Colonies of the hESC line MI01 contain 2 cell types distinguishable by laser-light scatter patterns, expression of pluripotent markers, and polyploidization in response to SAC activation. Morphology of typical (A) or atypical (B) MI01 colonies is shown. (C) Examples of microsurgical harvesting of typical colonies. Arrows indicate cut and lifted clumps of cells from colonies (Olympus S751; 10 × 20). (D) Flow cytometric analysis of laser-light scatter pattern and DNA content was used to distinguish 2 populations, hESC-A and hESC-B, in single-cell suspensions of harvested MI01 colonies. DNA content versus laser-light side-scatter is indicated. The R1 gate was used to separate viable cells from hypodiploid cells and cell debris. The ratio of percentages of hESC-A and hESC-B cells was 0.52 ± 0.21 for 6 separate experiments. Nocodazole treatment had no significant effect on this percent (P > .05; Figure S6). MI01 colonies were harvested, washed, and placed into human EB medium and cultured for 4 days, then hEBs were harvested and single cell suspensions were analyzed (E) as in panel D. Results are representative of 2 experiments. (F) Pluripotent marker expression of hESC-A and hESC-B. Isotype control antibody-staining intensity was below 10 fluorescence units (not shown). Data are representative of 2 experiments. hESC-A and hESC-B were treated with nocodazole or control solvent as in Figure 1 and harvested. hESC-A and hESC-B cells were gated as in Figure 5D. (G) Cell-cycle analysis was performed and DNA content is shown. (H) Percentage of polyploidy (mean ± 1SD) from 3 experiments. *Significant difference (P < .01) for nocodazole-treated hESC-Bs compared with control hESC-Bs.