Figure 4.

Characterization of the OSM activity. (A) Specificity of hematopoiesis-suppressing activity. Lin⁻Sca-1⁺c-Kit⁺ cells was co-cultured with hepatic stromal cells as in Fig. 3 in the presence of IL-6-family cytokine. In vitro hematopoiesis was inhibited specifically by OSM but not by IL-6, leukemia inhibitory factor, or IL-11. Addition of IL-6 produced 1.5- to ≈2× more floating cells than a control condition because IL-6 is a growth stimulator of hematopoietic cells. (B) Failure of the fetal hepatic culture derived from the E18.5-embryonic liver to expand hematopoietic cells from Lin⁻Sca-1⁺c-Kit⁺ cells, even in the presence of SCF. Each histogram represents the mean value of triplicate experiments ± SD of the absolute number of cells generated in 60-mm-diameter dishes. (C) Decrease of mRNA levels for hematopoietic cytokine (M-CSF) and chemokine macrophage chemoattractant protein 1 [(MCP-1)] in fetal hepatic cells in response to OSM and Dex. Fetal hepatic cells were incubated with or without OSM for indicated periods, and total RNA samples were extracted by the acid guanidinium thiocyanate/phenol/chloroform method. Ten micrograms of total RNA samples from cultured cells was analyzed by Northern blotting. The integrity and the amount of RNA samples were evaluated by ethidium bromide staining of the gel (data not shown). (D) Decrease of chemotactic activity produced in the conditioned media of the fetal hepatic culture. Various (100, 50, 25, and 0%) concentrations of culture media conditioned either in the presence or absence of OSM for 7 days were applied to an in vitro two-chamber migration assay as described in Materials and Methods.