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)
Thomson
B M
BM
Bone Growth and Metabolism Unit, Rowett Research Institute, Aberdeen, Scotland.
Dean
V
V
Farquharson
C
C
Noble
B S
BS
D000818:Animals; D006690:Bisbenzimidazole / Q000494:pharmacology; D001769:Blood; D001853:Bone Marrow / Q000187:drug effects / Q000378:metabolism; D001854:Bone Marrow Cells; D001973:Bromodeoxyuridine / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005347:Fibroblasts; D008027:Light; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013552:Swine
D003470:Culture Media; D016222:Fibroblast Growth Factor 2; D001973:Bromodeoxyuridine; D006690:Bisbenzimidazole