Hypoxic culture increases in stem cell properties. Cells were seeded at 50 cells/cm2 and expanded under normoxic and hypoxic conditions. (A) Flow cytometry for detecting surface CD markers. Cells under normoxic and hypoxic conditions have the same profile of surface CD markers. (B-C) Hypoxic cells increase in differentiation potential into osteoblasts, adipocytes and chondrocytes. (B) Normoxic and hypoxic cells at passage 6 were induced to differentiate to osteoblasts and adipocytes for 3 weeks, and stained by ARS and Oil Red O, respectively. Stained dye was extracted and OD values were measured. (C) Normoxic and hypoxic cells at passage 6 were induced to differentiate to chondrocytes for 3 weeks, and Alcian Blue staining and immunohistochemical study for collagen II and X were performed. Quantitative data performed by the computerized image analysis show hypoxic cells increase in Alcian Blue staining and IHC for collagen II and X. Values are mean + SD; *P < .05 and **P < .01 indicate significant variance (independent t test) between normoxic and hypoxic groups. Bar = 20 μm.
