Transcription profiling of human samples to investigate CD34 antigen role in haematopoietic commitment (GEO Super Series)
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Rossella Manfredini
PROVIDER: E-GEOD-8040 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:In order to investigate the role of CD34 antigen in haematopoietic commitment, we silenced the CD34 gene expression in CD34+ stem/progenitor cells using a siRNA approach. Experiment Overall Design: To maximize siRNA transfection efficiency, we utilized the NucleofectorTM technology (Amaxa). CD34+ cells were transfected with a mixture of 4 siRNAs targeting CD34 mRNA and with a non-targeting siRNA as a negative control. The expression level of CD34 antigen on control cells (MOCK and negative control treated cells) and CD34siRNA treated cells was assessed by immunofluorescence analysis at 24 and 48h post-nucleofection.
2008-06-18 | E-GEOD-8002 | biostudies-arrayexpress
Project description:In order to investigate the role of CD34 antigen in haematopoietic commitment, we overexpressed the human CD34 cDNA in human CD34+ cells by retroviral gene transfer. Experiment Overall Design: In a set of 10 independent experiments, gene transfer efficiency, assessed by flow cytometry analysis of ÎLNGFR positivity, ranged 15 to 30%. Transduced cells were than purified for NGFR expression at day 3 of liquid culture with the retroviral vector. Phenotypic analysis, performed on transduced/NGFR purified cells evidenced that the CD34 transgene was highly expressed at the protein level (80% of LCD34IÎN vs 30% of controls at 5 days post-infection) up to 14 days of liquid culture.
2008-06-18 | E-GEOD-8003 | biostudies-arrayexpress
Project description:The c-Myb transcription factor is highly expressed in immature hematopoietic cells and down-regulated during differentiation. To define the role of c-Myb in human hematopoietic lineage commitment, we studied the effects of its silencing during the commitment of human CD34+ Hematopoietic stem/progenitor cells. In CD34+ cells c-Myb silencing determined a cell cycle arrest in G0/G1 phase which strongly decreased the clonogenic efficiency, togheter with a reduction of erythroid colonies coupled with an increase of the macrophage and megakaryocyte ones. Moreover, morphological and flow cytometry data supported the preferential macrophage and megakaryocyte differentiation of c-Myb-silenced CD34+ cells. Taken together our data indicate that c-Myb is essential for the commitment along the erythroid and granulocyte lineages but not for the macrophage and megakaryocyte differentiation. Gene expression profiling of c-Myb-silenced CD34+ cells identified some potential c-Myb targets which can account for these effects, to study by Chromatin Immunoprecipitation and Luciferase Reporter Assay. To maximize siRNA transfection efficiency, we utilized the NucleofectorTM technology (Amaxa). CD34+ cells were transfected with a mixture of 3 siRNAs targeting c-Myb mRNA and with a non-targeting siRNA as a negative control. The expression level of c-Myb protein on control cells (MOCK and negative control treated cells) and MYBsiRNA treated cells was assessed by Western Blot at 24 and 48h post-nucleofection.
2010-07-14 | E-GEOD-13110 | biostudies-arrayexpress
Project description:Although a considerable number of reports indicate an involvement of the Hox-A10 gene in the molecular control of hematopoiesis, the conclusions of such studies are quite controversial since they support, in some cases, a role in the stimulation of stem cell self-renewal and myeloid progenitor expansion while, in others, implicate this transcription factor in the induction of monocyte - macrophage differentiation. To clarify this issue we analyzed the biological effects and the transcriptome changes determined in human primary CD34+ hematopoietic progenitors by retroviral transduction of a full length Hox-A10 cDNA. The results obtained clearly indicated that this homeogene is an inducer of monocyte differentiation, at least partly acting through the up-regulation of MafB gene, recently identified as master regulator of such maturation pathway. By using a combined approach based on computational analysis, EMSA experiments and luciferase assays, we were able to demonstrate the presence of a Hox-A10 binding site in the promoter region of the MafB gene, which suggested the likely molecular mechanism underlying the observed effect. Interestingly, stimulation of the same cells with the Vitamin D3 monocyte differentiation inducer resulted in a clear increase of Hox-A10 and MafB transcripts, indicating the existence of a precise transactivation cascade involving VDR, Hox-A10 and MafB transcription factors. Altogether these data allow to conclude that the Vitamin D3 / Hox-A10 pathway supports MafB function during the induction of monocyte differentiation. Experiment Overall Design: RNA pools (100 ng) of LXIDN and LHoxA10IDN transduced CD34+ cells, obtained from three independent experiments, were converted in labelled cRNA according with the âTwo cycleâ protocol advised by Affymetrix. cRNA has been used to hybridise Affymetrix HG-U133A GeneChip arrays. Images obtained by scanning chips of LXIDN and LHoxA10IDN transduced CD34+ cells were processed using the GeneChip Operating Software. Microarray analysis of Hox-A10 transduced CD34+ cells provided a substantial contribute for a better comprehension of the biological effects driven by this transcription factor in human primary hematopoietic stem / progenitor cells. Results of this analysis confirmed the stimulatory effect exerted by Hox-A10 on monocytopoiesis, disclosing an up-regulated expression of transcription factors and differentiation markers (CD antigens, granule proteins, cytokines / chemokines) that are typically associated with this maturation lineage. In addition, they also evidenced a decreased expression of genes related to erythroid and granulocyte differentiation programs. This last effect was also confirmed by cytochemical and morphological evaluation of Hox-A10 transduced CD34+ cells.
2008-10-21 | E-GEOD-12396 | biostudies-arrayexpress
Project description:The transcription factor cMyb plays a key role in human primary CD34+ hematopoietic progenitor cells (HPCs) lineage choice, by enhancing erythropoiesis at the expense of megakaryopoiesis. We previously demonstrated that cMyb affects erythroid versus megakaryocyte lineage decision in part by transactivating KLF1 and LMO2 expression. To further unravel the molecular mechanisms through which cmyb affects lineage fate decision, we profiled the miRNA and mRNA changes in myb-silenced CD34+ HPCs. mRNA and miRNA expression for each sample were profiled by Affymetrix GeneAtlas U219 strip array and Exiqon Human miRNome PCR Panel, respectively. miRNA/mRNA data were integrated by Ingenuity Pathway Analysis. The integrative analysis of miRNA/mRNA expression changes upon c-myb silencing in human CD34+ HPCs highlighted a set of 19 miRNA with 150 anticorrelated putative target mRNAs. Among the miRNAs downregulated in myb-silenced progenitors with the highest number of predicted target mRNAs, we selected hsa-miR-486-3p based on the in vitro effects of its overexpression on HPCs commitment. Indeed, morphological and flow cytometric analyses after liquid culture showed that hsa-miR-486-3p overexpression in HPCs enhanced erythroid and granulocyte differentiation while restraining megakaryocyte and macrophage differentiation. Moreover, collagen-based clonogenic assay demonstrated a strong impairement megakaryocyte commitment upon hsa-miR-486-3p overexpression in CD34+ cells. Gene expression profiling of hsa-miR-486-3p overexpressing CD34+ cells enabled us to identify a set of 8 genes downregulated and computationally predicted, putative hsa-miR-486-3p targets. Among them, we selected c-maf transcript as upregulated upon myb silencing. Worth of note, c-maf silencing in CD34+ progenitor cells was able to reverse the affects of myb silencing on erythroid versus megakaryocyte lineage choice. Integrative miRNA/mRNA analysis highlighted a set of miRNAs and anticorrelated putative target mRNAs modulated upon myb silencing, therefore potential players in myb-driven HPCs lineage choice. Among them, we demonstrated the hsa-miR-486-3p/c-maf pair as partially contributing to the effects of myb on HPCs commitment. Therefore, our data collectively identified myb-driven hsa-miR-486-3p upregulation and subsequent c-maf downregulation as a new molecular mechanism through which cMyb favours erythropoiesis while restraining megakaryopoiesis. RNA from CD34+ HPCs transfected with c-myb-targeting/non targeting control (NegCTR) synthetic siRNAs was collected 24 hours post-Nucleofection for a set of 5 independent experiments.
2015-08-07 | E-GEOD-60300 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2015-08-07 | E-GEOD-60301 | biostudies-arrayexpress
Project description:Umbilical cord blood (CB) is a non-invasive, convenient and broadly used source of hematopoietic stem cells (HSCs) for allogeneic stem cell transplantation. However, limiting numbers of HSCs remain a major constraint for its clinical application. One feasible option would be to expand HSCs to improve therapeutic outcome, however available protocols and the molecular mechanisms governing the self-renewal of HSC are unclear. Here we show that ectopic expression of a single miRNA, miR-125a, in purified murine and human multipotent progenitors (MPP) resulted in increased self-renewal and robust long-term multi-lineage repopulation in transplanted recipient mice. Using quantitative proteomics and Western blot analysis, we identified a restricted set of miR-125a targets which revealed the involvement of the MAP kinase signaling pathway in conferring long-term repopulating capacity to multipotent progenitors in human and mice. Our findings offer the innovative potential to use MPP with enhanced self-renewal activity to augment limited sources of HSC to improve clinical protocols.
2017-01-11 | PXD003931 | Pride
Project description:Dendritic Cell differentiation - CD molecule cluster follow up: The data files associated to this experiment show the gene expression levels for a subset of 152 transcripts (out of 12626 genes represented on Affymetrix Genechip HG_U95Av2) representing CD molecules specifically expressed in Dendritic Cells (DC) as assessed by the 9 conditions tested. Another subset of genes, corresponding to a cluster of Transcription regulators is available from E-MEXP-2 experiment.
2014-06-23 | E-MEXP-1 | biostudies-arrayexpress
Project description:Hematopoietic differentiation is strictly regulated by complex network of transcription factors that are controlled by ligands binding to cell surface receptors. Disruptions of the intricate sequences of transcriptional activation and suppression of multiple genes cause hematological diseases, such as leukemias, myelodysplastic syndromes or myeloproliferative syndromes. From a clinical standpoint, deciphering the pattern of gene expression during hematopoiesis may help unravel disease-specific mechanisms in hematopoietic malignancies. Herein, we describe a human in vitro hematopoietic model system where lineage specific differentiation of CD34+ cells was accomplished using specific cytokines. Microarray and RNAseq based whole transcriptome and exome analysis was performed on the differentiated erythropoietic, granulopoietic and megakaryopoietic cells to delineate changes in expression of whole transcripts and exons. Analysis on the Human 1.0 ST exon arrays indicated differential expression of 172 genes (P< 0.0000001) and significant splicing of 86 genes during differentiation. Pathway analysis identified these genes to be involved in Rac/RhoA signaling, Wnt/B-catenin signaling and alanine/aspartate metabolism. Comparison of the microarray data to next generation RNAseq analysis during erythroid differentiation demonstrated a high degree of correlation in gene (R= 0.72) and exon (R=0. 62) expression. Our data provides a molecular portrait of events that regulate differentiation of hematopoietic cells. Knowledge of molecular processes by which the cells acquire their cell specific fate would be beneficial in developing cell-based therapies for human diseases. Comparison of gene expression patterns between CD34, E, M and G groups
2011-11-22 | E-GEOD-29989 | biostudies-arrayexpress
Project description:Dendritic Cell differentiation - Transcription Regulator cluster follow-up: The data files associated to this experiment show gene expression levels for a subset of 481 transcripts (out of 12626 genes represented on Affymetrix Genechip HG_U95Av2) corresponding to Transcription Regulators whose expression is changed during the differentiation process of Dendritic Cells as assessed in the 9 conditions tested. Another subset of genes, corresponding to a cluster of CD molecules is available from E-MEXP-1 experiment.
2011-08-15 | E-MEXP-2 | biostudies-arrayexpress