcriptome analysis revealed a GM-CSF-driven gene expression repertoire that primes MPP for differentiation into inflammatory DC. Finally, TGF-beta1 induces expression of DC-lineage affiliated genes in MPP, including Flt3, Irf-4 and Irf-8. Under inflammatory conditions, however, the effect of TGF- beta1 is altered: Flt3 is not upregulated, indicating that an inflammatory environment inhibits steady state DC development. Altogether, our data indicate that distinct cytokine signals produced during steady state or inflammation have a different outcome on DC lineage commitment and differentiation. 6 samples in total. Multipotent progenitor - GM-MPP_1 - GM-MPP_2 Dendritic cell - GM-DC_1 - GM-DC_2 Dendritic cell plus TNFa - GM-TNFa-DC_1 - GM-TNFa-DC_2
Project description:miRNA profiling of CD34+ derived, in vitro-generated Langerhans cells (LCs) and interstitial-type dendritic cells (intDCs). Epidermal Langerhans cells (LCs) form a network of dendritic cells (DCs) in basal/suprabasal layers of the skin and are characterized by a unique phenotype (CD207+ CD1abright CD324+ CD11b-). Due to their specific location at body surfaces, LCs are constantly exposed to environmental stimuli. Conversely, interstitial-type DCs (intDCs) are located in adjacent tissues and can be discriminated from LCs phenotypically (CD207- CD1adim CD324- CD11b+). These DCs constitute a second line of defense against pathogens that have crossed the epithelial barrier. During development both DC subsets originally arise from myeloid progenitor cells via monocyte-committed intermediates in response to specific microenvironmental signals. Additionally certain pools of DCs can be replenished by tissue resident cells or monocytes in response to specific microenvironmental signals (2, 4, 5). In vitro generated GM-CSF/IL-4-dependent DCs derived either from CD14+ monocytes or via CD14+CD11b+ monocyte intermediates from CD34+ progenitor cells share many characteristics with intDCs in vivo. On the other hand, LCs generated from CD34+ cells under serum-free TGF-beta1-dependent conditions phenotypically resemble epidermal-resident LCs. Since these two DC subsets are of considerable interest for clinical cell therapy studies, an improved understanding of their development and function is of substantial relevance. To identify differentially expressed miRNAs in DC subsets, we performed a miRNA screen. Therefore, we generated LCs or intDCs from human CD34+ cord blood haematopoietic progenitor cells as described. For intDCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml) and TNFalpha (2.5 ng/ml) for 3-5 days and subsequently with GM-CSF (100 ng/ml) and IL-4 (2.5 ng/ml) for 4-5 days. For LCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml), TNFalpha (2.5 ng/ml) and TGF-beta1 (0.5 ng/ml). The well-established immunophenotype of LCs (CD1ahi CD11b- CD207+ CD324+) and intDCs (CD1a+ CD11b+ CD207- CD324-) was confirmed by FACS. These two DC subsets were purified and submitted to differential miRNA profiling. Two conditioned experiment LCs vs. intDCs. Biological replicates: 3 LCs, 3 intDCs, independently differentiated, harvested and purified. 3 replicates each were pooled and hybridized on one array. Supplementary files linked below.
Project description:Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) facilitate breast cancer (BC) metastasis, however stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, we sought to identify molecular markers that could distinguish tumor cells that had completed the EMT:MET cycle in the hopes of identifying and targeting unique aspects of metastatic tumor outgrowth.Therefore, normal murine mammary gland (NMumG) cells transformed by overexpression of EGFR (NME) cells were cultured in the presence of TGF-beta1 (5 ng/ml) for 4 weeks, at which point TGF-beta1 supplementation was discontinued and the cells were allowed to recover for an additional 4 weeks (Post-TGF-Rec). Total RNA was prepared from unstimulated cells (Pre-TGF) of similar passage and compared by microarray analysis. The two groups were analyzed in triplicate, three Pre-TGF samples and three Post-TGF-Rec samples.
Project description:We here compared gene expression profiles of primary murine hepatocytes (mPC) upon stimulation with 1 ng/ml TGF-beta1 for 20 min, 2 hours and 4 hours with untreated cells. Experiments were done in three independent replicates. The goal of this study was to determine genes regulated by TGF-beta1. 12 samples were hybridized to Affymetrix Mouse Gene 1.0 ST Array
Project description:This SuperSeries is composed of the following subset Series: GSE38133: TGF-beta1 effect on human osteosarcoma cell line MNNG/HOS GSE38134: Expression analysis of human osteosarcoma cell line MNNG/HOS sarcospheres, hypoxia-induced sarcospheres and TGF-beta1-induced sarcospheres Refer to individual Series