In utero transplantation of wild-type fetal liver cells rescues factor X-deficient mice from fatal neonatal bleeding diatheses

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.

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DNA Damage and Perturbed Topoisomerase IIα as a Target of 1,4-Benzoquinone Toxicity in Murine Fetal Liver Cells

Toxicological Sciences ◽

10.1093/toxsci/kfz158 ◽

2019 ◽

Vol 171 (2) ◽

pp. 339-346 ◽

Cited By ~ 1

Author(s):

Trent H Holmes ◽

Louise M Winn

Keyword(s):

Fetal Liver ◽

In Utero ◽

Liver Cells ◽

Environmental Pollutant ◽

Placental Barrier ◽

Topoisomerase Iiα ◽

Double Stranded Dna ◽

Fetal Liver Cells ◽

Dna Break ◽

Covalent Adducts

Abstract Benzene is a ubiquitous environmental pollutant. Recent studies have shown a link between the development of childhood leukemias and maternal benzene exposure, suggesting that these leukemias may be initiated in utero. Benzene crosses the placental barrier however the mechanisms behind in utero benzene toxicity have not been well elucidated. This study is the first to show that the benzene metabolite, benzoquinone (BQ), perturbs fetal topoisomerase IIα (Topo IIα), an enzyme essential for DNA repair. Using cultured murine CD-1 fetal liver cells, this study shows that Topo IIα activity decreases following 24 h of exposure to BQ (12.5 and 15.625 µM), with 12.5 µM confirmed to disrupt the c-kit+ Lin− Sca-1− Il7rα− population of cells in culture. Pretreatment with the antioxidant N-acetylcysteine did not prevent the inhibition of Topo IIα by BQ. An increase in Topo IIα-DNA covalent adducts was detected following 24-h exposure to BQ (12.5 and 50 µM). Interestingly, BQ (12.5 µM) exposure did not significantly increase levels of 4-hydroxynonenal (4-HNE), a marker of oxidative stress after 24 h. However, increased levels of the double-stranded DNA break marker γH2AX were detected following 24 h of BQ exposure, confirming that Topo IIα-induced breaks are increased in BQ-treated cells. This study shows that fetal Topo IIα is perturbed by BQ and suggests that this protein is a target of benzene and may be implicated with in utero benzene toxicity.

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

Blood ◽

10.1182/blood.v104.11.2161.2161 ◽

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.

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

Blood ◽

10.1182/blood.v120.21.610.610 ◽

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.

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

Blood ◽

10.1182/blood-2018-99-114144 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1269-1269 ◽

Cited By ~ 1

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.

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Roles of MOZ in Hematopoiesis.

Blood ◽

10.1182/blood.v106.11.2269.2269 ◽

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.

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A Differentiation Block in Erythroid Cells Lacking Erythroid Krupple-Like Factor (EKLF).

Blood ◽

10.1182/blood.v106.11.526.526 ◽

2005 ◽

Vol 106 (11) ◽

pp. 526-526

Author(s):

Patrick G. Gallagher ◽

Murat O. Arcasoy ◽

Serena E. Vayda ◽

Holly K. Dressman ◽

James J. Bieker ◽

...

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Fetal Liver ◽

Erythroid Differentiation ◽

Erythroid Cells ◽

Wild Type ◽

Fetal Liver Cells ◽

Differentiation Block ◽

Microarray Analyses ◽

Deficient Mice

Abstract Mice deficient in the erythroid specific zinc-finger transcription factor EKLF die ~d14-15 of gestation of severe anemia, attributed to decreased expression of β-globin. The morphology of fetal-liver derived erythroid cells in EKLF-deficient mice does not mimic that seen in thalassemia, but instead shows hemolysis with uniform, nucleated erythroid progenitor cells. This has led to the hypothesis that a block in erythroid differentiation contributes to the anemia in EKLF-deficient mice. To address this, we performed microarray analyses with Affymetrix GeneChip Mouse Genome 430 2.0 arrays and RNA from d13.5 fetal livers of wild type (WT) and EKLF-deficient mice. Three independent EKLF +/+ and −/− RNA samples were analyzed. Numerous genes were down regulated including AHSP, pyruvate kinase, ankyrin, β spectrin and band 3. Verification of reduced expression of selected genes demonstrated that expression levels of many genes identified as down regulated via microarray analyses were minimally reduced in EKLF −/− RNA (<20%) compared to normal (Rh 30, protein 4.2, protein 4.9, p55, AQP1, and ALAS-E). Flow cytometry of WT d14.5 fetal liver cells using TER 119 and CD71 was performed. In WT fetal livers, this identifies 5 populations, designated R1-R5, with R1/R2 composed of primitive progenitors and proerythroblasts and R3, R4, and R5 composed of more mature erythroblasts (Blood102:3938, 2003). In EKLF −/− fetal livers, R3, R4, and R5, populations involved in terminal erythroid differentiation, were completely absent, suggesting many of the genes identified by microarray analyses were differentially expressed because of a bias introduced by a differentiation block to more mature erythroid cells. Confirming this hypothesis, we demonstrated that genes with <20% difference in expression between WT and EKLF-deficient fetal liver mRNA had 4-fold or higher levels in wild type R3+R4+R5 RNA compared to R1+R2 RNA. To better understand how differentially expressed genes were integrated into specific regulatory and signaling pathway networks, we used Ingenuity Pathway Analysis. A subset of focus genes incorporated into a biological network with highly a significant scores (>40) was generated containing 35 focus genes. The biological function of this network involved cell cycle and DNA replication. At the central nodes of this network were E2F1 and E2F2, transcription factors involved in cell cycle control. Cell cycle analysis demonstrated that EKLF-deficient R1 cells exhibited a significant delay exiting G0+G1 and entering S phase and both R1 and R2 cells exhibited a defect in exiting S and entering G2+M. Colony assays with R1 and R2 cells revealed that EKLF-deficient fetal liver cells had decreased frequency of CFU-E, but similar absolute numbers of CFU-E as WT. As predicted by the cell cycle defect, EKLF−/− FL cells were severely (~10 fold) deficient in their ability to generate BFU-E. Flow cytometry with annexin V revealed no difference between WT and EKLF-deficient cells indicated that apoptosis was not contributing to the differentiation block. These results support the hypothesis that the failure of definitive erythropoiesis in EKLF deficient mice is due to decreased expression of many erythroid genes involved in erythroid differentiation, stabilization of α-globin protein, membrane stability, and glycolysis, not simply decreased transcription of the β-globin gene.

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