FIG. 1.

Loss of chondrocytic phenotype in culture-expanded cells. (A) Spindle-shaped morphology of P2 cells in monolayer (ML). n=5 experiments. (B) Gene expression profile of cells after each passage compared with P0 levels. A significant increase in Col1a1 was observed after the first passage along with downregulation of cartilage-associated gene aggrecan and cartilage oligomeric matrix protein (COMP) whereas a decrease in Col2a1 was seen only after the second passage. Genes expressed by interzone cells—TenacinC, Gli3, GDF5, and Wnt9a—were significantly upregulated in P2 cells compared with P0 or P1 cells. Data are expressed as mean with the uncertainty estimated by 95% confidence interval (95% CI; lower and upper limits are within brackets). n=5 experiments. P0, primary chondrocytes; P1-ML or P2-ML, passage 1 or 2 cells, respectively, harvested from ML. (C) Immunoblot analysis showed a decrease in Sox9 and p-ERK-1/2 levels in P2-ML compared with P0. n=3 experiments. (D) FACS analysis of surface markers showed 40% CD105⁺ and 99% CD44⁺ cells after two passages. n=3 experiments. ^Nonspecific background staining in P2 cells. (E) Photomicrographs of toluidine-blue-stained tissues formed by P2 cells cultured in serum containing three-dimensional conditions (SC3D) for 4 weeks show loss of capacity to accumulate cartilaginous matrix by P2 cells unlike P0 that retained abundant proteoglycan (PG)–rich matrix. n=5 experiments. ¶Collagen-type-II-coated membrane insert. Color images available online at www.liebertpub.com/tea