The in-vitro detection and quantitation of ovine bone marrow precursors of multipotential colony-forming cells.

The in-vitro detection and quantitation of ovine bone-marrow precursors of multipotential colony-forming cells (pre-multi CFC) is described. After 5 and 10 days in liquid culture containing medium conditioned by long term bone marrow stromal cell layers (SCM) along with lymph node-conditioned medium (LNCM) or recombinant ovine interleukin-3 (rov IL-3), increased numbers of multi CFC developed from bone marrow precursors as detected by subsequent soft agar clonogenic assay of mixed phenotype colonies. From a variety of cytokines and conditioned media (CM) tested that included recombinant ovine granulocyte-macrophage colony-stimulating factor (rov GM-CSF) and recombinant human macrophage colony-stimulating factor (rhu M-CSF), the combination of SCM plus LNCM or rov IL-3 supported the maximum numbers of multi-CFC in liquid culture. The development of multi CFC from precursors was demonstrated in bone marrow cells treated with a dose of mafosfamide that inactivated > 98% of clonogenic CFC. Quantitative limit dilution analysis of 10 bone marrow samples revealed an average of one pre-multi CFC per 34147 unfractionated cells (range 1:14230-81433). Pre-multi CFC were enriched 38-fold (average) in the 2.0% of bone marrow cells remaining after depletion of lymphocytes and myeloid/erythroid cells expressing the antigen recognized by monoclonal antibody 175. The quantitative assay also revealed preliminary evidence that the pre-multi CFC may consist of subpopulations differing in their sensitivity to mafosfamide.