Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA121571 | ENA |
REPOSITORIES: ENA
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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.
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Project description:Human cord blood CD34+ cells transduced with control of IK6 lentivirus were transplanted into NSG mice. Control and IK6 transduced CD34+38- progeny were acquired 10 weeks later by FACS Comparison of control and IK6+ human CD34+38- progenitor cells
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