Unknown,Transcriptomics,Genomics,Proteomics

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Differential gene expression between human Cord blood transduced with HPIP-wt, mutant NRPID and control YFP

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

SUBMITTER: Pawandeep Kaur

PROVIDER: E-GEOD-18640 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 CellGroÂ® 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 CellGroÂ® 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.

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Differential gene expression between human Cord blood transduced with HPIP-wt, mutant NRPID and control YFP

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