Dynamics of human erythroblast enucleation

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.

Red Blood Cell Generation by Three-Dimensional Aggregate Cultivation of Late Erythroblasts

“…4D). 10 These data indicate that erythroid cells are extremely vulnerable to media flow-induced shear stress and that they should be cultured without agitation, at least during the enucleation phase. Therefore, we confirmed that 3D culture with scaffolds can be achieved using a rotating plate or a stirring spinner flask, but shear stress needs to be minimized.…”

Dimethyl fumarate increases fetal hemoglobin, provides heme detoxification, and corrects anemia in sickle cell disease

“…Differentiation of erythroid progenitors from SCD PBMC. PBMC isolated from the whole blood of adult and pediatric SCD donors (Supplemental Methods 1 and Supplemental Table 1) were cultured and differentiated prior to exposure to test compounds using a 2-phase culture system (47). Compound incubations were performed either beginning from day 1 of culture or from day 8 of the culture, and cells were harvested for HbF induction by droplet digital PCR (ddPCR), flow cytometry, and HPLC analysis.…”

Signaling and cytoskeletal requirements in erythroblast enucleation

“…[1][2][3] After enucleation, the reticulocytes are released into the bloodstream, whereas the pyrenocytes expose apoptotic signals on their surface, resulting in engulfment and degradation by the central macrophage of the erythroblastic island. 3,4 The mechanism of enucleation has been a long-standing matter of investigation and remains controversial.Early electron microscopy studies suggested that enucleation may be analogous to cytokinesis, pointing to the resemblance of the cytoplasmic constriction between incipient reticulocyte and pyrenocyte to the cleavage furrow at the equatorial region of a mitotic cell. 5 Koury et al demonstrated with electron and immunofluorescent microscopy, using mouse splenocytes infected with the anemia-inducing strain of Friend virus, that F-actin bundles concentrate at the furrow behind the extruding nucleus, and that cytochalasin-D, a potent inhibitor of actin polymerization, inhibits enucleation.…”