Characterization and Expression of Senescence Marker in Prolonged Passages of Rat Bone Marrow-Derived Mesenchymal Stem Cells

Figure 4

Cellular senescence and cell cycle arrest of culture expanded rBM-MSCs. Cells from passages 1–5 were seeded at 5000 cells/well cultured in 96-well plate for 24 h, 48 h, and 72 h. Cultures were pulsed with 10 μL ³H-TdR at 24 hours prior to measurement. The rate of proliferation decreased as the passage increased (a). Cells at passage 2 and passage 5 were fixed with 70% ethanol for 10 minutes and stained with X-gal in the respective buffer overnight. Cells at passages 2 and 5 were stained positive with lysosomal β-gal (pH 4) staining showing that cells were expressing lysosomal β-gal in early passage as well as in late passage. Cells at passage 5 were stained positive for SA-β-gal (pH 6) staining as evidenced by blue precipitation but not at passage 2. Cells at passage 2 and passage 5 were cultured in 25 cm² flasks. Upon reaching 80%–90% confluency, cells were harvested and subjected to PI staining. Cells were analysed using flow cytometer. Results were representative of three independent experiments. Microscopic magnification: 200x.

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