Comparative transcriptome of CD34+ hematopoietic progenitors from myeloproliferative patients and control donors
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PROVIDER: EGAS00001005106 | EGA |
REPOSITORIES: EGA
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Project description:CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3
2011-01-22 | GSE26775 | GEO
Project description:CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3 CB CD34+ cells were isolated by Miltenyi miniMACS column. Cells were prestimulated in HPGM with 100 ng/ml KITL, FLT#L and TPO for 48 hrs. Cells were transduced with control MiNR1 or STAT5A-ER in three rounds over 48 hrs. Hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and megakaryocyte/erythroid progenitors (MEPs) were sorted (for details see Blood 2011, Fatrai et al). cells were stimulated with 100 ng/ml 4OHT for 24 hrs after which RNA was isolated for Illumina beadhchiop arrays HT12 v3
2011-01-22 | E-GEOD-26775 | biostudies-arrayexpress
Project description:RNA sequencing for enteroids treated with different concentrations of IL-17 (0 ng/ml. 1 ng/ml, 10 ng/ml and 100 ng/ml)
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Project description:CD34+ cells were isolated from leukopheresis preparations by Miltenyi positive selection kits for CD34 cells and cultured in stemPro medium+ supplement 2 culture conditions were compared, G1 = IL-3 at 5 ng/ml for the 17 days of culture or G3 = IL-3 at 5 ng/ml + SCF (stem cell factor) at 4 ng/ml + Flt3L at 8 ng/ml
2019-11-08 | GSE138608 | GEO
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.
2010-02-12 | GSE20215 | GEO
Project description:To evaluate the intestinal epithelial responses induced by IL-17, bulk RNA-sequencing (RNA-seq) was performed on the human small intestinal organoids (enteroids, n = 3) treated with different concentrations of IL-17 (0 ng/ml. 1 ng/ml, 10 ng/ml and 100 ng/ml) .
2021-08-20 | GSE182453 | GEO
Project description:Background: It has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival has also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34+ progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny. Methods: Hematopoietic CD34+ progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells. Results: In microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes have specific expression just at certain stages of ontogeny (STATs, GRB2, CREBB). Beside continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH). Conclusions: This broad overview of gene expression in erythropoiesis revealed transcription factors with a prevalence at certain stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway- coupled NFKBIA and YWHAH genes. Adult peripheral blood mononuclear cells were isolated from buffy coats of 3 healthy donors using Lymphocyte Separation Medium (BioWhittaker, Walkersville, MD). We washed mononuclear cells twice with Dulbecco's phosphate-buffered saline (PBS, Invitrogen Corporation, Carlsbad, CA), and CD34+ cells were purified by positive immunomagnetic selection using the MACS cell isolation system (Miltenyi Biotec, Auburn, CA). Fresh bone marrow CD34+ cells were collected (AllCells LLC, Berkeley, CA). Cord blood CD34+ cells (AllCells LLC) and fetal liver CD34+ cells (Cambrex Bio Science, Inc., Walkersville, MD) were collected and frozen. For analysis, CD34+ cells were resuspended in medium, which contained 30% FBS, 2 mmol/L glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% deionized BSA, 10 mmol/L beta-mercaptoethanol, 1 mmol/L dexamethasone, 33 µg/ml holo-transferrin, 10 ng/ml SCF, 1 ng/ml IL-3 and 1 ng/ml GM-CSF (Sigma, St. Louis, MO), and 1 U/ml human recombinant EPO (Amgen Inc, Thousand Oaks, CA) Erythroid progenitor cells differentiated from hematopoietic CD34+ progenitor cells of fetal liver, cord blood, bone marrow and peripheral blood origin Biological replicates: 2 fetal liver, 3 cord blood, 3 bone marrow, 3 peripheral blood origin
2012-05-31 | E-GEOD-37869 | biostudies-arrayexpress
Project description:Comparative transcriptome of CD34+ hematopoietic progenitors from 4 myeloproliferative patients (MPN) and 4 control donors performed by RNA-Sequencing.
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Project description:The aim of the present study was to clarify the role of IL-18 in mouse mast cell functions. Mouse mucosal mast cells (Balb/c) were differentiated in the presence of IL-3 (5 ng/ml), SCF (40 ng/ml), IL-9 (10 ng/ml) and human TGFbetaI (2 ng/ml) for >4 weeks. Cells were then stimulated by 100 ng/ml IL-18 for 2 hours to monitor the direct effect of IL-18. Treated samples were compared to the untreated ones in a paired experimental setting. Keywords: expression microarray, mast cell, IL-18
2008-09-26 | GSE9736 | GEO
Project description:Background Homeobox gene associated regulatory networks are among the key determinants of early hematopoietic development. Previously, the ‘hematopoietic PBX interacting protein’ (HPIP) has been identified as a novel interacting partner of the TALE homeodomain protein PBX1, forming a microtubule signalling complex. Expression of HPIP has been associated with increased tumorigenicity of the MCF7 breast cancer cell line. We now demonstrate that HPIP is a novel regulatory protein in human hematopoiesis: constitutive expression of HPIP in human umbilical cord blood derived CD34+ cells increased the absolute number of clonogenic progenitors in liquid expansion culture as well as in methylcellulose assays with a significantly enhanced formation of erythroid colonies compared to the control (p?0.01, n=6). Limiting dilution LTC-IC assays confirmed the hematopoietic activity of the protein on primitive human progenitor cells with an over 5fold increase in the absolute number of LTC-ICs compared to non-transduced cells (n=8; p<0.05). In vivo HPIP expression induced a significant shift towards myeloid engraftment (n=8;p<0.05) and doubled the proportion of hCD34+CD38+ human cells in transplanted mice (p?0.05, n=8). Structure – function analyses identified the C - terminal nuclear receptor/PBX interacting domain (NRPID; LXXLL domain) as a critical domain for the hematopoietic activity of HPIP. Gene expression data by microarray and Q-RT-PCR analysis demonstrated that HPIP induced particularly differential expression of genes involved in the MAPK pathway and cytokine-cytokine interaction. Taken together, these data demonstrate that proteins involved in the organization of microtubular signalling complexes such as HPIP can act as regulators of early human hematopoiesis. Stable PG13 packaging cell lines were used to perform transductions of human umbilical cord blood derived CD34+ cells. Phoenix amphotropic cells were used for transient transduction of PG13 with wt and ?NRPID mutant viruses. Both cell lines were cultured in DMEM with 10% fetal bovine serum and plated on corning dishes for transfections and transient transduction ( 2.5 x 106 cells per 10 cm plate) a day prior to the experiment. Transient transductions were performed as described before in literature. Briefly, cells at 2x10e5/mL were prestimulated for 48 hours in Iscove´s IMDM( GIBCO-Invitrogen, Karlsruhe, Germany) containing a serum substitute (BITTM, Stem Cell Technologies), 10-4M ? mercaptoethanol (Sigma-Aldrich, Taufkirchen, Germany), supplemented with the following recombinant human cytokines: 100 ng/mL Flt-3 Ligand, 100 ng/mL SF, 20 ng/mL IL-3, 20 ng/mL G-CSF, and 20 ng/mL IL-6 (Immunotools, Friesoythe, Germany). After 48 Hours, cells were resuspended in filtered virus-containing medium (VCM) supplemented with the same five cytokine cocktail and polybrene (5 µg/mL) on tissue culture dishes (Corning). The dishes were pre-loaded with VCM twice, each time for 45 mins. The procedure was repeated for a total of three infections. Fresh RNA was prepared using RNA easy micro kit (Sigma) with ? 2x105 retrovirally transduced human cord blood CD34+HPIP-WT-YFP+, CD34+?NRPID-HPIP-YFP+ or CD34+YFP+ cells. Total RNA was reverse transcribed (Superscript II, Invitrogen) by using oligo (dT) to prime cDNA synthesis. In vitro transcription was performed using a the GeneChip® Two-Cycle cDNA Synthesis Kit, uniquely configured and tested for GeneChip® target labeling. Two cycles of double-stranded cDNA synthesis containing the T7 promoter sequence were performed from 10 to 100 ng of total RNA using this Kit.
2010-09-30 | E-GEOD-18640 | biostudies-arrayexpress