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PubMed1980

Erythroid precursor cells in primary acquired and secondary sideroblastic anemia.

In order to study the changes in erythroid precursor cells in primary acquired and secondary sideroblastic anemia, bone marrow cells from 4 patients with primary acquired sideroblastic anemia (PASA) and 3 patients with refractory anemia with excess of myeloblasts (RAEM) or erythroleukemia associated with an excess of ringed sideroblasts were cultured for erythroid colony-forming units (CFU-E). The number of CFU-E was markedly decreased in all 7 cases, and erythroid colonies formed consisted exclusively of normal-appearing erythroblasts, while ringed sideroblasts were observed in scattered single erythroblasts or in small aggregates of erythroblasts in primary as well as in secondary sideroblastic anemia. These findings may indicate the presence of 2 populations of erythroid progenitor cells in the bone marrow of patients with primary acquired and secondary sideroblastic anemia. A slight to moderate decrease in granulocyte-macrophage colony-forming units (GM-CFU) was observed in 3 cases of PASA. The decrease in GM-CFU, however, was marked in sideroblastic anemia associated with RAEM or erythroleukemia.

Aged↗
PubMed1984

Cell kinetic behaviour of a synchronized population of erythroid precursor cells in vitro.

Erythropoiesis in vitro was studied with practically pure erythroid progenitor cells: CFU-E (colony-forming-units-erythroid). The isolation of CFU-e from spleens of thiamphenicol pretreated anaemic mice with the combined methods of centrifugal elutriation and Percoll density gradient centrifugation was monitored by flow cytometry. The ultimate CFU-e preparation with a density of 1.070 g/ml contained a high percentage of cells in the S phase of the cell cycle (80%). CFU-e occasionally found at a lower density of 1.065 g/ml were predominantly in the G2 + M and G1 phases. When CFU-e were cultured, the number of cells in the distinctive phases of the cell cycle changed periodically, so the cells were partly synchronized. Four periods up to 27 hr were observed by flow cytometrical screening of the cultured cells at hourly intervals. Cell-cycle times between 6 and 7 hr were found for all erythroid cell divisions. This was in agreement with results obtained from colony growth curves. Without the addition of erythropoietin cells start to degenerate after the second cell division. This experimental approach can be used for the cell kinetic modelling of erythropoiesis.

Animals↗
PubMed1996

CD4 Expression by erythroid precursor cells in human bone marrow.

Flow cytometry was used to assess CD4 expression in 62 consecutive bone marrow specimens from patients with a variety of clinical conditions. Using a lysed-whole-blood technique for labeling with monoclonal antibodies, two populations of CD4+ cells were identified within the lymphocyte/blast-cell fraction in 58 (94%) of these specimens. These consisted of (1) a population of T helper cells with high density expression of CD4 and (2) a second population of cells with low-density expression of CD4, which ranged from 1% to 36% of the gated cells. This latter population was present regardless of age, sex, or clinical condition including 21 of 21 specimens (100%) categorized as unremarkable bone marrows both morphologically and by flow cytometry and in four of four patients (100%) with human immunodeficiency virus-type 1 (HIV-1) infection. Coexpression of the erythroid lineage marker, glycophorin A, with the majority of cells in this second population was demonstrated in all 11 randomly selected samples using two-color flow cytometric analysis. These cells also expressed low levels of the myeloid markers, CD13 and CD33, but CD34 expression could not be demonstrated. These results provide evidence for expression of CD4 on cells of erythroid lineage in human marrow, and offer a potential mechanism for direct infection of erythroid precursor cells and deranged erythropoiesis in patients with HIV-1 infection.

Adult↗
PubMed1990

Pteridine modulation of lead inhibition of uroporphyrinogen synthesis in erythroid precursor cells.

The role of nutritional factors on heme synthesis and their influence on the development of anemia in the bone marrow during lead exposure is unclear. Previous reports suggested that pteridines could regulate the formation of uroporphyrinogen, a step midway along the heme synthetic pathway. Studies were undertaken to determine if pteridines could modulate lead inhibition of uroporphyrinogen synthesis in erythroid precursor cells. Pteroylpolyglutamates of various glutamate chain lengths were tested for the ability to protect against lead inhibition of uroporphyrinogen I (URO) synthase prepared from murine erythroleukemia cells (MELC). Pteroylpentaglutamate, the major endogenous polyglutamate form by chain length found to be present in MELC, afforded rapid and specific protection of URO synthase against lead inhibition. MELC are expected to be a useful in vitro model for studying the role of endogenous folates on uroporphyrinogen synthesis and heme formation in erythroid precursor cells following lead exposure.

Animals↗
PubMed2005

Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells.

Definitive erythropoiesis usually occurs in the bone marrow or fetal liver, where erythroblasts are associated with a central macrophage in anatomical units called 'blood islands'. Late in erythropoiesis, nuclei are expelled from the erythroid precursor cells and engulfed by the macrophages in the blood island. Here we show that the nuclei are engulfed by macrophages only after they are disconnected from reticulocytes, and that phosphatidylserine, which is often used as an 'eat me' signal for apoptotic cells, is also used for the engulfment of nuclei expelled from erythroblasts. We investigated the mechanism behind the enucleation and engulfment processes by isolating late-stage erythroblasts from the spleens of phlebotomized mice. When these erythroblasts were cultured, the nuclei protruded spontaneously from the erythroblasts. A weak physical force could disconnect the nuclei from the reticulocytes. The released nuclei contained an undetectable level of ATP, and quickly exposed phosphatidylserine on their surface. Fetal liver macrophages efficiently engulfed the nuclei; masking the phosphatidylserine on the nuclei with the dominant-negative form of milk-fat-globule EGF8 (MFG-E8) prevented this engulfment.

Adenosine Triphosphate↗
PubMed1994

Binding and uptake of exogenous isoferritins by cultured human erythroid precursor cells.

The interaction of extracellular human isoferritins with normal erythroid precursors developing in a two-phase liquid culture was studied. Cells at the stage of polychromatic normoblasts exhibited substantial specific binding of radioiodinated placental isoferritins. Considerably more acidic isoferritin was bound than basic isoferritin. The binding of ferritin was significantly higher at 37 degrees C than at 4 degrees C. All of the 125I-acidic isoferritin bound at 4 degrees C, but only part of that bound at 37 degrees C, could be dislodged by the addition of 500-fold excess of non-labelled acidic isoferritin. Acidic isoferritin displaced radio-iodinated acidic isoferritin from the erythroid cells more efficiently than intermediate or basic isoferritins. Kinetic analysis suggests a dissociation constant (Kd) of 3.9 x 10(-8) M for acidic ferritin and 3.7 x 10(-7) M for basic isoferritin. The average number of binding sites for acidic isoferritin was 1.3 x 10(5) per cell. The results point to specific binding and receptor-mediated internalization for predominantly acidic isoferritin by developing human erythroid cells.

Cells, Cultured↗
PubMed1987

Binding and internalization of recombinant human erythropoietin in murine erythroid precursor cells.

Erythropoietin (EPO) biosynthetically labelled with [35S]cysteine was produced from Chinese hamster ovary (CHO) cells containing amplified copies of human EPO cDNA. The glycosylated recombinant [35S]EPO, purified to virtual radiochemical homogeneity, was biologically active. We studied the interaction of this labeled recombinant EPO with erythroid precursor cells from mice made anemic with phenylhydrazine. The [35S]-labeled molecule bound to erythroid precursors in a time- and temperature-dependent manner. The binding was specific for EPO, and neither insulin, transferrin, epidermal growth factor, nor multiplication stimulating activity could compete for EPO binding sites. In the presence of 0.2% sodium azide, which blocks 80% to 90% of internalization, the recombinant molecule bound with an apparent Kd of 750 pmol/L and 100 to 200 binding sites per cell at 37 degrees C. Asialo-EPO was a more effective competitor than sialated EPO for the available binding sites. Thus, the enhanced biological specific activity of asialo-EPO could result from its enhanced binding affinity. We also studied recombinant human EPO labeled with 125I and found that it also bound to the erythroid cells in a saturable and specific manner. After 90 minutes of incubation at 37 degrees C, most of the bound [35S]EPO was internalized, whereas most of the [125I]EPO remained on the cell surface. The reduced internalization of the iodinated molecule could account for the previously reported functional deficit associated with iodination.

Animals↗
PubMed2001

Stem cell factor regulation of Fas-mediated apoptosis of human erythroid precursor cells.

Multiple cytokines regulate the development of erythrocytes. Increasing attention has been directed to the possible role of Fas and its cognate ligand (Fas-L), a subject of wide interest. Documentation of in vitro data supports the role of Fas and Fas-L in erythropoiesis. Several laboratories, including ours, investigated the opposing actions of erythropoietin (EPO) and stem cell factor (SCF) on Fas-mediated cell death of the erythroid cells. Only circumstantial in vivo evidence has accumulated concerning the issue. There are several reports suggesting that Fas-mediated cell death may have a role in some pathological conditions. Results of the accumulating findings and possible implications in clinical hematology are summarized in this review.

Apoptosis↗
PubMed1977

Characterization of an erythroid precursor cell of high proliferative capacity in normal human peripheral blood.

We have found that the peripheral blood of normal humans contains a significant number of committed erythroid stem cells of high proliferative capacity. These erythroid stem cells closely resemble the murine erythroid burst-forming unit (BFU-E) with respect to proliferative capacity, colony morphology, and erythropoietin requirement. BFU-E were isolated from the peripheral blood of normal individuals by Ficoll-Hypaque density gradient centrifugation and cultured in vitro using the plasma culture technique. Macroscopic erythroid colonies of between 100 and 1000 cells were observed after 10-14 days of culture in the presence of either sheep or human erythropoietin at 0.5-4 units/ml. Individual colonies contained between 3 and 20 subcolonies and reached a maximum mean size of approximately 500 cells. Colony number was linearly related to the cell input, suggesting that a single cellular entity was precursor to each colony. The frequency of cells capable of giving rise to an erythroid colony was at least 100 per ml of blood in a number of individuals tested. The ability to assay significant numbers of erythroid precursor cells of high proliferative capacity from normal peripheral blood should facilitate the study of both normal erythropoiesis and of disease states affecting erythropoiesis in which marrow samples are not available on a routine basis.

Blood Cells↗
PubMed2003

Mithramycin induces fetal hemoglobin production in normal and thalassemic human erythroid precursor cells.

We report in this paper that the DNA-binding drug mithramycin is a potent inducer of gamma-globin mRNA accumulation and fetal hemoglobin (HbF) production in erythroid cells from healthy human subjects and beta-thalassemia patients. Erythroid precursors derived from peripheral blood were grown in 2-phase liquid culture. In this procedure, early erythroid progenitors proliferate and differentiate during phase 1 (in the absence of erythropoietin) into late progenitors. In phase 2, in the presence of erythropoietin, the latter cells continue their proliferation and mature into Hb-containing orthochromatic normoblasts. Compounds were added on days 4 to 5 of phase 2 (when cells started to synthesize Hb), and cells were harvested on day 12. Accumulation of mRNAs for gamma-globin, beta-globin, alpha-globin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-actin were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR); induction of HbF was analyzed by high-performance liquid chromatography (HPLC) and, at cellular level, by flow cytometry. We demonstrated that mithramycin was able to up-regulate preferentially gamma-globin mRNA production and to increase HbF accumulation, the percentage of HbF-containing cells, and their HbF content. Mithramycin was more effective than hydroxyurea, being, in addition, not cytotoxic. This was shown by the lack of cytotoxicity on erythroid and myeloid in vitro primary cell cultures treated with mithramycin at concentrations effective for HbF induction. These results are of potential clinical significance because an increase of HbF alleviates the symptoms underlying beta-thalassemia and sickle cell anemia. The results of this report suggest that mithramycin and its analogs warrant further evaluation as potential therapeutic drugs.

Actins↗
PubMed1990

Transformation of early erythroid precursor cells (BFU-E) by a recombinant murine retrovirus containing v-erb-B.

Avian erythroblastosis virus (AEV) is a replication-defective retrovirus that transforms erythroid and fibroblast cells in vitro and in vivo. The transforming ability of AEV is due primarily to the oncogene v-erb-B. A recombinant murine retrovirus has been constructed by inserting a chimeric gag-v-erb-B gene into a Moloney murine leukemia virus based vector. This retrovirus was used to examine v-erb-B-induced transformation of murine hematopoietic cells. Infection of murine primary fetal liver, adult bone marrow or adult spleen cells with the recombinant virus generated large hemoglobinized erythroid colonies in the absence of exogenous growth factors. Generation of such colonies usually requires the presence of erythropoietin (Epo) and interleukin-3 (IL-3). These growth-factor independent colonies were shown to be derived from early (BFU-E) and not late (CFU-E) erythroid progenitor cells, and the effect was not attributable to growth factors elicited by the virus-producing cell lines. In order to confirm that the recombinant virus was responsible for this transformation of BFU-E to growth factor independence, bone marrow cells from post 5-fluorouracil treated mice were infected and used to repopulate lethally-irradiated mice. Growth factor-independent BFU-E were obtained in up to 30% of day-13 spleen colonies and it was shown by DNA analysis that cells from these colonies contained integrated provirus. Our results indicate that v-erb-B transforms early erythroid progenitors to growth factor independent growth and subsequent differentiation to erythrocytes -a process that normally requires Epo plus either IL-3 or granulocyte-macrophage colony stimulating factor (GM-CSF).

Alpharetrovirus↗
PubMed1990

Evolution and development of membrane components in erythroid precursor cells in newly-developed two phase culture system.

A selective two phase liquid culture system for human erythroid progenitors was established, utilizing mononuclear cells obtained from human peripheral blood. The culture was performed in the presence of PHA-leukocyte conditioned medium for 7 days as the first phase, and nonphagocytic cells were recultured under hypoxic culture conditions containing iron-saturated transferrin and erythropoietin. During the second phase of the culture, differentiation to orthochromatic erythroblasts was observed on day 4, and to denucleated cells on day 8 with 16.8 +/- 0.7 micrograms/10(6) cells of hemoglobin level, respectively. Flow cytometric analyses revealed that, on day 3, cells became type M-positive, and the expression of ABH increased gradually corresponding to the extent of erythroid maturation. Lewis antigens and P antigens were expressed at day 5. The system is beneficial for studies on human membrane disorders, because the erythroid progenitors in human peripheral blood were utilized and the reasonable number of erythroid cells (0.5-1.5 x 10(7] was obtained with good erythroid maturation, such as denucleated erythrocytes.

Antigens↗
PubMed1995

Mouse model for the lysosomal disorder galactosialidosis and correction of the phenotype with overexpressing erythroid precursor cells.

The lysosomal storage disorder galactosialidosis results from a primary deficiency of the protective protein/cathepsin A (PPCA), which in turn affects the activities of beta-galactosidase and neuraminidase. Mice homozygous for a null mutation at the PPCA locus present with signs of the disease shortly after birth and develop a phenotype closely resembling human patients with galactosialidosis. Most of their tissues show characteristic vacuolation of specific cells, attributable to lysosomal storage. Excessive excretion of sialyloligosaccharides in urine is diagnostic of the disease. Affected mice progressively deteriorate as a consequence of severe organ dysfunction, especially of the kidney. The deficient phenotype can be corrected by transplanting null mutants with bone marrow from a transgenic line overexpressing human PPCA in erythroid precursor cells. The transgenic bone marrow gives a more efficient and complete correction of the visceral organs than normal bone marrow. Our data demonstrate the usefulness of this animal model, very similar to the human disease, for experimenting therapeutic strategies aimed to deliver the functional protein or gene to affected organs. Furthermore, they suggest the feasibility of gene therapy for galactosialidosis and other disorders, using bone marrow cells engineered to overexpress and secrete the correcting lysosomal protein.

Abnormalities, Multiple↗
PubMed1987

Identification and characterization of an antigen specific for normal erythroid precursor cells and its application in diagnosis of erythroleukemia.

A monoclonal antibody (designated K:1-6F) generated by hybridization of mouse myeloma cells with spleen cells from mice immunized with the erythroleukemic cell line K562 was found by fluorescence-activated cell sorter analysis, dot-blot assay and electroimmunoblotting to bind to a majority of cells in the K562 and HEL erythroleukemic cell lines, to a subset of cells of the erythroid lineage from normal bone marrow, to a subset of cells in all analysed cases (total 10) of erythroleukemia, and weakly to cells from patients with myeloid leukemia. The antibody did not bind to normal erythrocytes, monocytes, T- and B lymphocytes or granulocytes, as well as a panel of human malignant cell lines of hemopoietic origin (HL60, U937, Daudi, Molt-3, RH-L4 and U266). Biochemical characterization of the antigen defined by the antibody suggests that eht epitope is defined by a carbohydrate structure alone or in combination with proteins. Four molecules with Mr 100 kD, 65 kD, 45 kD and 18 kD respectively were immunoprecipitated from Triton X-100 extract of K562 erythroleukemia cells. Neuraminidase did not affect the binding of the antibody, whereas tunicamycin reduced the K:1-6F expression. The K:1-6F Mab was in normal bone marrow found to be specific for erythroid precursor cells and may therefore be useful in examination of normal and leukemic erythropoiesis.

Animals↗
PubMed1976

Unbalanced globin chain synthesis in erythroid precursor cells of heterozygous alpha-thalassaemia.

Globin biosynthesis was studied in both erythroid precursors and reticulocytes of three individuals with heterozygous alpha-thalassaemia. In contrast to the finding of equal or nearly equal alpha and beta chain synthesis in the marrow of patients with heterozygous beta-thalassaemia previously examined, our studies showed equal degrees of unbalanced globin synthesis in both reticulocytes and nucleated-erythroid cells of alpha-thalassaemia heterozygotes. Greater stability and less susceptibility to proteolysis of the excess beta-chain formed in alpha-thalassaemia may explain our findings.

Anemia, Sickle Cell↗
PubMedDate not supplied

[Erythroid precursor cells in a tissue culture from subjects exposed to ionizing radiation].

Cloning efficiency of erythroid progenitor cells in gel diffusion chambers from patients irradiated as a result of the Chernobyl accident was studied. Stimulation of erythroid progenitor cells, activation of enzymatic activity in peripheral blood cells and an increase of 2,3-D Ph G were detected. These data supplement the notion about the development of adaptative processes in the erythron system as a response to effects of radiation.

2,3-Diphosphoglycerate↗
PubMed2004

A sustained and pancellular reversal of gamma-globin gene silencing in adult human erythroid precursor cells.

We systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factor beta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 +/- 1.0%; EPO + TGF-B = 10.8 +/- 1.9%; EPO + SCF = 19.1 +/- 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 +/- 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 +/- 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 +/- 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation.

Adult↗
PubMed1986

Mode of action of erythropoietin and glucocorticoids on the hepatic erythroid precursor cells: role of prostaglandins.

The hypothesis that prostaglandins, and especially PGE2, are the second messengers of erythropoietin (Ep) and that glucocorticoids inhibit Ep action by inhibiting PG synthesis was tested on the erythroid cell line from fetal rat liver. The optimal (10(-9) M) stimulatory concentration of PGE2 did not reproduce, by far, the maximal effect of Ep on the growth of CFUE erythroid colonies. Ep did not increase PGE2 release in liquid culture media of cell suspensions made of the whole erythroid line or enriched (over 85%) in precursor cells. Ep did not modify the turnover rate of arachidonate. Nevertheless, indomethacin partially inhibited Ep effect on CFUE development, and this inhibition was abolished by PGE2. These results suggest that PGE2 potentiates Ep action but is not its second messenger. Spontaneous PGE2 release in liquid culture media brought about concentrations of the order of 10(-9) M, and 10(-7) M dexamethasone completely inhibited this release. Part of (but not all) the anti-Ep effects of glucocorticoids might thus be mediated this way. Dexamethasone effects required previous protein synthesis.

Animals↗