Morphological characterization of stromal cell types in hematopoietically active long-term murine bone marrow cultures.

Accurate histological evaluation of stromal morphology is very difficult in cultures incubated in plastic flasks. Employing glass flasketts, we were able to characterize the morphology and immunocytochemistry of four marrow stromal cell types in a functionally intact microenvironment of murine long-term bone marrow cultures (LTBMCs). Fibroblastoid cells stained positively for collagen Type I and III, negatively for von Willebrand factor (vWf), the mouse macrophage F4/80 antigen, and the Bandeiraea simplicifolia lectin I isolectin B4 (BSL I-B4). Endothelial cells stained positively for vWf antigen and lectin BSL I-B4 but negatively for collagen Types I and III and for F4/80 antigen. Fat-containing cells had a dense, ovaloid, indented nucleus and fat-containing vacuoles. Macrophages were strongly positive for the F4/80 antigen and stained weakly with BSL I-B4. Between the fourth and ninth weeks after culture initiation, fibroblastoid and endothelial cells remained constant, between 21 +/- 2% and 24 +/- 2% and between 3 +/- 0.3% and 4 +/- 0.4%, respectively, of the total stromal cell population. By contrast, the percentage of fat-containing cells decreased significantly from 26 +/- 3% at Week 4 to 17 +/- 2% at Week 9, and macrophages increased significantly from 49 +/- 1% at Week 4 to 57 +/- 1% at Week 9. This characterization of the stromal cell types in functionally intact LTBMCs should assist in the study of the complex interactions among the marrow stroma, cytokine production, and hematopoiesis.