The influence of purified recombinant human heavy-subunit and light- subunit ferritins on colony formation in vitro by granulocyte- macrophage and erythroid progenitor cells

Abstract Purified recombinant human heavy subunit (rHF, acidic) and recombinant human light subunit (rLF, basic) ferritins were assessed for their effects in vitro on colony formation by normal human granulocyte- macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells. The purity of the samples was confirmed by electrophoresis in both nondenaturing and denaturing conditions and silver staining. Concentrations of 10(-8) to 10(-10) mol/L rHF caused an approximately 40% significant decrease in colony formation. Some significant activity was detected at 10(-11) mol/L, and activity was lost at 10(-12) mol/L. In contrast, rLF had no significant activity at 10(-8) to 10(-16) mol/L. rHF was significantly active against mouse bone marrow CFU-GM to concentrations as low as 10(- 8) to 10(-9) mol/L. The inhibitory activity of rHF was inactivated with three different monoclonal antibodies recognizing the heavy subunit of ferritin, but not with two monoclonal antibodies recognizing the light subunit of ferritin. The inhibitory activity of rHF was similar in the absence or presence of serum, monocytes, and T lymphocytes. We and others have shown an association of a glycosylated natural acidic isoferritin (AIF) with inhibitory activity, but since the rHF was expressed in Escherichia coli and did not bind to concanavalin A, glycosylation of AIF is not an absolute prerequisite for this activity. These results demonstrate that rHF has suppressive activity in vitro and substantiate our original observations using purified natural acidic isoferritins.

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The influence of purified recombinant human heavy-subunit and light- subunit ferritins on colony formation in vitro by granulocyte- macrophage and erythroid progenitor cells

Blood ◽

10.1182/blood.v68.6.1257.bloodjournal6861257 ◽

1986 ◽

Vol 68 (6) ◽

pp. 1257-1263 ◽

Cited By ~ 2

Author(s):

HE Broxmeyer ◽

L Lu ◽

DC Bicknell ◽

DE Williams ◽

S Cooper ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Progenitor Cells ◽

Inhibitory Activity ◽

Colony Formation ◽

Mouse Bone Marrow ◽

Significant Activity ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Normal Human

Purified recombinant human heavy subunit (rHF, acidic) and recombinant human light subunit (rLF, basic) ferritins were assessed for their effects in vitro on colony formation by normal human granulocyte- macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells. The purity of the samples was confirmed by electrophoresis in both nondenaturing and denaturing conditions and silver staining. Concentrations of 10(-8) to 10(-10) mol/L rHF caused an approximately 40% significant decrease in colony formation. Some significant activity was detected at 10(-11) mol/L, and activity was lost at 10(-12) mol/L. In contrast, rLF had no significant activity at 10(-8) to 10(-16) mol/L. rHF was significantly active against mouse bone marrow CFU-GM to concentrations as low as 10(- 8) to 10(-9) mol/L. The inhibitory activity of rHF was inactivated with three different monoclonal antibodies recognizing the heavy subunit of ferritin, but not with two monoclonal antibodies recognizing the light subunit of ferritin. The inhibitory activity of rHF was similar in the absence or presence of serum, monocytes, and T lymphocytes. We and others have shown an association of a glycosylated natural acidic isoferritin (AIF) with inhibitory activity, but since the rHF was expressed in Escherichia coli and did not bind to concanavalin A, glycosylation of AIF is not an absolute prerequisite for this activity. These results demonstrate that rHF has suppressive activity in vitro and substantiate our original observations using purified natural acidic isoferritins.

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Separation of erythroid progenitor cells in mouse bone marrow by isokinetic-gradient sedimentation

Blood ◽

10.1182/blood.v54.1.105.bloodjournal541105 ◽

1979 ◽

Vol 54 (1) ◽

pp. 105-116

Author(s):

J Misiti ◽

JL Spivak

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

The Other ◽

Mouse Bone Marrow ◽

Erythroid Progenitor ◽

Linear Gradient ◽

Erythroid Progenitor Cells ◽

Density Gradient Sedimentation ◽

Marrow Cells

Isokinetic-gradient sedimentation employing a shallow linear gradient of Ficoll in tissue culture medium was used to isolate erythroid progenitor cells (CFU-e) from mouse bone marrow. Following gradient sedimentation, 34% of the total nucleated cells and 48% of the CFU-e applied to the gradient were recovered, and three distinct modal populations of CFU-e could be distinguished. The slowest-migrating population did not require exposure to exogenous erythropoietin in order to form erythroid colonies in vitro. The other two modal populations of CFU-e required exposure to exogenous erythropoietin for differentiation. One of these, constituting 64% of the hormone- dependent CFU-e recovered, migrated with the bulk of the marrow cells, whereas the other migrated ahead of the bulk of the marrow cells. This latter population, which contained 34% of the CFU-e, was recovered with 11% of the marrow cells, representing a twofold to threefold enrichment. BFU-e migrated more slowly than the erythropoietin- dependent CFU-e. Resedimentation studies suggested that the two erythropoietin-dependent CFU-e populations were distinct modal populations. When cells from the fastest-migrating population of erythropoietin-dependent CFU-e were cocultured with unseparated marrow cells, a further twofold to threefold enhancement of erythroid colony formation was obtained. Comparison of isokinetic-gradient sedimentation with discontinuous and continuous albumin density-gradient sedimentation revealed that isokinetic-gradient sedimentation was a more efficient method than the former and a more rapid method than the latter for isolating CFU-e from mouse bone marrow.

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Separation of erythroid progenitor cells in mouse bone marrow by isokinetic-gradient sedimentation

Blood ◽

10.1182/blood.v54.1.105.105 ◽

1979 ◽

Vol 54 (1) ◽

pp. 105-116 ◽

Cited By ~ 7

Author(s):

J Misiti ◽

JL Spivak

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

The Other ◽

Mouse Bone Marrow ◽

Erythroid Progenitor ◽

Linear Gradient ◽

Erythroid Progenitor Cells ◽

Density Gradient Sedimentation ◽

Marrow Cells

Abstract Isokinetic-gradient sedimentation employing a shallow linear gradient of Ficoll in tissue culture medium was used to isolate erythroid progenitor cells (CFU-e) from mouse bone marrow. Following gradient sedimentation, 34% of the total nucleated cells and 48% of the CFU-e applied to the gradient were recovered, and three distinct modal populations of CFU-e could be distinguished. The slowest-migrating population did not require exposure to exogenous erythropoietin in order to form erythroid colonies in vitro. The other two modal populations of CFU-e required exposure to exogenous erythropoietin for differentiation. One of these, constituting 64% of the hormone- dependent CFU-e recovered, migrated with the bulk of the marrow cells, whereas the other migrated ahead of the bulk of the marrow cells. This latter population, which contained 34% of the CFU-e, was recovered with 11% of the marrow cells, representing a twofold to threefold enrichment. BFU-e migrated more slowly than the erythropoietin- dependent CFU-e. Resedimentation studies suggested that the two erythropoietin-dependent CFU-e populations were distinct modal populations. When cells from the fastest-migrating population of erythropoietin-dependent CFU-e were cocultured with unseparated marrow cells, a further twofold to threefold enhancement of erythroid colony formation was obtained. Comparison of isokinetic-gradient sedimentation with discontinuous and continuous albumin density-gradient sedimentation revealed that isokinetic-gradient sedimentation was a more efficient method than the former and a more rapid method than the latter for isolating CFU-e from mouse bone marrow.

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Human thymic epithelial cells maintain long-term survival of clonogenic myeloid and erythroid progenitor cells in vitro

British Journal of Haematology ◽

10.1046/j.1365-2141.2000.02337.x ◽

2000 ◽

Vol 111 (1) ◽

pp. 363-370 ◽

Cited By ~ 1

Author(s):

Katsuto Takenaka ◽

Mine Harada ◽

Tomoaki Fujisaki ◽

Koji Nagafuji ◽

Shinichi Mizuno ◽

...

Keyword(s):

Epithelial Cells ◽

Progenitor Cells ◽

Thymic Epithelial Cells ◽

Erythroid Progenitor ◽

Term Survival ◽

Long Term Survival ◽

Erythroid Progenitor Cells

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Fetal erythropoiesis in steel mutant mice. III. Defect in differentiation from BFU-E to CFU-E during early development

Blood ◽

10.1182/blood.v51.3.539.539 ◽

1978 ◽

Vol 51 (3) ◽

pp. 539-547 ◽

Cited By ~ 18

Author(s):

DH Chui ◽

SK Liao ◽

K Walker

Keyword(s):

Progenitor Cells ◽

Early Development ◽

The Other ◽

Mutant Mice ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells ◽

Other Hand ◽

Colony Forming Units ◽

Fetal Erythropoiesis

Abstract Erythroid progenitor cells in +/+ and Sl/Sld fetal livers manifested as burst-forming units-erythroid (BFU-E) and colony-forming units- erythroid (CFU-E) were assayed in vitro during early development. The proportion of BFU-E was higher as mutant than in normal fetal livers. On the other hand, the proportion of CFU-E was less in the mutant than in the normal. These results suggest that the defect in Sl/Sld fetal hepatic erythropoiesis is expressed at the steps of differentiation that effect the transition from BFU-E to CFU-E.

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Ligand-dependent repression of the erythroid transcription factor GATA-1 by the estrogen receptor.

Molecular and Cellular Biology ◽

10.1128/mcb.15.6.3147 ◽

1995 ◽

Vol 15 (6) ◽

pp. 3147-3153 ◽

Cited By ~ 88

Author(s):

G A Blobel ◽

C A Sieff ◽

S H Orkin

Keyword(s):

Bone Marrow ◽

Estrogen Receptor ◽

Transcription Factor ◽

Progenitor Cells ◽

Erythroid Cell ◽

High Dose ◽

Dependent Manner ◽

Erythroid Progenitor ◽

Erythroid Progenitor Cells

High-dose estrogen administration induces anemia in mammals. In chickens, estrogens stimulate outgrowth of bone marrow-derived erythroid progenitor cells and delay their maturation. This delay is associated with down-regulation of many erythroid cell-specific genes, including alpha- and beta-globin, band 3, band 4.1, and the erythroid cell-specific histone H5. We show here that estrogens also reduce the number of erythroid progenitor cells in primary human bone marrow cultures. To address potential mechanisms by which estrogens suppress erythropoiesis, we have examined their effects on GATA-1, an erythroid transcription factor that participates in the regulation of the majority of erythroid cell-specific genes and is necessary for full maturation of erythrocytes. We demonstrate that the transcriptional activity of GATA-1 is strongly repressed by the estrogen receptor (ER) in a ligand-dependent manner and that this repression is reversible in the presence of 4-hydroxytamoxifen. ER-mediated repression of GATA-1 activity occurs on an artificial promoter containing a single GATA-binding site, as well as in the context of an intact promoter which is normally regulated by GATA-1. GATA-1 and ER bind to each other in vitro in the absence of DNA. In coimmunoprecipitation experiments using transfected COS cells, GATA-1 and ER associate in a ligand-dependent manner. Mapping experiments indicate that GATA-1 and the ER form at least two contacts, which involve the finger region and the N-terminal activation domain of GATA-1. We speculate that estrogens exert effects on erythropoiesis by modulating GATA-1 activity through protein-protein interaction with the ER. Interference with GATA-binding proteins may be one mechanism by which steroid hormones modulate cellular differentiation.

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A role for calmodulin in the growth of human hematopoietic progenitor cells

Blood ◽

10.1182/blood.v75.7.1446.1446 ◽

1990 ◽

Vol 75 (7) ◽

pp. 1446-1454 ◽

Cited By ~ 11

Author(s):

N Katayama ◽

M Nishikawa ◽

F Komada ◽

N Minami ◽

S Shirakawa

Keyword(s):

Progenitor Cells ◽

Colony Formation ◽

Hematopoietic Progenitor Cells ◽

Dependent Manner ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Gm Csf ◽

Erythroid Progenitor Cells ◽

Dependent Inhibition ◽

Dose Dependent

Abstract A possible role for calmodulin in the colony growth of human hematopoietic progenitor cells was investigated using pharmacologic approaches. We obtained evidence for a dose-dependent inhibition of colony formation of myeloid progenitor cells (CFU-C) stimulated by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte CSF (G-CSF) by three calmodulin antagonists, N- (6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), N- (4-aminobutyl)-5-chloro-2-naphthalenesulfonamide hydrochloride (W-13), and trifluoperazine. Chlorine-deficient analogs of W-7 and W-13, with a lower affinity for calmodulin, did not inhibit the growth of CFU-C colonies. W-7, W-13, and trifluoperazine inhibited the colony formation of immature erythroid progenitor cells (BFU-E) stimulated by IL-3 plus erythropoietin (Ep) or GM-CSF plus Ep, in a dose-dependent manner, while they did not affect the colony formation of mature erythroid progenitor cells (CFU-E) induced by Ep. W-7, W-13, and trifluoperazine also led to a dose-dependent inhibition of GM-CSF-induced colony formation of KG-1 cells. Calmodulin-dependent kinase activity derived from the KG-1 cells was inhibited by these three calmodulin antagonists in a dose-dependent manner. These data suggest that calmodulin may play an important regulatory role via a common process in the growth of hematopoietic progenitor cells stimulated by IL-3, GM-CSF, and G-CSF. Mechanisms related to the growth signal of Ep apparently are not associated with calmodulin-mediated systems.

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Pregnancy-Secreted Acid Phosphatase, Uteroferrin, Enhances Fetal Erythropoiesis

Endocrinology ◽

10.1210/en.2014-1397 ◽

2014 ◽

Vol 155 (11) ◽

pp. 4521-4530 ◽

Cited By ~ 3

Author(s):

Wei Ying ◽

Haiqing Wang ◽

Fuller W. Bazer ◽

Beiyan Zhou

Keyword(s):

Acid Phosphatase ◽

Progenitor Cells ◽

Fetal Liver ◽

Hematopoietic Cells ◽

Erythroid Progenitor ◽

Colony Forming Unit ◽

Erythroid Progenitor Cells ◽

Uterine Glands ◽

Fetal Erythropoiesis

Abstract Uteroferrin (UF) is a progesterone-induced acid phosphatase produced by uterine glandular epithelia in mammals during pregnancy and targeted to sites of hematopoiesis throughout pregnancy. The expression pattern of UF is coordinated with early fetal hematopoietic development in the yolk sac and then liver, spleen, and bone to prevent anemia in fetuses. Our previous studies suggested that UF exerts stimulatory impacts on hematopoietic progenitor cells. However, the precise role and thereby the mechanism of action of UF on hematopoiesis have not been investigated previously. Here, we report that UF is a potent regulator that can greatly enhance fetal erythropoiesis. Using primary fetal liver hematopoietic cells, we observed a synergistic stimulatory effect of UF with erythropoietin and other growth factors on both burst-forming unit-erythroid and colony-forming unit-erythroid formation. Further, we demonstrated that UF enhanced erythropoiesis at terminal stages using an in vitro culture system. Surveying genes that are crucial for erythrocyte formation at various stages revealed that UF, along with erythropoietin, up-regulated transcription factors required for terminal erythrocyte differentiation and genes required for synthesis of hemoglobin. Collectively, our results demonstrate that UF is a cytokine secreted by uterine glands in response to progesterone that promotes fetal erythropoiesis at various stages of pregnancy, including burst-forming unit-erythroid and colony-forming unit-erythroid progenitor cells and terminal stages of differentiation of hematopoietic cells in the erythroid lineage.

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Constitutive Activation of STAT5 and Bcl-XL Overexpression Can Induce Endogenous Erythroid Colony Formation in Human Primary Cells.

Blood ◽

10.1182/blood.v106.11.3135.3135 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3135-3135

Author(s):

Loïc Garçon ◽

Chloe James ◽

Catherine Lacout ◽

Valérie Camara-Clayette ◽

Valérie Ugo ◽

...

Keyword(s):

Signaling Pathways ◽

Progenitor Cells ◽

Colony Formation ◽

Terminal Differentiation ◽

Erythroid Differentiation ◽

Primary Cells ◽

Erythroid Progenitor ◽

Constitutive Activation ◽

Human Primary Cells

Abstract In contrast with secondary erythrocytosis, progenitor cells from polycythemia vera (PV) patients can undergo in vitro erythroid differentiation despite absence of erythropoietin (EPO) and presence of such endogenous erythroid colonies (EEC) is routinely used as a diagnostic assay. Recent focus on the JAK2 mutation V617F in PV patients argue for a direct implication of JAK2 dependent signaling pathways in EEC formation. Because STAT5 is the principal target of JAK2 in erythroid cells, we investigated whether EEC formation was only dependent on STAT5 activation or required other signaling pathways that would be activated by JAK2. For this purpose, we transduced a retroviral vector coding for a constitutively active form of STAT5 (MIGR-STAT5CA) in UT7 cells, a leukemic cell line with erythroid properties. We observed in cells transduced with the MIGR-STAT5CA vector a spontaneous induction of erythroid differentiation in comparison with cells infected with the empty vector MIGR, as assessed by GPA staining. We next investigated effects of STAT5CA on erythroid differentiation of human primary progenitors. Purified CD34+ cells obtained from peripheral blood (PB) of patients treated with G-CSF were transduced with the STA5CA vector, the CD36+/GPA− erythroid progenitor cells were sorted and cultured in presence of SCF alone. When expressing STAT5CA, they both proliferate and undergo erythroid terminal differentiation despite the absence of EPO. We concluded that a phosphorylated form of STAT5 was sufficient to support in vitro erythroid differentiation of human primary cells. Because STAT5 has been shown to play a crucial role in erythropoiesis via induction of the antiapoptotic protein Bcl-xL, we next investigated whether effects of STAT5CA on erythroid maturation was dependent on Bcl-xL induction. Tansduction of human CD36+/GPA− cells with a retrovirus containing the coding sequence of human Bcl-xL progenitors allowed survival, proliferation and GPA acquisition despite the absence of EPO. We next investigated whether STAT5CA or Bcl-xL overexpression in normal primary cells could reproduce the malignant phenotype observed in PV patients, i.e. induction of EEC formation. CD36+/GPA− transduced with either the STAT5 CA or the Bcl-XL vectors were plated in methylcellulose in the absence of EPO. Bcl-xL as well as STAT5CA vectors could both induce endogenous erythroid colony formation. Regardless to these results, we hypothesized that the EEC formation observed in myeloproliferative disorders could be at least partially due to the JAK2 dependent activation of the STAT5/Bcl-XL pathway. Thus, both constitutive activation of STAT5 and Bcl-xL overexpression could substitute to EPO to induce terminal differentiation of human primary erythroid progenitors.

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UCP2 Modulates Cell Proliferation through the MAPK/ERK Pathway during Erythropoiesis and Has No Effect on Heme Biosynthesis

Blood ◽

10.1182/blood.v112.11.5372.5372 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5372-5372

Author(s):

Alvaro A Elorza ◽

Brigham B Hyde ◽

Hanna Mikkola ◽

Sheila Collins ◽

Orian S Shirihai

Keyword(s):

Cell Proliferation ◽

Progenitor Cells ◽

Fetal Liver ◽

Mitochondrial Protein ◽

Erythroid Progenitor ◽

Heme Synthesis ◽

Erythroid Progenitor Cells ◽

Deficient Mice

Abstract UCP2, an inner membrane mitochondrial protein, has been implicated in bioenergetics and Reactive Oxygen Species (ROS) modulation. UCP2 has been previously hypothesized to function as a facilitator of heme synthesis and iron metabolism by reducing ROS production. While UCP2 has been found to be induced by GATA1 during erythroid differentiation its role in erythropoiesis in vivo or in vitro has not been reported thus far. Here we report on the study of UCP2 role in erythropoiesis and the hematologic phenotype of UCP2 deficient mouse. In vivo we found that UCP2 protein peaks at early stages of erythroid maturation when cells are not fully committed in heme synthesis and then becomes undetectable at the reticulocyte stage. Iron incorporation into heme was unaltered in erythroid cells from UCP2 deficient mice. While heme synthesis was not influenced by UCP2 deficiency, mice lacking UCP2 had a delayed recovery from chemically induced hemolytic anemia. Analysis of the erythroid lineage from bone marrow and fetal liver revealed that in the UCP2 deficient mice the R3 (CD71high/Ter119high) population was reduced by 24%. The count of BFU-E and CFU-E colonies, scored in an erythroid colony assay, was unaffected, indicating an equivalent number of early erythroid progenitor cells in both UCP2 deficient and control cells. Ex-vivo differentiation assay revealed that UCP2 deficient c-kit+ progenitor cells expansion was overall reduced by 14% with population analysis determining that the main effect is at the R3 stage. No increased rate of apoptosis was found indicating that expansion rather than cell death is being compromised. Reduced expansion of c-kit+ cells was accompanied by 30% reduction in the phosphorylated form of ERK, a ROS dependent cytosolic regulator of cell proliferation. Analysis of ROS in UCP2 null erythroid progenitors revealed altered distribution of ROS resulting in 14% decrease in cytosolic and 32% increase in mitochondrial ROS. Restoration of the cytosolic oxidative state of erythroid progenitor cells by the pro-oxidant Paraquat reversed the effect of UCP2 deficiency on cell proliferation in in vitro differentiation assays. Together, these results indicate that UCP2 is a regulator of erythropoiesis and suggests that inhibition of UCP2 function may contribute to the development of anemia.

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