Single-cell sorting. (A) CFSE-labeled (green fluorescence, arrow) single cell seeded on an irradiated murine feeder layer (phase contrast). (B) Doublet of CFSE-labeled cells originated from a single cell 4 days after sorting (arrowheads). (C) X (spectrum green fluorescence) and Y (spectrum orange fluorescence) human chromosomes revealed in hMASC nuclei (blue fluorescence) by FISH analysis. (D) Antihuman lamin A/C antibody decorates (A488, green fluorescence) decorates the nuclei of expanded clones. Blue fluorescence of DAPI identifies nuclei. Scale bars represent 10 μm. (E) Epifluorescence image of oct4 protein expression (A488; green fluorescence) in a hMASC single-cell clone. (F) Epifluorescence image of nanog protein expression (A488; white fluorescence) in a hMASC single-cell clone. Nuclei are depicted by the blue fluorescence of DAPI. (G) Telomerase activity products of a hMASC single-cell clone (C) display 6-bp periodicity. Cells treated with RNase (Ci) were used as negative control. TSR8 lane represents the telomerase quantitation control template. (H) Representative karyotype of single-cell–derived expanded clones. (A,B,E,F) Epifluorescence and phase contrast images were obtained using a live cell imaging dedicated system consisting of a Leica DMI 6000B microscope connected to a Leica DFC350FX camera (Leica Microsystems) equipped with a 5 × dry objective (numeric aperture: 0.12; B), a 10 × dry objective (numeric aperture: 0.25; A), and a 63 × oil immersion objective (numeric aperture: 1.4; E,F). (C,D) Image acquisition was carried out, at room temperature (RT), by a confocal laser microscope (Leica TCS-SP2), using a 40 × oil immersion objective (numeric aperture: 1.25). Adobe Photoshop software was used to combine RGB channels, overlay the images, and adjust contrast. (G) Developed autoradiographic film was acquired by SnapScan 1236 (Agfa) connected to a Macintosh G3 Computer (Apple) equipped with ScanWise software (v1.2.1; Agfa).