Abstract
One characteristic of the erythroid terminal differentiation is hemoglobin expression and enucleation. In the present study, by using real-time cultured Friend Virus Anemia-inducing (FVA) cells, scanning electron microscopy (SEM), immunofluorescence combined with laser scanning confocal microscopy (LSCM), the FVA cells induced by Erythropoietin (EPO) for were used for: 1) to study the shape and proportion of cells at different differentiating stages, enucleation process, the formation of blood islands, and engulfment of nuclei by macrophages in real time, 2) to quantitatively analyze erythroid cell surface markers including CD71 and Ter119, and cytoskeletal-associated proteins (stathmin, septin8 and RBBP4). The results of real-time monitoring of enucleation indicated that it took about 7 to 8 hours to extrude the nuclei from karyopyknosis (polychromatic erythroblasts). It further showed that the macrophages engulfed the expelled erythroid nuclei. SEM showed a variety of shapes of the nascent reticulocytes. In the process of erythroid differentiation, expressiosn of both the transferring receptors CD71 and Ter119 were higher than that of adult blood cells, whereas, cytoskeletal-associated proteins (stathmin, septin8 and RBBP4) decreased gradually. Therefore, systematic observation of the process of differentiation and enucleation will provide a deeper understanding of cellular and molecular mechanisms for erythroid differentiation and carcinogenesis.
Highlights
Erythroid differentiation is a complex process, and the terminally differentiated cells are characterized by shrinkage of the cells, karyopyknosis, enucleation, and differentiating into mature red blood cells
Mice were injected with Friend virus anemia-inducing (FVA), and the erythroid cells from their spleen were synchronized as proerythroblast and retained the differentiation characteristics of Colony Forming Unit-Erythroid (CFU-E) cells [1,2,3]
In the early stages of erythroid differentiation, the macrophage functions as a “nurse” cell for forming blood islands [7,8,9], the direct contact of macrophages and erythroid cells improves the proliferation of erythroid cells [10,11,12,13], and provides conditions for erythrocyte formation [14], the nuclei are engulfed by macrophages through related molecules on the nucleus that interact with macrophages [15]
Summary
Erythroid differentiation is a complex process, and the terminally differentiated cells are characterized by shrinkage of the cells, karyopyknosis, enucleation, and differentiating into mature red blood cells. There were many changes in the cell morphology, haemoglobin accumulation, the expelled nuclei were wrapped by small amount of cytoplasm and plasma membrane, and formed reticulocytes [5,6] in the process of differentiation. In the early stages of erythroid differentiation, the macrophage functions as a “nurse” cell for forming blood islands [7,8,9], the direct contact of macrophages and erythroid cells improves the proliferation of erythroid cells [10,11,12,13], and provides conditions for erythrocyte formation [14], the nuclei are engulfed by macrophages through related molecules on the nucleus that interact with macrophages [15]. There is still a lack of systematic study about cell morphology at various differentiation stages, surface markers, changes in cell number, the time needed for enucleation and polymorphism of the nascent reticulocytes
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