Visualisation and selective elimination of a subpopulation of mitogen-responsive bone marrow stromal cells using bromodeoxyuridine and photo-induced cell killing

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.(ABSTRACT TRUNCATED AT 250 WORDS)