A CYTOADHESION ASSAY FOR THE BINDING OF CLONED HEMATOPOIETIC PROGENITOR CELLS TO STROMA

Cell adhesion molecules responsible for the specific recognition and a dhesion that necessarily occur between hemopoietic progenitor cells (H PC) and stromal cells within the bone marrow are likely of multiple na ture in the cell membrane. Systems less complex than intact bone marro w, in which the interactions between adhesion molecules and their liga nds may be studied, is greatly needed. Using 4 cloned murine IL-3-depe ndent HPC lines, B6Sut, FDCP-1, FDCP-2 and FDCP-Mix, a system of co-cu lture has been established and standardized with 2 murine stromal cell lines, GB1/6 and 3T3. HPC were radiolabeled with Cr-51, and an optima l set of conditions was established for the adherence of HPC to stroma l cells. It was found that a source of IL-3, whether supplied as recom binant murine IL-3 or WEHI-conditioned medium, was a necessary compone nt of the labeling and assay medium to achieve maximal adherence to st roma. Likewise, the presence of serum also resulted in overall better cytoadhesion than did serum-free conditions. All 4 cell lines bound GB 1/6 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. App roximately 25 to 30% of the cell populations were not able to adhere t o stroma, indicating a fairly constant degree of heterogeneity with re spect to expression of adhesion molecules. Cytoadhesion was found to h ave 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 inde pendently 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 lin es.