FIG. 4.

Cells cultured in 6% oxygen maintain mesenchymal stem cell markers and mutilineage potential. (A) Cells cultured at 21% (left panels) or 6% oxygen (right panels) were analyzed for mesenchymal stem cell markers CD105 coexpressed with CD44 (top) and CD90 coexpressed with CD 73 (bottom). (B) Perivascular stem cells were cultured in 6% (left) or 21% (right) oxygen and tested for the ability to differentiate. Cells were maintained in adipogenic medium, fixed, and stained for lipids with oil red O (Top). Stem cells were centrifuged into a pellet and cultured in the chondrogenic medium containing TGF-β1. Pellets were then sectioned, and stained with Alcian blue and nuclear fast red for detection of sulfated glycosaminoglycans and nuclei, respectively (middle). Cells cultivated in the osteogenic medium were stained with alizarin red at pH 4.2, staining calcium deposits red (bottom). The high degree of differentiation is shown by the almost solid blue (sulfated glycosaminoglycans, middle image) and solid red (calcium, bottom image) observed. Images at 40 ×.