A cytoadhesion assay for the binding of cloned hemopoietic progenitor cells to stroma

Cell adhesion molecules responsible for the specific recognition and adhesion that necessarily occur between hemopoietic progenitor cells (HPC) and stromal cells within the bone marrow are likely of multiple nature in the cell membrane. Systems less complex than intact bone marrow, in which the interactions between adhesion molecules and their ligands may be studied, is greatly needed. Using 4 cloned murine IL-3-dependent HPC lines, B6Sut, FDCP-1, FDCP-2 and FDCP-Mix, a system of co-culture has been established and standardized with 2 murine stromal cell lines, GB1/6 and 3T3. HPC were radiolabeled with 51Cr, and an optimal set of conditions was established for the adherence of HPC to stromal cells. It was found that a source of IL-3, whether supplied as recombinant murine IL-3 or WEHI-conditioned medium, was a necessary component of the labeling and assay medium to achieve maximal adherence to stroma. Likewise, the presence of serum also resulted in overall better cytoadhesion than did serum-free conditions. All 4 cell lines bound GB1/6 in a reproducible manner from approximately 63% for FDCP-1 to 20% for FDCP-Mix; binding to 3T3 was higher than to GB1/6 for all HPC. Approximately 25 to 30% of the cell populations were not able to adhere to stroma, indicating a fairly constant degree of heterogeneity with respect to expression of adhesion molecules. Cytoadhesion was found to have at least one component that was temperature-sensitive, as adhesion of FDCP-1 to GB1/6 was 41.5 +/- 1.3% at 4 degrees C compared with 63.2 +/- 1.1% at 37 degrees C. The adhesion reaction itself occurred independently of metabolic energy production and relied on the presence of receptor-ligand molecules. This very standard and reproducible system should allow closer examination of the individual cytoadhesive events that occur between HPC and marrow stromal cells using cloned cell lines.