Arsenite exposure inhibits the erythroid differentiation of human hematopoietic progenitor CD34+ cells and causes decreased levels of hemoglobin

AbstractArsenic exposure poses numerous threats to human health. Our previous work in mice has shown that arsenic causes anemia by inhibiting erythropoiesis. However, the impacts of arsenic exposure on human erythropoiesis remain largely unclear. We report here that low-dose arsenic exposure inhibits the erythroid differentiation of human hematopoietic progenitor cells (HPCs). The impacts of arsenic (in the form of arsenite; As3+) on red blood cell (RBC) development was evaluated using a long-term culture of normal human bone marrow CD34+-HPCs stimulated in vitro to undergo erythropoiesis. Over the time course studied, we analyzed the expression of the cell surface antigens CD34, CD71 and CD235a, which are markers commonly used to monitor the progression of HPCs through the stages of erythropoiesis. Simultaneously, we measured hemoglobin content, which is an important criterion used clinically for diagnosing anemia. As compared to control, low-dose As3+ exposure (100 nM and 500 nM) inhibited the expansion of CD34+-HPCs over the time course investigated; decreased the number of committed erythroid progenitors (BFU-E and CFU-E) and erythroblast differentiation in the subsequent stages; and caused a reduction of hemoglobin content. These findings demonstrate that low-dose arsenic exposure impairs human erythropoiesis, likely by combined effects on various stages of RBC formation.

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Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Blood ◽

10.1182/blood.v63.6.1376.bloodjournal6361376 ◽

1984 ◽

Vol 63 (6) ◽

pp. 1376-1384 ◽

Cited By ~ 1

Author(s):

T Yokochi ◽

M Brice ◽

PS Rabinovitch ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Monoclonal Antibodies ◽

K562 Cells ◽

Erythroid Differentiation ◽

Antigenic Determinants ◽

Erythroid Progenitors ◽

Cell Surface Antigens ◽

Cytotoxicity Assays ◽

Complement Dependent Cytotoxicity

Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

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Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells: from the erythroid committed progenitor level to the mature erythroblast

Blood ◽

10.1182/blood.v63.6.1376.1376 ◽

1984 ◽

Vol 63 (6) ◽

pp. 1376-1384 ◽

Cited By ~ 20

Author(s):

T Yokochi ◽

M Brice ◽

PS Rabinovitch ◽

T Papayannopoulou ◽

G Stamatoyannopoulos

Keyword(s):

Monoclonal Antibodies ◽

K562 Cells ◽

Erythroid Differentiation ◽

Antigenic Determinants ◽

Erythroid Progenitors ◽

Cell Surface Antigens ◽

Cytotoxicity Assays ◽

Complement Dependent Cytotoxicity

Abstract Two new cell surface antigens specific for the erythroid lineage were defined with cytotoxic IgM monoclonal antibodies (McAb) (EP-1; EP-2) that were produced using BFU-E-derived colonies as immunogens. These two antigens are expressed on in vivo and in vitro derived adult and fetal erythroblasts, but not on erythrocytes. They are not detectable on resting lymphocytes, concanavalin-A (Con-A) activated lymphoblasts, granulocytes, and monocytes or granulocytic cells or macrophages present in peripheral blood or harvested from CFU-GM cultures. Cell line and tissue distributions distinguish McAb EP-1 and EP-2 from all previously described monoclonal antibodies. McAb EP-1 (for erythropoietic antigen-1) inhibits the formation of BFU-E and CFU-E, but not CFU-GM, colonies in complement-dependent cytotoxicity assays. By cell sorting analysis, about 90% of erythroid progenitors (CFU-E, BFU-E) were recovered in the antigen-positive fraction. Seven percent of the cells in this fraction were progenitors (versus 0.1% in the negative fraction). The expression of EP-1 antigen is greatly enhanced in K562 cells, using inducers of hemoglobin synthesis. McAb EP-2 fails to inhibit BFU-E and CFU-E colony formation in complement-dependent cytotoxicity assays. EP-2 antigen is predominantly expressed on in vitro derived immature erythroblasts, and it is weakly expressed on mature erythroblasts. The findings with McAb EP-1 provide evidence that erythroid progenitors (BFU-E and CFU-E) express determinants that fail to be expressed on other progenitor cells and hence appear to be unique to the erythroid lineage. McAb EP-1 and EP-2 are potentially useful for studies of erythroid differentiation and progenitor cell isolation.

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Complexing of the CD-3 subunit by a monoclonal antibody activates a microtubule-associated protein 2 (MAP-2) serine kinase in Jurkat cells

Biochemical Journal ◽

10.1042/bj2620449 ◽

1989 ◽

Vol 262 (2) ◽

pp. 449-456 ◽

Cited By ~ 20

Author(s):

C Hanekom ◽

A Nel ◽

C Gittinger ◽

A Rheeder ◽

G Landreth

Keyword(s):

T Cells ◽

Time Course ◽

Gel Filtration ◽

Maximal Activity ◽

Jurkat Cells ◽

Serine Kinase ◽

Transient Activation ◽

Normal Human ◽

Dose Dependent

Treatment of Jurkat T-cells with anti-CD-3 monoclonal antibodies resulted in the rapid and transient activation of a serine kinase which utilized the microtubule-associated protein, MAP-2, as a substrate in vitro. The kinase was also activated on treatment of Jurkat cells with phytohaemagglutinin, but with a different time course. The activation of the MAP-2 kinase by anti-CD-3 antibodies was dose-dependent, with maximal activity observed at concentrations of greater than 500 ng/ml. Normal human E-rosette-positive T-cells also exhibited induction of MAP-2 kinase activity during anti-CD-3 treatment. The enzyme was optimally active in the presence of 2 mM-Mn2+; lower levels of activity were observed with Mg2+, even at concentrations up to 20 mM. The kinase was partially purified by passage over DE-52 Sephacel with the activity eluting as a single peak at 0.25 M-NaCl. The molecular mass was estimated to be 45 kDa by gel filtration. The activation of the MAP-2 kinase was probably due to phosphorylation of this enzyme as treatment with alkaline phosphatase diminished its activity. These data demonstrate that the stimulation of T-cells through the CD-3 complex results in the activation of a novel serine kinase which may be critically involved in signal transduction in these cells.

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Myeloperoxidase expression in CD34+ normal human hematopoietic cells

Blood ◽

10.1182/blood.v82.7.2069.2069 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2069-2078 ◽

Cited By ~ 6

Author(s):

H Strobl ◽

M Takimoto ◽

O Majdic ◽

G Fritsch ◽

C Scheinecker ◽

...

Keyword(s):

Progenitor Cells ◽

Transferrin Receptor ◽

Staining Pattern ◽

Hematopoietic Progenitor ◽

Colony Forming Unit ◽

Cd34 Cells ◽

Normal Human ◽

Colony Forming Capacity ◽

Adult Peripheral Blood

Abstract Bone marrow (BM), adult peripheral blood (aPB), and umbilical cord blood (CB) samples contain small proportions of CD34+ cells that include virtually all hematopoietic progenitor cells. Myeloperoxidase (MPO) is considered to be selectively expressed in cells committed to granulomonocytic differentiation. Using flow cytometry and an antibody against MPO, we studied at which stage of normal hematopoietic differentiation CD34+ cells being to express MPO. We consistently observed a characteristic MPO/CD34 staining pattern and found that 35% +/- 9% of CD34+ BM cells express MPO. The MPO+ CD34+ subset and the CD33+ CD34+ subset were of similar size and overlapped considerably. MPO+ CD34+ cells expressed high levels of HLA-D molecules, were weakly CD71/transferrin receptor positive to negative, were CD45RA+ and lacked the CD45RO isoform of the leukocyte common antigen. Additionally, MPO+ CD34+ cells were on average larger in size than MPO- CD34+ cells. Virtually identical phenotypic features have previously been described for in vitro colony-forming granulomonocytic progenitor cells. In vitro clonogenic assays performed with MPO-enriched and MPO-depleted fractions of CD34+ BM cells performed by us also suggest, but do not formally prove, that at least a portion of MPO+ CD34+ cells have in vitro cluster (10 to 50 cells/colony) or colony-forming unit granulocyte-macrophage (> or = 50 cells/colony) forming capacity. CD34+ cells from CB and aPB resembled CD34+ BM cells in that considerable proportions of them coexpressed CD33. However, in contrast to BM, CD34+ cells from CB and aPB samples lacked significant MPO expression and, in line with this, the majority of them (CB, 59% +/- 7%; aPB, 66% +/- 5%) coexpressed CD45RO.

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Ranitidine Fails to Suppress the Growth in vitro of Haemopoietic Progenitors from Human Peripheral Blood or Bone Marrow

Human Toxicology ◽

10.1177/096032718900800104 ◽

1989 ◽

Vol 8 (1) ◽

pp. 19-22

Author(s):

C.D.L. Reid ◽

A. Kirk

Keyword(s):

Bone Marrow ◽

Drug Concentration ◽

Peripheral Blood ◽

Human Peripheral Blood ◽

Erythroid Progenitors ◽

Drug Concentrations ◽

Normal Human ◽

Normal Human Peripheral Blood ◽

Clonal Assays

Ranitidine was added in various concentrations (25-1600 ng/ml) to clonal assays of haemopoietic progenitors of normal human peripheral blood or bone marrow. Although a significant reduction in colonies forming from granulocyte-macrophage progenitors (CFU-GM) was demonstrated at the lowest drug concentration, no significant growth suppression was seen at higher concentrations. There was no evidence for growth inhibition of either erythroid progenitors (BFU-E) or pluripotent progenitors (CFU-mix) at any of the drug concentrations studied. A direct toxic effect of ranitidine on normal haemopoietic progenitors thus appears an unlikely cause of cytopenias observed during treatment.

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Erythropoiesis in Polycythemia Vera Is Hyper-Proliferative and Has Accelerated Differentiation during In Vitro Erythroid Expansion and Is Associated with Higher Expressions of cMYB and EPOR at Early Erythroid Progenitors

Blood ◽

10.1182/blood.v110.11.1549.1549 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1549-1549

Author(s):

Hana Bruchova ◽

Donghoon Yoon ◽

Archana Agarwal ◽

Eva Otahalova ◽

Hyojin Kim ◽

...

Keyword(s):

Polycythemia Vera ◽

Mononuclear Cells ◽

Early Stage ◽

Severe Disease ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Differentiation Stage

Abstract Erythroid differentiation is a dynamic process leading to the production of mature red blood cells. Even small variations in this process may result in severe disease phenotype. To study this process, we used a three-phase erythroid expansion system to expand homogeneous erythroid progenitors (EPs) from peripheral blood mononuclear cells (PB-MNCs) (Bruchova H. et al, 2007, Exp. Hematology, in press). We then characterized the expanded EPs from polycythemia vera (PV) patients and healthy donors at various points of maturation comparing cell proliferation and differentiation stage. EPs from PV patients outgrew controls up to day 14 (∼12 fold for PV and ∼4 fold for control compared to day 1). Differentiation was analyzed using both FACS analysis (with CD71/CD235a staining) and morphological evaluation (Wright-Giemsa staining), and demonstrated a more rapid differentiation of PV EPs when compared to controls up to day 14. We then evaluated apoptosis/cell cycle analysis by propidium iodide staining. Although PV EPs contained larger S phase population (45%) than controls (34%) at day 11, the apoptosis proportion of PV EPs was increased ∼2 fold to control from day 14. To understand the molecular mechanism of these differences between PV and controls, we analyzed the gene expression of several known regulators in erythropoiesis - BCL2, EPOR, cMYB, p27. Two transcripts (EPOR and cMYB) showed unique profiles on PV EPs. The EPOR transcript increased earlier in PV; i.e. from day 7 until day 21 and reached a plateau at day 11, compared to day 9 until day 19 and plateau at day 14 in controls. In addition, PV EPs contained higher levels of EPOR transcripts than control on most of timepoints. Interestingly, cMYB, which is known to augment early progenitor proliferation, was highly expressed from day 7 in PV, through day 11. Control EPs also expressed cMYB from day 9 through day 11; however, cMYB levels from any stages of control EPs were markedly lower than PV EPs at day 7. In this study, we demonstrate that PV erythropoiesis has unique features of hyperproliferation and an accelerated differentiation. These features are associated with earlier and higher expressions of cMYB and EPOR at the early stage of erythropoiesis.

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The Erythroid Phenotype of EKLF-Null Mice: Defects in Hemoglobin Metabolism and Membrane Stability

Molecular and Cellular Biology ◽

10.1128/mcb.25.12.5205-5214.2005 ◽

2005 ◽

Vol 25 (12) ◽

pp. 5205-5214 ◽

Cited By ~ 111

Author(s):

Roy Drissen ◽

Marieke von Lindern ◽

Andrea Kolbus ◽

Siska Driegen ◽

Peter Steinlein ◽

...

Keyword(s):

Blood Cells ◽

Target Genes ◽

Expression Profiles ◽

Critical Role ◽

Gene Expression Profiles ◽

Erythroid Differentiation ◽

Membrane Stability ◽

Erythroid Progenitors ◽

Hemoglobin Metabolism

ABSTRACT Development of red blood cells requires the correct regulation of cellular processes including changes in cell morphology, globin expression and heme synthesis. Transcription factors such as erythroid Krüppel-like factor EKLF (Klf1) play a critical role in erythropoiesis. Mice lacking EKLF die around embryonic day 14 because of defective definitive erythropoiesis, partly caused by a deficit in β-globin expression. To identify additional target genes, we analyzed the phenotype and gene expression profiles of wild-type and EKLF null primary erythroid progenitors that were differentiated synchronously in vitro. We show that EKLF is dispensable for expansion of erythroid progenitors, but required for the last steps of erythroid differentiation. We identify EKLF-dependent genes involved in hemoglobin metabolism and membrane stability. Strikingly, expression of these genes is also EKLF-dependent in primitive, yolk sac-derived, blood cells. Consistent with lack of upregulation of these genes we find previously undetected morphological abnormalities in EKLF-null primitive cells. Our data provide an explanation for the hitherto unexplained severity of the EKLF null phenotype in erythropoiesis.

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Myeloperoxidase expression in CD34+ normal human hematopoietic cells

Blood ◽

10.1182/blood.v82.7.2069.bloodjournal8272069 ◽

1993 ◽

Vol 82 (7) ◽

pp. 2069-2078 ◽

Cited By ~ 48

Author(s):

H Strobl ◽

M Takimoto ◽

O Majdic ◽

G Fritsch ◽

C Scheinecker ◽

...

Keyword(s):

Progenitor Cells ◽

Transferrin Receptor ◽

Staining Pattern ◽

Hematopoietic Progenitor ◽

Colony Forming Unit ◽

Cd34 Cells ◽

Normal Human ◽

Colony Forming Capacity ◽

Adult Peripheral Blood

Bone marrow (BM), adult peripheral blood (aPB), and umbilical cord blood (CB) samples contain small proportions of CD34+ cells that include virtually all hematopoietic progenitor cells. Myeloperoxidase (MPO) is considered to be selectively expressed in cells committed to granulomonocytic differentiation. Using flow cytometry and an antibody against MPO, we studied at which stage of normal hematopoietic differentiation CD34+ cells being to express MPO. We consistently observed a characteristic MPO/CD34 staining pattern and found that 35% +/- 9% of CD34+ BM cells express MPO. The MPO+ CD34+ subset and the CD33+ CD34+ subset were of similar size and overlapped considerably. MPO+ CD34+ cells expressed high levels of HLA-D molecules, were weakly CD71/transferrin receptor positive to negative, were CD45RA+ and lacked the CD45RO isoform of the leukocyte common antigen. Additionally, MPO+ CD34+ cells were on average larger in size than MPO- CD34+ cells. Virtually identical phenotypic features have previously been described for in vitro colony-forming granulomonocytic progenitor cells. In vitro clonogenic assays performed with MPO-enriched and MPO-depleted fractions of CD34+ BM cells performed by us also suggest, but do not formally prove, that at least a portion of MPO+ CD34+ cells have in vitro cluster (10 to 50 cells/colony) or colony-forming unit granulocyte-macrophage (> or = 50 cells/colony) forming capacity. CD34+ cells from CB and aPB resembled CD34+ BM cells in that considerable proportions of them coexpressed CD33. However, in contrast to BM, CD34+ cells from CB and aPB samples lacked significant MPO expression and, in line with this, the majority of them (CB, 59% +/- 7%; aPB, 66% +/- 5%) coexpressed CD45RO.

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Effect of modulators of erythropoiesis on the hemoglobinization of human erythroid cell cultures

Blood ◽

10.1182/blood.v60.2.346.bloodjournal602346 ◽

1982 ◽

Vol 60 (2) ◽

pp. 346-351

Author(s):

BJ Clarke ◽

AM Brickenden ◽

RA Ives ◽

DH Chui

Keyword(s):

Human Peripheral Blood ◽

Spectrophotometric Assay ◽

Conditioned Media ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

Hemoglobin Content ◽

Plasma Clot ◽

Total Hemoglobin ◽

Valuable Adjunct

A rapid spectrophotometric assay capable of detecting the hemoglobin content of 1000 mature erythrocytes has been utilized to quantitate the total hemoglobin synthesized by the progeny of circulating human erythroid progenitors in both the plasma clot and methylcellulose culture systems. The pronounced variation in the effect of different erythropoietin preparations on the hemoglobin content of cultured human peripheral blood bursts, previously described in a subjective manner, has been objectively quantitated. Further experiments demonstrated that both lymphocyte conditioned media and dexamethasone substantially increased the total hemoglobin synthesized by the progeny of cultured erythroid progenitors. The elevated amount of hemoglobin present in erythroid cultures containing either lymphocyte conditioned media and/or dexamethasone was due to both increased colony numbers and colony size. Assay of the total hemoglobin content per erythroid culture is an accurate, sensitive, measure of erythropoiesis in vitro and should be a valuable adjunct to the enumeration of BFU-E-derived erythroid colonies.

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Influence of the endothelial cells on the erythroid differentiation of umbilical cord blood hematopoietic stem cells in vitro

Proceedings of the National Academy of Sciences of Belarus Medical series ◽

10.29235/1814-6023-2020-17-1-78-86 ◽

2020 ◽

Vol 17 (1) ◽

pp. 78-86

Author(s):

A. S. Voytehovich ◽

E. V. Vasina ◽

V. S. Kastsiunina ◽

I. N. Seviaryn ◽

N. V. Petyovka

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Erythroid Differentiation ◽

Erythroid Progenitors ◽

Hematopoietic Stem ◽

Vascular Niche

The objective is to study the effect of umbilical cord blood endothelial cells on the hematopoietic cells growth and the maturation in the erythroid direction in co-culture, as well as the expression of adult and fetal hemoglobin genes during erythroid differentiation under the conditions of vascular niche modeling in vitro. We used the following research methods: cultural, flow cytometry, real-time PCR and morphological analysis. We have developed the method of hematopoietic cord blood stem cells erythroid differentiation in co-culture using cord blood endothelial cell progenitors. CD34+CD31+CD144+CD105+CD90–CD45– progenitors of endothelial cells stimulate the erythroid differentiation of hematopoietic CD34+ cord blood cells and the growth of erythroid progenitors in co-culture from the 4th to 11th day in the presence of the stem cell factor, the erythropoietin and the fibroblast growth factor-2. The in vitro modeling of the vascular niche increases the mature CD36–CD235a+ erythroid cells 2.5 times higher than those in the liquid culture. The microenvironment of endothelial cells does not affect the level and expression ratio of fetal and adult hemoglobin during the erythroid differentiation in vitro.

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