Abstract
How human erythroblasts enucleate remains obscure, and some investigators suspect the effect of mechanical forces on enucleation in vitro. We determined the dynamics of the enucleation process of highly purified human erythroblasts and whether enucleation can occur without external mechanical forces. Highly purified human CD34+ cells were cultured in liquid phase with interleukin-3, stem cell factor and erythropoietin (EPO) for 7 days and the generated erythroblasts were replaced in the same medium with EPO alone. In some experiments, the enucleating cells were processed without centrifugation and pipette aspiration to avoid physical stress and were directly observed by differential interference contrast (DIC) microscopy. Enucleation initiated at day 12 and the enucleation ratio (percent of enucleated reticulocytes in total cells) reached a maximum at day 14 with a value of 63 ± 7%. The direct observation by DIC microscopy showed 61 ± 9% of enucleation ratio at day 14. The human erythroblasts enucleated without contact with macrophage. The time required for enucleation was 8.4 ± 3.4 min. The enucleation rate was 1.16 ± 0.42%/h at day 12 and then decreased with a time dependent manner. The expelled nucleus was connected to the reticulocyte through plasma membrane and associated cytoskeletal elements, and spontaneous separation of the extruded nucleus from reticulocyte was extremely rare. In conclusion, human erythroblasts enucleate in a relatively short period without contact with macrophages, but nascent reticulocytes fail to completely separate from nuclei in the absence of macrophages, unless some physical force is applied to them.
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Acknowledgments
Supported in part by Grants-in-Aid (17659286) and funds from the “Global Center of Excellence Program (COE)” of the Ministry of Education, Science, Technology, Sports, and Culture of Japan, and a research grant from the Idiopathic Disorders of Hematopoietic Organs Research Committee of the Ministry of Health, Labour and Welfare of Japan. Author contributions: M. H. designed and performed experiments, analyzed data, wrote manuscript; Y. M. G., K. Saito, H. W., A. K., N. F., N. T., T. T., W. N. Y. T. and T. S. analyzed and interpreted data, helped write manuscript, K. Sawada designed experiments, interpreted data, wrote manuscript. The authors are grateful to Dr. Mark J Koury for helpful discussions and comments on this paper and to H. Kataho, E. Kobayashi and E. Kikuchi (Internal Medicine III, School of Medicine, Akita University) for their valuable technical assistance.
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Supplementary movie 1
Day 9 cells that stained with SYTO21 were cultured with EPO alone until day 13. The cells were immobilized on the Petri dish using anti-GPA antibody and were directly observed using DIC microscopy, every 60 seconds for 60 minutes, at 37°C. (MOV 534 kb)
Supplementary movie 2
Direct DIC imaging of day 12 cells that were not immobilized on the Petri dish, every 60 seconds for 71 minutes, at 37°C, by transmitted light. Arrow indicates the erythroblast in enucleation process. (MOV 335 kb)
Supplementary movie 3
Direct DIC imaging of day 15 cells that were not immobilized on the Petri dish, every 60 seconds for 64 minutes, at 37°C, by transmitted light. Reticulocyte attached the expelled nucleus (a-d). Erythroblasts in the prolonged stages of enucleation with an active cytoplasmic movement (e-h). Erythroblasts lost cytoplasmic elasticity becoming a thin and stretched during the period when enucleation was achieved in other cells that underwent successful enucleation. (i-l). The macrophage (M) engulfed the erythroblasts that failed to enucleate (o), nucleus connected to reticulocyte (n and m) and a reticulocyte (p), but released the reticulocyte (p). White and yellow arrows indicate thread-like and pseudopodium-like structures between the cells, respectively. (MOV 2,007 kb)
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Hebiguchi, M., Hirokawa, M., Guo, YM. et al. Dynamics of human erythroblast enucleation. Int J Hematol 88, 498–507 (2008). https://doi.org/10.1007/s12185-008-0200-6
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DOI: https://doi.org/10.1007/s12185-008-0200-6