Stem Cell Research (2013) 10, 442–453 Available online at www.sciencedirect.com www.elsevier.com/locate/scr Autonomous control of terminal erythropoiesis via physical interactions among erythroid cells Hye Sook Choi a, Eun Mi Lee a,HyunOkKimb, Moon-Il Park c, Eun Jung Baek a,⁎ a Department of Laboratory Medicine, School of Medicine, Hanyang University, Seoul, Republic of Korea b Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Republic of Korea c Department of Obstetrics and Gynecology, School of Medicine, Hanyang University, Seoul, Republic of Korea Received 27 November 2012; received in revised form 25 January 2013; accepted 2 February 2013 Available online 13 February 2013 Abstract In vitro erythropoiesis has been studied extensively for its application in the manufacture of transfusable erythrocytes. Unfortunately, culture conditions have not been as effective as in vivo growth conditions, where bone marrow macrophages are known to be a key regulator of erythropoiesis. This study focused on the fact that some erythroblasts are detached from macrophages and only contact other erythroblasts. We hypothesized that additional factors regulate erythroblasts, likely through either physical contact or secreted factors. To further elucidate these critical factors, human erythroblasts derived from cord blood were cultured at high density to mimic marrow conditions. This growth condition resulted in a significantly increased erythroid enucleation rate and viability. We found several novel contact-related signals in erythroblasts: intercellular adhesion molecule-4 (ICAM-4) and deleted in liver cancer-1 (DLC-1). DLC-1, a Rho-GTPase-activating protein, has not previously been reported in erythroid cells, but its interaction with ICAM-4 was demonstrated here. We further confirmed the presence of a secreted form of human ICAM-4 for the first time. When soluble ICAM-4 was added to media, cell viability and enucleation increased with decreased nuclear dysplasia, suggesting that ICAM-4 is a key factor in contact between cells. These results highlight potential new mechanisms for autonomous control of erythropoiesis. The application of these procedures to erythrocyte manufacturing could enhance in vitro erythrocyte production for clinical use. © 2013 Elsevier B.V. All rights reserved. Introduction niches called erythroblastic islands plays a critical role in controlling the maturation, differentiation, and enucleation Definitive erythropoiesis occurs in the bone marrow, where a of erythroid cells (Bessis, 1958; Chasis and Mohandas, 2008). set of complex interactions encourages red blood cell (RBC) It is known that the main route of signal transport between production. The central macrophage in the erythropoietic macrophages and erythroid cells is physical contact via several ligands and receptors (Rhodes et al., 2008; Soni et al., 2006; Spring and Parsons, 2000; Telen, 2000). Various adhesion Abbreviations: Hb, hemoglobin; ICAM-4, intercellular adhesion molecule-4; DLC-1, deleted in liver cancer-1. molecules responsible for maintaining this cell-to-cell contact ⁎ Corresponding author at: Department of Laboratory Medicine, within the erythroid islands have been identified, mainly School of Medicine, Hanyang University, 471-701, Gyomun-dong, through the relationship between macrophages and erythroid Guri-si, Gyeonggi-do, Republic of Korea. Fax: +82 31 560 2166. cells. However, not every erythroid cell is attached to a E-mail address: [email protected] (E.J. Baek). macrophage (Rhodes et al., 2008), and some erythroid cells 1873-5061/$ - see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scr.2013.02.003 Erythropoietic niche for RBC production in vitro 443 contact only other erythroid cells. Erythroid cells seem to be adhesion protein. Furthermore, we found that ICAM-4 and the able to autonomously regulate erythropoiesis, albeit with Rho-GTPase-activating protein DLC-1 are binding counterparts reduced efficiency (Chasis and Mohandas, 2008). Based on in erythroid cells. Addition of recombinant ICAM-4 protein to these observations, we speculated that there must be factors culture media of low-density cells reproduced the results regulating erythroid cells that are not based on direct contact observed at higher densities, such as enhanced viability and with macrophages. enucleation. One potential explanation is autonomous regulation among In this study, we demonstrate that ICAM-4 is related to erythroid cells through direct contact, as they reside in the erythropoietic niche between erythroid cells via both clusters within the bone marrow (Neu et al., 2003). However, direct contact and through its secreted form. This study no secreted factors regulating erythropoiesis or signal trans- provides new mechanisms by which autonomous control of duction between mature erythroid cells have been identified. erythropoiesis in vivo occurs and could improve in vitro RBC Fas-FasL is known to be involved in the apoptotic control of production for clinical use. pro-erythroblasts via mature polychromatic erythroblasts (Allen and Dexter, 1982; Manwani and Bieker, 2008). However, Materials and methods 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 Isolation of CD34 cells and culture for knowntobeinvolvedinmacrophage–erythroblast attachment erythroid differentiation and possibly in homotypic erythroblast attachment, but there have been no data establishing this link (Soni et al., 2006). Cord blood (CB) was collected from healthy pregnant women Additionally, there have been no reports that erythropoiesis is after obtaining their written informed consent. CD34+ cells controlled via an auto-paracrine system (Manwani and Bieker, were isolated from the CB using the EasySep CD34 isolation 2008). kit (StemCell Technologies, Vancouver, Canada). The cul- Elucidation of the diverse systems regulating erythroid cells ture methods are described in a previous report (Baek et al., is important not only for understanding basic and pathologic 2009, 2010). Several cytokines were added to stroma- and erythropoiesis, but also for developing strategies for generating serum-free media in a stage-specific manner to induce human RBCs in vitro for transfusion. The severe blood supply CD34+ cells to differentiate to a purely erythroid lineage in shortage and the ongoing need for safe blood have underscored the absence of feeder cells and serum/plasma. Live cells efforts to develop RBCs in vitro (Chasis, 2006; Sato et al., were counted by trypan blue staining. 1979). However, the process of obtaining final RBC products is still inefficient, mainly due to a low enucleation rate and Cell lines and culture conditions low viability in the final maturation step, particularly in stromal-cell-free conditions. The enucleation process, where The human erythroleukemic cell line K562 was purchased from immature erythroid cells extrude their nuclei, is a critical step a Korean cell line bank. This line was grown in RPMI 1640 towards those cells becoming reticulocytes and finally mature medium (Gibco, Invitrogen, Carlsbad, CA) supplemented with RBCs (Sato et al., 1979). Our knowledge regarding which 100 IU/ml penicillin, 100 mg/ml streptomycin, and 10% fetal mechanisms regulate these final processes and why enucle- bovine serum (FBS; Gibco) in a 5% CO2 incubator at 37 °C. ation and viability in vitro are so markedly low is lacking. In order to elucidate the missing mechanisms of the Cell-density-dependent growth analysis 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 The cells were found to expand too rapidly to maintain a compact clusters. In conventional culture densities (less than constant density. Therefore, cells were maintained at a low 50% confluency) (Miharada et al., 2006), the erythroid cells cell density from the beginning of culture until day 17. On day repel each other in suspension due to strong negative charges 17 of culture, polychromatic and orthochromatic erythroblasts on their surface membranes, and do not easily attach as other were seeded to reach 50% and 100% confluences in order to adhesive cells do. To mimic bone marrow conditions, homog- mimic the packed cellular environment of bone marrow (Fig. 1A). Fifty percent and 100% confluences were designated enous erythroid cells were cultured at a high density to increase 6 2 the chance of contact; the results revealed a significantly as optimal (2.5×10 cells/1 ml/2 cm )orsupraoptimal 6 2 – increased enucleation rate and viability. (5.0×10 cells/2 ml/2 cm ), respectively (Figs. 1B D). The This prompted us to hypothesize that there are adhesion cells were kept at the same cell density in the media to factors that control terminal maturation and enucleation maintain the same level of nutrients and waste. Cultures were among erythroid cells in the absence of macrophages. To observed under phase contrast microscopy to confirm their identify candidate genes affecting erythroid cell attachment confluence, which was determined by cells flocking to the and enucleation, we searched for genes showing marked center of the plate. Unless otherwise stated, media were changes before and after enucleation via microarray. By replenished every other day and cell morphology was verified – comparing the candidate gene