One potential explanation is autonomous

One potential explanation is autonomous regulation among erythroid phorbol myristate acetate through direct contact, as they reside in clusters within the bone marrow (Neu et al., 2003). However, no secreted factors regulating erythropoiesis or signal transduction between mature erythroid cells have been identified. Fas-FasL is known to be involved in the apoptotic control of pro-erythroblasts via mature polychromatic erythroblasts (Allen and Dexter, 1982; Manwani and Bieker, 2008). However, the role of this ligand and receptor has not been studied with regard to the final maturation or enucleation of mature erythroid cells. Erythroblast macrophage protein (Emp) is known to be involved in macrophage–erythroblast attachment and possibly in homotypic erythroblast attachment, but there have been no data establishing this link (Soni et al., 2006). Additionally, there have been no reports that erythropoiesis is controlled via an auto-paracrine system (Manwani and Bieker, 2008). Elucidation of the diverse systems regulating erythroid cells is important not only for understanding basic and pathologic erythropoiesis, but also for developing strategies for generating human RBCs in vitro for transfusion. The severe blood supply shortage and the ongoing need for safe blood have underscored efforts to develop RBCs in vitro (Chasis, 2006; Sato et al., 1979). However, the process of obtaining final RBC products is still inefficient, mainly due to a low enucleation rate and low viability in the final maturation step, particularly in stromal-cell-free conditions. The enucleation process, where immature erythroid cells extrude their nuclei, is a critical step towards those cells becoming reticulocytes and finally mature RBCs (Sato et al., 1979). Our knowledge regarding which mechanisms regulate these final processes and why enucleation and viability in vitro are so markedly low is lacking. In order to elucidate the missing mechanisms of the regulatory system of erythroid cells and to identify methods to increase RBC production, we focused on the bone marrow microenvironment, in which mature erythroid cells reside in compact clusters. In conventional culture densities (less than 50% confluency) (Miharada et al., 2006), the erythroid cells repel each other in suspension due to strong negative charges on their surface membranes, and do not easily attach as other adhesive cells do. To mimic bone marrow conditions, homogenous erythroid cells were cultured at a high density to increase the chance of contact; the results revealed a significantly increased enucleation rate and viability. This prompted us to hypothesize that there are adhesion factors that control terminal maturation and enucleation among erythroid cells in the absence of macrophages. To identify candidate genes affecting erythroid cell attachment and enucleation, we searched for genes showing marked changes before and after enucleation via microarray. By comparing the candidate gene profiles of erythroid cells cultured at different densities, several density-evoked signals were demonstrated for the first time. Two of these genes are deleted in liver cancer-1 (DLC-1), which has never been studied in primary cells or erythroid cells, and intercellular adhesion molecule-4 (ICAM-4), which is an erythroid cell adhesion protein. Furthermore, we found that ICAM-4 and the Rho-GTPase-activating protein DLC-1 are binding counterparts in erythroid cells. Addition of recombinant ICAM-4 protein to culture media of low-density cells reproduced the results observed at higher densities, such as enhanced viability and enucleation. In this study, we demonstrate that ICAM-4 is related to the erythropoietic niche between erythroid cells via both direct contact and through its secreted form. This study provides new mechanisms by which autonomous control of erythropoiesis in vivo occurs and could improve in vitro RBC production for clinical use.
Materials and methods
Results
Discussion Cell-to-cell communication through the attachment of erythroid cells to macrophages is critical for erythroid cell differentiation and maturation (Chasis and Mohandas, 2008). However, during the maturation period, erythroid cells at the outer edge of an erythroblastic island progressively lose their adhesion ligands to the adjacent macrophage (Mohandas and Chasis, 2010). Moreover, some erythroid cells exist outside of these islands and form clusters by themselves (Chasis, 2006). There have been some studies on the interactions between immature erythroid cells (via Emp) (Soni et al., 2006) and between immature and mature erythroid cells (via ICAM-4-VLA-4 and Fas-FasL proteins) (Chasis, 2006; Manwani and Bieker, 2008; Soni et al., 2006; Hermand et al., 2000; Spring et al., 2001). However, the ligands related to communication within erythroid cells in the late stages of maturation (polychromatic and orthochromatic erythroblasts) have not been studied, nor has any mechanism for autonomous regulation of enucleation and viability been shown. Published reports on this topic are rare, probably due to difficulties in culturing mature human erythroblasts without feeder stromal cells.