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.
Moredun Research Institute, Edinburgh, UK.
D M
DM
Haig
J
J
Thomson
A
A
Percival
D000276:Adjuvants, Immunologic; D000818:Animals; D001853:Bone Marrow / Q000187:drug effects; D001854:Bone Marrow Cells; D002452:Cell Count; D002478:Cells, Cultured; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned; D003520:Cyclophosphamide / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D006133:Growth Substances / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects; D007377:Interleukin-3 / Q000494:pharmacology; D011994:Recombinant Proteins; D012756:Sheep
D000276:Adjuvants, Immunologic; D017077:Culture Media, Conditioned; D006133:Growth Substances; D007377:Interleukin-3; D011994:Recombinant Proteins; C048341:mafosfamide; D003520:Cyclophosphamide