Clonal analysis of the SSEA-1⁺ population. (A) Flow histograms showing levels of expression of Oct-4 and Nanog in the clone-3–derived population. SSEA-1⁻ MCs and ES cells were used as negative and positive controls, respectively. The numbers indicate the ratio of MFI values from the specific staining over those from the isotype controls. (B) Multicolor immunostaining performed on clone-3 cells at 45 doublings showing nuclear expression of Nanog, Rex-1, and Oct-4. Immnunostainings were performed using Alexa 488– and Alexa 594–conjugated secondary antibodies. Appropriate rabbit IgGs and mouse IgG1 isotype controls were also used and cells were counterstained with DAPI. (C) Representative diploid metaphase spread of clone-3 population. Original magnifications, 40×/0.75 NA dry objective (C) and 100×/0.4 NA oil objective (D).