Nucleated Red Cell Precursors Research Articles

Maturation-associated gene expression profiles during normal human bone marrow erythropoiesis

Erythropoiesis has been extensively studied using in vitro and in vivo animal models. Despite this, there is still limited data about the gene expression profiles (GEP) of primary (ex vivo) normal human bone marrow (BM) erythroid maturation. We investigated the GEP of nucleated red blood cell (NRBC) precursors during normal human BM erythropoiesis. Three maturation-associated populations of NRBC were identified and purified from (fresh) normal human BM by flow cytometry and the GEP of each purified cell population directly analyzed using DNA-oligonucleotide microarrays. Overall, 6569 genes (19% of the genes investigated) were expressed in ≥1 stage of BM erythropoiesis at stable (e.g., genes involved in DNA process, cell signaling, protein organization and hemoglobin production) or variable amounts (e.g., genes related to cell differentiation, apoptosis, metabolism), the latter showing a tendency to either decrease from stage 1 to 3 (genes associated with regulation of erythroid differentiation and survival, e.g., SPI1, STAT5A) or increase from stage 2 to stage 3 (genes associated with autophagy, erythroid functions such as heme production, e.g., ALAS1, ALAS2), iron metabolism (e.g., ISCA1, SLC11A2), protection from oxidative stress (e.g., UCP2, PARK7), and NRBC enucleation (e.g., ID2, RB1). Interestingly, genes involved in apoptosis (e.g., CASP8, P2RX1) and immune response (e.g., FOXO3, TRAF6) were also upregulated in the last stage (stage 3) of maturation of NRBC precursors. Our results confirm and extend on previous observations and providing a frame of reference for better understanding the critical steps of human erythroid maturation and its potential alteration in patients with different clonal and non-clonal erythropoietic disorders.

Newly diagnosed adult AML and MPAL patients frequently show clonal residual hematopoiesis

Adult acute myeloid leukemia (AML) is a highly heterogeneous stem cell malignancy characterized by the clonal expansion of immature myeloid precursors. AML may emerge de novo, following other hematopoietic malignancies or after cytotoxic therapy for other disorders. Here, we investigated the clonal vs reactive nature of residual maturing bone marrow cells in 59 newly diagnosed adult AML and mixed phenotype acute leukemia (MPAL) patients as assessed by interphase fluorescence in situ hybridization analysis of AML and myelodysplastic syndrome-associated cytogenetic alterations and/or the pattern of chromosome X inactivation, in females. In addition, we investigated the potential association between the degree of molecular/genetic involvement of hematopoiesis and coexistence of altered immunophenotypes by flow cytometry. Our results indicate that residual maturing neutrophils, monocytes and nucleated red cell precursors from the great majority of newly diagnosed AML and MPAL cases show a clonal pattern of involvement of residual maturing hematopoietic cells, in association with a greater number of altered immunophenotypes. These findings are consistent with the replacement of normal/reactive hematopoiesis by clonal myelopoiesis and/or erythropoiesis in most newly diagnosed AML and MPAL cases, supporting the notion that in most adults presenting with de novo AML, accumulation of blast cells could occur over a pre-existing clonal hematopoiesis.

Cell‐cycle distribution of different cell compartments in normal versus reactive bone marrow: A frame of reference for the study of dysplastic hematopoiesis

Limited information is currently available about the proliferation activity and cell-cycle distribution of different bone marrow (BM) cell subsets defined according to their lineage and maturation stage in normal versus cytopenia-associated reactive BM samples. Here, we report a three-color flow cytometry approach to investigate the cell-cycle distribution of different BM cell compartments-CD34(+) hematopoietic progenitor and precursor cells (HPC), maturing neutrophils and monocytic cells, mature lymphocytes, eosinophils, and nucleated red blood cell precursors (NRBC)-from normal (n = 47) versus cytopenia-associated reactive (n = 47) BM samples. Highly similar proliferation profiles were detected in normal versus reactive BM, with a higher proliferation index (PI) for the more immature CD34(+) HPC, CD11b(-) maturing neutrophils and NRBC versus other BM cell compartments. The only differences observed between normal and reactive BM were restricted to the more mature (CD13(hi) /CD11b(+) ) bands/neutrophils and to monocytic cells, which showed an increased PI (0.9% ± 0.8% vs. 0.6% ± 0.5% and 6 ± 3.6 vs. 4.6 ± 4.5, respectively) at the expense of a lower PI of CD34(+) HPC in reactive conditions. Of note, bands/mature neutrophils and mature lymphocytes showed either residual numbers or absence of S + G₂ /M-phase cells in both normal and reactive BM. Our results suggest that a slight shift of proliferation from the early precursors to the more mature granulomonocytic compartment occurs in reactive BM, which could reflect an attempt of the hematopoietic system to rapidly produce functional neutrophils and monocytes, at the expense of a lower expansion of the minor compartments of CD34(+) HPC.

Inner calvarial erosion from traumatic subdural haematoma in infancy

This is a report of a case of a subdural haematoma in infancy of possible non-accidental aetiology with raised pericerebral pressure, which we postulate has eroded the inner table of the cranial bones and resulted in leakage of marrow precursor cells into the extradural space. Subdural tapping via the fontanelle has created a channel allowing subsequent ingress of nucleated red cell precursors into the subdural space. This addition to the subdural collection has prolonged its course necessitating subduro-peritoneal shunting.

Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors.

The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with [35S]methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells (Blikstad, I., W. J. Nelson, R. T. Moon, and E. Lazarides, 1983. Cell, 32:1081-1091). Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.

Lack of Rhesus antigen expression by human committed erythroid progenitors

The D antigen of the Rhesus blood group, an erythroid-specific cell surface marker, is expressed by all morphologically recognizable human nucleated red blood cell precursors including, in low density, the pronormoblast. The object of the present study was to determine the expression of the D antigen by committed erythroid progenitors. Under conditions that produced complete inhibition of BFU-E and CFU-E by known cytotoxic antisera, no significant inhibition was produced by anti-D. Use of anti-human IgG (rabbit) to increase sensitivity and trypsinization to reveal cryptic Rh determinants were both without inhibitory effect. Erythroid bursts and colonies grew normally in methylcellulose that contained anti-D. The addition of anti-D to day 7 BFU-E did not inhibit their proliferation to mature bursts at day 14. These results suggest that the D antigen is not expressed by human committed erythroid progenitor cells. The D antigen is therefore an erythroid-specific differentiation marker, rather than an erythroid- lineage-specific antigen. The development of expression of the D antigen during erythropoiesis parallels that of band 3 protein, to which anti-D has been reported to bind. Lack of Rh expression by committed erythroid progenitors is consistent with the rarity of red cell aplasia in Rhesus hemolytic disease of the newborn and in idiopathic and drug-induced autoimmune hemolytic anemia in which the autoantibodies have apparent Rh specificity. These results imply that Rh compatibility is not a contraindication to human bone marrow transplantation.

Lack of Rhesus Antigen Expression by Human Committed Erythroid Progenitors

The D antigen of the Rhesus blood group, an erythroid-specific cell surface marker, is expressed by all morphologically recognizable human nucleated red blood cell precursors including, in low density, the pronormoblast. The object of the present study was to determine the expression of the D antigen by committed erythroid progenitors. Under conditions that produced complete inhibition of BFU-E and CFU-E by known cytotoxic antisera, no significant inhibition was produced by anti-D. Use of anti-human IgG (rabbit) to increase sensitivity and trypsinization to reveal cryptic Rh determinants were both without inhibitory effect. Erythroid bursts and colonies grew normally in methylcellulose that contained anti-D. The addition of anti-D to day 7 BFU-E did not inhibit their proliferation to mature bursts at day 14. These results suggest that the D antigen is not expressed by human committed erythroid progenitor cells. The D antigen is therefore an erythroid-specific differentiation marker, rather than an erythroid-lineage-specific antigen. The development of expression of the D antigen during erythropoiesis parallels that of band 3 protein, to which anti-D has been reported to bind. Lack of Rh expression by committed erythroid progenitors is consistent with the rarity of red cell aplasia in Rhesus hemolytic disease of the newborn and in idiopathic and drug-induced autoimmune hemolytic anemia in which the autoantibodies have apparent Rh specificity. These results imply that Rh compatibility is not a contraindication to human bone marrow transplantation.

Protease modification enhances anti-D binding to nucleated red blood cell precursors.

Protease modification of nucleated red blood cell precursors resulted in significant enhancement of anti-D binding, indicating the presence of cryptic Rh determinants within the plasma membrane of the developing erythroid cell. Protease-modified nucleated precursor cells bound less anti-D than protease-modified mature red blood cells, however, suggesting that the total content of D antigen in the developing erythropoietic plasma membrane is reduced. These findings support the hypothesis that the D antigen, an integral membrane protein, is progressively synthesized during erythropoiesis.

Protease Modification Enhances Anti-D Binding to Nucleated Red Blood Cell Precursors

Protease Modification Enhances Anti-D Binding to Nucleated Red Blood Cell Precursors

Blood group D antigen content of nucleated red cell precursors

The D antigen content of nucleated red cell precursors in human bone marrow was estimated using autoradiography and 125I-anti-D. D antigen first appeared in the pronormoblast, and the quantity of antigen progressively increased during red cell maturation. Maximal anti-D binding occurred on mature red blood cells. Pronormoblasts, basophilic normoblasts, polychromatophilic normoblasts, and orthochromatic normoblasts, respectively, had approximately 1/4, 1/2, 2/3, and 3/4 the quantity of antigen found on mature red cells. None of the other cell types were found in bone marrow labeled with anti-D.

Blood group D antigen content of nucleated red cell precursors

Abstract The D antigen content of nucleated red cell precursors in human bone marrow was estimated using autoradiography and 125I-anti-D. D antigen first appeared in the pronormoblast, and the quantity of antigen progressively increased during red cell maturation. Maximal anti-D binding occurred on mature red blood cells. Pronormoblasts, basophilic normoblasts, polychromatophilic normoblasts, and orthochromatic normoblasts, respectively, had approximately 1/4, 1/2, 2/3, and 3/4 the quantity of antigen found on mature red cells. None of the other cell types were found in bone marrow labeled with anti-D.

Decreased αGlobin mRNA in Nucleated Red Cell Precursors in αThalassemia

Abstract The alpha thalassemias are associated with a decrease in alpha chain synthesis. Hemoglobin H (HbH) disease is a moderately severe form of alpha thalassemia characterized by the production of 5%--20% of HbH, while alpha thalassemia trait is a milder form of alpha thalassemia. In two patients with HbH disease, the ratio of alpha chain synthesis to beta chain synthesis (alpha/beta ratio) was decreased in both bone marrow cells and reticulocytes. When isolated mRNA from bone marrow cells and reticulocytes was translated in a heterologous cell-free system, the alpha/beta ratios were lower than the intact cell ratios. These findings were confirmed by hybridization of the mRNA of both marrow cells and reticulocytes using purified alpha and beta cDNA probes. In the intact cells of two patients with alpha thalassemia trait, the alpha/beta ratios were also decreased and were similar in marrow cells and reticulocytes. Cell-free studies of translatable mRNA also demonstrated decreased alpha/beta ratios, but, unlike the HbH studies, the cell-free alpha/beta ratios were similar to the intact cell ratios. One hybridization study utilizing peripheral blood mRNA had an alpha/beta ratio consistent with the cell-free ratios. These results indicated that, in both HbH disease and alpha thalassemia trait, there was decreased alpha globin mRNA present in both nucleated red cell precursors and reticulocytes. In addition, the data suggested that there may be translational mechanisms that operate in intact HbH cells which attempt to balance globin chain production. In alpha thalassemia trait cells, no such controls appeared to be active and globin chain synthesis was directly proportional to the amount of alpha and beta globin mRNA in the cells.

Decreased alpha globine mRNA in nucleated red cell precursors in alpha thalassemia

The alpha thalassemias are associated with a decrease in alpha chain synthesis. Hemoglobin H (HbH) disease is a moderately severe form of alpha thalassemia characterized by the production of 5%--20% of HbH, while alpha thalassemia trait is a milder form of alpha thalassemia. In two patients with HbH disease, the ratio of alpha chain synthesis to beta chain synthesis (alpha/beta ratio) was decreased in both bone marrow cells and reticulocytes. When isolated mRNA from bone marrow cells and reticulocytes was translated in a heterologous cell-free system, the alpha/beta ratios were lower than the intact cell ratios. These findings were confirmed by hybridization of the mRNA of both marrow cells and reticulocytes using purified alpha and beta cDNA probes. In the intact cells of two patients with alpha thalassemia trait, the alpha/beta ratios were also decreased and were similar in marrow cells and reticulocytes. Cell-free studies of translatable mRNA also demonstrated decreased alpha/beta ratios, but, unlike the HbH studies, the cell-free alpha/beta ratios were similar to the intact cell ratios. One hybridization study utilizing peripheral blood mRNA had an alpha/beta ratio consistent with the cell-free ratios. These results indicated that, in both HbH disease and alpha thalassemia trait, there was decreased alpha globin mRNA present in both nucleated red cell precursors and reticulocytes. In addition, the data suggested that there may be translational mechanisms that operate in intact HbH cells which attempt to balance globin chain production. In alpha thalassemia trait cells, no such controls appeared to be active and globin chain synthesis was directly proportional to the amount of alpha and beta globin mRNA in the cells.

Growth of Human Bone Marrow in Liquid Culture

Abstract A system for the cultivation of normal and malignant human bone marrow cells in liquid medium is described. The apparatus used is an in vitro diffusion chamber in which cells grow in suspension and upon a dialysis membrane. Proliferation and maturation of normal granulocytes and macrophages were sustained in culture for several weeks in the absence of exogenous stimulating substances. Cell replication was documented by a rise in viable counts, 3H-thymidine incorporation, and labeled mitoses. The entire maturational sequence of the granulocyte and macrophage series was identified in culture, and the cells were characterized by light and electron microscopy, cytochemical properties, phagocytic ability, and the presence of surface receptors for IgG. Nucleated red cell precursors were observed in some cultures for up to 11 days. Malignant cells from patients with various hematologic neoplasms were also successfully cultured. With this system human myeloma cells were maintained in culture for up to 3 wk on primary explant, and continued to synthesize immunoglobulin. The in vitro diffusion chamber technique permits the cultivation of normal human bone marrow cells in liquid medium and provides a convenient means for studying normal and neoplastic hematopoietic cell differentiation and function in short-term culture.

Heterozygous Beta Thalassemia: Balanced Globin Synthesis in Bone Marrow Cells

In two patients with heterozygous beta thalassemia the rates of synthesis of the alpha and beta chains of hemoglobin were equal in nucleated red cell precursors, although beta chain synthesis was reduced in peripheral blood reticulocytes. This finding suggests a relative instability of beta chain messenger RNA in beta thalassemia.

Haem synthetase activity of human blood cells

Specimens of human blood were selected from patients with a wide variety of diseases, which contained an absolute enrichement of different cell types. Increased haem synthetase activity was associated with increases in white blood cells or nucleated red cell precursors but not with increases in reticulocytes. The cell sap obtained from specimens of blood containing increased numbers of nucleated red cell precursors shows haem synthetase activity and this is greatly stimulated by ether. The significance of these findings is discussed with respect to the cellular distribution of the enzyme in normal and pathological blood.

Mitotic Indices of Human Bone Marrow Cells. III. Duration of Some Phases of Erythrocytic and Granulocytic Proliferation Computed from Mitotic Indices

Abstract 1. Weighted average mitotic time of human bone marrow cells is estimated. Mitotic time is about 0.75 hours in red cell precursors and about 0.58 hours in neutrophil precursors. 2. Estimated weighted average DNA-synthesis time is about 9 hours in red cell precursors and about 24 hours in neutrophil precursors. 3. Upper and lower limits for compartment transit times of proerythroblasts, basophilic normoblasts, polychromatic normoblasts, myeloblasts, promyelocytes and myelocytes are presented (table 2). 4. Total mass of nucleated red cell precursors is estimated at 3.5 x 109/kg. and total mass of neutrophils and their precursors in the marrow at 8.8 x 109/kg.