Transferrin binding capacity as a marker of differentiation and maturation of rat erythroid cells fractionated by counter current distribution in aqueous polymer two-phase systems

Rat bone marrow cell populations, containing different proportions of erythroid cells, have been fractionated by counter-current distribution in the non-charge-sensitive dextran/polyethyleneglycol two-phase systems on the basis of hydrophobic cell surface properties. Cell fractions with a low distribution coefficient, which contain non-erythroid cells and early erythoblasts, showed a low transferrin binding capacity and a low haemoglobin/cell ratio whereas cell fractions with a high distribution coefficient, which contain intermediate-late erythroblasts and mature red cells, showed an elevated transferrin binding capacity and the highest haemoglobin/cell ratio. These results support transferrin binding capacity as a good marker parameter for the erythroid bone marrow cell differentiation and maturation processes.     

Changes in enzyme activities involved in the degradation of 1,3-bisphosphoglycerate during erythropoiesis in rat bone marrow

Rat bone marrow cells have been fractionated by density gradient in Percoll. Differential counting of erythroid cells, haemoglobin concentration and bisphosphoglycerate mutase and phosphoglycerate kinase activities have been determined in cellular fractions. As shown by means of a statistical approach, an increase in bisphosphoglycerate mutase activity and a slight decrease in phosphoglycerate kinase activity is found in erythroid cells as their haemoglobin content increases. Our results suggest that there is a synthesis of 2,3-bisphosphoglycerate during the erythropoietic process which parallels the synthesis of haemoglobin.     

Biosynthesis of rat hemoglobin fractions under normal and increased erythropoiesis]

Studies of erythroid elements of hematopoietic organs and peripheric blood and bone marrow cell populations fractionated by precipitation in an albumin density gradient and synchronized by actinomycin D revealed that the rate of synthesis of individual haemoglobin fractions in the course of erythropoiesis is changed. The proliferating cells are characterized by a predominant accumulation of haemoglobin in fractions with beta c-and in the maturing cells--with beta b-chains. The radioactivity in the whole population of hematopoietic organs under phenylhydrazine anemia is mainly increased in the beta c-chains, whereas under the effects of erythropoietic factors (erythropoietin, animal sera)--that of beta b-chains is increased. In early erythroblasts the erythropoietic factors activate the formation of beta c-chains and in late ones--that of beta b-chains. Similar time dependence was observed in case of synthesis and activation of alpha b- and alpha a-chains. The specific cell responses in the erythroid series of bone marrow, spleen and blood to the effects of erythropoietic factors and anemia were established. The molecular mechanisms involved in the switch-over of the synthesis of various haemoglobin chains are discussed.     

Terminal differentiation of yolk-sac erythroid cells of the Syrian hamster in vitro

Summary The developmental fate of Syrian hamster yolk-sac (primitive) erythroid cells was examined in vitro. Highly purified yolk-sac erythroid cells at the polychromatophilic stage, obtained from the peripheral blood of embryos at day 10 of gestation, showed morphological and biochemical changes in our modified semi-solid culture system. Several morphological changes observed in the primitive erythroid cell cultures, such as nuclear condensation, approach of nuclei to the periphery of cells, development by cells of an extended pear-like shape, enucleation, and an increase in haemoglobin content, were quite similar to those of the terminal differentiation of fetal liver or adult bone marrow (definitive) erythroid cells. In addition, the transition of molecular species of haemoglobin from the embryonic to the fetal/adult pattern was also observed in our culture system. Thus we provide evidence, by the in vitro culture of yolk-sac erythroid cells, that primitive erythroid cells undergo terminal differentiation in a manner similar to that of definitive erythroid cells.     

Antianaemic properties of ayurvedic drugs, raktavardhak, punarnavasav and navayas louh in albino rats during phenylhydrazine induced haemolytic anaemia

Single injection of phenylhydrazine[PH] reduced the number of RBC and haemoglobin content; decreased myeloid; erythroid cell ratio in bone marrow and increased Cathepsin D activity in spleen of rats. Ayurvedic drugs raktavardhak, punarnavasav and navayas louh recovered the number of RBC and haemoglobin content and raised myeloid: erythroid cell ratio and normalised cathepsin D activities by counteracting the action phenyl hydrazine. The results confirm the claims of ayurveda that these drugs possess the potency to cure anaemia through protection of RBCs from haemolysis and simultaneously lowering cathepsin D activities from the spleen.     

The effect of haemorrhage on the cell populations of the thymus and bone marrow in wild starlings (Sturnus vulgaris)

Summary Following the withdrawal of blood from the brachial vein of adult wild starlings (Sturnus vulgaris) changes in the cell populations within the bone marrow and thymus were observed over an eight day period. The packed cell volume, haemoglobin content and reticulocyte count of the peripheral blood was determined before and after haemorrhage.The maximum effect of the haemorrhage was observed in the bone marrow after four days when the population of small lymphocytes, and basophilic erythroid precursors were reduced to less than 1%. At the same time the percentage of another line of erythroid cells increased to 68%. This second erythroid lineage was the major erythroid line in the thymus, and again maximum representation occurred at 4 days post haemorrhage. After this the thymus became predominantly lymphoid and started to increase in size.The two erythroid lines are described and their status with regard to avian thrombocytes is also discussed.The peripheral blood had not attained the pre-haemorrhagic values for reticulocyte counts by eight days although the packed cell volumes and haemoglobin contents were similar.I would like to thank Dr. Peter Ward of the Institute of Terrestrial Ecology for help in obtaining the starlings. Thanks are also due to the staff of the Anatomy Department of St. Thomas's Hospital Medical School, and in particular Mr. Watson. This and other work on the thymus is possible due to the support of the Research (Endowments) Committee of St. Thomas's Hospital     

Comparative distribution of marrow CFU-e and CFU-gm progenitors in different anatomic sites in the dog

The interpretation of marrow cloning activity, particularly in serial cultures, is greatly influenced by the reproducibility of the collected marrow samples. In order to establish whether bone marrow cloning activities and precision of the cloning assays are influenced by the site of bone marrow collection in the dog, we studied the incidence of marrow erythroid (CFU-e) and granulocyte-macrophage (CFU-gm) progenitor cells in the iliac crest, sternum, vertebrae, femur, and humerus, using microplasma clot and soft agar culture systems. Marrow samples obtained from the femur and humerus revealed consistently higher cell concentrations than those from the iliac crest, vertebrae, or sternum. Those aspirated from the sternum and vertebrae had lower cell concentrations and were less reproducible. Statistical analysis revealed no significant differences in the incidence of marrow CFU-e and CFU-gm progenitor cells between the femur, humerus, iliac crest or vertebrae. With multiple sampling, the marrow cloning efficiency was consistent and reproducible within the individual dogs. We conclude that the distribution of CFU-e and CFU-gm is comparable throughout the active marrow in the dog and that these sites may be used interchangeably for multiple quantitative analysis of marrow hematopoietic progenitor cells.     

Morphogenetic shift of hemoglobin types in the ontogeny of rats

The embryonic haemoglobin represented by 4 fractions was shown to form in the nucleate erythroid cells of the yolk sac. The dynamics of their quantitative changes was determined in 10 to 14 days old embryos. Anuclear red cells of the subsequent haemopoietic centers synthesize the heterogenous haemoglobin system (fractions 1--6). It includes components such as HbF (fractions 5, 6) the maximal contents of which is timed to a later embryonic stage. Fractions 3 and 4 constitute HbA haemoglobin. These fractions were shown to differ by their resistance against acids and to be unequally distributed among the red blood cells. In the blood of foetuses and phenyl hydrasine anemic animals the amount of cells with predominance of fractions 5 and 6 increases. Under these conditions the level of alkali resistant haemoglobin also increases.     

Biological effect of human erythropoietin in transgenic mice]

Transgenic mice were obtained inheriting the human erythropoietin gene under the control of viral regulatory elements. The reliable difference in haematocrit, the content of haemoglobin and percentage of reticulocytes in peripheral blood were not revealed. The level of serum erythropoietin in transgenic mice is several fold higher than in control mice. The increased pool of erythroid cells was observed in the bone marrow of transgenic mice, especially of normoblasts (3-fold) and reticulocytes (4,5-fold).     

Characteristics of the pathways of erythroid cell differentiation in birds in anemia

A comparative study has been made of erythroid cell development pathways in the peripheral blood of pigeons during severe, moderate and weak forms of anaemia. Three modes of erythrocyte formation from bone marrow precursor are described: 1. A reserve erythropoiesis--the principal process during severe anaemia; the bone marrow precursors are basophylic erythroblasts which are reversibly blocked in phase G2 of the cell cycle; in results the rapid, increase of erythrocyte population above the normal level, although the cells have 25-30 per cent deficiency in haemoglobin content. 2) A mode of erythropoiesis, whose precursors are proliferating polychromatophylic erythroblasts; this is the principal mode of erythropoiesis at the moderate anaemia, leading to restoration of the normal quantity of erythrocytes with a normal haemoglobin content. 3) A mode of erythropoiesis with proliferating orthochromatic erythroblasts being precursors (which do not divide normally); this is the principal mode during the weak anaemia to result in a slow restoration of the number of erythrocytes with an excess in haemoglobin content. It is shown that regulation of the restoration processes during anaemia are characterized by a specific combination of cell proliferation and differentiation.     

Effect of blood transfusion on erythropoiesis of homozygotic beta-thalassemia patients

The influence of blood transfusion on erythropoiesis (bone marrow erythroblasts, peripheral blood erythroblasts and reticulocytes) has been studied in 20 non splenectomized homozygous beta thalassaemia patients aged 3 to 16 years and in 10 splenectomized patients aged 8 to 24 years affected with the same disease. The number of reticulocytes was the same in the two groups but the number of erythroblasts in the splenectomized group was higher than in the other group. There was no correlation between the erythroblasts and the reticulocytes of the peripheral blood on one hand and the haemoglobin level proceeding from the same sample on the other hand. In the non splenectomized group of patients, an inverse relationship was found between the percentage of bone marrow erythroblasts and the mean annual haemoglobin level (r = -0.71; p less than 0.01). These results demonstrate the effect of blood transfusion on the erythroid cell line in homozygous beta thalassaemia and the delay between the transfusions and the medullary erythroblastic response.     

Ultrastructural Aspects of Erythropoietic Differentiation in Long-term Bone Marrow Culture

Long-term liquid cultures of mouse bone marrow produce stem cells (CFU-S) and differentiated granulocytes for many months. Addition of AMS (anaemic mouse serum) to the cultures almost entirely eliminates the granulopoietic activity and stimulates erythropoiesis, with full erythroid maturation and the production of adult haemoglobin. Ultrastructural analsysis of in situ fixed material reveals the cell shape and surface morphology of the erythroid maturation series, and the generation of erythroblastic islands in vitro. Each erythroblastic island consists of one or more synchronously maturing cohorts of erythroid cells undergoing four or five divisions between proerythroblast and normoblast. Each island is centered on a macrophage, which interacts with the developing erythroid population in several ways. Expelled nuclei are phagocytosed by the macrophage, which also has large areas of closely apposed membrane with the erythroid cells, gap junctions, and possible reciprocal vesicular activity. Changes in the adherent layer (stromal cells) also occur with the transition from granulopoiesis to erythropoiesis. There is a reduction in the endothelial cell cover, and mobilisation of lipid from the granulopoietic associated apidocytes.     

Hereditary erythroblastic anaemia in the laboratory mouse

Hereditary erythroblastic anaemia was found in a homozygous mutant (hea/hea) of an inbred strain CFO, which originated from noninbred CF#1 mice from Carworth Inc. This newly-described anaemia is inherited as an autosomal recessive and is lethal at 15-25 days of age. Erythrocytes of anaemic mice show striking polychromasia, anisocytosis, and poikilocytosis. One of the most marked features of this anaemia is the presence of large numbers of nucleated cells, mainly orthochromatophilic erythroblasts and myeloid cells, in the circulation. They also include immature erythroid and myeloid cells. Many naked nuclei appear on smears from circulating blood of anaemic infants. Erythrocytes, haematocrit percentage, and haemoglobin content of older anaemic infants were only about 50% of those of the normal. Formation of erythroid, myeloid cells, and megakaryocytes in the bone marrow seems to be progressively affected by mutant alleles in the anaemic infants.     

Differential analysis of animal bone marrow by flow cytometry.

A simple procedure was developed for rapid analysis of animal bone marrow by flow cytometry using the lipophilic cationic dye 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)]. The batch process allows differentiation of bone marrow cells into lymphoid, erythroid, and myeloid populations and enables classification of erythroid and myeloid cells into proliferating and maturing subpopulations. From these data, myeloid:erythroid (M:E) ratios and maturation indices for erythroid and myeloid cells (EMI and MMI, respectively) can be derived. This procedure provides the opportunity to analyze bone marrow quantitatively and offers distinct advantages to current manual methods in terms of simplicity, throughput, and reproducibility. The method has been tested successfully using marrow from Wistar rats, B6C3F1 mice, beagle dogs, and cynomolgus monkeys. This technique facilitates the evaluation of bone marrow samples taken from preclinical safety studies or from animal colonies of large size.     

Stilbestrol as a factor modifying the toxicity of ekatin for the pharaoh quail (Coturnix coturnix pharaoh). I. Young birds

1. The noted fall of the erythrocyte count, haemoglobin and haematocrit levels after stilbestrol indicate that this hormone disturbs erythropoiesis, probably by depressing the maturation of erythroid cells in the bone marrow,2. The reaction of the erythrocyte system of birds intoxicated with Ekatin indicates that this pesticide has a haemolytic influence on the morphotic elements of the blood.3. Estrogenisation enhances the action of Ekatin on the erythrocyte system by as it seems increasing the susceptibility of the red blood cells to the haemolytic action of this pesticide.4. The high heterophilic leucocytosis in birds receiving stilbestrol is evidence that this hormone causes by an increased release of corticoids, hyperplasia of the granulopoietic tissue in the bone marrow and depresses the marrow barrier for heterophils.5. Changes in the leucocyte system of quails intoxicated with Ekatin indicate a stressogenic action of this poison.6. The reaction of the latter system to Ekatin administered after estrogenisation seems to be an indication that stilbestrol reduces the susceptibility of the quails to the stressogenic action of the pesticide.     

Stilbestrol as a factor modifying the toxicity of Ekatin for the Pharaoh quail (Coturnix coturnix Pharaoh). I. Young birds.

1. The noted fall of the erythrocyte count, haemoglobin and haematocrit levels after stilbestrol indicate that this hormone disturbs erythropoiesis, probably by depressing the maturation of erythroid cells in the bone marrow. 2. The reaction of the erythrocyte system of birds intoxicated with Ekatin indicates that this pesticide has a haemolytic influence on the morphotic elements of the blood. 3. Estrogenisation enhances the action of Ekatin on the erythrocyte system by as it seems increasing the susceptibility of the red blood cells to the haemolytic action of this pesticide. 4. The high heterophilic leucocytosis in birds receiving stilbestrol is evidence that this hormone causes by an increased release of corticoids, hyperplasia of the granulopoietic tissue in the bone marrow and depresses the marrow barrier for heterophils. 5. Changes in the leucocyte system of quails intoxicated with Ekatin indicate a stressogenic action of this poison. 6. The reaction of the latter system to Ekatin administered after estrogenisation seems to be an indication that stilbestrol reduces the susceptibility of the quails to the stressogenic action of the pesticide.     

Differentiation of stem cells from different parts of the hematopoietic system of adult thymectomized CBA mice stimulated with thymarin or cortexine

Essential differences were detected in differentiation of GFUs from bone marrow and peripheral blood. It was shown that as a result of thymectomy of adult animals the ability of bone marrow CFUs to form granulocytic colonies decreased and that of splenic CFUs to form erythroid colonies increased. The immunostimulating low-molecular-weight polypeptides, thymarin and cortexine , normalized the differentiation of CFUs from bone marrow and spleen but interfered with the formation of erythroid colonies from CFUs of peripheral blood of thymectomized mice.     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.