Figure 2.
Cell competition in hPSC cultures is cell-contact mediated
(A) Percentage of caspase-3-positive cells in Transwell cultures of different sublines.
(B) Effect of increasing the ratio of variant-GFP cells in co-cultures with wild-type cells on the ratio of wild-type cells at day 3.
(C) Effect of increasing plating cell density of co-cultures on the number of wild-type cells.
(D and E) Cell-confrontation assay of wild-type-RFP and variant-GFP cells (D) and wild-type and wild-type-RFP (E) cells at 48 h post contact. (D) Top panel: nuclei of wild-type-RFP and variant-GFP cells represented as blue and green dots, respectively. Top panel (E): nuclei of wild-type and wild-type-RFP cells represented as blue and red dots, respectively. Middle panel (D and E), cleaved caspase-3-positive cells represented as purple dots. Bottom panels: Percentage of cleaved caspase-3-positive cells calculated as the number of cleaved caspase-3-positive cells in the total cell number within a defined area of a cell insert. The width of the bar corresponds to the analyzed area of the insert shown in the middle panel above.
(F and G) Time-lapse images of cell-confrontation assay of wild-type-RFP (red) and variant-GFP (green) cells (F) or wild-type and wild-type-RFP (red) cells (G). Left panel: inserts at 4 h before contact. Middle panel: inserts at the time when cells first come into contact (denoted as 0 h). Right panel: inserts at 48 h post contact. Dashed white line indicates the position on the insert where the two different populations first met at 0 h.
(H) Time-lapse images of cell-confrontation assay of wild-type and variant-GFP cells with live caspase-3/7 dye. Left panel: region of the inserts where wild-type and variant-GFP cells come into contact. Dashed red line indicates the boundary between the two different populations. Right panel: middle region of the wild-type cell population on the inserts. Scale bar: 10 μm.
Data represent the mean of three independent experiments ± SD (A–C). Statistical analysis was performed by Student’s t test (A) or two-way ANOVA, followed by Holm-Sidak’s multiple comparisons test (B); n.s. nonsignificant; ∗∗∗∗p < 0.0001.