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Results for “Erythroid Precursor Cells”

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At least 19 recordsLinked to original sources
PubMed1974

The effect of erythropoietin on colonial growth of erythroid precursor cells in vitro.

A method is described for the colonial growth, in semi-solid medium, of erythropoietin-responsive erythroid cell precursors. The erythroid cell precursors were isolated by immune hemolysis from fetal mouse liver. Both the number of precursor cells triggered to proliferate and differentiate, and the size of the erythropoietic colonies formed, are directly dependent upon the concentration of erythropoietin included in the culture.

Animals↗
PubMed1989

Cyclic changes in fluorescence polarization of a membrane probe during the cell cycle of an erythroid precursor cell line.

The membrane lipid bilayer of the K562 cell line undergoes marked changes during cell cycling. These changes can be detected by measuring the fluorescence polarization of the rod-like hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene. Single-cell measurements were performed by flow microfluorometry on synchronized K562 cells. The fluorescence polarization of the probe increased after cell division and was maximal during the S phase. Concomitant with the transition from S to G2 and M phase was a decrease in fluorescence polarization. The data are interpreted as reflecting a minimal membrane lipid fluidity during the S phase and a maximal fluidity during the G2/M-phase of the above erythroid precursor cell line.

Cell Cycle↗
PubMed1972

Globin messenger RNA activity in erythroid precursor cells and the effect of erythropoietin.

Cell populations enriched for erythroid precursor cells were fractionated from 13-day fetal-mouse livers by a method of immune hemolysis. These preparations of precursors, contaminated by less than 7% hemoglobinized erythroblasts, synthesize globin at a rate less than 6% that of the unfractionated liver erythroid cell population. RNA was isolated from these precursor cells and assayed for globin mRNA activity in a cell-free system from Krebs ascites tumor. The 6-16S RNA fraction from precursor cells has less than 5% of the globin mRNA activity of RNA isolated from unfractionated populations. Precursor cells incubated with erythropoietin show an increment in the rate of synthesis of globin only after 5-10 hr of incubation. After 10 hr of culture with this hormone, precursor cells show a 6- to 10-fold increase in globin mRNA activity. These results suggest that the precursor cells of hepatic erythropoiesis, responsive to erythropoietin, do not contain globin mRNA in a biologically active form. Erythropoietin-induced differentiation of these cells to erythroblasts is associated with an increase in globin mRNA.

Animals↗
PubMed1989

Folate (pteroylglutamate) uptake in human red blood cells, erythroid precursors and KB cells at high extracellular folate concentrations. Evidence against a role for specific folate-binding and transport proteins.

Membrane-associated folate (pteroylglutamate, PteGlu)-binding proteins (FBPs) play an important role as PteGlu-transport proteins in malignant and normal human cells. Since high extracellular folate (PteGlu) concentrations (EFC) profoundly influenced uptake and toxicity of the anti-PteGlu methotrexate in malignant KB cells, we studied human cells to determine additional mechanisms for PteGlu uptake when the EFC was varied. At low EFC (less than 10 nM), the predominant mechanism for folate uptake in mature erythrocytes was through binding to externally oriented FBPs which were quantitatively insignificant (4-6 orders of magnitude lower) and of no apparent physiological relevance when compared with KB cells. However, the predominant mechanism of PteGlu accumulation at high EFC [10-250 nM] in intact erythrocytes and sealed right-side-out (RSO) ghosts was not FBP-mediated and non-specific. This conclusion was based on the findings that radiolabelled PteGlu uptake: (i) continued even in the presence of a 1000-fold excess of unlabelled PteGlu and was linear and not saturable up to 250 nM; (ii) was two-fold higher at pH 4.5 than 7.5; (iii) was less than 2-fold increased at 37 degrees C compared with 4 degrees C; and (iv) was unaffected after trypsin-mediated proteolysis of greater than 75% FBPs. The [3H]PteGlu and 125I-PteGlu (histamine derivative) accumulated intracellularly through the non-specific PteGlu-uptake mechanism was unaltered biochemically and in a soluble compartment. Raising the EFC 500-fold higher than controls during erythropoiesis in vitro resulted in reversal of the expected anti-(placental folate-receptor)-antiserum-induced megaloblastic changes in orthochromatic normoblasts derived from burst-forming unit-erythroid colonies. Furthermore, at EFC greater than 0.1 microM, KB-cell accumulation of [3H]PteGlu was also predominantly through a mechanism that did not involve specific FBPs. Thus, at high EFC, a major component of PteGlu transport in human cells is not mediated through FBPs and is likely to be a passive diffusion process.

Carrier Proteins↗
PubMed1976

Isolation and in vitro differentiation of human erythroid precursor cells.

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

Anemia, Sickle Cell↗
PubMed1983

Isolation and induction of erythroleukemic cell lines with properties of erythroid progenitor burst-forming cell (BFU-E) and erythroid precursor cell (CFU-E).

We isolated erythroleukemic cell lines arrested at different levels of the erythroid differentiation pathway. One cell line (CB5), established from mice infected with the helper-independent Friend murine leukemia virus (F-MuLV), exhibited properties similar to those of the normal erythroid progenitor burst-forming cell (BFU-E). Six erythroleukemic cell lines, which were established from the anemia-inducing Friend virus complex (FV-A)-infected mice, formed erythroid colonies similar to the erythroid colony-forming precursor cell (CFU-E) after induction with dimethyl sulfoxide or erythropoietin. Three lines that were established from the polycythemia-inducing Friend virus complex (FV-P)-infected mice also formed low proportions (2-5%) of CFU-E-like colonies after induction by these same inducers. These data, together with the earlier findings that F-MuLV induces an increase in the levels of BFU-E and that FV-A or FV-P stimulates enhancement of CFU-E early after infection, indicate that the erythroleukemic cell lines isolated late in the diseases are at the same levels of differentiation as the leukemic cells in the corresponding initial stages. These cell lines with properties of BFU-E and CFU-E can be induced to differentiate in culture and should add to our understanding of the nature of erythroid progenitor cells and their early differentiation programs.

Animals↗
PubMed1973

Early stimulation of RNA synthesis by erythropoietin in cultures of erythroid precursor cells.

The effect of erythropoietin on cultured erythroid precursor cells from 13-day mouse-fetal livers was examined. Within 1 hr, erythropoietin causes a 2- to 3-fold stimulation of uridine incorporation into RNA by these cells. The types of RNA preferentially stimulated by erythropoietin during the first hour of exposure of the cells to the hormone include ribosomal RNAs and their precursors, as well as 4-5S RNA. No unique RNA species, not present in control cells, could be detected by sucrose gradient sedimentation or gel electrophoresis. Inhibition of protein synthesis for up to 1 hr does not abolish the stimulatory effect of erythropoietin on RNA synthesis, suggesting that the effect of the hormone on RNA synthesis is not mediated by a newly synthesized protein.

Animals↗
PubMed1983

Inhibition of the growth and differentiation of erythroid precursor cells by an endotoxin-induced mediator from peritoneal macrophages.

Conditioned medium from cultures of mouse macrophages incubated with endotoxin in a serum-free medium contains an inhibitor of the growth and differentiation of erythroid precursor cells of mouse Friend virus-transformed erythroleukemia cells. Endotoxin itself has no inhibitory effect. The endotoxin-induced macrophage mediator inhibits the growth and differentiation of dimethyl sulfoxide-, hexamethylenebisacetamide-, butyric acid-, and hypoxanthine-induced cells but has no effect on hemin-induced cells. The conditioned medium has its maximal inhibitory effect on committed erythroid precursor cells, a decreased effect on uncommitted stem cells, and no effect on fully differentiated erythroid cells. These results demonstrate that endotoxin stimulation of macrophages leads to the production of a humoral factor(s) which is critical for the growth and differentiation of erythroid precursor cells.

Animals↗
PubMed1980

Expression of red cell membrane proteins in erythroid precursor cells.

Specific antibodies to human glycophorin A and spectrin were used to study the expression of these membrane proteins in normal and pathologic human bone marrow. In immunofluorescence experiments spectrin and glycophorin A are found in 50-60% of the nucleated cells in normal bone marrow. These two proteins are expressed at all stages of red cell differentiation and can be traced at least to the earliest morphologically recognizable nucleated red cell precursor, the proerythroblast; the two proteins are specific for cells of the red cell series and are not found to be expressed in lymphocytic, granulocytic cells or platelets. These conclusions were drawn from studies on bone marrow in patients with a temporary block in erythropoiesis at the level of stem cells or of the pronormoblast. Bone marrow from these individuals either lacked all nucleated cells stainable for glycophorin A and spectrin or contained only pronormoblasts. Similar findings were obtained on spleen cells from mice which were made severely anemic by multiple injections with N-acetyl-phenylhydrazine. Antibodies to a sialoglycoprotein isolated from mouse red cell membranes stain 70-80% of all cells in the spleen of anemic animals, while only 1-2% of such cells are seen in the spleen of normal animals. Spectrin and glycophorin A could be labeled metabolically and isolated using specific antibodies. The human tumor cell line K562 expresses both membrane proteins, but induction experiments with various agents thus far have failed to change their expression.

Adult↗
PubMed1980

Fluorescence microphotometric studies of the transferrin receptor in human erythroid precursor cells.

The receptor site for transferrin in normal human erythroid precursor cells was studied by fluorescence microscopy. F-transferrin saturated with iron was used as probe of the available receptor sites on reticulocytes and nucleated red cells. In a series of experiments specificity and certain structural details of the ligand site were evaluated. Hydrolytic cleavage of exposed carbohydrate moieties by purified glycosidases revealed increased fluorescence after treatment of fixed cells by neuramindase, no perceptible change after N-acetylhexosaminidase treatment, but a pronounced decrease after exposure to beta-galactosidase. Inhibitor studies with monosaccharides and tryptic glycopeptides of normal reticulocytes complemented and amplified the results obtained with enzymes. The data suggest that an oligosaccharide chain is essential for specific transferrin binding to erythroid precursors. N-acetyl-neuraminic acid, galactose, N-acetylgalactosamine, and fucose appear to be saccharides on the receptor. These studies also demonstrate the applicability of fluorescence microscopic methods to qualitative structural analysis of receptor biochemistry.

Binding Sites↗
PubMed1981

Harvey and Kirsten sarcoma viruses promote the growth and differentiation of erythroid precursor cells in vitro.

Harvey and Kirsten murine sarcoma viruses have previously been shown to transform fibroblastic cells in culture, and type C virus pseudotypes of these viruses cause erythroleukemia in susceptible mice. We report a cell culture assay for quantitating the growth-promoting effect of Harvey and Kirsten viruses on erythroid cells. Murine hemopoietic cells were infected in vitro with Harvey or Kirsten sarcoma virus, and then cultured in methylcellulose in the presence of relatively low concentrations of erythropoietin. Under these conditions, large colonies of erythroid cells form in the semi-solid culture media 6 to 8 days after infection. The induction of erythroid bursts was not caused by the murine type C helper viruses used to pseudotype either Ha-MuSV or Ki-MuSV, or by media from cells carrying the Ki-MuSV and Ha-MuSV genomes. Induction of the erythroid colonies is under genetic control at the Fv1 susceptibility locus, but not at the Fv2 susceptibility locus. A striking feature of the erythroid colonies induced by the Harvey and Kirsten viruses was that they not only proliferated to large size but also differentiated along the erythroid lineage and synthesized hemoglobin. The results indicate that Ha-MuSV and Ki-MuSV can induce proliferation of erythroid precursor cells apparently without interfering with the differentiation program of the cells. The relation between the growth-promotion effect of these viruses on erythroid precursor cells and their ability to induce erythroleukemia is discussed.

Animals↗
PubMed1992

Protein deficiency impairs erythropoiesis in rats by reducing serum erythropoietin concentration and the population size of erythroid precursor cells.

Erythropoietin (EPO), a glycoprotein produced mainly by the kidney, is the major physiological regulator of erythropoiesis. We developed a sensitive and rapid ELISA for measurement of rat serum EPO with two monoclonal antibodies that recognize different epitopes. To understand the mechanism by which erythropoiesis is impaired in rats deficient in dietary protein, we investigated the levels of the immunoreactive EPO (iEPO) in serums and erythroid precursor cells in hemopoietic tissues during protein deprivation. The iEPO level of 32-d-old rats fed a protein-free diet was lowered to one-third that of rats fed 20% casein at 6 h after protein deprivation began. Protein deprivation decreased the number of EPO-responsive cells in spleen. These results indicate that the impairment of erythropoiesis during protein deficiency is caused by the decrease in serum EPO and the subsequent reduction of the population size of erythroid precursor cells in spleen.

Animals↗
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↗
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↗