Porcine bone marrow stromal cells (PBMSC) are a source of skeletogenic mesenchymal progenitor cells. Unfortunately, the heterogeneous nature of these cells in culture complicates the interpretation of their growth-factor responsiveness. We have therefore pulse-labelled mitogen-stimulated PBMSC with bromodeoxyuridine (BrdU) in order to study individual, growth-factor responsive cells in the presence of large numbers of nonresponsive PBMSC. Transfer of growing cells to low serum medium reduced BrdU labelling from 35% to 4% over a period of 24 hours. Subsequent addition of foetal calf serum (FCS) to serum-arrested cultures increased the number of BrdU positive cells to 54% by 16 hours. Addition of basic fibroblast growth factor (bFGF) to serum-arrested cultures induced DNA synthesis in 28% of cell by 16 hours after stimulation. In order to selectively eliminate mitogen responsive cells from mixed cultures, BrdU-substituted cells were photosensitised with bisbenzimide and exposed to bright light. BrdU-labelled PBMSC died within 20–40 hours of bisbenzimide treatment and subsequent illumination, whereas BrdU-labelled cells survived in the dark despite treatment with bisbenzimide. The photokilling procedure appeared to have no long term effect upon the viability of non-BrdU-labelled cells because if subconfluent cells were brought to serum arrest prior to photokilling, no change in DNA content relative to controls was observed after a subsequent 4 or 7 day incubation in Dulbecco's modified Eagle's medium (DMEM)/ 10% FCS. In contrast, subconfluent PBMSC growing in DMEM/10% FCS (LI [labelling index] 24.6 ± 2.5) showed a 60% reduction in cell numbers relative to controls after photokilling and a subsequent 4 day incubation in DMEM/10% FCS. Cells stimulated to enter S-phase by bFGF also died within 20 hours of photokilling, whereas BrdU-labelled, bisbenzimide-treated, bFGF-responsive cells remained viable when maintained in the dark. Elimination of this cohort of bFGF-responsive cells abolished subsequent response to bFGF. These methods should assist in the characterisation of minor stromal cell subpopulations by allowing unique markers to be associated with specific patterns of mitogen responsiveness. |