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PubMed2004

Assessment of efficacy of cell separation techniques used in the enrichment of foetal erythroblasts from maternal blood: triple density gradient vs. single density gradient.

The aim of this study was to determine the efficacy of cell separation with single density and triple density-gradient techniques in the yield of foetal erythroblasts isolated from maternal blood. Maternal blood was obtained from 20 singleton pregnancies at 11-14 weeks of gestation immediately before foetal karyotyping by chorionic villus sampling. In each woman, the blood sample was divided into two portions; one portion was used for single density-gradient separation and the other, for triple density-gradient separation. Magnetic cell sorting (MACS) was subsequently performed with anti-CD71/antiglycophorin-A. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the gamma, epsilon and zeta globin chains. The percentage of foetal cells positive for each stain was calculated. Fluorescence in situ hybridization (FISH) for X- and Y-chromosomes was also performed. Comparison was made in the proportion of enriched foetal cells between the two separation methods for each CD71 and glycophorin-A (GPA) antibody. The percentage of erythroblasts enriched from maternal blood that stained positive for gamma, epsilon and zeta globin chains and with Kleihauer-Giemsa was significantly higher in the triple density-gradient separation fractions compared with the single density-gradient fractions with both anti-CD71 and GPA MACS. FISH analysis for the Y-chromosome confirmed the increase in foetal cell proportion in the triple density-gradient samples. Isolation of foetal erythroblasts from maternal blood using triple density-gradient separation and MACS is more effective with regard to foetal cell yield and purity than single density-gradient separation and MACS.

Cell Separation↗
PubMed1991

Erythroblast- and erythrocyte-bound antibodies in alpha and beta thalassaemia syndromes.

Thirty-five Thai patients with various alpha-thalassaemia (alpha-thal 1/alpha-thal 2, alpha-thal 1/HbCS, HbCS/HbCS) and beta-thalassaemia (beta-thal/HbE, severe and mild form, HbE/HbE) syndromes were examined for the presence of immunoglobulins and C3d on o-tolidine positive erythroblasts in the bone marrow, and for the amounts of IgG of some specificities bound to circulating erythrocytes. In mild, but not in severe beta-thal/HbE and in alpha-thalassaemia, the percentages of Ig-positive erythroblasts were significantly higher than in controls and correlated well with the percentage of IgG-positive erythroblasts. By contrast, the percentages of IgM and C3d positive erythroblasts were low and similar in thalassaemic and control marrows. A substantial proportion of thalassaemic patients showed more erythrocyte-bound IgG than controls, but statistically significant elevations were seen only in severe beta-thal/HbE. Within a particular syndrome erythrocyte-bound IgG was more abundant in splenectomized than non-splenectomized subjects. It showed specificity for spectrin in some beta-thalassaemic patients and for band 3 protein in several individuals with alpha- or beta-thalassaemia. The results suggest that IgG antibodies play a role in the haemolysis of thalassaemia and that they are likely to be involved in the ineffective erythropoiesis in at least some of the syndromes studied.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1993

Erythroblasts as an index of initial stage of 2,7-FAA, ENU and BNU-induced rat leukemia.

The appearance of orthochromatic erythroblasts in the peripheral blood was evaluated as an index of the initial stage of leukemia induced in rats by the oral administration of N,N'-2,7-fluorenylenebisacetamide, 1-ethyl-1-nitrosourea and 1-butyl-1-nitrosourea. Solitary leukemia lesions found mainly in the bone marrow and composed of one or a few nodular foci were found by using erythroblasts as an index. The time interval from the first appearance of erythroblasts to autopsy was short in solitary lesions, slightly longer in scattered lesions, and even longer in diffuse leukemia lesions. Although the appearance of orthochromatic erythroblasts in the peripheral blood was due to several causes other than leukemia, this appearance was useful as an index of the initial stage of chemically induced leukemia in rats.

2-Acetylaminofluorene↗
PubMed1993

The Ets-related transcription factor PU.1 immortalizes erythroblasts.

In vivo studies of Friend virus erythroleukemia have implied that proviral integrations adjacent to the gene for the Ets-related transcription factor PU.1 may inhibit the commitment of erythroblasts to differentiate and cause their capability for indefinite transplantation (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 62:4129-4135, 1988; R. Paul, S. Schuetze, S. L. Kozak, C. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test this hypothesis, we ligated PU.1 cDNA into a retroviral vector and studied its effects on cultured cells. Infection of fibroblasts with PU.1-encoding retrovirus resulted in PU.1 synthesis followed by nuclear pyknosis, cell rounding, and degeneration. In contrast, in long-term bone marrow cultures, erythroblasts were efficiently and rapidly immortalized. The resulting cell lines were polyclonal populations that contained PU.1, were morphologically blast-like, required erythropoietin and bone marrow stromal cells for survival and proliferation, and spontaneously differentiated at low frequency to synthesize hemoglobin. After 9 months in culture, erythroblasts became stroma independent, and they then grew as clonal cell lines. We conclude that PU.1 perturbs the pathway(s) that controls potential for indefinite proliferation and that it can be used to generate permanent erythroblast cell lines.

3T3 Cells↗
PubMed1986

Studies of erythroblast function in congenital dyserythropoietic anaemia, type I: evidence of impaired DNA, RNA, and protein synthesis and unbalanced globin chain synthesis in ultrastructurally abnormal cells.

Two patients with congenital dyserythropoietic anaemia, type I (CDA) were studied. Their blood reticulocytes showed unbalanced globin chain synthesis with increased alpha:beta globin chain synthesis ratios. A high proportion of the erythroblasts displayed the characteristic "Swiss cheese" abnormality of the nuclear chromatin and some also showed cytoplasmic intrusions lined with nuclear membrane within the nucleus. Occasional erythroblast profiles contained intracytoplasmic inclusions that were ultrastructurally indistinguishable from precipitated alpha chains. The technique of combined Feulgen microspectrophotometry and 3H-thymidine autoradiography showed gross abnormalities of proliferation in the early polychromatic erythroblasts. The proliferative abnormalities included an arrest of DNA synthesis after the progress of cells through part of the S phase and the formation of several mononucleate and binucleate cells with hypertetraploid total DNA contents. The bone marrow cells gave a normal deoxyuridine suppressed value, indicating that there was no impairment of the methylation of deoxyuridylate. Electron microscope autoradiographic studies showed that a high proportion of the erythroblasts with the "Swiss cheese" nuclear abnormality suffered from a severe impairment, or arrest of DNA, RNA, and protein synthesis.

Adult↗
PubMed1997

The expression of transferrin receptors on erythroblasts in anaemia of chronic disease, myelodysplastic syndromes and iron deficiency.

The presence of transferrin receptors on erythroblasts in patients with iron deficiency, anaemia of chronic disease (ACD) and myelodysplastic syndrome (MDS) was studied by two-colour analysis on a flow cytometer. CD 71 was used to quantify the number of transferrin receptors and GLY-A to identify erythroblasts. In cases of iron deficiency, the number of transferrin receptors was increased on part of the erythroblasts thus facilitating iron uptake by the cells. In patients with ACD or MDS, a decrease of the number of transferrin receptors on erythroblasts was found. This leads to the conclusion that the ineffective response to iron therapy in cases of ACD and MSD can be explained by a decline of transferrin receptors on the red cells.

Anemia↗
PubMed1976

Erythroblastic islands and extra-medullary erythropoiesis in chronic myeloid leukaemia.

Aspirates from bone marrow, spleen and liver were morphologically analysed in 15 untreated patients wich chronic myeloid leukaemia. Megaloblastic changes of the erythroblasts were found to be more common in the spleen and liver than in the bone marrow. A significant increase of 'erythroblastic islands', i.e. erythroblasts in contact with reticulum cells, were recorded in the leukaemia patients compared to 15 healthy controls. It is suggested that the presence of such formations may indicate an increased death rate of defective erythroblasts followed by phagocytosis into reticuloendothelial cells.

Bone Marrow↗
PubMed1992

Ion channels in human erythroblasts. Modulation by erythropoietin.

To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].

Adult↗
PubMed2005

Core erythropoietin receptor signals for late erythroblast development.

Critical signals for erythroblast formation are transduced by activated, tyrosine-phosphorylated erythropoietin receptor (EpoR) complexes. Nonetheless, steady-state erythropoiesis is supported effectively by EpoR alleles that are deficient in cytoplasmic phosphotyrosine sites. To better define core EpoR action mechanisms, signaling capacities of minimal PY-null (EpoR-HM) and PY343-retaining (EpoR-H) alleles were analyzed for the first time in bone marrow-derived erythroblasts. Jak2 activation via each allele was comparable. Stat5 (and several Stat5-response genes) were induced via EpoR-H but not via EpoR-HM. Stat1 and Stat3 activation was nominal for all EpoR forms. For both EpoR-HM and EpoR-H, Akt and p70S6-kinase activation was decreased multifold, and JNK activation was minimal. ERKs, however, were hyperactivated uniquely via EpoR-HM. In vivo, Epo expression in EpoR-HM mice was elevated, while Epo-induced reticulocyte production was diminished. In vitro, EpoR-HM erythroblast maturation also was attenuated (based on DNA content, forward-angle light scatter, and hemoglobinization). These EpoR-HM-specific defects were corrected not only upon PY343 site restoration in EpoR-H, but also upon MEK1,2 inhibition. Core EpoR PY site-independent signals for erythroblast formation therefore appear to be Stat5, Stat1, Stat3, p70S6-kinase, and JNK independent, but ERK dependent. Wild-type signaling capacities, however, depend further upon signals provided via an EpoR/PY343/Stat5 axis.

Animals↗
PubMed2006

Lyn kinase promotes erythroblast expansion and late-stage development.

Lyn kinase is known to modulate the formation and function of B cells, monocytes, and mast cells. However, Lyn-/- mice also develop erythrosplenomegaly, and cases for both negative and positive erythropoietic actions of Lyn recently have been outlined. In phenylhydrazine-treated Lyn-/- mice, extramedullary splenic erythropoiesis was hyperactivated, but this did not lead to accelerated recovery from anemia. Furthermore, ex vivo analyses of the development of bone marrow-derived Lyn-/- erythroblasts in unique primary culture systems indicated positive roles for Lyn at 2 stages. Late-stage Lyn-/- erythroblasts exhibited deficit Ter119(pos) cell formation, and this was paralleled by increased apoptosis (and decreased Bcl-xL expression). During early development, Lyn-/- erythroblasts accumulated at a Kit(pos)CD71(high) stage, possessed decreased proliferative capacity, and were attenuated in entering an apparent G1/S cell-cycle phase. In proposed compensatory responses, Lyn-/- erythroblasts expressed increased levels of activated Akt and p60-Src and decreased levels of death-associated protein kinase-2. Stat5 activation and Bcl-xL expression, in contrast, were significantly decreased in keeping with decreased survival and developmental potentials. Lyn, therefore, is proposed to function via erythroid cell-intrinsic mechanisms to promote progenitor cell expansion beyond a Kit(pos)CD71(high) stage and to support subsequent late-stage development.

Animals↗
PubMed2001

Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts.

Erythropoietin (Epo) controls red cell production in the basal state and during stress. Epo binding to its receptor, EpoR, on erythroid progenitors leads to rapid activation of the transcription factor Stat5. Previously, fetal anemia and increased apoptosis of fetal liver erythroid progenitors were found in Stat5a(-/-)5b(-/-) mice. However, the role of Stat5 in adult erythropoiesis was not clear. The present study shows that some adult Stat5a(-/-)5b(-/-) mice have a near-normal hematocrit but are deficient in generating high erythropoietic rates in response to stress. Further, many adult Stat5a(-/-)5b(-/-) mice have persistent anemia despite a marked compensatory expansion in their erythropoietic tissue. Analysis of erythroblast maturation in Stat5a(-/-)5b(-/-) hematopoietic tissue shows a dramatic increase in early erythroblast numbers, but these fail to progress in differentiation. Decreased expression of bcl-x(L) and increased apoptosis in Stat5a(-/-)5b(-/-) early erythroblasts correlate with the degree of anemia. Hence, Stat5 controls a rate-determining step regulating early erythroblast survival.

Anemia↗
PubMed2002

No apoptotic cell death of erythroid cells of erythroblastic islands in bone marrow of healthy rats.

A possibility of apoptotic cell death in erythropoietic regulation was examined by means of detailed light microscopical histoplanimetry, electron microscopy, the in situ nick-end labeling method, and an immunohistological method in the rat bone marrow. Serum erythropoietin concentrations were shown at normal levels. The erythroid series on a mature process presented several morphological features of apoptosis, i.e. the shrinkage of both nuclei and cytoplasm and the chromatin condensation. In the light microscopical histoplanimetry, however, morphological signs of final apoptotic cell death were never found in any erythroid cell within the erythroblastic islands. This finding was also supported by detailed ultrastructural observation: No erythroid cell bodies were trapped and degraded by the central macrophages of the erythroblastic islands, while the denucleated nuclei with small amount of cytoplasm of late erythroblasts were often trapped and degraded in the macrophages. Nuclear DNA fragmentation was not detected in any erythroblasts, but was detected in the lysosomes of the central macrophages. These findings suggest that erythropoiesis is regulated by other regulatory mechanisms than apoptotic cell death. An additional ultrastructural finding shows that the reticulocytes anchored to the central macrophages are transported into the peripheral blood circulation.

Animals↗
PubMed2005

The fetal erythroblast is not the optimal target for non-invasive prenatal diagnosis: preliminary results.

Fetal cells, present in the blood of pregnant women, are potential targets for non-invasive prenatal diagnosis. The fetal erythroblast has been the favorite target cell type. We investigated four methods of enrichment for fetal erythroblasts, identifying only three fetal erythroblasts in 573 ml of maternal blood. This is much less than the expected two to six fetal cells per ml of maternal blood. Hamada and Krabchi used a cell type-independent marker, i.e., the Y chromosome in maternal blood from male pregnancies after Carnoy fixation, leaving the nuclei for hybridization with X-and Y-chromosome-specific probes. We found with a similar technique 28 fetal cells in 15 ml of maternal blood. The fetal origin of cells was confirmed by hybridizing the nuclei with X- and Y-chromosome-specific probes, using two consecutive hybridizations with the two probes in opposite colors (reverse FISH). Candidate fetal cells were inspected after each hybridization. Only cells that were found to change the color of both probe signals from first to second hybridization were diagnosed as fetal. To reduce the labor-intensive slide screening load, we used semiautomated scanning microscopy to search for candidate cells. We conclude that erythroblasts form only a small fraction of fetal cells present in maternal blood.

Blood Cell Count↗
PubMed1975

Effects of heterologous anti-erythrocyte antibodies on the generation cycle of erythroblasts in rats.

An injection of anti-rat erythrocyte rabbit serum or its 7S globulin fraction into rats resulted in immunohemolytic anemia associated with a positive anti-gloublin test examined with anti-rabbit gammaglobulin serum. However, an injection of 19S globulin fraction from the anti-erythrocyte serum failed to cause immunohemolytic anemia, although it induced a transient decrease in red cell count. These results indicate that 7S antibodies are responsible for the induction of anti-globulin test positive, experimental immunohemolytic anemia. The ferrokinetic and stathmokinetic studies revealed the prolongation of generation time of basophilic and polychromatic erythroblasts in rats injected with anti-erythrocyte serum or its 7S globulin fraction. The results from in vitro [3H] thymidine incorporation experiments also confirmed this prolongation. The analysis of in vivo [3H] thymidine labeling of erythroblasts in rats which were given the 7S GLOBULIN ANTIBODIES SUGGESTED THE PROLONGATION OF THE G1 time of the erythroblasts. These results suggest that the effects of anti-erythrocyte antibodies on the generation cycle of erythroblasts are to prolong their G1 time and keep them dormant.

Anemia, Hemolytic↗
PubMed1980

The effect of chloramphenicol and thiamphenicol on the ultrastructure of the erythroblasts in mouse splenic colonies.

Lethally irradiated mice were injected with 1 x 10(5) normal bone marrow cells taken from mice of the same strain. They were divided into two groups: One was given chloramphenicol (CAP) or thiamphenicol (TAP) in an early stage after marrow transfusion and the other in a later stage. In both groups, more prominently in the former group, some ultrastructural changes were recognized in mitochondria of the early erythroblasts. Mitochondria increased in number and became swollen with electron lucent matrix and paucity of cristae. Condensed mitochondria described in earlier reports were never observed. Intermediate or late erythroblasts seen only in the latter group did not show such alterations. Mitochondrial changes in the early erythroblasts were considered to reflect the dose-related toxic effects of CAP and TAP, and it is assumed that these drugs affect the development of erythroblasts in a very early stage.

Animals↗
PubMed1983

A quantitative morphology of nucleoli of erythroblasts in the mouse spleen: an electron microscopic study.

Nucleolar changes during the differentiation of erythroblasts in the mouse spleen were examined quantitatively by electron microscopy. On the basis of a three-dimensional nuclear analysis, as reported previously, the erythroblasts could be classified into four types: small (S), medium (M), large (L) and extra-large (EL). Extra-large and large erythroblasts have large prominent nucleoli which are usually attached to the nuclear membrane. Medium and small erythroblasts have small nucleoli which are generally separated from the nuclear margin. The volumetric ratio of nucleoli to nucleus obtained by a point-counting method is 0.157 +/- 0.016 in EL; 0.135 +/- 0.011 in L; 0.035 +/- 0.004 in M and 0.015 +/- 0.004 in S, and the volume of nucleoli is 37.8 microns3 in EL; 17.3 microns3 in L; 2.3 microns3 in M; 0.5 microns3 in S, respectively. The number of nucleoli per nucleus is largest (3.9) in EL and smallest (0.6) in S. The nucleolar changes are discussed in relation to the developmental sequence of the erythroid series.

Aging↗
PubMed2000

Involvement of Gialpha2 in sodium butyrate-induced erythroblastic differentiation of K562 cells.

The chronic myelogenous leukaemia cell line K562 can be triggered in culture to differentiate along the erythrocytic pathway in response to a variety of stimulatory agents. In the presence of sodium butyrate, these cells differentiate to erythroblasts and acquire the capability to synthesize haemoglobin. We used this cell system to study alterations in the levels of several G-protein subunits during the cell differentiation programme and to assess the involvement of G(i)alpha2 in this process. Western immunoblot analysis revealed the presence of G(s)alpha1, G(s)alpha2, G(i)alpha2, G(q)alpha, Galpha(12), Gbeta1 and Gbeta2 in K562 cells. G(o)alpha, G(z)alpha, Galpha(13) and Galpha(16) were not detected. Although the levels of several G-protein subunits were altered after treatment with sodium butyrate, the most striking change was the robust increase in the levels of G(i)alpha2, which was accompanied by an increase in the mRNA for G(i)alpha2. Inactivation of G(i)alpha2 by adding Bordetella pertussis toxin to the cultures inhibited erythroblastic differentiation by as much as 62%, as measured by haemoglobin accumulation. Furthermore, the addition of an oligonucleotide anti-sense to G(i)alpha2 inhibited the sodium butyrate-induced robust increase in G(i)alpha2 levels, decreasing it to the basal levels seen in control cells; this treatment decreased the erythroblastic differentiation of the cells (as measured by haemoglobin expression) by 50%. Taken together, these findings imply that increased levels of G(i)alpha2 contribute to the sodium butyrate-induced erythroblastic differentiation of K562 cells.

Butyrates↗
PubMed2000

Fetal and embryonic hemoglobins in erythroblasts of chromosomally normal and abnormal fetuses at 10-40 weeks of gestation.

BACKGROUND AND OBJECTIVES: During fetal development a change in erythropoiesis from hepatic to medullary site occurs. In chromosomally abnormal fetuses this change is delayed. Hemoglobin production also undergoes developmental switches from embryonic to fetal hemoglobins in the first trimester of pregnancy. The aim of study was to determine the proportion of embryonic and fetal hemoglobins in fetal erythroblasts of chromosomally normal and abnormal fetuses at 10-40 weeks of gestation. DESIGN AND METHODS: Fetal blood was obtained from 93 chromosomally normal and 19 abnormal fetuses at 10-40 weeks of gestation. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody. Fluorescent antibodies were used to immuno-stain for zeta (zeta), epsilon (epsilon) and gamma (gamma) hemoglobin chains. RESULTS: The percentages of the positively stained cells were calculated. In chromosomally normal fetuses the percentage of erythroblasts expressing the zeta chain was 25% at 10 weeks but this decreased exponentially with gestation to less than 1% by 17 weeks. Similarly, the percentage of cells expressing the epsilon chain decreased from 97% at 10 weeks to less than 1% by 25 weeks. In contrast, expression of the gamma chain increased from about 30% at 10 weeks to 90% by 16 weeks and decreased thereafter to 60% at 40 weeks. In the abnormal fetuses, the percentage of erythroblasts expressing the zeta chain and the epsilon chain decreased to less than 1% by 23 and 28 weeks respectively, while maximum expression of the gamma chain was at about 22 weeks. INTERPRETATION AND CONCLUSIONS: In the chromosomally abnormal group the pattern of change in the expression of the various hemoglobin chains during gestation was similar to that in the normal fetuses but was delayed by three to six weeks. These findings suggest that in fetuses with chromosomal abnormalities there is a developmental delay in the switch from embryonic to fetal hemoglobin chains.

Adult↗