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. 2007 Jun 22;358(1):99-103.
doi: 10.1016/j.bbrc.2007.04.142. Epub 2007 Apr 30.

Identification of superficial zone articular chondrocyte stem/progenitor cells

Shintaro Hattori  1 , Carol Oxford, A Hari Reddi
Affiliations

Identification of superficial zone articular chondrocyte stem/progenitor cells

Shintaro Hattori et al. Biochem Biophys Res Commun. .

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-beta1 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.

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Figures

Fig 1
Fig 1. Flow cytometry analysis
The distribution of chondrocytes by flow cytometry from the surface, middle and deep zone. (a) surface zone: we identified the side population cells. (b) addition of verapamil prevented the fractionation of SP cells. (c),(d) The middle zone and deep zone were devoid of SP cells.
Fig 2
Fig 2. Alcian blue staining of the masses and intensity
(a) primary (5 days culture of monolayer) (b) SP (were derived by sorting the primary cells and then expanding the culture for 7 days); The absorbance was dramatically higher with BMPs treatment. (c) The non-SP (NSP) cells were cultured similarly.
Fig 3
Fig 3. Immunoblotting for SZP
SZP expression was dramatically increased with BMP-7 and TGF-β1 treatment in primary, SP and non-SP cells.
Fig 4
Fig 4. Histology and Immunohistochemistry
The SP masses were stained with toluidine blue. In controls, there was no metachromasia with toluidine blue (A). The SP masses with BMP-7 treatment was thicker than with TGF-β1 treatment masses, and many round shape cells like a chondrocyte were observed (D and G). For SZP expression, the SP masses with TGF-β1 treatment was stronger than BMP-7 treatment (E, H). However, for collagen type 2 expression, there is no difference with BMP-7 and TGF-β1 treatment (F, I). Bar is 50μm.
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References

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