Characterization of factor-producing and factor-dependent clones from long-term hamster bone marrow suspension...

Long-term hamster bone marrow (BM) cultures produce stem cells that can be grown in the absence of an adherent layer and without addition of exogenous growth factors or hormones. Cloning of these three- to five-month-old suspension cultures by the limiting dilution method generated both factor-dependent (FD) and factor-producing (FP) cell lines. The FP clones have been in culture for two years and are composed of macrophages by morphologic and histochemical criteria. FP serum-free conditioned medium (FPCM) produces both stimulatory and inhibitory effects on hematopoiesis. Addition of 1%-5% FPCM to fresh or cultured hamster BM cells stimulates both CFU-C and erythropoietin (epo)-dependent BFU-E colony formation but not CFU-GEMM in semisolid media, as well as increased numbers of differentiating myeloid and erythroid cells in suspension. In contrast, addition of 10%-15% FPCM produces substantial inhibition of epo-stimulated erythropoiesis. FD cells have been in culture for over 12 months. They exhibit an absolute requirement for the continued presence of 5% hamster spleen conditioned medium (SCM). The clones generate varying numbers of "blast" cells, myeloid and macrophage elements, and occasional mastlike cells. Addition of increasing amounts of SCM produces a dose-dependent proliferation and differentiation of FD cells and also stimulates CFU-C but not BFU-E or CFU-GEMM colony formation. FD cells will also respond to FPCM, but concomitant addition of SCM and FPCM generates four times more cells than either CM alone. These results suggest that the hamster suspension cultures contain both stem cells and distinct regulatory cells that stimulate the growth and differentiation of myelopoiesis.