▪ Abstract Erythropoiesis is the process in which new erythrocytes are produced. These new erythrocytes replace the oldest erythrocytes (normally about one percent) that are phagocytosed and destroyed each day. Folate, vitamin B₁₂, and iron have crucial roles in erythropoiesis. Erythroblasts require folate and vitamin B₁₂ for proliferation during their differentiation. Deficiency of folate or vitamin B₁₂ inhibits purine and thymidylate syntheses, impairs DNA synthesis, and causes erythroblast apoptosis, resulting in anemia from ineffective erythropoiesis. Erythroblasts require large amounts of iron for hemoglobin synthesis. Large amounts of iron are recycled daily with hemoglobin breakdown from destroyed old erythrocytes. Many recently identified proteins are involved in absorption, storage, and cellular export of nonheme iron and in erythroblast uptake and utilization of iron. Erythroblast heme levels regulate uptake of iron and globin synthesis such that iron deficiency causes anemia by retarded production rates with smaller, less hemoglobinized erythrocytes.

Erythroblasts adhere to central macrophages forming erythroblastic islands in hematopoietic tissues, but the function of these islands is not understood. Murine erythroblastic islands were reconstituted in vitro with macrophages and developmentally synchronous proerythroblasts. Erythroblasts cocultured with macrophages proliferated 3-fold greater than erythroblasts cultured alone. Direct contact with the macrophages was necessary for this enhanced erythroblast proliferation, which resulted from decreased transit time in the G0/G1 phase of cell cycle. Increased erythroblast proliferation in erythroblastic islands occurred over a wide range of erythropoietin concentrations and was the result of a mechanism different from the antiapoptotic effect of erythropoietin. Erythroblasts adherent to macrophages had slightly delayed enucleation, but otherwise differentiation was similar to erythroblasts cultured alone or those that became nonadherent in cocultures. These results suggest a mechanism for the development of anemias associated with abnormal macrophage function and for reduced responsiveness of those anemias to erythropoietin therapy.

Most studies of mammalian reticulocyte maturation have used blood reticulocytes.Nascent reticulocytes, as found in bone marrow, have not been available in developmentally synchronized populations. Nascent murine reticulocytes formed in vitro by enucleation of Friend virus–infected erythroblasts were purified and recultured for 110 hours. At 0 hours, all recultured cells were lobulated and contained dense, centralized reticulin. By 110 hours, about 20% to 25% of the cells became biconcave erythrocytes. Most ribosomes and cellular RNAs were degraded within 20 hours, and during that period, heme synthesis declined from a rate equal to that of late erythroblasts to less than 10% of that rate. Many mitochondria appeared normal until they showed outer membrane swelling, degradation, and apparent fusion with intracellular vacuoles at 40 hours of culture. During the period of mitochondrial loss, Bcl-XL, an antiapoptotic protein that accumulates during erythroblast differentiation and maintains mitochondrial membrane integrity, demonstrated progressive decreases and changes consistent with deamidation. Nevertheless, the reticulocytes did not undergo apoptosis, because their apoptotic machinery was degraded. This experimental system that provides a developmentally synchronized population of nascent murine reticulocytes that mature into biconcave erythrocytes in vitro should be useful in further investigations of the cellular events involved in reticulocyte maturation.

The erythroid terminal differentiation program couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. Here we use Epor^−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. We find that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. EpoR-regulation of cell size is independent of established red cell size regulation by iron. High erythropoietin (Epo) increases red cell size in wild-type mice and in human volunteers. The increase in mean corpuscular volume (MCV) outlasts the duration of Epo treatment and is not the result of increased reticulocyte number. Our work shows that EpoR signaling alters the relationship between cycling and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress.