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PubMed2001

Erythroblasts are a source of angiogenic factors.

In adult bone marrow, mature erythroblasts are produced within structures called erythroblastic islands and then cross the endothelial barrier to reach circulation. Erythroblastic islands are composed of a central macrophage surrounded by maturing erythroblasts. In this study, it is shown that erythroid cells, but not the other mature hematopoietic cells, coexpress 2 angiogenic factors, vascular endothelial growth factor A (VEGF-A) and placenta growth factor (PlGF). Secretion of both VEGF-A and PlGF increases during in vitro erythroid differentiation. Erythroblast-conditioned medium can induce both migration of monocytes and endothelial cells and the permeability of endothelial cells. These effects are inhibited by anti-PlGF and/or anti-VEGF antibodies. Finally, it is shown that VEGF-A and PlGF proteins are expressed by bone marrow erythroblasts in vivo. Angiogenic factors secreted by erythroblasts may promote interactions either with macrophages in erythroblastic islands or with endothelial cells that would facilitate the passage of erythroid cells through the endothelial barrier. (Blood. 2001;97:1968-1974)

Animals↗

PubMed1984

Beta globin messenger RNA content of bone marrow erythroblasts in heterozygous beta-thalassemia.

RNA from bone marrow erythroblasts and peripheral blood reticulocytes of patients with heterozygous beta-thalassemia was analyzed for relative content of alpha and beta globin messenger RNA by molecular hybrization. Erythroblasts from nonthalassemic patients exhibited approximately the same alpha and beta globin mRNA content (beta/alpha mRNA ratio = 0.8-1.0) as circulating reticulocytes (beta/alpha mRNA ratio = 0.74-1.2). The mRNA ratios corresponded well to levels of globin synthesis observed in bone marrow and peripheral blood. Erythroblasts from four patients with heterozygous beta-thalassemia also exhibited approximately the same beta/alpha mRNA ratios in bone marrow erythroblasts (0.34-0.59) as in reticulocytes (0.34-0.4): beta globin mRNA was clearly deficient in bone marrow erythroblasts. Globin biosynthesis by erythroblasts of beta-thalassemia heterozygotes was balanced despite the mRNA deficiency (beta/alpha = 0.9-1.0), suggesting that post-translational phenoma (eg, proteolysis of free globin chains), rather than instability of beta mRNA, accounts for the balanced globin chain synthesis frequently observed in bone marrow erythroblasts of patients with beta-thalassemia trait.

Adult↗

PubMed2000

An examination of different Percoll density gradients and magnetic activated cell sorting (MACS) for the enrichment of fetal erythroblasts from maternal blood.

OBJECTIVE: To increase the yield of fetal erythroblasts from the blood of pregnant women by optimising and simplifying the enrichment procedure. METHODS: By using an experimental system whereby cord blood was mixed with normal adult blood, we evaluated the recovery of fetal erythroblasts using different single or double percoll density gradients with or without subsequent enrichment by Magnetic Activated Cell Sorting (MACS). RESULTS: The recovery of erythroblasts on single density gradients increased from 3%-68% of the input erythroblasts with an increase in the density of percoll from 1075 g/l-1098 g/l. Although the use of a double density gradient did eliminate most lymphocytes, it was accompanied by a significant loss of erythroblasts. The combination with MACS enrichment, however, lead to a almost 200 fold increase in the purity of erythroblasts. CONCLUSIONS: Erythroblasts are preferentially retained by higher density gradients. To enhance their purity a subsequent more specific enrichment step, such as MACS is advisable.

Blood Cell Count↗

PubMed2003

Fetal erythroblasts in maternal blood in relation to gestational age.

OBJECTIVES: To determine the distribution of fetal erythroblasts in the maternal circulation at different gestations. MATERIALS AND METHODS: Maternal blood was obtained from 152 normal singleton pregnancies at 11-40 weeks of gestation. Fetal erythroblasts were isolated using triple density gradient separation and anti-CD71 magnetic cell sorting techniques. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the zeta (zeta), epsilon (epsilon) and gamma (gamma) globin chains. The percentage of fetal cells positive for each stain was calculated. Fluorescence in situ hybridization for X and Y chromosomes was also performed. Comparison was made in the proportion of positive fetal erythroblasts among the different gestational ages. RESULTS: The proportion of erythroblasts stained positive with gamma-globin chain and Kleihauer-Giemsa decreased with gestation from a median of 2% at 11 weeks to 0.5% at 40 weeks. Similarly, there was a decrease in the percentage of Y-signal-positive cells from 1% at 11 weeks to 0.3% at 40 weeks. The proportion of enriched fetal erythroblasts stained positive with zeta- and epsilon-globin chains decreased exponentially from respective medians of 0.6% and 1.5% at 11 weeks to zero after 19 weeks and 24 weeks. CONCLUSION: In normal singleton pregnancy the percentage of fetal erythroblasts enriched from maternal blood decreases with gestation.

Adolescent↗

PubMed1975

Erythroblastic islands and ineffective erythropoiesis in acute myeloid leukaemia.

The erythropoietic part of the bone marrow has been morphologically analysed in 34 untreated, anaemic patients with acute myeloid leukaemia and in 17 healthy controls. In the patients with leukaemia the percentage of basophilic erythroblasts was abnormally high and elevated mitotic indices of the erythroblasts were recorded. The accumulation of basophilic erythroblasts and a high frequency of megaloblastoid changes of the cells may indicate ineffective erythropoiesis. A threefold increase of "erythroblastic islands", i.e. erythroblasts in contact with reticulum cells were recorded in the patients with erythroleukaemia and a twofold increase in the patients with acute myeloid leukaemia. The erythroblastic islands may indicate phagocytosis of defective erythroblasts and an intramedullary haemolysis may thus contribute to the development of anaemia.

Adolescent↗

PubMed2002

[Biological characterization of mouse erythroblastic leukemia cells in haploiden tical mice].

Using transplantable erythroblastic leukemia cells of EL9611(H-2d), the cells were inoculated to CB6F(1)(H-2d/b) generation of BALB/c x C57BL/6 mouse, the biological characterization of erythroblastic leukemia in haploidentical mouse was studied, that provides an experimental model for the study of graft-versus leukemia (GVL) with bone marrow or stem cell transplantation. When 10(3) - 10(8) of the spleen cells of EL9611(H-2d) had been intravenously inoculated to CB6F(1) mouse, the erythroblastic leukemia cells were transplanted successively and the F(1) generation of erythroblastic leukemia model in mice was established with 100% incidence of erythroblastic leukemia. There was a linear relationship between the survival time and the number of leukemic cell. The survival time of the mice was (9.6 +/- 0.8) days when 10(6) cells were inoculated. If the CB6F(1) mouse was transplanted successively for four generations, the incidence was 100%. The main targets for the leukemic EL9611(H-2d) cells were liver, spleen and marrow. The reaction of the erythroblastic leukemia cells for hemoglobin staining was positive, while the peroxidase reaction was negative. These cells were sensitive to some chemotherapeutic drugs, such as cytosine arabinoside and cyclophosphamide. This study presents the convenience for the studies on the GVL with haplo-allogeneic transplantation, in the F(1) generation of erythroblastic leukemia model of the commonly-used CD57BL/6 x BALB/c mouse.

Animals↗

PubMed2005

Numerous erythroblasts in maternal blood are impervious to fluorescent in situ hybridization analysis, a feature related to a dense compact nucleus with apoptotic character.

BACKGROUND AND OBJECTIVES: The analysis by fluorescence in situ hybridization (FISH) of fetal erythroblasts enriched from maternal blood remains an attractive alternative for risk-free prenatal diagnosis of aneuploidies. However, current results are discouraging because of the low levels of sensitivity or the inability to detect fetal erythroblasts by FISH. DESIGN AND METHODS: Erythroblasts were enriched from 35 maternal blood samples by magnetic cell sorting (MACS), identified morphologically following May-Grünwald Giemsa staining and examined by FISH for chromosomes X, Y and 18. RESULTS: We observed that circulating erythroblasts comprised two distinct groups: one was clearly of maternal origin and could be reliably analyzed by FISH, whereas the other, which appeared to be of fetal origin, was largely impervious to FISH analysis. This latter feature seemed to be related to an abnormally dense nucleus with an apoptotic character. Since the oxygen tension in the maternal circulation is higher than that in the fetus, we cultured fetal cord blood erythroblasts in conditions mimicking this difference in oxygen concentrations and found that high oxygen concentrations rapidly induced shrinkage of the erythroblast nucleus, rendering it impervious to FISH analysis. INTERPRETATION AND CONCLUSIONS: Our data show that circulating erythroblasts of presumed fetal origin cannot be reliably analyzed by FISH because of an abnormally dense nucleus. This nuclear phenotype appears to be induced by the higher oxygen tension present in the maternal circulation than in fetal blood.

Aneuploidy↗

PubMedDate not supplied

[Comparative investigation of the non-histone proteins of chromatin from pigeon erythroblasts and erythrocytes].

Chromosomal non-histone proteins are obtained from nuclei of two types of pigeon erythroid cells: erythroblasts (cells active in RNA synthesis) and erythrocytes (cells with repressed RNA synthesis). They are well soluble in solutions of low ionic strength. Electrophoretic separation of the obtained non-histone proteins in polyacrylamide gels with urea and SDS shows the presence of qualitative differences in the pattern of non-histone proteins of chromatine from erythroblasts and erythrocytes. By electrophoresis in urea some protein bands of non-histone proteins of chromatine from erythroblasts were found which disappear with the aging of cells. At the same time two protein fractions were observed in chromatine from erythrocytes which were absent in that of erythroblasts. Disappearance of some high molecular weight protein fractions from erythrocyte chromatine as compared to erythroblasts was observed by separation of the non-histone proteins in the presence of SDS. These fractions of the non-histone proteins disappearing during aging of cells are well extractable from erythroblast chromatine by 0.35 M NaCl solution. In the in vitro system with E. coli RNA polymerase addition of non-histone proteins of chromatine from erythroblasts to chromatine from erythrocytes increases RNA synthesis 2--3 times. At the same time addition of non-histone proteins from erythrocytes is either without any influence on this process or somewhat inhibiting.

Animals↗

PubMed1993

Long-term time-lapse cinemicrography of human erythroblasts under compression and without compression--direct measurement of generation time of each maturation stage.

The in vitro proliferation and the successive morphological alterations of single human erythroblasts were recorded for 5 days by time-lapse, phase-contrast cinemicrography. To visualize their fine structures more clearly, we introduced a new device to compress the erythroblasts under a cover slide. Appropriate compression did not significantly affect the capacity of the erythroblasts to proliferate and differentiate, although compression produced a slight tendency towards shorter generation time. The directly measured generation time was 24.5 +/- 4.5 h (mean +/- S.D.) for generation -1 (one generation prior to the orthochromatic erythroblast stage); 18.4 +/- 3.0 h for generation -2; and 18.3 +/- 4.1 h for generation -3 [corrected], these being arranged in inverse order of cell lineage. The cell size became progressively smaller and the nucleus-to-cytoplasm ratio decreased along with the maturation of the erythroblasts, showing a distinctive distribution range for each generation. Progression of hemoglobinization was simultaneously recorded qualitatively by Soret band absorption micrography. Cell movements characteristic for each maturation stage, e.g. perimitotic bleb-throwing of immature erythroblasts and vigorous movements of orthochromatic erythroblasts, heralding enucleation, were also clearly recorded. The method described here will be useful for the direct study of the dynamic morphology of the erythron in normal and abnormal states.

Cell Differentiation↗

PubMed1999

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro.

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.

Blood Sedimentation↗

PubMed2001

Use of the Kleihauer test to detect fetal erythroblasts in the maternal circulation.

Non-invasive prenatal diagnosis of aneuploidies on fetal nucleated erythrocytes present in the maternal circulation is hampered by the extremely small cell number of uncertain origin (70% of erythroblasts circulating during pregnancy have a maternal origin). Therefore, a method allowing selection of the fetal cells among the maternal cells is indispensable after the erythroblast enrichment step. In the present study, after an erythroblast enrichment step on a ficoll gradient followed by a positive immuno-magnetic selection with anti-CD71 or anti-GPA antibodies, a rapid, simple and direct chemical staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and adult haemoglobin (Hb). The fetal cells appear with an intense pink cytoplasmic staining while maternal cells with adult haemoglobin are colourless. Preservation of the cytoplasmic integrity allows one to distinguish morphological characteristics and to visualize simultaneously nuclear hybridization signal by FISH (fluorescent in situ hybridization). This approach was tested by FISH analysis using dual-colour X- and Y-specific DNA probes on blood samples from 15 pregnant women, with the results being compared to cytogenetic or sonographic sex determination. For 12 pregnancies fetal sex was determined successfully (5 XY/7 XX), in two cases in situ hybridization failed, and in one case no fetal erythroblast was observed after the Kleihauer test. The selection method was applied to a pregnancy at risk for cystic fibrosis (CF). After a Kleihauer test, fetal erythroblasts were collected by microdissection, whole genomic DNA was amplified by primer extension pre-amplification (PEP) followed by a nested CF PCR. The fetal genotype was successfully characterized and confirmed by conventional prenatal diagnosis.

Antibodies↗

PubMed1975

Nucleolar changes in maturing avian erythroblasts and erythrocytes.

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.

Animals↗

PubMed1982

A quantitative ultrastructural study of normal rat erythroblasts and reticulocytes.

Early, intermediate and late erythroblasts and reticulocytes were studied by electron microscopic morphometry. The volumes of mitochondria, Golgi zone and autophagosomes, as well as the surface areas of membranes of rough endoplasmic reticulum (RER) and of mitochondrial cristae and the numbers of ribosomes per unit volume cytoplasm were calculated. the results revealed 3 phases in erythroid maturation: (1) early and intermediate erythroblasts, (2) late erythroblasts, and (4) reticulocytes. The only significant difference between early and intermediate proliferating erythroblasts was a decrease in the surface area of the RER in the latter. After the last mitotic division in late erythroblasts significant reductions occurred in the RER, the Golgi apparatus, in the mitochondria and the number of ribosomes. The numbers of mitochondria and ribosomes were further reduced at the reticulocyte stage (clustered ribosomes more rapidly than single ones). Morphometric analysis showed no evidence of degradation of erythroid mitochondria whilst they are free in the cytoplasm, but there was some evidence of degradation after their uptake into autophagosomes in late erythroblasts and in reticulocytes.

Animals↗

PubMed1991

Ultrastructural study of a cytoplasmic bridge connecting a pair of erythroblasts in mice.

A unique cytoplasmic connection between erythroblasts was studied by electron microscopy in mouse hemopoietic tissues (fetal liver, fetal and neonatal spleen and adult bone marrow). Many pairs of interphase erythroblasts were connected by a "cytoplasmic bridge" that was very thin and sometimes long in comparison with telophase bridges. The stage of maturation of the cells in a pair was similar. Small numbers of microtubules ran along the cytoplasmic bridge; a mid-body was not seen. The plasma membrane at approximately the middle of the bridge bulged to form a ring-shaped ridge filled with dense amorphous substances; this was called a "bulging ring." Thus, the cytoplasmic bridge between erythroblasts did not morphologically correspond to the telophase bridge in the usual cytokinesis. Cytoplasmic bridges were observed in various differentiating stages of erythroblasts, whereas other cell types of the hemopoietic lineage did not have such a bridge. The cytoplasmic bridge is unique to erythroblasts and provides an evidence for the atypical cytokinesis of the erythroblastic lineage.

Animals↗

PubMed1986

Iron uptake and ferritin synthesis in human erythroblasts.

The erythroblasts from four normal bone marrows were enriched by using an anti-myeloid monoclonal antibody (TG-1), and a polyclonal antibody against mononuclear cells to effect complement mediated lysis of unwanted cells. The erythroblasts were cultured for 2 h in medium containing [59Fe]transferrin and [3H]-leucine, then fractionated on Percoll gradients according to their density. Whole cell iron uptake by early erythroblasts was greater than in the dense late erythroblasts. In all marrows, iron uptake into ferritin was highest in fractions containing the earliest erythroblasts and decreased with increasing erythroblast maturity. In three of the four marrows this paralleled the pattern of ferritin synthesis. It seems likely that apoferritin is synthesized in response to the amount of iron taken into the cell and this iron is incorporated within the protein shell to form ferritin.

Bone Marrow↗

PubMed1980

Membrane differentiation in human erythroid cells: unique profiles of cell surface glycoproteins expressed in erythroblasts in vitro from three ontogenic stages.

HUMAN ERYTHROBLASTS IN CULTURE, IRRESPECTIVE OF THE ONTOGENIC STAGE OF THEIR PROGENITORS, ARE CHARACTERIZED BY: (i) the barely detectable amount of band 3 glycoprotein, (ii) the presence of two glycoproteins with molecular weights 105,000 and 95,000, (iii) the high concentration of glycophorin, and (iv) a minimum quantity of the carbohydrate chain susceptible to endo-beta-galactosidase ("polylactosaminoglycan"). In contrast, mature erythrocytes, whether of fetal, neonatal, or adult origin, are characterized by a high concentration of band 3 glycoprotein, polylactosaminoglycan, and glycophorins, but do not contain 105- and 95-kilodalton-glycoproteins. Thus, the process of erythroid maturation from erythroblasts to erythrocytes is accompanied by the appearance of band 3, the disappearance of 105- and 95-kilodalton glycoproteins, and a great increase in the quantity of polylactosaminoglycan. The structure of polylactosaminoglycan may not be different between mature erythrocytes and erythroblasts from the same ontogenic stage, but it is distinctively different from one stage to the other. The profiles of oligosaccharides released by endo-beta-galactosidase and immunofluorescence studies with anti-Ii antibodies indicated that a linear polylactosaminoglycan structure was present in erythroblasts as well as in erythrocytes of the fetal and newborn stage, whereas a branched polylactosaminoglycan structure was present in erythroblasts as well as erythrocytes of adult blood. Thus, two membrane characteristics are closely associated with the process of erythroid cell development; one-the membrane proteins band 3, band 4.5, and 95- and 105-kilodalton glycoproteins-determines the degree of maturation, and the other-polylactosaminoglycan-may determine the ontogenic stage of the erythroblast progenitors.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1990

Impaired iron uptake and transferrin binding by erythroblasts in the anaemia of rheumatoid arthritis.

Serum and bone marrow from 18 patients with rheumatoid arthritis (RA) and five healthy controls were studied in order to establish a possible role of impaired iron uptake and transferrin binding by erythroblasts in the pathophysiology of anaemia of chronic disease (ACD) in RA. Iron incorporation into erythroblasts was reduced in patients with ACD using a method based on incubation of erythroblasts with radiolabelled 59Fe-125I-transferrin. It correlated negatively with C-reactive protein (CRP). In iron deficient RA patients it tended to be reduced as well. These patients had the same level of RA disease activity as in ACD. Transferrin binding by erythroblasts was significantly impaired in ACD compared to controls, although it tended to be reduced in all RA groups. These findings suggest that impaired iron uptake by erythroblasts, probably due to decreased transferrin binding to erythroblasts, might be a pathophysiological factor in ACD in RA.

Anemia↗

PubMed1982

Erythroblast iron metabolism in sideroblastic marrows.

The uptake of iron by bone marrow erythroblasts and its intracellular distribution have been studied in 23 patients with primary sideroblastic anaemia (SA), five patients with secondary SA and one patient with only non-ringed sideroblasts. EM of erythroblasts from 18 cases showed both mitochondrial iron deposits and cytoplasmic ferritin aggregates in all cases except the patient with only non-ringed sideroblasts. Iron uptake by erythroblasts in whole bone marrow was normal but there was a decreased incorporation into haem and an increased incorporation into cell stroma. Age matching of erythroblasts using Percoll density gradient centrifugation indicated that stromal iron incorporation was high at all stages of erythroblast development even before haem synthesis had become a major metabolic activity and in intermediate and late erythroblasts a real decrease in haem synthesis appeared less certain. These observations, together with the inability to correct the abnormality in vitro with either pyridoxal phosphate or delta amino-laevulinic acid suggest that the primary defect in SA may be an abnormality of mitochondrial iron metabolism rather than an abnormality of haem synthesis.

Adult↗