Selection of two human bone marrow stromal cell subpopulations with different effects on the maintenance and differentiation of myeloid progenitor cells

The simple selection of two human bone marrow stromal cell populations is described. Adherent stromal cell layers were formed in primary cultures of low-density marrow cells. At time of confluence, persistent nonadherent cells were collected and transferred to new culture flasks, where they formed a secondary stromal layer. These primary and secondary stromal layers differed in their ability to support myelopoiesis, as tested by progenitor cell production after inoculation with fresh bone marrow cells. In the presence of primary stromal layers the number of granulocyte-macrophage colony-forming cells (GM-CFC) decreased gradually, but in the presence of secondary layers production of GM-CFC was evident during the first 3 weeks. The regulation of the two stromal types on hemopoietic cell proliferation and differentiation was investigated by determining the kinetics of the transitions within the differentiation sequence of three myeloid progenitor cells. Pre-CFC, day-14 CFC, and day-7 CFC were fractionated by cell sorting on the basis of forward light scatter and cocultured with the two stromal layer types. It was found that the decrease in CFC numbers in the presence of primary stromal layers could be explained by the stimulation of hemopoietic cells into rapid differentiation with loss of proliferative capacity at an early stage of culture. Secondary layers appeared to promote survival and self-renewal of later types of progenitor cells and to trigger more immature cells to proliferate and differentiate at a later time of culture.