CHARACTERIZATION OF STROMA-ADHERENT COLONY-FORMING CELLS - A CLONOGENIC-ASSAY FOR EARLY HEMATOPOIETIC-CELLS

Hemopoietic cells that adhered to preformed selected marrow stromal ce ll layers were characterized on the basis of progenitor cell productio n capacity, colony-formation kinetics and forward light scatter (FLS) properties. It was shown that early hemopoietic cells attached to the stromal layers within 2 hours of incubation, and were responsible for the initial production of the more differentiated granulocyte-macropha ge colony-forming cells (day 14 GM-CFC) in longterm cultures (LTC). In a clonogenic assay system, hemopoietic cells that adhere to stromal l ayers can be detected by the formation of small colonies of blast-like cells and are designated as stroma-adherent CFC. Cell fractionation o n the basis of FLS and counting colonies on days 5, 14 and 21 revealed that there was a succession of colony formation, indicating that the stroma-adherent CFC consisted of subpopulations with different lag-pha ses before initiation of proliferation. Day 5, day 14 and day 21 strom a-adherent CFC were shown to have a high, intermediate and low FLS, re spectively. The cells that produced GM-CFC by day 21 showed FLS proper ties similar to those of day 21 stroma-adherent CFC, suggesting a corr elation between day 21 stroma-adherent CFC and CFC-producing cells in LTC. The CFC present on day 21 required the synergistic action of GM-C SF + IL-3 + stem cell factor (SCF) for optimal proliferation. The prol onged lag-phase, the low FLS and the multifactor-responsive progeny ar e properties similar to those reported for other early cells, and it i s proposed that day 21 stroma-adherent CFC represent an early hemopoie tic cell type whereas day 5 stroma-adherent CFC represent a more matur e stage of differentiation.