Fig. 3. Telomeres form aggregates during cellular senescence in hMSCs. (A) Telomeres form aggregates in cells at passage 12. Cells at passage 3 and 12 were labeled with PNA probes to identify telomeres. 3D image processing and quantitative image analysis of PNA fluorescence was carried out using TeloView. (Ai) Maximum projections of PNA hybridization from representative nuclei at passage 3 (a) and 12 (b and c). A nucleus with mainly telomere aggregates (c) represents a class of cells found at passage 12, which were excluded from statistical analyses. (Aii) Telomere fluorescence intensity in cells at passage 3 and 12. The PNA fluorescent dots were sorted and plotted according to their intensity. Shown are the intensity plots obtained from a typical cell in passage 3 (open squares) and 12 (closed triangles). The thresholds for definition of small and big telomere aggregates are indicated with straight or dashed red lines, respectively. (Aiii) B-box plots of the ratio of big telomere intensities (i>T) or small telomere intensities (t<i<T) to all telomeres in cells at passage 3 and passage 12. (Aiv) B-box plots of the ratio in the fluorescent intensity between telomere aggregates (i>t) (agg.) to normal telomeres, and the ratio between the number of spots found in aggregates to total PNA spots. Statistical analyses represent 1400 PNA fluorescent dots obtained from 20 cells per passage. Based on fluorescence intensity, the PNA fluorescent dots are calculated as equivalent to 90-100% of expected telomeres. Cells with less telomeres (Fig. 2Aic) were excluded from statistical analyses. (B) Telomere organization correlates with changes in nuclear shape. hMSCs at PS 9 were labeled with PNA followed by immunofluorescence with anti lamin B antibody (visualized in green). 3D reconstructions of confocal images were carried out using Teloview. Based on lamin B organization and the nuclear depth, cells were grouped into normal and senescent groups. Maximum projections of the x-y and y-z axis show representative normal or senescent cells B-box plots on the right show the ratio in the fluorescent intensity between telomere aggregates (i>t) (agg.) to normal telomeres, and the ratio between the number of spots found in aggregates and total PNA spots. Statistical analysis involved 1050 PNA fluorescent dots obtained from 15 cells per group.