scholarly journals Constitutive in vivo cytokine and hematopoietic growth factor gene expression in the bone marrow and peripheral blood of healthy individuals

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2038-2044 ◽  
Author(s):  
FH Cluitmans ◽  
BH Esendam ◽  
JE Landegent ◽  
R Willemze ◽  
JH Falkenburg
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Steady State ◽  
Growth Factor ◽  
Tnf Alpha ◽  
Hematopoietic Growth Factor ◽  
Healthy Individuals ◽  
Paracrine Regulation

We investigated hematopoietic growth factor (HGF) and cytokine gene expression in the bone marrow (BM) and peripheral blood (PB) of healthy individuals as a starting point for delineating the physiologic role of cytokines in steady state hematopoiesis. BM biopsy specimens and PB samples from 7 healthy individuals were analyzed by polymerase chain reaction amplification of reverse-transcribed RNA using gene-specific primer sets. Consistent gene expression in the BM of all 7 individuals was detected for macrophage colony-stimulating factor (CSF), stem cell factor, interleukin-6 (IL-6), IL-7, erythroid-potentiating factor, erythroid-differentiating factor, and insulinlike growth factor 1, all cytokines with reported direct stimulatory effects on in vitro hematopoiesis. Of these, erythroid-potentiating factor and erythroid-differentiating factor appeared to be the only stimulating factors that were also expressed in the PB. Among the cytokines with inhibitory effects on in vitro hematopoiesis IL-4, tumor necrosis factor-alpha (TNF-alpha), TNF-beta, transforming growth factor-beta, and macrophage inflammatory protein-1 alpha were expressed in the BM of the 7 individuals. Except for TNF-alpha, the latter cytokines were also expressed in the PB. Consistent expression in the BM and PB of all tested individuals was also observed for IL-1 beta, IL-1 receptor antagonist, and IL-1 beta converting enzyme, which are all members of the IL-1 family with a possible indirect effect on hematopoiesis. Remarkably, no expression of granulocyte CSF, granulocyte-macrophage CSF, and IL-3 was found in the BM or PB of all investigated individuals (n = 15). This was also the case for IL-1 alpha, IL-2, IL-5, IL-9, IL-12, IL-13, leukemia-inhibiting factor, interferon-gamma, and inhibin. Weak IL-8 and IL-10 expression was found in the BM and/or PB of a minority of investigated individuals. These findings provide insight into which cytokines or HGFs potentially are involved in the autocrine or paracrine regulation of in vivo steady state hematopoiesis. The absence of expression of granulocyte CSF, granulocyte-macrophage CSF, and IL-3 in the BM of healthy individuals implicates that it is highly unlikely that these HGFs are involved in the autocrine or paracrine regulation of constitutive hematopoiesis.

In Vivo Expression of Murine Platelet Glycoprotein Ibα

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 488-495 ◽  
Author(s):  
Hiroyuki Fujita ◽  
Yoshimi Hashimoto ◽  
Susan Russell ◽  
Barbara Zieger ◽  
Jerry Ware
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Luciferase Activity ◽  
Blood Platelet ◽  
Northern Analysis ◽  
Platelet Glycoprotein ◽  
Heterologous Proteins ◽  
In Vivo Evaluation

Abstract We have performed a systematic in vivo evaluation of gene expression for the glycoprotein (GP) Ibα subunit of the murine platelet adhesion receptor, GP Ib-IX-V. This study is warranted by in vitro observations of human GP Ibα expression in cells of nonhematopoietic lineage and reports of regulation of the GP Ibα gene by cytokines. However, an in vivo role for a GP Ib-IX-V receptor has not been established beyond that described for normal megakaryocyte/platelet physiology and hemostasis. Our Northern analysis of mouse organs showed high levels of GP Ibα mRNA in bone marrow with a similar expression pattern recapitulated in mice containing a luciferase transgene under the control of the murine GP Ibα promoter. Consistently high levels of luciferase activity were observed in the two hematopoietic organs of mice, bone marrow (1,400 relative light units/μg of protein [RLUs]) and spleen (500 RLUs). Reproducible, but low-levels of luciferase activity were observed in heart, aorta, and lung (30 to 60 RLUs). Among circulating blood cells, the luciferase activity was exclusively localized in platelets. No increase in GP Ibα mRNA or luciferase activity was observed after treatment of mice with lipopolysaccharides (LPS) or tumor necrosis factor-α (TNF-α). We conclude the murine GP Ibα promoter supports a high level of gene expression in megakaryocytes and can express heterologous proteins allowing an in vivo manipulation of platelet-specific proteins in the unique environment of a blood platelet.

scholarly journals The immunosuppressant rapamycin blocks in vitro responses to hematopoietic cytokines and inhibits recovering but not steady-state hematopoiesis in vivo

Blood ◽  
1994 ◽  
Vol 84 (5) ◽  
pp. 1543-1552 ◽  
Author(s):  
VF Quesniaux ◽  
S Wehrli ◽  
C Steiner ◽  
J Joergensen ◽  
HJ Schuurman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
T Cell ◽  
T Cell Response ◽  
Colony Stimulating Factor ◽  
Gm Csf ◽  
Hematopoietic Recovery ◽  
Stimulating Factor

Abstract The immunosuppressive drug rapamycin suppresses T-cell activation by impairing the T-cell response to lymphokines such as interleukin-2 (IL- 2) and interleukin-4 (IL-4). In addition, rapamycin blocks the proliferative response of cell lines to a variety of hematopoietic growth factors, including interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage- colony stimulating factor (GM-CSF), and kit ligand (KL), suggesting that it should be a strong inhibitor of hematopoiesis. In this report, we studied the effects of rapamycin on different hematopoietic cell populations in vitro and in vivo. In vitro, rapamycin inhibited the proliferation of primary bone marrow cells induced by IL-3, GM-CSF, KL, or a complex mixture of factors present in cell-conditioned media. Rapamycin also inhibited the multiplication of colony-forming cells in suspension cultures containing IL-3 plus interleukin-1 (IL-1) or interleukin-11 (IL-11) plus KL. In vivo, treatment for 10 to 28 days with high doses of rapamycin (50 mg/kg/d, orally) had no effect on myelopoiesis in normal mice, as measured by bone marrow cellularity, proliferative capacity, and number of colony-forming progenitors. In contrast, the same treatment strongly suppressed the hematopoietic recovery normally seen 10 days after an injection of 5-fluorouracil (5- FU; 150 mg/kg, intravenously [i.v.]). Thus, rapamycin may be detrimental in myelocompromised individuals. In addition, the results suggest that the rapamycin-sensitive cytokine-driven pathways are essential for hematopoietic recovery after myelodepression, but not for steady-state hematopoiesis.

Thrombopoietin Regulates Proliferation and Maturation of Murine Mast Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1707-1707
Author(s):  
Giovanni Migliaccio ◽  
Barbara Ghinassi ◽  
Lucia Centurione ◽  
Maria Zingariello ◽  
Lucia Bianchi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Mast Cells ◽  
Wild Type ◽  
Connective Tissues ◽  
Immature Cells

Abstract Megakaryocytopoiesis is regulated by extrinsic (interaction of the growth factor thrombopoietin, TPO with its receptor Mpl) and intrinsic (interaction between the trascription factors GATA-1 and Fog-1) factors. The observation that mice impaired for GATA-1 expression (i.e. harbouring the GATA-1low mutation) are defective not only in megakaryocyte maturation but also in mast cell differentiation (Migliaccio et al. J Exp Med197:281, 2003), led us to investigate whether TPO might control mast cell differentiation as well. We first observed that mice genetically unable to responde to TPO (Mplnull mice) express in the connective tissues 5 times more mast cells than their normal littermates. Then, we analysed the effects on mast cell differentiation of in vivo treatment with TPO. Normal mice, and their GATA-1low littermates, were injected i.p. with TPO (100 μg/kg/day per 5 days, kindly provided by Kirin Brewery, Japan) and the number of immature (Toluidinepos) and mature (AlcianBlue/Saphraninepos) mast cells present in the connective tissues of the animals, as well as the frequency of GATA-1pos and TUNELpos mast cells, was evaluated 14 days after treatment. In wild-type animals, TPO reduced the presence of GATA-1 in mast cells (by immuno-histochemistry) and increased the number of immature cells (from 320±28 to 852±60) and of those undergoing apoptosis (from 16±1 to 600±43). In contrast, in GATA-1low animals, TPO-treatment induced the expression of GATA-1 in mast cells while decreased the number of immature cells (from 1100±72 to 427±29) as well as that of apoptotic cells (from 600±45 to 60±2). The role of TPO on mast cell differentiation were further confirmed by the analysis of the effects exerted by the growth factor on in vitro differentiation of bone marrow derived mast cells (BMMC). In these experiments, wild type bone marrow and spleen cells were cultured for 21 days with SCF and IL-3 with or without TPO and BMMC differentiation measured on the basis of the number of cells expressing the phenotype c-kithigh/CD34high and FcεRIpos. In cultures stimulated with SCF and IL-3, all the cells expressed the phenotype c-kithigh/CD34high and FcεRIpos. In contrast, in cultures supplemented also with SCF, IL-3 and TPO, only 25% of the cells were c-kithigh/CD34high and none of them was FcεRIpos. These results establish a role for TPO in the control of mast cell differentiation (possibly by modulating the GATA-1 content of the cells) and unveil further similarities between the mechanism(s) controlling megakaryocyte and mast cell differentiation.

Novel Murine Model To Study Modulation of Genes and Molecular Pathways Induced Following In Vivo Interaction between Multiple Myeloma Cells and Human BM Milieu.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3409-3409
Author(s):  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Masood Shammas ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Murine Model ◽  
Cell Signalling

Abstract Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

Enhanced Erythroblast Mobilization in Nix-Deficient Mice Confers Resistance to Phenylhydrazine-Induced Anemia Despite Accelerated Erythrocyte Turnover.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3659-3659
Author(s):  
Abhinav Diwan ◽  
Andrew G. Koesters ◽  
Amy M. Odley ◽  
Theodosia A. Kalfa ◽  
Gerald W. Dorn
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Half Life ◽  
Hematopoietic Cells ◽  
Dynamic Regulation ◽  
Genetic Ablation ◽  
Deficient Mice ◽  
Erythrocyte Fragility

Abstract Steady-state and dynamic regulation of erythrocyte production occurs by altering the balance of cell-survival versus apoptosis signaling in maturing erythroblasts. Previously, the pro-apoptotic factor Nix was identified as a critical death signal in normal erythropoietic homeostasis, acting in opposition to erythroblast-survival signaling by erythropoietin and Bcl-xl. However, the role of Nix in stress-erythropoiesis is not known. Here, by comparing the consequences of erythropoietin administration, acute phenylhydrazine-induced anemia, and aging in wild-type and Nix-deficient mice, we show that complete absence of Nix, or its genetic ablation specifically in hematopoietic cells, mimics the effects of erythropoietin (Epo). Both Nix ablation and Epo treatment increase early erythroblasts in spleen and bone marrow and increase the number of circulating reticulocytes, while maintaining a pool of mature erythroblasts as an “erythropoietic reserve”. As compared with WT, Nix null mice develop polycythemia more rapidly after Epo treatment, consistent with enhanced sensitivity to erythropoietin observed in vitro. After phenylhydrazine administration, anemia in Nix-deficient mice is less severe and recovers more rapidly than in WT mice, despite lower endogenous Epo levels. Anemic stress depletes mature erythroblasts in both WT and Nix null mice, but Nix null mice with basal erythroblastosis are resistant to anemic stress. These findings show that Nix null mice have greatly expanded erythroblast reserve and respond normally to Epo- and anemia-stimulated induction of erythropoiesis. However, the hematocrits of young adult Nix null mice are not elevated, and these mice paradoxically develop anemia as they age with decreased hemoglobin content (10g/dl) and hematocrit (36%; at 80±3 weeks of age) compared to WT mice (13g/dl and 46%; 82±5 weeks of age), inspite of persistent erythoblastosis observed in the bone marrow and spleen. Nix null erythrocytes, which are macrocytic and exhibit membrane abnormalities typically seen in immature cells or with accelerated erythropoiesis, demonstrate shorter life span with a half life of 5.2±0.6 days in the peripheral circulation by in vivo biotin labeling (as compared with a half life of 11.7±0.9 days in WT), and increased osmotic fragility as compared with normal erythrocytes. This suggests that production and release of large numbers of reticulocytes in Nix null mice can decrease erythrocyte survival. To rule out a non-hematopoietic consequence of Nix ablation that contributes to or causes increased erythrocyte fragility and in vivo consumption, such as primary hypersplenism, we undertook Tie2-Cre mediated conditional Nix gene ablation. Nixfl/fl + Tie2-Cre mice (hematopoietic-cell specific Nix null) develop erythroblastosis with splenomegaly, reticulocytosis, absence of polycythemia and increased erythrocyte fragility; suggesting that erythroblastosis and accelerated erythrocyte turnover are a primary consequence of Nix ablation in hematopoietic cells. Hence, dis-inhibition of erythropoietin-mediated erythroblast survival pathways by Nix ablation enhances steady-state and stress-mediated erythropoiesis.

scholarly journals ASXL2 Is Recurrently Mutated in t(8;21) AML and Regulates Hematopoietic Development

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1051-1051
Author(s):  
Vikas Madan ◽  
Lin Han ◽  
Norimichi Hattori ◽  
Anand Mayakonda ◽  
Qiao-Yang Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Research Funding ◽  
Hematopoietic Differentiation ◽  
Wild Type ◽  
Flow Cytometric ◽  
Deficient Mice

Abstract Chromosomal translocation t(8;21) (q22;q22) leading to generation of oncogenic RUNX1-RUNX1T1 fusion is a cytogenetic abnormality observed in about 10% of acute myelogenous leukemia (AML). Studies in animal models and recent next generation sequencing approaches have suggested cooperativity of secondary genetic lesions with t(8;21) in inducing leukemogenesis. In this study, we used targeted and whole exome sequencing of 93 cases (including 30 with matched relapse samples) to profile the mutational landscape of t(8;21) AML at initial diagnosis and post-therapy relapse. We identified recurrent mutations of KIT, TET2, MGA, FLT3, NRAS, DHX15, ASXL1 and KMT2Dgenes in this subtype of AML. In addition, high frequency of truncating alterations in ASXL2 gene (19%) also occurred in our cohort. ASXL2 is a member of mammalian ASXL family involved in epigenetic regulation through recruitment of polycomb or trithorax complexes. Unlike its closely related homolog ASXL1, which is mutated in several hematological malignancies including AML, MDS, MPN and others; mutations of ASXL2 occur specifically in t(8;21) AML. We observed that lentiviral shRNA-mediated silencing of ASXL2 impaired in vitro differentiation of t(8;21) AML cell line, Kasumi-1, and enhanced its colony forming ability. Gene expression analysis uncovered dysregulated expression of several key hematopoiesis genes such as IKZF2, JAG1, TAL1 and ARID5B in ASXL2 knockdown Kasumi-1 cells. Further, to investigate implications of loss of ASXL2 in vivo, we examined hematopoiesis in Asxl2 deficient mice. We observed an age-dependent increase in white blood cell count in the peripheral blood of Asxl2 KO mice. Myeloid progenitors from Asxl2 deficient mice possessed higher re-plating ability and displayed altered differentiation potential in vitro. Flow cytometric analysis of >1 year old mice revealed increased proportion of Lin-Sca1+Kit+ (LSK) cells in the bone marrow of Asxl2 deficient mice, while the overall bone marrow cellularity was significantly reduced. In vivo 5-bromo-2'-deoxyuridine incorporation assay showed increased cycling of LSK cells in mice lacking Asxl2. Asxl2 deficiency also led to perturbed maturation of myeloid and erythroid precursors in the bone marrow, which resulted in altered proportions of mature myeloid populations in spleen and peripheral blood. Further, splenomegaly was observed in old ASXL2 KO mice and histological and flow cytometric examination of ASXL2 deficient spleens demonstrated increased extramedullary hematopoiesis and myeloproliferation compared with the wild-type controls. Surprisingly, loss of ASXL2 also led to impaired T cell development as indicated by severe block in maturation of CD4-CD8- double negative (DN) population in mice >1 year old. These findings established a critical role of Asxl2 in maintaining steady state hematopoiesis. To gain mechanistic insights into its role during hematopoietic differentiation, we investigated changes in histone marks and gene expression affected by loss of Asxl2. Whole transcriptome sequencing of LSK population revealed dysregulated expression of key myeloid-specific genes including Mpo, Ltf, Ngp Ctsg, Camp and Csf1rin cells lacking Asxl2 compared to wild-type control. Asxl2 deficiency also caused changes in histone modifications, specifically H3K27 trimethylation levels were decreased and H2AK119 ubiquitination levels were increased in Asxl2 KO bone marrow cells. Global changes in histone marks in control and Asxl2 deficient mice are being investigated using ChIP-Sequencing. Finally, to examine cooperativity between the loss of Asxl2 and RUNX1-RUNX1T1 in leukemogenesis, KO and wild-type fetal liver cells were transduced with retrovirus expressing AML1-ETO 9a oncogene and transplanted into irradiated recipient mice, the results of this ongoing study will be discussed. Overall, our sequencing studies have identified ASXL2 as a gene frequently altered in t(8;21) AML. Functional studies in mouse model reveal that loss of ASXL2 causes defects in hematopoietic differentiation and leads to myeloproliferation, suggesting an essential role of ASXL2 in normal and malignant hematopoiesis. *LH and NH contributed equally Disclosures Ogawa: Takeda Pharmaceuticals: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Kan research institute: Consultancy, Research Funding.

Novel Model To Evaluate Changes in Gene Expression Profile of Myeloma Cells In Vivo Following Interaction with Human BM Microenvironment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2490-2490
Author(s):  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Masood A. Shammas ◽  
Daniel R. Carrasco ◽  
Renate Burger ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Growth ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Stromal Cells ◽  
Novel Agents ◽  
Bone Chip

Abstract Multiple Myeloma (MM) cells interact with bone marrow (BM) microenvironment leading to induction of adhesion-mediated and cytokine mediated cell signalling which plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. We have previously evaluated gene expression changes following interaction between MM cells and BM stromal cells in vitro. However, the interaction between MM cells and microenvironment cells within the bone marrow is unique and its understanding is critical in evaluating effects of novel agents. We here describe a unique model that allows us to analyse in vivo expression changes in MM cells within the human BM milieu; and present preliminary results of expression changes following these in vivo interactions. In this model, BM stromal and IL-6-dependent human MM cell line INA-6 tranduced with GFP (green fluorescent protein) was injected in human fetal bone chip transplanted into SCID mice (SCID-hu mice). The MM cells were allowed to interact with the bone marrow for variable length of time, the bone chip was then retrieved, cells flashed out and GFP+ MM cells were separated by flow cytometry. The GFP negative fraction, containing stromal elements was also separated. Similar flow isolation process was used on INA-6GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affimetrix). We report that interaction between INA-6 cells and the BM microenvironment in vivo induced significant changes in expression profile. In particular, we observed up-regulation of genes implicated in regulation of cell proliferation (RGS 1 and 2, FOS, FOSB, S100A4); DNA transcription (AP1, SWI/SNF related member 1); chromosome organization (Histone1, 2 and 3); cellular trafficking and transport (ARFGEF2, Aquarin 3 and ATPase 4B); and signal transduction (Chemokine ligand 2, 3 and 15, Chemokine receptor 1, 2 and 4, Dual specificity phosphatase 1 and 4, Protein tyrosine phosphatase 1, PIP5-kinase 1A and ZAP70). We also observed down-regulation of genes involved in apoptosis (BCL2-interacting killer, APC, E1A binding protein p300, Fas-associated via death domain, Caspase-activated Dnase, Raf1); and cell-cell adhesion molecules (Cadherin 15, Leupakin, Neurekin, CD44, ICAM2 and PECAM-1a). Although some similarities were observed in gene profile changes following in vitro and in vivo interaction with microenvironment cells, differences were also found. We are now evaluating the effects of interaction on expression profile of stromal cells as well as duration of interaction. Taken together these data confirm the ability of BM microenvironment to modulate gene expression profile of the MM cells in vivo to mediate the MM cell growth, survival and migration. This model now provides us with an opportunity to study effects of novel agents on MM cells expression profile in vivo to pre-clinically characterize their activity.

Frequent in Vivo redundancy of C/EBPβ and C/EBPε during Myeloid Development

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2440-2440
Author(s):  
Nils Heinrich Thoennissen ◽  
Tadayuki Akagi ◽  
Sam Abbassi ◽  
Daniel Nowak ◽  
Ann George ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcription Factors ◽  
Bone Marrow Cells ◽  
Double Knockout ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Marrow Cells

Abstract CCAAT/enhancer binding protein (C/EBP) transcription factors are involved in a variety of cellular responses including proliferation and differentiation. Although C/EBPβ and C/EBPε are believed to be most important for macrophage and granulocyte activity, respectively, experiments by others and ourselves suggest a possible overlap in their function in myelopoiesis. In order to explore further this potential redundancy, we assessed the in vivo and in vitro function of both transcription factors by generating a double knockout (KO) germline murine model (C/EBPβ/ε−/−/−/−) and compared their hematopoiesis to those of single deficient (C/EBPβ−/−, C/EBPε−/−) and wild-type (WT) mice. Gene expression analysis of bone marrow cells showed expression of C/EBPβ in C/EBPε−/− and WT mice, and vice versa. The weight of the double-KO mice was significantly less as measured at 4 weeks of age (11.5 ± 0.9 g) compared to WT (13.4 ± 0.6 g), C/EBPβ−/− (14.5 ± 1.4 g), and C/EBPε−/− mice (15.4 ± 2.3 g) (p < 0.05). The double-KO mice were prone to infections of the eyes, lungs, liver, and peritoneum. In contrast, C/EBPβ−/−, C/EBPε−/− and WT mice demonstrated no signs of infection. Microscopic imaging of peripheral blood showed metamyelocytes and myelocytes in the double-KO mice. FACS analysis found that the fraction of bone marrow cells which were Lin(−) (no expression of B220, CD3, Gr1, Ter119, and Mac1) were modestly elevated in double-KO and C/EBPβ−/− mice (8.42 % and 8.1 %, respectively) compared to C/EBPε−/− (4.24 %) and WT (3.93 %) mice. A subanalysis highlighted an elevated level of B220(−)/Gr1(−) bone marrow cells in the double-KO mice (54 %) compared to the levels in the C/EBPβ−/− (31 %), C/EBPε−/− (33 %) and WT (21.5 %) mice. Moreover, the proportion of hematopoietic stem cells in the bone marrow were significantly increased in the hematopoietic stem cell compartment [Sca1(+)/c-Kit(+)] in the double-KO mice (20.8 %) compared to the C/EBPβ−/− (6.9 %), C/EBPε−/− (5.9 %) and WT (6.9 %) mice. When given a cytotoxic stress (5-FU) to kill cycling hematopoietic progenitor cells, the mean neutrophil count at their nadir (day 4) was 0.14 × 109 cells/L in the double-KO mice compared to 0.71 × 109 cells/L in the WT mice (p < 0.001); both reached normal values again on day 10. Taken together, these results indicated a relatively higher percentage of immature hematopoietic cells in the double-KO mice compared to the WT mice. Nevertheless, clonogenic assays in methylcellulose using bone marrow cells of the double-KO showed a significant decreased number of myeloid colonies. For example, in the presence of G-CSF, GM-CSF, and SCF, a mean of 83 ± 10 hematopoietic colonies formed in the double-KO mice compared to 135 ± 6 in C/EBPβ−/−, 159 ± 12 in C/EBPε−/− and 165 ± 2 in WT mice (p < 0.001, double-KO vs. WT). Similar clonogenic results occurred when bone marrow cells were stimulated with either G-CSF, GM-CSF or SCF/G-CSF alone. Although our in vitro experiments suggested that double-KO mice had a decreased clonogenic response to G-CSF, their bone marrow cells had normal levels of phosphorylated STAT3 protein when stimulated with G-CSF. Hence, the G-CSFR and its secondary signaling pathway seemed to be intact. In further experiments, downstream targets of the C/EBP transcription factors were examined. Bone marrow macrophages activated with LPS and IFNγ from both double-KO and C/EBPβ−/− mice had decreased gene expression of IL6, IL12p35, TNFα, and G-CSF compared to the levels detected in macrophages of C/EBPε−/− and WT. Interestingly, expression levels of cathelicidin antimicrobial peptide (CAMP) were similarly robust in the macrophages from C/EBPβ−/−, C/EBPε−/−, and WT mice. In sharp contrast, CAMP expression was undetectable in the activated macrophages of the double-KO mice. In conclusion, the phenotype of the double-KO mice was often distinct from the C/EBPβ−/− and C/EBPε−/− mice suggesting a redundancy of activity of both transcription factors in myeloid hematopoiesis.

Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3414-3414 ◽  
Author(s):  
Marijke W Maijenburg ◽  
Marion Kleijer ◽  
Kim Vermeul ◽  
Erik P.J. Mul ◽  
Floris P.J. van Alphen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Wnt Proteins

Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

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