Murine fetal liver macrophages bind developing erythroblasts by a divalent cation-dependent hemagglutinin

During mammalian development the fetal liver plays an important role in hematopoiesis. Studies with the macrophage (M phi)-specific mAb F4/80 have revealed an extensive network of M phi plasma membranes interspersed between developing erythroid cells in fetal liver. To investigate the interactions between erythroid cells and stromal M phi, we isolated hematopoietic cell clusters from embryonic day-14 murine fetal liver by collagenase digestion and adherence. Clusters of erythroid cells adhered to glass mainly via M phi, 94% of which bound 19 +/- 11 erythroblasts (Eb) per cell. Bound Eb proliferated vigorously on the surface of fetal liver M phi, with little evidence of ingestion. The M phi could be stripped of their associated Eb and the clusters then reconstituted by incubation with Eb in the presence of divalent cations. The interaction required less Ca++ than Mg++, 100 vs. 250 microM for half-maximal binding, and was mediated by a trypsin-sensitive hemagglutinin on the M phi surface. After trypsin treatment fetal liver M phi recovered the ability to bind Eb and this process could be selectively inhibited by cycloheximide. Inhibition tests showed that the Eb receptor differs from known M phi plasma membrane receptors and fetal liver M phi did not bind sheep erythrocytes, a ligand for a distinct M phi hemagglutinin. We propose that fetal liver M phi interact with developing erythroid cells by a novel nonphagocytic surface hemagglutinin which is specific for a ligand found on Eb and not on mature red cells.     

Proliferation kinetics of bone marrow cells in congenital dyserythropoietic anemia type II

The proliferation kinetics of erythropoiesis and of myelopoiesis have been studied in a case of congenital dyserythropoietic anemia type II (HEMPAS) by means of quantitative 14-C autoradiography, Feulgen cytophotometry and 59-Fe ferrokinetics. Increased total erythropoietic activity and ineffective erythropoiesis was demonstrated by ferrokinetics. Quantitative 14-C autoradiography showed a generally delayed proliferation rate of erythroid cells, most evident in the polychromatic compartment. A deficiency of cell production of 25% was detected among the polychromatic erythroblasts. Part of this fraction is represented by cells still capable of passing to the successive stages of maturation. We conclude that only part of the deficiency of cell production in the polychromatic compartment represents real cell destruction. Most of the measured ineffectiveness is confined to later stages of maturation, such as orthochromatic erythroblasts and marrow reticulocytes.     

Endothelial cells create a hematopoietic inductive microenvironment preferential to erythropoiesis in the mouse spleen

The spleen is an erythropoietic organ in mouse. To reconstruct a microenvironment essential for erythropoiesis in vitro, the stroma (MSS31) cell line had been established from a newborn mouse spleens. MSS31 cells exhibited properties of endothelial cells: (a) the cells showed the activity to uptake acetylated low-density lipoprotein (Ac-LDL) and (b) the cells can form a capillarylike structure by a phenotypic modulation in collagen matrices. MSS31 cells selectively supported the proliferation and differentiation of the erythroid progenitor cells by direct cell-to-cell contact in a semisolid medium in the presence of erythropoietin. These layers also supported erythrocyte maturation and enucleation of erythroblasts. This suggests that spleen endothelial cells are a new type of stromal cell with erythropoietic stimulation activity and may have a critical function in the hemopoietic inductive microenvironment of the mouse spleen.     

Carbon monoxide induced erythroid differentiation of K562 cells mimics the central macrophage milieu in erythroblastic islands

Growing evidence supports the role of erythroblastic islands (EI) as microenvironmental niches within bone marrow (BM), where cell-cell attachments are suggested as crucial for erythroid maturation. The inducible form of the enzyme heme oxygenase, HO-1, which conducts heme degradation, is absent in erythroblasts where hemoglobin (Hb) is synthesized. Yet, the central macrophage, which retains high HO-1 activity, might be suitable to take over degradation of extra, harmful, Hb heme. Of these enzymatic products, only the hydrophobic gas molecule - CO can transfer from the macrophage to surrounding erythroblasts directly via their tightly attached membranes in the terminal differentiation stage.Based on the above, the study hypothesized CO to have a role in erythroid maturation. Thus, the effect of CO gas as a potential erythroid differentiation inducer on the common model for erythroid progenitors, K562 cells, was explored. Cells were kept under oxygen lacking environment to mimic BM conditions. Nitrogen anaerobic atmosphere (N₂A) served as control for CO atmosphere (COA). Under both atmospheres cells proliferation ceased: in N₂A due to cell death, while in COA as a result of erythroid differentiation. Maturation was evaluated by increased glycophorin A expression and Hb concentration. Addition of 1%CO only to N₂A, was adequate for maintaining cell viability. Yet, the average Hb concentration was low as compared to COA. This was validated to be the outcome of diversified maturation stages of the progenitor''s population.In fact, the above scenario mimics the in vivo EI conditions, where at any given moment only a minute portion of the progenitors proceeds into terminal differentiation. Hence, this model might provide a basis for further molecular investigations of the EI structure/function relationship.     

Classification of erythroblastic islets of the bone marrow and the study of their cellular composition

Cell composition of erythroblastic islands (EI), normal and at the phase of stimulation and inhibition of erythropoiesis at modelling of heat adaptation has been studied on hemopoietic tissue of 108 rats. In the hemopoietic tissue, together with EI of the 1st, 2nd and 3d classes of maturity, that have in their composition correspondingly up to 8, from 9 up to 16 and above 16 nuclei-containing erythroid cells, there are involutive and reconstructing EI. The involutive EI are presented as nuclei-containing erythroid cells, that are not able to divide and are at late stages of differentiation: polychromatophilic and oxyphilic normoblasts, as well as reticulocytes. The reconstructing EI have in their composition both erythroid cells, that are not able to divide, and erythroid cells (pro-, erythroblasts and basophilic normoblasts), that are at early stages of differentiation. Application of the EI classification suggested, takes into account the rate, with which the erythroid cell-predecessors are drawn into erythropoiesis and intensity of erythroid differentiation. Therefore, it is possible to obtain earlier and more exact data on the state of hemopoiesis in comparison with traditional hematological methods.     

TRAIL promotes a pro-survival signal in erythropoietin-deprived human erythroblasts through the activation of an NF-kB/IkBalpha pathway

The biological activity of TNF-related apoptosis inducing ligand (TRAIL) was analyzed in primary human erythroblasts derived from mononuclear cells of blood donors, kept in culture in the presence of 20 percent foetal calf serum, growth factors (EPO, SCF, IL-3) and glucocorticoids (10-6 M dexamethasone, 10-6 M oestradiol) or under growth factor and serum starvation. In the presence of growth factors and serum, primary erythroblasts showed a differential expression of TRAIL-Receptors (Rs) at various degrees of maturation and responded to TRAIL treatment with a mild cytotoxicity. On the other hand, in the absence of serum and growth factors, TRAIL treatment unexpectedly up-regulated TRAIL-R4 decoy receptor and promoted erythroblast survival. The concomitant activation of NF-kB/IkB survival pathway was detected with Western blotting and immunofluorescence procedures and confirmed by experiments performed with SN50, a pharmacological inhibitor of the NF-kB/IkB pathway. Our study indicates that TRAIL has a twofold activity on erythroid lineages: it induces a mild erythroid cell cytotoxicity in the presence of serum and growth factors, while it promotes erythroid cell survival through the activation of the NF-kB/IkB pathway under starvation conditions.     

Transitional change of colony stimulating factor requirements for erythroid progenitors.

The course of the differentiation and proliferation of the human erythroid burst-forming units (BFU-E) to colony-forming units (CFU-E) was directly investigated using a combination of highly purified BFU-E, a liquid culture system, and the following clonal assay. Highly purified human blood BFU-E with a purity of 45-79% were cultured in liquid medium with recombinant human erythropoietin (rEP) and recombinant human interleukin-3 (rIL-3) to generate more differentiated erythroid progenitors. The cultured cells were collected daily for investigating the morphology, the increment in the number of cells and the clonality. Ninety percent of purified BFU-E required not only rEP but also rIL-3 for clonal development. By 7 days of liquid culture, the total cell number increased 237 +/- 20-fold above the starting cells, while erythroid progenitors increased 156 +/- 74-fold. As the incubation time in liquid culture increased, the cells continuously differentiated in morphology. Replating experiments with rEP combined with or without rIL-3 showed the following: 1) The number of erythroblasts that were part of erythroid colonies decreased with accompanying erythroid progenitor differentiation and proliferation. 2) As the incubation time in liquid culture increased, erythroid progenitors had a graded loss of their dependency on rIL-3 and a complete loss of dependency was observed after 3 days of liquid culture. At that time 85% of the erythroid progenitors gave rise to colonies of more than 100 erythroblasts which were equivalent to mature BFU-E. These studies provide a quantitative assessment of the loss of IL-3 dependency by BFU-E and indicate that the size of the generated erythroid colonies and their IL-3 requirement correlate with the erythroid differentiated state.     

Stimulatory effects of androgens on normal children's bone marrow in culture: effects on BFU-E, CFU-E, and uroporphyrinogen I synthase activity

We studied the effect of natural and synthetic androgens on children's erythropoietic precursor cells in culture. Cultures of normal marrow were carried out according to a miniaturized methylcellulose method in the presence of erythropoietin. We then evaluated the effects of testosterone, nortestosterone, fluoxymesterone and etiocholanolone (10(-9)-10(-6) M) on erythroid colony-forming units (CFU-E) and burst-forming units (BFU-E). Androgen-induced growth of erythroid progenitors was quantified by directly scoring colonies and by a biochemical determination of the uroporphyrinogen I synthase activity (UROS). We observed a significant increase (p less than or equal to 0.05) in the number of CFU-E and BFU-E and in the UROS activity of derived colonies in the presence of androgens (10(-8) or 10(-7)M). This microculture assay could be useful not only to study the effect of androgens on erythroid progenitor cells in culture, but also to predict the best androgenic treatment of anemia in children and adults.     

小鼠胎肝红系细胞在分化过程中形态学观察及时序性表达分析

小鼠胎肝是小鼠发育早期主要的造血器官,红系细胞在胎肝造血过程中形态特征和组成成分等方面发生了明显变化。根据红系细胞体积的变化,利用Countstar细胞计数仪对小鼠E12．5-E17．5胎肝中直径8-14岬细胞进行数量统计,再结合观测到的红系细胞的形态特征和血红蛋白表达量的不同,将E9．5．E17．5胎肝中的细胞分为10类。统计结果显示,随着胎肝造血系统的发育,哺乳类红系细胞在终末分化时出现细胞体积减小、细胞核固缩、排核和血红蛋白表达量增加等时序性变化。红系细胞表面特异标志Terll9和CD71在EryD中高表达而在成体骨髓细胞和外周血细胞中表达较低的结果表明胎肝中红系细胞具有较高的分化能力。这些数据为研究红系分化、克隆红系分化相关基因及探讨红白血病发生的机制提供了理论依据。     

Membrane-bound glycoconjugates of fetal mouse erythropoietic cells with special reference to phagocytosis by hepatic macrophages

Using lectin and colloidal iron (CI) stainings in combination with neuraminidase digestion, glycoconjugates on the surface of erythropoietic cells of the yolk sac and liver in fetal mice were examined. Fetal hepatic macrophages were capable of distinguishing between phagocytozed and non-phagocytozed erythroid elements as described in our previous study. Marked differences between these two elements could be ultrahistochemically detected on their cell surface. The phagocytozed elements, such as nuclei expelled from erythroblasts and degenerating primitive erythroblasts, faintly bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a weak peanut agglutinin (PNA) binding. In contrast, erythroblasts at various maturation stages, erythrocytes and normal primitive erythroblasts heavily bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a moderate PNA binding. No differences in binding of either concanavalin agglutinin,Ricinus communis agglutinin-I or PNA were noted between phagocytozed and non-phagocytozed erythroid elements. Desialylation appears to be one of the most important signs for the recognition mechanism of fetal macrophage phagocytosis. During maturation of hepatic erythroblasts, sialic acid changes its affinity forLimax flavus agglutinin from strong to weak, and soybean agglutinin binding sites disappear at the basophilic erythroblast stage. Glycoconjugates on polychromatophilic erythroblasts acquire similar compositions to those of erythrocytes.     

Effects of Hypoxia on Heterotypic Macrophage Interactions

The role of macrophages in modulating the systemic response to hypoxia and oxidative stress is emerging from basic biological processes, such as the regulation of red blood cell production, and from analysis of tumor progression, as a key factor determining whether cells survive, proliferate or differentiate under micro-environmental pressures. Our recent work identified a novel role for macrophages in promoting expansion of erythroid progenitors in vitro while confirming previous data that macrophages are not required for red cell enucleation. This work emerged from analyses of hypoxia and cell death in the Rb null fetal liver where we demonstrated that defects in erythropoietic islands were due to deterioration in the fetal liver microenvironment that disrupted heterotypic interactions of macrophages with erythroblasts and not to intrinsic defects in Rb null macrophages. The significance of these findings for the effect of hypoxia on macrophage interactions and activity during tumor progression is also discussed.     

Cyclic AMP phosphodiesterase activity during differentiation of rabbit erythroid bone marrow cells.

Changes in the activity of cyclic AMP phosphodiesterase during differentiation of rabbit bone marrow erythroid cells were investigated. The cells were separated by velocity sedimentation at unit gravity into six fractions corresponding to different stages of development: proerythroblasts, basophilic cells, polychromatic cells, early orthochromatic and late orthochromatic cells and reticulocytes. Cyclic AMP phosphodiesterase was found to be very active in the most immature cells, the proerythroblasts, which also have the highest content of cyclic AMP. After differentiation into basophilic erythroblasts, a 4-fold decrease in cyclic AMP phosphodiesterase activity was observed. In these cells the amount of cyclic AMP was about 80% lower than that in proerythroblasts. In polychromatic cells a further drop in phosphodiesterase activity occurred. After the final cell division the enzyme activity was very low and the levels of cyclic AMP in the early and late orthochromatic cells remained constant. Kinetic studies demonstrated a heterogeneity of erythroid cell cyclic AMP phosphodiesterase: high affinity, low-Km (5.5 X 10(-6) M) and low affinity, high-Km (0.1 X 10(-3) M) enzymes were found. The phosphodiesterase activity was dependent on the presence of Mg2+ and was activated by Ca2+ at low Mg2+ concentrations (1 mM). The changes in cyclic AMP phosphodiesterase activity during differentiation and maturation of erythroid cells suggest the possible importance of this enzyme in the physiological control of cyclic AMP concentrations in developing erythroblasts. The loss of cyclic AMP phosphodiesterase activity after cessation of cell division supports the concept of the significance of the final cell division in erythroblast differentiation.     

Production of IL-10, TNF-alpha, IFN-gamma, TGF-beta1 by different populations of erythroid cells derived from human embryonal liver

It has previously been determined that erythroid cells of mice are capable of expressing such cytokines as interleukin (IL) 1 alpha and beta, IL-4, IL-6, interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta). It has been shown that glycophorin A(+) (GlA(+)) and antigen erythroblasts (AG-EB(+)) (both human erythroid cells of embryonic origin) are also capable of producing a series of cytokines such as IL-1 beta, IL-2, IL-4 and IL-6. The aim of this work was to study the capacity of erythroid cells from human embryonic liver to produce such cytokines as IFN-gamma, TGF-beta1, tumour necrosis factor alpha (TNF-alpha) and IL-10. The erythroid cells were isolated by means of antibodies specific to erythroblasts (GlA and AG-EB), as well as those from single erythroid colonies. The production level of some cytokines varies insignificantly under the action of erythropoietin (Epo) and quantitatively differs in GlA(+) and AG-EB(+) erythroid cells. Hence, the erythroid cells express IFN-gamma, TGF-beta1, TNF-alpha and IL-10. The erythroid cells could be involved through the production of these cytokines in the regulation of such processes as self-renewal, proliferation and differentiation of cells of other blood-forming sites.     

Synthesis and expression of cell-surface glycoproteins during chick erythroid differentiation

Abstract. A panel of monoclonal antibodies to differentiation antigens on avian erythroid cells was used to study the reprogramming of protein synthesis during erythroid differentiation at the molecular level. This panel detected five distinct cell-surface glycoproteins on immature leukemic erythroblasts, all of which were initially synthesised as smaller intracellular precursors. Two distinct in vitro differentiation systems (erythroblasts transformed by ts mutants of the erb-B and sea retroviral oncogenes, in which the synchronous terminal differentiation of CFU-E-like precursors is induced by simple elevation of temperature) were used to study cell-surface expression and the biosynthesis of each protein during erythroid cell maturation. For four glycoproteins, both cell-surface expression and biosynthesis decreased between the erythroblast and erythrocyte stages, although with widely different time courses. The fifth glycoprotein, which is reticulocyte specific on normal erythroid progenitors and is aberrantly expressed in onco-gene-transformed erythroblasts, rapidly disappeared shortly after differentiation induction but was then re-expressed on reticulocytes with the same time course as that seen during normal erythroid differentiation. This indicates that ts erb-B- and ts sea -transformed erythroblasts revert to a normal precursor phenotype before undergoing temperature-induced differentiation.     

DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU-E-derived colonies, late CFU-E-derived colonies and mixed hematopoietic (CFU-GEMM-derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time-dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.     

Morphology of interactions of the haematopoietic microenvironment with the haematopoietic cells in erythroid spleen colonies

The morphology of the interactions of the cells of the haematopoietic microenvironment with haematopoietic cells was studied in exogenous erythroid spleen colonies formed in mice, on the fifth and eighth day after their irradiation with a lethal dose of gamma rays and bone marrow transplantation. The characteristic type of stromal cell interacting with less mature cells of the erythroid series was a dark, branching reticular cell. The typical structural interaction of the reticular cells with erythroblasts was the formation of very long, fine cytoplasmic processes by the reticular cells. The processes were in close contact with the erythroblasts and formed a three-dimensional network stretching long distances from the nucleus of the reticular cells. Other cells of the haematopoietic microenvironment, in contact chiefly with poly- and ortochromic erythroblasts, were macrophages and the two together formed typical erythroblastic islands. In places, the macrophages and erythroblasts formed close, firm contacts by means of their cytoplasmic membranes. These morphological observations support the conception that close functional cooperation exists between the cells of the haematopoietic microenvironment and the haematopoietic cells in differentiation and proliferation processes in the haematopoietic tissue of spleen colonies.     

The effect of bone marrow depletion on prostaglandin E-producing suppressor macrophages in mouse spleen

The i.p. injection of Corynebacterium parvum (CP) into CBA/J mice effected increases in macrophage colony-forming cells (M-CFC) when spleen cells were cultured with L cell culture filtrate as a source of colony-stimulating factor. Significant increases in phagocytic macrophages (M phi) with Fc receptors for IgG2a and IgG2b immune complexes were additionally noted among the spleen cells in these mice. These M phi effectively inhibited Con A-induced lymphocyte proliferation, probably reflecting a 10-fold increase above normal controls in prostaglandin E to 47 ng/3 X 10(6) spleen cells/ml. To determine whether the suppressor M phi are immediate derivatives of splenic M-CFC, we tried to induce suppressor M phi by the injection of CP into mice depleted of bone marrow M-CFC by the earlier administration of the bone-seeking isotope, 89Sr. This procedure reduced M-CFC in the bone marrow to less than 1% of normal for more than 30 days. Monocytes in the blood fell to 5% of normal by day 10 and were 30% on day 30. Levels of resident peritoneal M phi showed relatively little change in this period. By contrast, splenic M-CFC increased to 20-fold higher than the "cold" 88Sr controls. CP-induced suppressor M phi activity, however, was sharply reduced in 89Sr marrow-depleted mice on day 10, despite the striking increase in M-CFC. There was a threefold increase in the number of phagocytic M phi binding IgG2a immune complexes, with no significant increase in IgG2b binding M phi. The kinetics of recovery of suppressor M phi activity showed that on days 20, 30, and 50 after 89Sr injection the activities reached 20%, 30%, and 70% of the "cold" control, respectively, and correlated with the recovery of significant levels of M-CFC in the bone marrow. Taken together, these observations suggest that splenic M-CFC are not an immediate source of PGE-suppressor M phi in vivo. It appears more likely that the CP-inducible suppressor M phi, in particular, originate from radiosensitive bone marrow cells or require for differentiation a microenvironment provided by bone marrow cells. The data also suggest that the expression of the Fc gamma 2b receptor and of suppressor activity by CP-induced splenic M phi are related phenomena.     

Synergy between erythropoietin and stem cell factor during erythropoiesis can be quantitatively described without co-signaling effects

Synergistic interactions between cytokines underlie developmental processes fundamental to tissue and cellular engineering. However, a mechanistic understanding of the cell-specific and population-mediated effects is often lacking. In this study, we have investigated the synergistic generation of erythroid cells in response to erythropoietin (EPO) and stem cell factor (SCF). We have used a quantitative approach to determine if the effects of EPO and SCF superpose in a supra-additive fashion on the cell proliferation rate or on the death rate, suggesting a contribution from a joint cytokine effect (co-signaling). Primary mouse bone marrow hematopoietic cells and the stem cell-like FDCP-mix cell line were used to investigate the effects of EPO and SCF (individually or in combination) on erythroid output. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cell-division tracking and mathematical modeling were used to measure cell type-specific proliferation and death rates. We observed a significant synergistic effect of EPO and SCF on the net generation of benzidine positive (erythroid) colony-forming cells, CD71++ (early erythroblasts) cells and TER-119+ (late erythroblasts and reticulocytes) cells in culture. When the observed increases in cell number were decomposed into proliferation and death rates, the cytokines were shown to act independently at different stages of erythroid development; SCF promoted the early proliferation of primitive cells, while EPO primarily promoted the survival of differentiating erythroid progenitor cells. Our analysis demonstrates that EPO and SCF have distinct and predominantly sequential effects on erythroid differentiation. This study emphasizes the necessity to separate proliferation rates from death rates to understand apparent cytokine synergies.     

In vitro apoptotic cell death during erythroid differentiation

Erythropoiesis occurs in bone marrow and it has been shown that during in vivo erythroid differentiation some immature erythroblasts undergo apoptosis. In this regard, it is known that immature erythroblasts are FasL- and TRAIL-sensitive and can be killed by cells expressing these ligand molecules. In the present study, we have investigated the cell death phenomenon that occurs during a common unilineage model of erythroid development. Purified CD34+ human haemopoietic progenitors were cultured in vitro in the presence of SCF, IL-3 and erythropoietin. Their differentiation stages and apoptosis were followed by multiple technical approaches. Flow cytometric evaluation of surface and intracellular molecules revealed that glycophorin A appeared at day 3-4 of incubation and about 75% of viable cells co-expressed high density glycophorin A (Gly(bright)) and adult haemoglobin at day 14 of culture, indicating that this system reasonably recapitulates in vivo normal erythropoiesis. Interestingly, when mature (Gly(bright)) erythroid cells reached their higher percentages (day 14) almost half of cultured cells were apoptotic. Morphological studies indicated that the majority of dead cells contained cytoplasmic granular material typical of basophilic stage, and DNA analysis by flow cytometry and TUNEL reaction revealed nuclear fragmentation. These observations indicate that in vitro unilineage erythroid differentiation, as in vivo, is associated with apoptotic cell death of cells with characteristics of basophilic erythroblasts. We suggest that the interactions between different death receptors on immature basophilic erythroblasts with their ligands on more mature erythroblasts may contribute to induce apoptosis in vitro.