scholarly journals What Links Neutropenia to Immature Cardiolipin in Patients with Barth Syndrome (tafazzin-deficiency)?

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3579-3579
Author(s):  
Jihee Sohn ◽  
Thomas Brouse ◽  
Najihah Aziz ◽  
David B Sykes
Keyword(s):  
Er Stress ◽  
Knockout Mouse ◽  
Mitochondrial Membrane ◽  
Fetal Liver ◽  
Liver Cells ◽  
Barth Syndrome ◽  
Developmental Defect ◽  
Wild Type ◽  
Inner Mitochondrial Membrane ◽  
Fetal Liver Cells

Barth syndrome is an inherited X-linked disorder characterized by cardiomyopathy, skeletal muscle myopathy, and neutropenia. The syndrome arises because of inherited mutations in the gene TAZ, resulting in a loss of function of the protein tafazzin. Of note, a group of investigators recently described how tafazzin can regulate 'stemness' in models of acute myeloid leukemia (Cell Stem Cell, 2019). Tafazzin is an enzyme that processes the final step of cardiolipin maturation, replacing saturated with unsaturated acyl chains. Cardiolipin is a 4-tailed phospholipid that is almost-exclusively found in the inner membrane of the mitochondria. The lack of tafazzin activity results in a cardiolipin pool that contains more highly saturated lipid tails and it is this lack of unsaturated cardiolipins that contributes to a disorganized inner mitochondrial membrane. The link between tafazzin-deficiency and myopathy is generally explained by the dependence of muscle cells on mitochondrial function as well as oxidative respiration. The components of the electron transport chain are co-localized with cardiolipin in the inner mitochondrial membrane, and it is felt that their appropriate organization within the membrane lipid bilayer is dependent on the presence of mature cardiolipin which is lacking in those individuals with Barth syndrome. The link between tafazzin-deficiency and neutropenia is less clear. Neutrophils are terminally-differentiated effector cells of the innate immune system. They are critical for protection against bacterial and fungal pathogens and patients without sufficient neutrophils are among the most immunocompromised and at risk of lethal infection. Neutrophils have few mitochondria at baseline and are generally believed to rely primarily on glycolysis for energy production. It is not known if the mechanism of neutropenia in Barth syndrome is due to a lack of production or due to increased clearance (e.g. more prone to apoptosis). We undertook the study of tafazzin-deficient neutrophils to try to elucidate the mechanism of neutropenia in patients with Barth syndrome. We took advantage of an existing tafazzin-knockout mouse and a system of conditional immortalization of granulocyte-monocyte progenitors (GMP) using the ER-Hoxb8 system pioneered in our laboratory. This ER-Hoxb8 system allows for the unlimited ex vivo expansion of myeloid progenitors in the presence of estradiol and active Hoxb8. Once estradiol is removed from culture media, the Hoxb8 protein is inactive and the cells undergo normal, synchronous and terminal neutrophilic differentiation. In this manner, we were able to generate tafazzin-wild-type and knockout GMP lines from murine fetal liver cells. Analysis of the myeloid progenitor compartment in fetal liver cells (d14.5-d16.5) showed no difference between wild-type and knockout mice, arguing against a developmental defect (E15 results shown in PANEL A). Furthermore, the tafazzin-deficient ER-Hoxb8 GMPs and neutrophils were remarkably normal when tested across a variety of assays including phagocytosis, cytokine production and ROS generation (ROS by H2DCFDA shown in PANEL B). We hypothesized that the unpredictable neutropenia in patients with Barth Syndrome might be due to an increased proclivity to apoptosis because of the mitochondrial membrane defect. Indeed, the tafazzin-deficient GMPs showed an increased sensitivity to Bcl2-inhibition following treatment with ABT199 (PANEL C). Two lines of evidence have suggested that the increased tendency towards apoptosis may be due to endoplasmic-reticulum (ER) stress. (1) Transmission electron microscopy demonstrated 'swollen' ER in the tafazzin-deficient cells (not shown) and (2) a comparison of gene expression patterns demonstrated an increased expression of ATF4 and CHOP (DDIT3) in the tafazzin-deficient cells (PANEL D). We are now focused on validating these findings and in establishing models to confirm the ER-stress phenotype in vivo in the TAZ-knockout mouse model as well as primary samples from patients with Barth Syndrome. We hope that this line of work will confirm the mechanism of neutropenia and shed light on potential targets for therapeutic intervention. In addition, this very rare disorder has provided insight into a previously-unexpected link between neutrophil survival and the membrane integrity of the inner mitochondrial membrane. Figure Disclosures Sykes: Clear Creek Bio: Equity Ownership, Other: Co-Founder.

Basis of hematopoietic defects in platelet-derived growth factor (PDGF)-B and PDGF β-receptor null mice

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 1990-1998 ◽  
Author(s):  
Wolfgang E. Kaminski ◽  
Per Lindahl ◽  
Nancy L. Lin ◽  
Virginia C. Broudy ◽  
Jeffrey R. Crosby ◽  
...  
Keyword(s):  
Growth Factor ◽  
Fetal Liver ◽  
Liver Cells ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Liver Growth ◽  
Hematologic Disorders ◽  
Fetal Liver Cells ◽  
Β Receptor

Abstract Platelet-derived growth factor (PDGF)-B and PDGF β-receptor (PDGFRβ) deficiency in mice is embryonic lethal and results in cardiovascular, renal, placental, and hematologic disorders. The hematologic disorders are described, and a correlation with hepatic hypocellularity is demonstrated. To explore possible causes, the colony-forming activity of fetal liver cells in vitro was assessed, and hematopoietic chimeras were demonstrated by the transplantation of mutant fetal liver cells into lethally irradiated recipients. It was found that mutant colony formation is equivalent to that of wild-type controls. Hematopoietic chimeras reconstituted with PDGF-B−/−, PDGFRβ−/−, or wild-type fetal liver cells show complete engraftment (greater than 98%) with donor granulocytes, monocytes, B cells, and T cells and display none of the cardiovascular or hematologic abnormalities seen in mutants. In mouse embryos, PDGF-B is expressed by vascular endothelial cells and megakaryocytes. After birth, expression is seen in macrophages and neurons. This study demonstrates that hematopoietic PDGF-B or PDGFRβ expression is not required for hematopoiesis or integrity of the cardiovascular system. It is argued that metabolic stress arising from mutant defects in the placenta, heart, or blood vessels may lead to impaired liver growth and decreased production of blood cells. The chimera models in this study will serve as valuable tools to test the role of PDGF in inflammatory and immune responses.

Stress Erythropoiesis Elicits a Program of Gene Regulation That Overlaps with Interferon-γ.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2161-2161
Author(s):  
Kai Huang ◽  
Monica L. Bailey ◽  
Dwayne L. Barber
Keyword(s):  
Fetal Liver ◽  
Liver Cells ◽  
Early Gene ◽  
Deubiquitinating Enzyme ◽  
Wild Type ◽  
Data Set ◽  
Time Points ◽  
Stress Erythropoiesis ◽  
Adult Mice ◽  
Fetal Liver Cells

Abstract Erythropoietin (EPO), the primary cytokine regulator of red blood cell production, acts through binding to its cognate receptor (EPO-R), which is primarily expressed on erythroid precursors. Knockout studies have illustrated a critical role for EPO, EPO-R and the downstream tyrosine kinase JAK2 in embryogenesis as mice lacking any of these components die from a fatal anemia at E13.5. These data suggest that EPO-R and/or JAK2 are required to promote erythropoiesis in vivo. EPO provides mitogenic, differentiative and cell survival signals to erythroid progenitors. We have performed microarray studies to identify target genes regulated by EPO in cell lines and primary cells. We utilized an erythroid cell line (HCD-57), a myeloid cell line stably expressing the EPO-R (Ba/F3-EPO-R), fetal liver cells isolated from E13.5 mice as well as splenocytes isolated from Phenylhydrazine (PHZ)-primed adult mice. Fetal liver cells permit the study of normal erythropoiesis in a fetal setting whereas the PHZ-primed erythroblasts permit analysis of stress erythropoiesis in adult mice. We harvested cells at 1, 8, 12 and 24 hr after EPO stimulation which correspond to immediate early gene induction (1 hr), S phase entry (8 hr) and G2/M (24 hr) time points. RNA was prepared and hybridized to the Affymetrix U74A mouse chip. Data was analyzed and only those genes with statistical significance (p < 0.05) were considered for further characterization. Analysis of the 1 hr time points has revealed that six genes are co-regulated by EPO in all four cellular environments. Included within this co-hort are the Suppressor of Cytokine Signaling genes (Cis, SOCS-1 and SOCS-3) and Myc, as well as two novel genes. We compared our datasets with other published analyses. The Williams laboratory has identified an Interferon-Stimulated Gene “ISG” data set corresponding to genes induced by Type I or Type II Interferon’s. We queried our PHZ-primed erythroblast data set against the Williams ISG database. Of the 305 human genes in the ISG database, 218 are expressed on the Affymetrix chip. We searched our dataset for genes that are induced 1.5-fold or greater at 2 of 4, 3 of 4 or 4 of 4 time points. Thirty-four genes are also stimulated by EPO in PHZ-primed erythroblasts including classical IFN-regulated genes such as Interferon-regulator factor-1 (IRF-1), Interferon-stimulated gene-15 (ISG-15), Interferon-induced transmembrane protein 3-like (IFITM-3l), Protein Kinase R (PKR) and Signal Transducer and Activator of Transcription-1 (STAT1). We have previously demonstrated that STAT1 is a negative regulator of murine erythropoiesis utilizing STAT1-deficient mice. We also analyzed immediate early gene regulation in fetal liver cells and PHZ-primed erythroblasts isolated from STAT1-deficient mice stimulated with EPO for 1 hr. These data were compared with the relevant wild type data sets. EPO stimulates the induction of the ubiquitin-like protein, ISG-15 in both wild type and STAT1−/− erythroblasts. Several signaling proteins have been shown to be covalently modified by ISG-15 including STAT1. ISG-15 is removed from ISGylated products by the deubiquitinating enzyme, Ubp43. EPO stimulates a rapid accumulation of Ubp43 in wild type cells, however, EPO fails to induce Ubp43 mRNA in STAT1-deficient fetal liver and PHZ-primed erythroblasts. Experiments are underway to confirm that the mechanism by which STAT1 exerts negative regulation of erythropoiesis is via upregulation of the deubiquitinating enzyme, Ubp43.

An Anti-Apoptotic Molecule, Anamorsin, Is Essential for Erythropoiesis Through the Regulation of Cellular Labile Iron Pool

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 610-610
Author(s):  
Akira Tanimura ◽  
Yuri Hamanaka ◽  
Natsuko Fujita ◽  
Yukiko Doi ◽  
Tomohiko Ishibashi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Iron Homeostasis ◽  
Fetal Liver ◽  
Liver Cells ◽  
Binding Activity ◽  
Wild Type ◽  
Labile Iron Pool ◽  
Cellular Iron ◽  
Fetal Liver Cells ◽  
Induction Of Apoptosis

Abstract Abstract 610 Introduction: Iron has crucial roles in many cellular biological processes. Cellular iron uptake and export must be tightly regulated. Insufficient iron concentrations impair the function of numerous iron proteins, whereas excess free iron can oxidize and damage the contents of cells. Anamorsin (AM, also called CIAPIN-1) is an anti-apoptotic factor, which we originally isolated as a molecule that confers factor-independent survival of hematopoietic cells. AM-deficient mice are embryonic lethal at late gestation due to the defect of definitive hematopoiesis. It is thought that AM plays a crucial role in hematopoiesis, however its precise biological mechanisms remain unclear. Recently, it was reported that the yeast AM homolog, Dre2, was implicated in cytosolic iron-sulfur (Fe/S) cluster assembly (Zhang Y., et al. Mol.Cell.Biol. 28:5569–5582, 2008). The AM carries conserved cysteine motifs (CX2CXC and twin CX2C) at its C termini, which may form iron binding sites. In this study, we have focused on the possibility that AM may be involved in the maturation of Fe/S cluster and the cellular iron homeostasis, especially, the regulation of labile iron pool (LIP) and that AM may affect the accumulation of reactive oxygen species (ROS), leading to impaired erythropoiesis. Methods and Results: To analyze the function of Fe/S protein, we established wild-type cell lines (AMWT) and AM-deficient cell lines (AMKO) from wild-type and AM-deficient fetal liver (14.5dpc) respectively by using SV40 large T antigen. Iron regulatory protein 1 (IRP1) is a well-known Fe/S protein with dual functions. In the presence of Fe/S cluster, IRP1 functions as a cytosolic aconitase. While, in the absence of Fe/S cluster, IRP1 stabilizes the transferrin receptor (TfR) mRNA by binding to the iron responsive element (IRE). We compared the aconitase activity and the IRE binding activity of IRP1 between AMWT and AMKO. The results showed that the cytosolic aconitase activity in AMKO decreased approximately 30% compared to AMWT and the IRE binding activity of IRP1 in AMKO increased 3-fold compared to AMWT. Furthermore, we compared the iron homeostasis. In the presence of iron chelator, desferrioxamine, the expression of TfR in AMWT was markedly elevated, while it was hardly elevated in AMKO. The LIP is a pool of chelatable and redox-active iron, which serves as a crossroad of cell iron metabolism. The measurement of LIP with the metal-sensitive sensor calcein acetoxymethyl ester showed that AMKO had 5-fold higher cellular LIP than AMWT. Moreover we evaluated the accumulation of ROS and the induction of apoptosis by extracellular iron uptake between AMWT and AMKO. The results showed the accumulation of ROS and the induction of apoptosis in AMKO were enhanced about twice as much as in AMWT. These enhancements could be restored by transduction of AM expressing retrovirus vector to AMKO. We also evaluated the effects of AM-deficiency on erythroid differentiation. Fetal liver cells from wild-type or AM-deficient embryos (14.5dpc) were divided into primitive and more matured erythroid populations based on their expression of CD71 and Ter119 by FACS analysis. AM-deficient fetal liver cells had a significant increase in the CD71low TER119low population, containing primitive erythroid progenitors, compared to wild-type (9.4±2.1% vs. 5.2±1.1%, P<0.05). Conversely, the CD71lowTER119highpopulation, comprised of late orthochromatophilic erythroblasts and reticulocytes, decreased in AM-deficient fetal liver cells compared to wild-type cells (2.3±0.8% vs. 7.4±1.3%, P < 0.05). Moreover we studied LIP in wild-type or AM-deficient embryo fetal liver cells. In accordance with the cell lines, the LIP in AM-deficient fetal liver cells increased 3 to 5-fold more than in wild-type fetal liver cells. The accumulation of ROS and the number of apoptotic cells also increased 2 to 5-fold in AM- deficient fetal liver cells compared to wild-type fetal liver cells. Thus, it was showed that AM deficiency impaired the iron homeostasis and conferred low sensitivity for iron concentration, resulting in the increase of LIP, the accumulation of ROS and the induction of apoptosis. Furthermore, dysregulation of cellular iron homeostasis was thought to be the cause of the embryonic lethal due to AM deficiency. Conclusion: Our current findings indicate that AM functions in cytosolic Fe/S cluster biogenesis and iron homeostasis and is essential for erythropoiesis. Disclosures: Kanakura: Shire: Consultancy.

scholarly journals Bclaf1 Promotes Maintenance and Self-Renewal of Fetal Hematopoietic Stem Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1269-1269 ◽  
Author(s):  
Lynn S. White ◽  
Deepti Soodgupta ◽  
Rachel L. Johnston ◽  
Jeffrey A. Magee ◽  
Jeffrey J. Bednarski
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Lower Percentage ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Hematopoietic stem cells (HSC) persist throughout life by undergoing extensive self-renewal divisions while maintaining an undifferentiated state. The mechanisms that support HSC self-renewal change throughout the course of development as temporal changes in transcriptional regulators coordinate distinct genetic programs in fetal, post-natal and adult HSCs. These self-renewal programs are often ectopically activated in leukemia cells to drive neoplastic proliferation and high expression of HSC-associated genes predicts a poor prognosis in acute myelogenous leukemia (AML). In this regard, it was recently shown that expression of the transcriptional regulator BCLAF1 (Bcl2 associated transcription factor 1) is increased in AML blasts relative to normal precursor populations and suppression of BCLAF1 causes reduced proliferation and induction of differentiation to a dendritic cell fate. These findings raise the question of whether BCLAF1 may regulate normal as well as neoplastic self-renewal programs. We find that Bclaf1 is highly expressed in HSCs versus committed bone marrow populations consistent with a potential role for this gene in HSC functions. To test whether BCLAF1 regulates HSC development and hematopoiesis, we used germline loss of function mice. Bclaf1-/- mice succumb to pulmonary failure shortly after birth due to poor lung development, so we assessed prenatal hematopoiesis. Bclaf1-deficient mice had significantly reduced HSC and hematopoietic progenitor cell (HPC) frequencies and numbers despite normal fetal liver cellularity. To determine if Bclaf1 is required for HSC function during fetal development, we performed competitive reconstitution assays. Fetal liver cells from Bclaf1+/+or Bclaf1-/-mice were transplanted along with wild-type competitor bone marrow cells into lethally irradiated recipient mice. Compared to recipients of Bclaf1+/+fetal liver cells, recipients of Bclaf1-/-cells had a significantly lower percentage of donor-derived leukocytes at all time points after transplantation as well as reduced percentage of donor HSCs at 16 weeks post-transplant. Notably, all leukocyte populations (B cells, T cells, granulocytes and macrophages) from Bclaf1-/-donors were reduced consistent with an abnormality in HSC repopulating activity rather than a defect in a specific differentiation pathway. Consistent with these findings, Bclaf-deficient cells did not engraft in competitive transplants with limiting numbers of sorted fetal liver HSCs whereas sorted wild-type Bclaf1+/+cells effectively reconstituted hematopoiesis in recipient mice. In addition, Vav-cre:Bclaf1flox/floxmice, which have selective deletion of Bclaf1 in hematopoietic cells, have reduced frequencies and numbers of fetal liver HSCs identical to the findings observed in germline Bclaf1-/-mice. These results show that loss of Bclaf1 leads to defective development and repopulating activity of fetal HSCs. Interestingly, when adult mice are successfully engrafted with Bclaf1-deficient HSCs, the donor HSCs suffer no additional functional impairment. Furthermore, in secondary transplant experiments Bclaf1-deficient HSCs maintain long-term repopulating activity. Thus, Bclaf1 may have distinct functions in fetal versus adult HSC self-renewal. Collectively, our findings reveal Bclaf1 is a novel regulator of fetal HSC function and suggest that it may have distinct functions in different developmental contexts. Disclosures No relevant conflicts of interest to declare.

Roles of MOZ in Hematopoiesis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2269-2269
Author(s):  
Takuo Katsumoto ◽  
Yukiko Aikawa ◽  
Takahiro Ochiya ◽  
Issay Kitabayashi
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Fetal Liver ◽  
Liver Cells ◽  
Recipient Mouse ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Homozygous Mutant ◽  
Fetal Liver Cells ◽  
Acute Myeloid

Abstract The AML1-CBFβ transcription factor complex is the most frequent target of specific chromosome translocations in acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene, which encodes a MYST-type histone acetyltransferase (HAT), is also involved in leukemia-associated translocations such as t(8;16), t(8;22) and inv(8), which are associated with acute myeloid leukemia with M4/5 subtypes. We previously found that MOZ functions as a potent coactivator for AML1. To investigate roles of MOZ in normal hematopoiesis, we generated MOZ-deficient mice using gene-targeting method. MOZ homozygous mutant is embryonic lethal and it died between days 14 and 15 of gestation. In fetal liver of MOZ-deficient E14.5 embryos, the total cell numbers and the colony-forming cells (CFCs) in a methylcellulose medium were remarkably reduced when compared with wild-type littermates. Flow cytometry analysis indicated that hematopoietic stem cells (HSCs) and progenitors of both myeloid and lymphoid lineages were severely reduced in MOZ-deficient embryos. Especially, the levels of c-kit expression were strongly reduced in lineage-negative cells. Differentiation arrest of erythroid progenitors at a terminal stage and increase in the numbers of Mac-1 and Gr-1 positive cells suggest that MOZ also plays roles in cell differentiation of erythroid, monocytic and granulocytic lineages. In E12.5 MOZ deficient fetal liver cells, expression profile analysis revealed decreases in expressions of thrombopoietin receptor c-mpl, Wnt related ligand dkk2 and HoxA9 and increase in HoxA5 expression. To further determine roles of MOZ in HSCs functions and their progenitors differentiation ability, competitive reconstitution assays were performed. Ly5.2+ fetal liver cells from wild-type, heterozygous or homozygous mutant embryos together with Ly5.1+ competitor fetal liver cells were transplanted into γ-irradiated Ly5.1+/Ly5.2+ recipient mouse. Ly5.2+ wild-type cells were observed in recipient mice after transplantation. However, cells derived from MOZ homozygous mutant embryos were not detected in peripheral blood, bone marrow, spleen and thymus. Reduced population of cells derived from heterozygous mutant embryos were observed. These data suggest that MOZ is required for lymphoid and myeloid development and for self-renewal of HSCs.

Roles of Histone Acetyltransferase MORF and MOZ in Hematopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2525-2525
Author(s):  
Takuo Katsumoto ◽  
Issay Kitabayashi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Liver Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract Abstract 2525 Poster Board II-502 MOZ (MOnocytic leukemia Zinc finger protein) and MORF (MOz Related Factor), Myst-type histone acetyltransferases, are involved in chromosome translocations associated with FAB-M4/5 subtypes of acute myeloid leukemia. We have reported that MOZ is essential for hematopoietic cell development and self-renewal of hematopoietic stem cells. To explore the possibility MORF also plays important roles in hematopoiesis, we generated Morf-deficient mice with homologous recombination methods. Morf−/− mice were smaller than their wildtype littermates and died within 4 weeks after birth on C57BL/6 background. In MORF−/− fetal liver, Flt3-negative KSL (c-Kit+ Sca-1+ Lineage-) cells containing hematopoietic stem cells were decreased. When fetal liver cells were transplanted into irradiated recipient mice, MORF−/− cells less efficiently reconstituted hematopoiesis than wild-type cells. Additionally, bone marrow cells reconstituted with MORF−/− cells rarely contributed to hematopoiesis in secondary transplants. To reveal relationship between MORF and MOZ in hematopoiesis, we generated double heterozygous (Moz+/− Morf+/−) mouse. Double heterozygous mice were smaller than wild-type littermates and died at least 4 weeks after birth. Numbers of KSL cells, especially Flt3- KSL cells and common myeloid progenitors were decreased in the double heterozygous embryos. The double heterozygous fetal liver cells also displayed less activity to reconstitute hematopoiesis than MOZ+/− or MORF+/− cells. Since MORF−/− mice and MOZ/MORF double heterozygous mice were alive at adult on a mixed C57BL/6/DBA2 genetic background, we investigated adult hematopoiesis in these mice. MORF−/− or MOZ/MORF double heterozygous mice were smaller than their wild-type littermates and had small numbers of thymocytes and splenocytes. However, there were no significant differences in number of bone marrow cells and hematopoietic lineage population in MORF−/− or MOZ/MORF double heterozygous mice. These results suggest that MORF as well as MOZ plays important roles in self-renewal of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Impaired Repopulating Activity of Fus-Deficient Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2222-2222
Author(s):  
Takeaki Sugawara ◽  
Atsushi Iwama
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Chromosomal Translocation ◽  
Liver Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Fetal Liver Cells

Abstract RNA-binding protein FUS (also known as TLS) was originally identified in chromosomal translocation in human myxoid liposarcoma. The FUS gene is also translocated with the transcription factor gene ERG in human myeloid leukemia with recurrent chromosomal translocation t(16;21). Multiple data suggest that wild-type FUS is also involved in the development of leukemia as one of the downstream targets for oncoproteins including BCR-ABL. However, little is known about the role of FUS in the normal hematopoiesis. The previous report demonstrated that Fus-deficient (Fus−/−) newborn mice, which die shortly after birth because they cannot suckle, have a non-cell-autonomous defect in B lymphocyte development. No cell-autonomous defect of Fus−/− hematopoietic cells has been documented. Here we report the detailed analyses of the Fus−/− fetal liver hematopoietic stem cells (HSCs). Fus−/− fetal livers at embryonic day 14.5 were smaller in size and exhibited a significant reduction in hematopoietic cell numbers by 60% compared with the wild type (WT). Nonetheless, no significant difference was observed in the proportion of stem/progenitor cell fraction (lineage-marker-c-Kit+Sca-1+; KSL) as well as colony-forming cells between WT and Fus−/− fetal livers. Fus−/− KSL cells proliferated and differentiated almost normally in vitro. To examine in vivo repopulating activity, we transplanted fetal liver cells to lethally irradiated CD45.1 recipients with competitor bone marrow cells. Fus−/− fetal liver donor cells reconstituted recipients’ hematopoiesis for the long term and contributed to all cell lineages including B lymphocytes. In contrast to the in vitro results, however, the chimerism of donor-derived cells was significantly lower in recipients receiving Fus−/− fetal liver cells compared with WT controls (approximately 2-fold reduction). This trend was reproducible with both unfractionated and purified KSL fetal liver test cells. Our data demonstrated that the proto-oncogene Fus is involved in the maintenance of normal HSC functions. Detailed analyses on the underlying mechanisms are in progress.

Gi-Coupled GPCR Signaling Is Required at Multiple Levels and In Different Lineages In Hematopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2635-2635
Author(s):  
Marie Terpager ◽  
Hiroshi Kataoka ◽  
Ivo Cornelissen ◽  
Shaun R. Coughlin
Keyword(s):  
Bone Marrow ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Liver Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells ◽  
Multiple Levels

Abstract Abstract 2635 G protein-coupled receptors (GPCRs) can regulate cell migration, survival, proliferation and differentiation –– key processes in hematopoiesis. The Gi-coupled receptor CXCR4 plays a key role in hematopoiesis, suggesting that related receptors might also contribute. Because all Gi family members except Gz are inhibited by pertussis toxin (PTX), we utilized a ROSA26-CreOnPTX mouse line that expresses PTX in a Cre-dependent manner to broadly probe the role of Gi signaling in hematopoiesis. Mice hemizygous for the hematopoietic lineage-specific Cre transgene Vav-iCre were crossed with ROSA26-CreOnPTX/CreOnPTX mice to generate offspring expressing PTX in hematopoietic lineages (Vav-PTX) and Cre-negative controls in which the PTX allele remained silent. Vav-PTX mice were born at the expected Mendelian rate, and except for a smaller thymus, were grossly normal, but all died with pneumonia between days 2 and 14. Bone marrow in 3 day-old Vav-PTX mice was hypocellular with significant underrepresentation of granulocytic and lymphocytic lineages as well as hematopoietic stem and progenitor cells (lin-, c-kit+, Sca1+). In bone marrow reconstitution studies, cells from Vav-PTX fetal livers (E14.5) showed impaired short-term and no long-term repopulating activity. Additionally, Vav-PTX fetal liver cells were significantly impaired in their ability to form granulocyte/macrophage and erythroid colonies in vitro. Interestingly, when wild-type E14.5 fetal liver cells were grown in vitro in presence of exogenous PTX, only erythroid colony formation was impaired, and flow cytometric analysis of the progenitor populations of Vav-PTX fetal liver revealed a significant decrease in granulocyte-macrophage progenitors (GMPs) as well as in common myeloid progenitors (CMPs) but not in megakaryocyte/erythroid progenitors (MEPs). Thus, reduced progenitor populations may account for reduced granulocyte/macrophage colony-forming activity in fetal liver cell cultures but does not account for reduced erythroid colony-forming activity. Indeed, normal MEP numbers in Vav-PTX livers and the ability of exogenous PTX to inhibit formation of erythroid colonies in wild-type fetal liver cultures suggests that Gi signaling in MEPs or their progeny may contribute to erythropoiesis in fetal liver. Several of the necessary roles of Gi signaling identified above are not accounted for by the function of CXCR4, and, taken together, our data suggest that Gi-coupled GPCRs likely contribute to hematopoiesis at multiple levels and in different lineages. An effort to identify GPCRs that contribute to erythropoiesis is underway. Disclosures: No relevant conflicts of interest to declare.

Long-Term Cell Autonomous Effect of Tet2 Loss in Hematopoietic Cells in Mice.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2416-2416
Author(s):  
Tomoya Muto ◽  
Goro Sashida ◽  
Motohiko Oshima ◽  
Chiaki Nakaseko ◽  
Kotaro Yokote ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Extramedullary Hematopoiesis ◽  
Liver Cells ◽  
Severe Anemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Fetal Liver Cells

Abstract Abstract 2416 TET2 mutations are frequently observed in myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN. Several groups have already reported that deletion of Tet2 in mice leads to dysregulated hematopoietic stem cell self-renewal and subsequent development of myeloid malignancies. Of note, mice hypomorphic or heterozygous for the Tet2 allele have been reported to show similar phenotypes as those of Tet2-null mice, suggesting that haploinsufficiency of Tet2 plays a role in the development of myeloid malignancies. However, little is known about long-term cell autonomous effects of Tet2 loss in hematopoietic cells: most of the reports were based on relatively short-term observations or did not exclude the influence of Tet2 loss in the niche cells. To study long-term cell autonomous effect of Tet2 loss in hematopoietic cells, we analyzed the hematopoiesis of wild-type recipient mice reconstituted with fetal liver cells from Tet2 hypomorphic mice for a longer period up to 1 year. Tet2 gene trap mice (Tet2trap/trap), in which the gene trap vector was inserted into the exon 2 of Tet2 just before the first coding exon, express Tet2 mRNA at the level approximately 20% of that of the wild-type (WT) mice (Shide et al. Leukemia 2012). We transplanted fetal liver cells from E14.5 WT or Tet2trap/trap mice into lethally irradiated recipient mice. At 4 months after transplantation, the recipient mice reconstituted with Tet2trap/trap cells showed a significantly increased proportion of monocytes in peripheral blood (PB) compared with those with WT cells (WT=5.59±2.57%, Tet2trap/trap=12.67±7.45%, p=0.01). While there were no significant differences between the two groups in the bone marrow (BM) compartments including the numbers of Lineage−Sca-1+c-Kit+ (LSK) hematopoietic stem/progenitor cells, extramedullary hematopoiesis in the spleen was markedly enhanced in the recipients with Tet2trap/trap cells. The proportion of LSK, granulocyte/macrophage progenitors (GMPs) and megakaryocyte/erythroid progenitors (MEPs) in the spleen of recipient mice reconstituted with WT and Tet2trap/trap cells were 0.002±0.001% vs 0.006±0.001% (p<0.01), 0.007±0.004% vs 0.029±0.01% (p=0.026) and 0.084±0.024% vs 0.25±0.044% (p<0.01), respectively. These findings were compatible with those reported previously and indicated that recipient mice reconstituted with Tet2trap/trapcells induce chronic myelomonocytic leukemia (CMML)-like disease. Of note, after a long observation period, particularly after 9 months post-transplantation, mice reconstituted with Tet2trap/trap cells developed advanced hematological disease and 53.3% (8 of 15) died or were killed because of their moribund condition by 11 months after transplantation. Detailed analysis on moribund as well as surviving mice reconstituted with Tet2trap/trap cells (n=3 each) revealed that the 2/3 of the mice developed CMML-like disease with monocytosis in PB and evident extramedullary hematopoiesis harboring increased number of LSK and GMPs in spleen, whereas the remaining 1/3 of mice developed MDS/MPN-like disease with severe anemia. The latter mice did not show monocytosis in PB and BM, but displayed dyserythropoiesis accompanied by massive extramedullary erythropoiesis, as we saw increased number of LSK and MEPs, but not GMPs, in spleen. The proportion of Annexin V+ cells in Ter119highCD71higherythroblasts in the BM of WT mice, CMML-like disease-carrying mice and MDS/MPN-like disease-carrying mice were 9.46±0.53%, 10.71±0.36%, and 19.04±0%, respectively, suggesting that the enhanced apoptosis led to the severe anemia seen in the MDS/MPN-like disease-carrying mice. Thus, decreased expression of Tet2 is sufficient to promote not only CMML-like disease, but also an MDS/MPN-like disease as well. Our findings confirmed a long-term cell autonomous effect from insufficient function of Tet2 in the development of hematological diseases. Interested in the molecular mechanism of disease progression, we have identified 1,642 differentially methylated regions (DMRs) in Tet2trap/trap GMPs compared with WT GMPs by ChIP-sequencing. We are now working to understand how these DMRs are involved in the development of the two distinct diseases associated with hypomorphic Tet2. Disclosures: No relevant conflicts of interest to declare.

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