Increased binucleation upon increasing passage number. To facilitate cell staining and counting of nuclei, cells were grown on glass coverslips and stained using wheat-germ agglutinin (a cell membranes), acridine orange (b cytoplasm) and DAPI (c nuclei), shown in ×100 magnification. d Overlay with cell membrane in red, cytoplasm in green and nuclei in blue. A representative example of a high passage binucleate cell is shown. Note also the presence of a micronucleus (panel
c). e Upon increasing passage number, more binucleate cells were noted, but no differences were seen between healthy donor-derived and osteosarcoma (OS) patient-derived MSCs (Kruskal–Wallis test P value <0.0001, Dunn’s post-test compared to early passage cells; P value **<0.01; ***<0.001.