Identification of superficial zone articular chondrocyte stem/progenitor cells☆,☆☆
Section snippets
Materials and methods
Harvest of bovine articular cartilage and isolation of the cells. Surface zone of bovine articular cartilage was obtained from the calf knee joint as described previously in our laboratory by Khalafi et al. [13]. The bovine articular cartilage was divided into three zones (surface, middle, and deep). The total thickness of articular cartilage was about 5 mm. The surface zone of approximately 100 μm thickness was obtained by dermatome. After washing with medium, they were minced and digested with
Flow cytometry analysis
The superficial zone of bovine articular cartilage was digested in collagenase-P for 3 h and the released cells were cultured as monolayer on 100 mm culture dish. The cells were analyzed by flow cytometry and we identified a population which effluxed Hoechst 33342 dye. This fraction is designated as side population (SP) cells. (Fig. 1A). The proportion of the SP population relative to total number of cells was about 0.1%. The SP population was not distinguished when surface zone cells were
Conclusion
There is a growing realization of the presence of stem/progenitor cells in a variety of tissues. In this investigation, we set out to determine the utility of Hoechst 33342 dye in the identification of stem/progenitor cells in the articular cartilage. The efflux of the dye by stem/progenitor cells has permitted the isolation of a “Side Population” (SP) of cells with stem cell properties [4]. It is noteworthy that in the surface zone of articular cartilage, there are cells with stem/progenitor
Acknowledgments
The authors thank D. Raunschweig for technical support in FACS analysis and cell sorting. We thank our colleagues for helpful comments.
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This study was funded by NIH, the Lawrence Ellison Chair Endowment and The Musculoskeletal Transplant Foundation.
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There are no conflicts of interest for the authors.