Isolation and Short-term Culture of Mouse Splenic Erythroblastic Islands

Abstract

We isolated and cultured erythroblastic islands (EI) from the spleens of phlebotomized mice using a combination of collagenase digestion, unit gravity sedimentation, and Percoll density gradients separation. The isolated El were composed of surrounding erythroid cells and central stromal macrophages (M⊘), which were identified by Forssman antigen. While 60% of the erythroblasts incorporated bromodeoxyuridine, the M⊘ did not. El could be maintained on a plastic dish for a short period in the presence of erythropoietin. Two hours later, the central M⊘ spread well and bound to erythroblasts via cytoplasmic processes. One day later, erythropoietic activity on the M⊘ surface continued, although their processes had retracted. Some El showed synchronized expansion of erythroblasts and others showed differentiation to reticulocytes. Two days later, about 50% of the El still showed erythropoietic activity and most erythroblasts differentiated to the orthochromatic stage. On the other hand, the M⊘ secreted colony-stimulating activity during the culture. It was infrequently observed that erythroid and myeloid populations simultaneously expanded on a central M⊘. These results indicate that this El culture system is useful for studying interactions between the stomal and hematopoietic cells. © 1990, Japan Society for Cell Biology. All rights reserved.