Phenotypical characteristics of replicative senescent MSCs. (A) MSCs derived from the bone marrow of 2 healthy individuals were culture-passaged at the indicated time points. Doubling time was calculated based on the cell numbers at seeding and harvesting time points and duration of the culture. (B) MSCs from replicative fit and senescent phases were seeded at similar density in the 96-well plates. MTT assay was performed at the indicated time points to determine the growths of MSCs. Results are plotted as mean ± standard deviation (SD). (C) Cell-cycle analysis was performed on fit and senescent MSCs through propidium iodide staining and subjected to flow cytometry. (D) Size and granularity of fit and senescent MSCs were determined by forward and side scatter analysis by flow cytometry. (E) Fit and senescent MSCs were subjected to senescence-associated lysosomal β-galactosidase staining. Dark gray staining represents β-galactosidase staining. Scale bars represent 400 μm. (F) Replicative fit and senescent MSCs were subjected to staining for MSC markers as defined by the ISCT and acquired through flow cytometry. Open and gray histograms represent marker and isotype controls, respectively. Similar results were obtained in a repeat experiment with an additional 1 or 2 fit and senescent MSC donor pairs. (G) Fold difference in the P16INK4A, PERP, LAMP, and LY96 mRNA of fit and senescent MSCs, relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were determined in quantitative sybr green real-time PCR. Cumulative is shown from 2 independent donors.