A quantitative assay that evaluates the capacity of human stromal cells to support granulomonopoiesis in situ

Abstract

We describe an assay that makes possible the observation of granulomonocytic colonies grown on allogeneic stromal layers and the quantification of the stroma-adherent colony-forming cells (CFC). Stromal layers were generated from Stro-1 positive cells isolated from adherent layers of primary long-term marrow cultures using magnetic beads coated with the Stro-1 antibody. The stromal layers consisted mainly of myofibroblastic cells. Marrow fractions depleted of cells bearing receptors for soybean agglutinin (SBA) and enriched in CD34+ cells were obtained by panning. SBA-, CD34+ marrow cells were seeded onto stromal cells grown in 96-well plates. After four weeks, a mixture of cytokines was added (granulocyte-macrophage colony-stimulating factor [GM-CSF]: 25 U/ml, interleukin [IL]-3: 4 ng/ml, Steel factor: 5 ng/ml and growth factors provided by 3% conditioned medium from the 5637 cell line). Wells with large colonies (containing 10(3) to 10(4) cells) were scored after 14 days. Limiting dilution analysis of data revealed a Poisson distribution of the stroma-adherent CFC. There was an average of one stroma-adherent CFC per 167 CD34+ enriched marrow cells, which gave an estimated frequency of one CFC per 10(5) unfractionated bone marrow cells. Colonies contained cells that gave rise to CFU-GM after replating in agar (5-40 CFU-GM were provided per each stroma-adherent CFC), but not cells with self-renewal ability (as indicated by negative results after replating single colonies onto secondary adherent layers). Colonies usually formed from a cobblestone-area and developed in intimate contact with alpha SM actin positive stromal cells. Some of the stromal cells were located above granulocytic cells, corresponding to the description of "blanket cells." This assay should allow the study of colony-formation on marrow stroma without disrupting the hemopoietic niche.