Development of a murine hematopoietic progenitor complementary DNA microarray using a subtracted complementary DNA library

Abstract With the goal of creating a resource for in-depth study of myelopoiesis, we have executed a 2-pronged strategy to obtain a complementary DNA (cDNA) clone set enriched in hematopoietic genes. One aspect is a library subtraction to enrich for underrepresented transcripts present at early stages of hematopoiesis. For this, a hematopoietic cDNA library from primary murine bone marrow cells enriched for primitive progenitors was used as tester. The subtraction used 10 000 known genes and expressed sequence tags (ESTs) as driver. The 2304 randomly picked clones from the subtracted cDNA libraries represent 1255 distinct genes, of which 622 (50%) are named genes, 386 (30%) match uncharacterized ESTs, and 247 (20%) are novel. The second aspect of our strategy was to complement this subtracted library with genes known to be involved in myeloid cell differentiation and function. The resulting cDNAs were arrayed on polylysine-coated glass slides. The microarrays were used to analyze gene expression in primary and cultured murine bone marrow–derived progenitors. We found expression of various types of genes, including regulatory cytokines and their receptors, signal transduction genes, and transcription factors. To assess gene expression during myeloid differentiation, we examined patterns of change during induced differentiation of EML cells. Several hundred of the genes underwent fluctuations in expression level during myeloid cell differentiation. The complete database, accessible on the World Wide Web at http://yale130132115135.med.yale.edu/, allows for retrieval of information regarding these genes. Our microarray allows for genomewide expression analysis of myeloid stem cells, which will help in defining the regulatory mechanisms of stem cell differentiation.

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T-cell growth factor P40 promotes the proliferation of myeloid cell lines and enhances erythroid burst formation by normal murine bone marrow cells in vitro

Blood ◽

10.1182/blood.v76.5.906.bloodjournal765906 ◽

1990 ◽

Vol 76 (5) ◽

pp. 906-911 ◽

Cited By ~ 1

Author(s):

DE Williams ◽

PJ Morrissey ◽

DY Mochizuki ◽

P de Vries ◽

D Anderson ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Growth Factor ◽

Cell Growth ◽

Cell Lines ◽

Bone Marrow Cells ◽

Myeloid Cell ◽

Murine Bone Marrow ◽

T Cell Growth Factor ◽

Marrow Cells

T-cell growth factor P40 was examined for possible effects on murine interleukin-3 (IL-3)-dependent myeloid cell lines and freshly isolated murine bone marrow cells. The results showed that P40 stimulated the proliferation of some IL-3-dependent myeloid cell lines of both early myeloid and mast cell phenotype and synergized with IL-3. P40 did not promote proliferation of fresh bone marrow cells, bone marrow enriched for early myeloid cells by 5-fluorouracil treatment, or bone marrow derived mast cells as assessed in 3H-TdR incorporation assays. P40 did not influence the growth of murine colony-forming unit granulocyte- macrophage in agar cultures, either alone or in the presence of optimal or sub-optimal concentrations of CSF-1, GM-colony-stimulating factor, or IL-3. P40 did potentiate burst-forming unit-erythroid (BFU-E) formation in the presence of erythropoietin; however, this was dependent on the cell plating density, suggesting an indirect stimulation of BFU-E by P40. The indirect nature of P40 action on BFU-E was further demonstrated in cell separation experiments and indicated that the effect was mediated by T cells. These data expand the repertoire of cells that P40 influences.

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MDS1-EVI1 and EVI1 Overexpression Results in Changes in the Behavior of Murine Hematopoietic Cells.

Blood ◽

10.1182/blood.v108.11.1452.1452 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1452-1452

Author(s):

Jean-Yves Metais ◽

Rotraud Wieser ◽

Cynthia E. Dunbar

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Hematopoietic Cells ◽

Gene Products ◽

Gene Complex ◽

Murine Bone Marrow ◽

And Control ◽

The Impact ◽

Marrow Cells

Abstract We have found non-random patterns of retroviral integration in long-term hematopoietic repopulating cells in the rhesus macaque, with frequent integration events of MLV vectors into the MDS1-EVI1 gene complex. These findings, along with reports regarding frequent integration events in the same gene complex in patients with chronic granulomatous disease receiving MLV-transduced hematopoietic cells in a clinical trial and the ability of MLV vectors activating expression of this gene via integration to immortalize primary murine bone marrow cells, suggests these gene products could have important roles in normal and leukemic hematopoiesis. Expression from this gene complex can result in translation of at least three distinct proteins: MDS1, EVI1, and MDS1-EVI1. EVI1 has been the most studied protein of this locus. Its overexpression, as a consequence of chromosomal rearrangement or viral integration, is associated with leukemia. MDS1-EVI1 contains a PR domain that is lacking in EVI1 and is thought to possibly be antagonistic to EVI1, however the location of the integrations in our prior rhesus studies would indicate that overexpression of either gene product could be immortalizing. Both proteins share the same expression profile in normal tissues as well as most reports of myeloid leukemias. To investigate the impact of the three gene products on hematopoietic cells, we cloned murine mds1, evi1, and mds1-evi1 into the pMIEV-GFP retroviral vector and produced ecotropic vector particles. These were used to transduce the murine BaF3 hematopoietic cell line as a model to study the impact of expression of these various gene products. Gene expression analysis using Afflymetrix arrays demonstrated that both EVI1 and MDS1-EVI1 expression produced dramatic changes in gene expression profiles of these cells, compared to MDS1 and control vector. For instance, EVI1 transduced cells overexpressed oncogenes such as small G proteins belonging to the RAS family. There was modulation of genes implied in hematopoiesis, apoptosis, TGF beta signaling, and cell cycle. To assess changes in cell cycling of transduced BaF3 cells we used a flow cytometric assay, which unraveled an arrest in G1 phase only when EVI1 was overexpressed. These changes were concomitant to an increased metabolic activity as measured by an MTT assay. Further studies of these different pathways have to be performed in order to confirm the results obtained by the DNA chips analysis. Primary murine bone marrow cells could be immortalized after transduction by both EVI1 and MDS1-EVI1 vectors, compared to MDS1 and control vectors. Mice have been transplanted with primary bone marrow cells transduced with all vectors, and are being followed for hematopoietic changes or leukemia. In conclusion, both MDS1-EVI1 and EVI1 overexpression appear to result in marked changes in the behavior of primitive hematopoietic cells.

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Role of pluripoietins in murine bone marrow stem cell differentiation

Leukemia Research ◽

10.1016/0145-2126(80)90036-3 ◽

1980 ◽

Vol 4 (3) ◽

pp. 287-299 ◽

Cited By ~ 24

Author(s):

Emilia Frindel ◽

Dominique Dumenil ◽

Françoise Sainteny

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Cell Differentiation ◽

Stem Cell Differentiation ◽

Bone Marrow Stem Cell ◽

Murine Bone Marrow ◽

Marrow Stem Cell

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Redundant Leukemogenicity of NUP98-HOX Fusion Genes in Primary Murine Bone Marrow Cells Correlates with Gene Expression Changes Consistent with Common Key Target Genes.

Blood ◽

10.1182/blood.v104.11.1134.1134 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1134-1134

Author(s):

Lars Palmqvist ◽

Nicolas Pineault ◽

Patricia Rosten ◽

Keith R. Humphries

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Target Genes ◽

Bone Marrow Cells ◽

Human Leukemia ◽

Fusion Partner ◽

Fusion Genes ◽

Leukemic Transformation ◽

Murine Bone Marrow ◽

Marrow Cells

Abstract Several Abd-B HOX genes have been found in translocations with the nucleoporin gene NUP98 in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We have previously tested both known and engineered NUP98-HOX fusions in the murine bone marrow transplantation model (N. Pineault et al., MCB24:1907, 2004). Strikingly, an engineered NUP98-HOXA10 (NA10) fusion, not observed in patients, and the AML-associated fusion gene NUP98-HOXD13 (ND13) have a virtually indistinguishable ability to transform myeloid progenitor cells and to induce leukemia in collaboration with MEIS1. Importantly, their transforming ability is lost when the DNA-binding homeodomain is mutated. This functional overlap provides a potentially powerful strategy to identify key genes/pathways mediating HOX-induced leukemias by looking for overlapping gene expression changes induced by different NUP98-HOX fusion genes. 5-FU bone marrow cells were transduced with retroviral vectors encoding for the leukemogenic ND13 or NA10 fusion genes or a non-leukemogenic ND13 gene with a N51S homeodomain mutation or the empty MIG vector. RNA was extracted from transduced GFP+ Sca1+ Lin- cells and linear RNA amplification was performed before the analysis on the Affymetrix GeneChip MOE430. Three independent experiments were conducted and analyzed. Correlation analysis showed a high degree of similarity between ND13 and NA10 in their overall gene expression profiles, compared to the N51S mutant or the MIG control. Validation with real-time quantitative RT-PCR on non-amplified RNA revealed good agreement between the gene array and the PCR, with a tendency for bigger fold-changes with the PCR method. Close to 500 genes were found differentially expressed (changed ≥2-fold vs. MIG ctrl and t-test p-value <0.05) and some 100 of these were changed by both ND13 and NA10 but not by the N51S homeodomain mutant. These genes are strong candidates as direct and/or immediate downstream targets involved in leukemic transformation. Remarkably, among these were genes previously identified as a NUP98 fusion partner in human leukemia (DEAD-box protein, Ddx10), or part of the same family of genes found in NUP98-fusions (Ddx4 and the paired mesoderm homeobox gene, Pmx2). This suggests a possible molecular link in leukemogenicity between HOX- and non-HOX-NUP98 fusions. Other interesting genes that were induced by ND13 and NA10, but not by the N51S homeodomain mutant, were genes previously implicated in leukemia (e.g. Flt3, Evi1) as well as Hox-related genes, such as the Hox cofactor Pbx3 and several Hox A cluster members. Furthermore, approximately one third were ESTs or genes with unknown function. In conclusion, our results document similar changes in gene expression induced by functionally redundant but different NUP98-HOX fusions and should facilitate the identification of common target genes involved in leukemic transformation.

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Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transplantation factor NGFI-A (EGR1).

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4566 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4566-4572 ◽

Cited By ~ 67

Author(s):

S L Lee ◽

Y Wang ◽

J Milbrandt

Keyword(s):

Bone Marrow ◽

Cell Differentiation ◽

Zinc Finger ◽

Bone Marrow Cells ◽

Myeloid Cell ◽

Hematopoietic Progenitor Cell ◽

Wild Type ◽

Granulocytic Differentiation ◽

Macrophage Differentiation ◽

Marrow Cells

The zinc finger protein NGFI-A (also called EGR1, Krox24, or zif268) is a candidate regulator of myeloid cell differentiation. Evidence supporting this hypothesis is twofold. First, NGFI-A antisense oligonucleotides prevent macrophage differentiation in HL-60 and U937 myeloid leukemia cell lines and in normal bone marrow cells. Second, enforced expression of NGFI-A blocks granulocytic differentiation and promotes macrophage differentiation in HL-60 cells and in the hematopoietic progenitor cell line 32D. We sought to determine the effect of NGFI-A deficiency on macrophage differentiation and function in vivo by examining native bone marrow cells from mice homozygous for a disrupted allele of NGFI-A derived from gene-targeted ES cells. Macrophages were observed in peripheral blood and several tissues, indicating that NGFI-A was not required for the formation of a variety of macrophage compartments. No differences in myeloid cell differentiation were observed between wild-type and NGFI-A-/- bone marrow cells cultured in the presence of macrophage, granulocyte-macrophage, or granulocyte colony-stimulating factor (M-CSF, GM-CSF, or G-CSF). Activation of NGFI-A-/- macrophages was comparable to that of wild-type macrophages as determined by nitric oxide production and increased cell surface expression of class II major histocompatibility complex molecules. Moreover, NGFI-A-/- mice showed no increased mortality or bacteria] burden when challenged with Listeria monocytogenes. Together, these results indicate that NGFI-A is not required for macrophage differentiation or activation.

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T-cell growth factor P40 promotes the proliferation of myeloid cell lines and enhances erythroid burst formation by normal murine bone marrow cells in vitro

Blood ◽

10.1182/blood.v76.5.906.906 ◽

1990 ◽

Vol 76 (5) ◽

pp. 906-911 ◽

Cited By ~ 37

Author(s):

DE Williams ◽

PJ Morrissey ◽

DY Mochizuki ◽

P de Vries ◽

D Anderson ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Growth Factor ◽

Cell Growth ◽

Cell Lines ◽

Bone Marrow Cells ◽

Myeloid Cell ◽

Murine Bone Marrow ◽

T Cell Growth Factor ◽

Marrow Cells

Abstract T-cell growth factor P40 was examined for possible effects on murine interleukin-3 (IL-3)-dependent myeloid cell lines and freshly isolated murine bone marrow cells. The results showed that P40 stimulated the proliferation of some IL-3-dependent myeloid cell lines of both early myeloid and mast cell phenotype and synergized with IL-3. P40 did not promote proliferation of fresh bone marrow cells, bone marrow enriched for early myeloid cells by 5-fluorouracil treatment, or bone marrow derived mast cells as assessed in 3H-TdR incorporation assays. P40 did not influence the growth of murine colony-forming unit granulocyte- macrophage in agar cultures, either alone or in the presence of optimal or sub-optimal concentrations of CSF-1, GM-colony-stimulating factor, or IL-3. P40 did potentiate burst-forming unit-erythroid (BFU-E) formation in the presence of erythropoietin; however, this was dependent on the cell plating density, suggesting an indirect stimulation of BFU-E by P40. The indirect nature of P40 action on BFU-E was further demonstrated in cell separation experiments and indicated that the effect was mediated by T cells. These data expand the repertoire of cells that P40 influences.

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Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.6046 ◽

1990 ◽

Vol 10 (11) ◽

pp. 6046-6050 ◽

Cited By ~ 36

Author(s):

D Patinkin ◽

S Seidman ◽

F Eckstein ◽

F Benseler ◽

H Zakut ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Conditioned Medium ◽

Xenopus Oocytes ◽

Bone Marrow Cells ◽

Murine Bone Marrow ◽

Marrow Cells

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.

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Zidovudine (AZT) treatment suppresses granulocyte-monocyte colony stimulating factor receptor type alpha (GM-CSFRα) gene expression in murine bone marrow cells

Life Sciences ◽

10.1016/s0024-3205(02)01790-3 ◽

2002 ◽

Vol 71 (8) ◽

pp. 967-978 ◽

Cited By ~ 16

Author(s):

Shilpa Chitnis ◽

Debasis Mondal ◽

Krishna C Agrawal

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Bone Marrow Cells ◽

Receptor Type ◽

Colony Stimulating Factor ◽

Murine Bone Marrow ◽

Stimulating Factor ◽

Marrow Cells

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Tissue-Specific Gene Expression of Marrow Cells Co-Cultured with Various Murine Organs

Blood ◽

10.1182/blood.v112.11.388.388 ◽

2008 ◽

Vol 112 (11) ◽

pp. 388-388

Author(s):

Jason M. Aliotta ◽

Mandy Pereira ◽

Peter J. Quesenberry

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Troponin I ◽

Bone Marrow Cells ◽

Fold Increase ◽

Specific Gene ◽

Rt Pcr ◽

Tissue Specific ◽

Murine Bone Marrow ◽

Marrow Cells

Abstract We have previously demonstrated that murine bone marrow cells co-cultured with lung opposite a cell-impermeable membrane express high levels of lung cell-specific genes (Aliotta et al, Stem Cells, 2007;25(9):2245–56). Greater changes in gene expression were noted in marrow cells co-cultured with radiation-injured lung. A factor smaller than the pore size of the cell-impermeable membrane (0.4μm) is responsible for these changes as cell-free conditioned media (CM) had a similar effect on co-cultured marrow cells. Lung-derived microvesicles were found to enter marrow cells in culture and may be among the factors responsible for these phenotypic changes. We wished to determine if these observations could be generalized to co-cultures using other murine organs and whether these changes in gene expression are tissue-specific. Whole bone marrow (WBM) cells were isolated from C57BL/6 mice and co-cultured with lung, liver, heart and WBM cells from C57BL/6 mice exposed to 1200 centigrey (cGy) of total body irradiation (TBI) or no radiation. Control WBM cells were co-cultured with no tissue (control). Co-cultured WBM cells were analyzed 7 or 14 days later by Real Time RT-PCR and fold difference in target gene expression was determined (relative to control cells). In addition, cell-free CM made from the same organs was co-cultured for 7 or 14 days with WBM cells which were then analyzed by RT-PCR. Alternatively, CM was analyzed for the presence of microvesicles by electron microscopy (EM) of ultracentrifuged (UCF) pelleted material. WBM co-cultured only with lung had increased gene expression of surfactant proteins A (Sp-A) and C (Sp-C, 89 and 334-fold increase vs. control, respectively) whereas WBM co-cultured only with brain had increased gene expression of Glial Fibrillary Acidic Protein (GFAP, 4.6-fold increase vs. control). Slight increases in Albumin expression were seen in all co-culture groups but expression was markedly elevated in WBM co-cultured with liver (162,657-fold increase vs. control). Expression of heart-specific markers, including Troponin I and T2, was seen in WBM co-cultured with heart but these levels were not significantly different from those of other co-culture groups. Radiation injury augmented expression of certain genes in co-cultured WBM, including Sp-A (1019 vs. 89-fold increase) in lung co-cultures and GFAP (24 vs. 4.6-fold increase) in brain co-cultures. WBM co-cultured with CM from all organs demonstrated similar changes in gene expression. In addition, pelleted material from UCF CM contained RNA that was specific to the tissue from which the CM was made. EM of UCF CM demonstrated numerous membranebound particles 50–200nm in size that were typical of microvesicles in appearance. These data suggest that changes seen in gene expression of co-cultured WBM are largely tissue-specific, depending on the tissue they are co-cultured with. Microvesicles released by various tissues in co-culture may be among the mediators responsible for the changes seen in WBM gene expression.

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