Cellular interactions in erythroblastic islands in long-term bone marrow cultures, as studied by time-lapse video

Long-term bone marrow cultures (LTBMC) are readily converted from the usual granulopoietic to erythropoietic production by the addition of anemic mouse serum (AMS). The "statics" of proliferation and maturation, previously shown by ultrastructural methods to closely mirror the in vivo situation, were studied dynamically using a time-lapse video system. Several cell pedigrees were followed, but the most complete series showed three successive divisions and subsequent enucleations in the progeny of three synchronously mitotic cells observed in the culture; this is indicative of a five division sequence in the erythron. As in erythroblastic islets observed in marrow in vivo, the striking synchrony of maturation was maintained in vitro. Furthermore, when some of the erythroid progeny became displaced to other macrophages, the synchrony, which was maintained by the original erythroid group on the original erythroblastic islet macrophage, was lost. Time-lapse video, which is inexpensive to run and can be maintained in continuous recording for many weeks, is an ideal technique for recording both erythroid cell pedigrees, and the initial events leading to the formation of an erythroblastic islet in vitro after stimulation with AMS.     

Association between leukemic erythroid progenitors and bone marrow macrophages

Previous ultrastructural investigations have shown that the erythroblastic island is composed of erythroblasts at different stages of maturation which are intimately associated with a central macrophage. However, it is still unclear at which stage of erythroid differentiation this interaction occurs, mainly because of the lack of purified populations of normal erythroid progenitors [erythroid colony-forming units (CFU-E) and erythroid burst-forming units (BFU-E)] and early precursor cells (proerythroblasts) and because of our limited knowledge of their ultrastructural characteristics. In the present work we analyzed the ultrastructure of CFU-E enriched from normal human bone marrow by avidin-biotin immune rosetting and leukemic blasts of erythroid origin from two patients. Normal and leukemic CFU-Es were defined as glycophorin A (GPA)-negative blasts, devoid of rhopheocytosis, containing some ferritin molecules, either free in the cytoplasm or associated with theta-granules (theta-Gr) in the Golgi zone. Peroxidase activity was detected in the endoplasmic reticulum of these blasts. A preproerythroblast stage was identified, which corresponded to an intermediate phenotype with few GPA sites and rhopheocytosis. In contrast to hemoglobin synthesis, which was absolutely dependent on the presence of erythropoietin (Epo) during culture for 24 hours, ferritin molecules accumulated in the absence of Epo. Interestingly, leukemic CFU-E-like blasts were always in contact with bone marrow macrophages and adhesion between these cell types resisted mechanical dissociation. This result suggests that erythroid progenitors may be part of the erythroblastic island. The mechanisms involved in erythroblast-macrophage binding are still unknown, but the expression by macrophages and erythroid progenitors of receptors for fibronectin and thrombospondin (TSP), as well as their respective ligands in the case of macrophages, suggests that these molecules could be involved in the formation of the erythroblastic island.     

Erythropoietin and phagocyte activity of central macrophage of the erythroblastic island in rats

Administration of erythropoietin enhanced the intensity of the phagocytosis in the erythroblastic island. The effect was more evident in case of normal erythropoiesis. The phagocyte activation of the central macrophage erythroblastic island was shown to depend on the condition of erythropoiesis in the erythroblastic island crown.     

Cell-cell interactions and erythropoiesis

The erythroblastic island, a distinct anatomical unit consisting of a central macrophage surrounded by a ring of erythroblasts, is a key feature of erythropoiesis in the bone marrow. While a number of functional sequelae for the interaction between the erythroblasts and macrophage have been suggested, much remains to be learned. We suggest that this interaction may play a role in regulated assembly of membrane proteins during erythroid differentiation.     

Klf1 affects DNase II-alpha expression in the central macrophage of a fetal liver erythroblastic island: a non-cell-autonomous role in definitive erythropoiesis

A key regulatory gene in definitive erythropoiesis is the erythroid Kruppel-like factor (Eklf or Klf1). Klf1 knockout (KO) mice die in utero due to severe anemia, while residual circulating red blood cells retain their nuclei. Dnase2a is another critical gene in definitive erythropoiesis. Dnase2a KO mice are also affected by severe anemia and die in utero. DNase II-alpha is expressed in the central macrophage of erythroblastic islands (CMEIs) of murine fetal liver. Its main role is to digest the DNA of the extruded nuclei of red blood cells during maturation. Circulating erythrocytes retain their nuclei in Dnase2a KO mice. Here, we show that Klf1 is expressed in CMEIs and that it binds and activates the promoter of Dnase2a. We further show that Dnase2a is severely downregulated in the Klf1 KO fetal liver. We propose that this downregulation of Dnase2a in the CMEI contributes to the Klf1 KO phenotype by a non-cell-autonomous mechanism.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

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.     

the effect of colonystimulating macrophage factor on activity of the nuclear organisers in central macrophages of the cultures of the bone marrow erythroblastic islands

Erythroblastic islands of the bone marrow are morpho-functional units of erythropoiesis. The functional state of erythroblastic islands' cells of the bone marrow was for the first time defined by estimation of activity of the nuclear organisers of the central macrophages in the erythroblastic islands cultivated for 24 hrs in presence of various doses of the colony-stimulating macrophage factor. The findings indicate that increased doses of the colonystimulating macrophage factor was accompanied by a respective enhancement of the activity of nucleolar organisers in central macrophages of erythroblastic islands.     

Absence of erythroblast macrophage protein (Emp) leads to failure of erythroblast nuclear extrusion

In mammals, the functional unit for definitive erythropoiesis is the erythroblastic island, a multicellular structure composed of a central macrophage surrounded by developing erythroblasts. Erythroblast-macrophage interactions play a central role in the terminal maturation of erythroblasts, including enucleation. One possible mediator of this cell-cell interaction is the protein Emp (erythroblast macrophage protein). We used targeted gene inactivation to define the function of Emp during hematopoiesis. Emp null embryos die perinatally and show profound alterations in the hematopoietic system. A dramatic increase in the number of nucleated, immature erythrocytes is seen in the peripheral blood of Emp null fetuses. In the fetal liver virtually no erythroblastic islands are observed, and the number of F4/80-positive macrophages is substantially reduced. Those present lack cytoplasmic projections and are unable to interact with erythroblasts. Interestingly, wild type macrophages can bind Emp-deficient erythroblasts, but these erythroblasts do not extrude their nuclei, suggesting that Emp impacts enucleation in a cell autonomous fashion. Previous studies have implicated the actin cytoskeleton and its reorganization in both erythroblast enucleation as well as in macrophage development. We demonstrate that Emp associates with F-actin and that this interaction is important in the normal distribution of F-actin in both erythroblasts and macrophages. Thus, Emp appears to be required for erythroblast enucleation and in the development of the mature macrophages. The availability of an Emp null model provides a unique experimental system to study the enucleation process and to evaluate the function of macrophages in definitive erythropoiesis.     

Dual biological function of a T-cell-derived murine immunoenhancing factor.

Murine enhancing factor (MEF), derived from the culture fluid of mixtures of histoincompatible spleen cells, was found to have two apparently different, but perhaps closely related, biological activities. First, MEF can functionally replace T cells in nonspecifically augmenting the anti-sheep erythrocyte plaque-forming cell response of T-cell-depleted, mouse splenic B-cell cultures. Second, the mediator acts similarly to colony stimulating factor from human urine in promoting the formation of colony-forming units (CFU) in soft agar bone marrow cell cultures. This latter function of MEF was manifest in the absence of detectable increases in the level of incorporation of [³H]thymidine by cultured bone marrow cells. Morphologically, the cells comprising the CFU were macrophage-like in appearance. The data suggest that MEF may function as a differentiation signal for the maturation of antigen-activated B lymphocytes into immunoglobulin-secreting cells, as well as for the modulation of hematopoietic or granulopoietic macrophage stem cells into mature, functional macrophages.     

Hybrid Model of Erythropoiesis

A hybrid model of cell dynamics is presented. It is illustrated by model examples and applied to study erythropoiesis (red blood cell production). In this approach, cells are considered as discrete objects while intra-cellular proteins and extra-cellular biochemical substances are described with continuous models. Spatial organization of erythropoiesis occurring in specific structures of the bone marrow, called erythroblastic island, is investigated.     

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.     

KINETICS OF DEVELOPMENT OF EMBRYONIC ERYTHROID CELLS

Embryonic erythropoiesis is an intrinsically non-steady-state process. A method of non-steady-state analysis is employed to approximately determine the kinetics of maturation of embryonic erythroid cells during the hepatic phase of erythropoiesis in the mouse. It appears from this analysis that embryonic erythroid cells have significantly shorter maturation times than their adult counterparts. In the embryo, there is insufficient time for more than three divisions between the proerythroblast and the orthochromatic erythroblast.     

Long-term survival of murine erythroid progenitors in long-term bone marrow culture and stromal cell culture: differentiation in peritoneal diffusion chamber culture

Bone marrow cells of normal and cytosine-arabinoside (Ara-C) treated C57B1 mice were cultured in primary long-term culture (LTBMC) for a period of eight weeks. Non-adherent cells collected at weekly culture feedings consisted of neutrophils, macrophages and megakaryocytes. These were transferred into a) secondary peritoneal diffusion chamber cultures (DC) and b) secondary stromal cell cultures (SCC) first, and then into tertiary DC cultures. While in LTBMC and SCC there was no evidence of erythropoiesis, many erythroid colonies developed in DC cultures. It appears that undifferentiated erythroid progenitors may have a long survival in LTBMC and SCC devoid of erythropoietin and then differentiate in vivo in DC cultures in host mice without specific erythropoietic stimuli. Terminal differentiation and maturation of erythroid progenitors occurs to a limited extent in conventional DC cultures. The large number of erythroid colonies in DC observed in the present study could be due to increased sensitivity of undifferentiated erythroid progenitors from LTBMC to physiological levels of Epo in host mice of DC.     

Absence of erythroblastic islands in plasma clot culture and their possible reconstitution after clot lysis

Ultrastructural studies of erythroid colonies derived from human peripheral blood and growing in plasma clot culture have confirmed the absence of a macrophage inside each colony of erythroblasts. However, when macrophages and erythroblasts were liberated from the semisolid media by clot lysis, these two types of cells rapidly acquired intimate contacts, suggesting the reconstitution of any erythroblastic island. The possible significance of this phenomenon is discussed.     

Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.

In this study, we have mapped the onset of hematopoietic development in the mouse embryo using colony-forming progenitor assays and PCR-based gene expression analysis. With this approach, we demonstrate that commitment of embryonic cells to hematopoietic fates begins in proximal regions of the egg cylinder at the mid-primitive streak stage (E7.0) with the simultaneous appearance of primitive erythroid and macrophage progenitors. Development of these progenitors was associated with the expression of SCL/tal-1 and GATA-1, genes known to be involved in the development and maturation of the hematopoietic system. Kinetic analysis revealed the transient nature of the primitive erythroid lineage, as progenitors increased in number in the developing yolk sac until early somite-pair stages of development (E8.25) and then declined sharply to undetectable levels by 20 somite pairs (E9.0). Primitive erythroid progenitors were not detected in any other tissue at any stage of embryonic development. The early wave of primitive erythropoiesis was followed by the appearance of definitive erythroid progenitors (BFU-E) that were first detectable at 1-7 somite pairs (E8.25) exclusively within the yolk sac. The appearance of BFU-E was followed by the development of later stage definitive erythroid (CFU-E), mast cell and bipotential granulocyte/macrophage progenitors in the yolk sac. C-myb, a gene essential for definitive hematopoiesis, was expressed at low levels in the yolk sac just prior to and during the early development of these definitive erythroid progenitors. All hematopoietic activity was localized to the yolk sac until circulation was established (E8.5) at which time progenitors from all lineages were detected in the bloodstream and subsequently in the fetal liver following its development. This pattern of development suggests that definitive hematopoietic progenitors arise in the yolk sac, migrate through the bloodstream and seed the fetal liver to rapidly initiate the first phase of intraembryonic hematopoiesis. Together, these findings demonstrate that commitment to hematopoietic fates begins in early gastrulation, that the yolk sac is the only site of primitive erythropoiesis and that the yolk sac serves as the first source of definitive hematopoietic progenitors during embryonic development.     

Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models.

Hematopoietic cells of various lineages are organized in distinct cellular architectures in the bone marrow hematopoietic compartment (BMHC). The homogeneous Kroghian model, which deals only with a single cell type, may not be sufficient to accurately describe oxygen transfer in the BMHC. Thus, for cellular architectures of physiological significance, more complex biophysical-transport models were considered and compared against simulations using the homogeneous Kroghian model. The effects of the heterogeneity of model parameters on the oxygen tension (pO(2)) distribution were examined using the multilayer Kroghian model. We have also developed two-dimensional Kroghian models to simulate several cellular architectures in which a cell cluster (erythroid cluster) or an individual cell (megakaryocyte or adipocyte) is located in the BMHC predominantly occupied by mature granulocytes. pO(2) distributions in colony-type cellular arrangements (erythroblastic islets, granulopoietic loci, and lymphocytic nodules) in the BMHC were also evaluated by modifying the multilayer Kroghian model. The simulated results indicate that most hematopoietic progenitors experience low pO(2) values, which agrees with the finding that low pO(2) promotes the expansion of various hematopoietic progenitors. These results suggest that the most primitive stem cells, which are located even further away from BM sinuses, are likely located in a very low pO(2) environment.