Morphological studies on long-term culture of marrow cells: characterization of the adherent stromal cells and their interactions in maintaining the proliferation of hemopoietic stem cells.

Abstract

In long-term cultures of bone marrow, the adherent stromal cells provide support for the proliferation and maintenance of hemopoietic stem cells. These stromal cells and their interactions were characterized by means of scanning (SEM) and transmission (TEM) electron microscopy in correlation with functional studies. Cultures were initiated by establishing the adherent stromal layer as a "soil" which was then "seeded" after 3 weeks by the addition of another marrow-cell suspension. Clonal assay of the supernatant demonstrated the continuous proliferation of the hemopoietic stem cell. The stroma essentially consisted of two cell types, macrophages and epithelioid cells. Macrophages were smaller, 10-15 microns, phagocytosed latex and carbon particles, and contained lysosomes. Their surface did not stain with polycationic ferritin (PCF). Epithelioid cells were much larger, more than 100 microns; contained numerous thin, elongated mitochondria; did not phagocytose latex particles; but did display strong surface staining with PCF. The appearance of epithelioid cells in TEM depended on their state of development and whether the section was parallel or perpendicular to the substratum. Epithelioid cells displayed a maturational spectrum, at two ends of which were synthetic and storage phases. In the synthetic phase, the cell contained numerous profiles of rough endoplasmic reticulum, and in the storage phase, numerous storage granules. These two phases were best appreciated in sections perpendicular to the substratum, demonstrating synthetic cells on top settling over the substratum upon maturation into the storage cells. Both macrophages and epithelioid cells contained fat globules which increased in number and size with the addition of hydrocortisone to the culture medium. A distinct fat-cell type, as has been claimed, was not found in this study. Granulopoiesis was observed in the culture system in the absence of colony-stimulating activity in the supernatant, suggesting direct cellular interaction or short-range factors in the induction of granulopoiesis. Widespread cellular interactions were noted between macrophages and epithelioid cells, the latter often completely embracing the former and both extending cytoplasmic processes toward each other. This is reminiscent of the cooperative interaction of endoderm and mesoderm in chick embryo hemopoiesis and may be necessary for the maintenance of stem cells in these cultures.