Ovine bone-marrow stromal cell-dependent myelopoiesis

Abstract

Conditions for the development of ovine bone marrow stromal cell-dependent LTBMC (long term bone marrow cultures) are described. After the addition of freshly isolated allogeneic bone marrow cells to a fully developed adherent stromal cell layer, myelopoiesis in the absence of erythropoiesis was sustained for up to 6 months. Macrophages and/or mast cells predominated in the later stages of all cultures. Stromal cell conditioned medium (SCM) and lymph node conditioned medium (LNCM) both support the development of a similar range of colony-forming cell (CFC) types in soft agar clonogenic assays, including multipotential-CFCs capable of development into more than two cell lineages, haemopoietic blast-CFCs (undifferentiated cells), mast-CFCs, macrophage-CFCs, eosinophil-CFCs and neutrophil-CFCs. However SCM supported the development of larger colonies containing more cells than those supported by LNCM. Furthermore SCM together with LNCM supported increased numbers of all colony types compared with the numbers supported by SCM or LNCM alone. In both liquid and semisolid agar cultures of bone marrow cells, SCM together with ovine interleukin-3 (IL-3) augmented the development of mast cells in particular, a cell type supported by IL-3 alone, whereas SCM together with ovine granulocyte-macrophage colony stimulating factor (GM-CSF) supported increased numbers of neutrophils and macrophages, which were cell types supported by GM-CSF alone. Bone marrow stromal cells and medium conditioned from these cells are therefore able to support myelopoiesis and the development of inflammatory cell types in culture. SCM in combination with IL-3 or GM-CSF is able to amplify the development of particular cell lineages supported by either IL-3 or GM-CSF alone.