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| Status |
Public on Sep 30, 2010 |
| Title |
Differential gene expression between human Cord blood transduced with HPIP-wt, mutant NRPID and control YFP |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
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.
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| Overall design |
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 β 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.
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| Citation missing |
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| Submission date |
Oct 20, 2009 |
| Last update date |
Mar 25, 2019 |
| Contact name |
Pawandeep Kaur |
| E-mail(s) |
pmroke@yahoo.com
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| Phone |
0049897099425
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| Organization name |
Helmholtz-Muenchen
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| Department |
Hematology
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| Lab |
Human Stem Cell
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| Street address |
Marchioninstrasse 25
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| City |
Muenchen |
| State/province |
Bavaria |
| ZIP/Postal code |
83177 |
| Country |
Germany |
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| Platforms (1) |
| GPL570 |
[HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array |
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| Samples (3) |
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| Relations |
| BioProject |
PRJNA121571 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE18640_RAW.tar |
24.2 Mb |
(http)(custom) |
TAR (of CEL) |
| Processed data included within Sample table |
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