Identification of superficial zone articular chondrocyte stem/progenitor cells,☆☆

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Abstract

Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of “side population” (SP) stem cells identified by the Hoechst 33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for Alcian blue—a marker of chondrogenesis. However, with BMP-7 treatment, Alcian blue staining was increased. Superficial zone protein—a specific marker for articular cartilage superficial zone chondrocytes—increased with BMP-7 and/or TGF-β1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte.

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.

☆☆

There are no conflicts of interest for the authors.

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