A QUANTITATIVE ASSAY THAT EVALUATES THE CAPACITY OF HUMAN STROMAL CELLS TO SUPPORT GRANULOMONOPOIESIS IN-SITU

We describe an assay that makes possible the observation of granulomon ocytic colonies grown on allogeneic stromal layers and the quantificat ion of the stroma-adherent colony-forming cells (CFC). Stromal layers were generated from Stro-1 positive cells isolated from adherent layer s of primary long-term marrow cultures using magnetic beads coated wit h the Stro-1 antibody. The stromal layers consisted mainly of myofibro blastic cells. Marrow fractions depleted of cells bearing receptors fo r soybean agglutinin (SBA) and enriched in CD34+ cells were obtained b y panning. SBA-, CD34+ marrow cells were seeded onto stromal cells gro wn in 96-well plates. After four weeks, a mixture of cytokines was add ed (granulocyte-macrophage colony-stimulating factor [GM-CSF]: 25 U/ml , interleukin [IL]-3: 4 ng/ml, Steel factor: 5 ng/ml and growth factor s provided by 3% conditioned medium from the 5637 cell line). Wells wi th large colonies (containing 10(3) to 10(4) cells) were scored after 14 days. Limiting dilution analysis of data revealed a Poisson distrib ution of the stroma-adherent CFC. There was an average of one stroma-a dherent CFC per 167 CD34+ enriched marrow cells, which gave an estimat ed frequency of one CFC per 10(5) unfractionated bone marrow cells. Co lonies contained cells that gave rise to CFU-GM after replating in aga r (5-40 CFU-GM were provided per each stroma-adherent CFC), but not ce lls with self-renewal ability (as indicated by negative results after replating single colonies onto secondary adherent layers). Colonies us ually formed from a cobblestone-area and developed in intimate contact with alphaSM actin positive stromal cells. Some of the stromal cells were located above granulocytic cells, corresponding to the descriptio n of ''blanket cells.'' This assay should allow the study of colony-fo rmation on marrow stroma without disrupting the hemopoietic niche.