High OCT4 and Low Expressions Determine In Vitro Lifespan of Mesenchymal Stem Cells

Figure 2

Morphological and molecular changes of senescent BM-MSCs. (a) Representative images of cells at early passage (5th passage) and late/senescent passage. Most early passage cells showed spindle-like morphology and few cells stained positive for the senescence-associated β-galactosidase marker. Most senescent passage cells showed enlarged and flattened morphology and an increased number of cells stained for β-galactosidase (arrows). (b, c) The protein expression levels of and in early and late passage cells from the same donor were analyzed by western blot. Band intensities were densitometrically evaluated and and protein levels were quantified and normalized to the corresponding β-actin levels (loading control). Subsequently, the normalized values obtained were then used to calculate the fold change in expression between late and early passage cells (bar graphs). These results are representative of three independent experiments. Most senescent cells showed increased expression levels. On the contrary, most senescent cells showed decreased expression levels. (d, e) The levels of secreted IL6 and IL8 in early and late passage cells from the same donor were determined using a CBA proinflammatory kit. Values obtained (expressed as pg/mL, bottom) were then used to calculate the fold change in secreted protein levels between late and early passage cells (bar graphs). These results were obtained in triplicate and are representative of two independent experiments. Most senescent BM-MSCs showed enhanced secretion of these proinflammatory cytokines.

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