NOTCH4in28 deletion affects lymphoid potential of hPSCs. (A-B) Flow cytometric analysis of T-cell differentiation from WT and 2 NOTCH4^{Δin28−/Δin28−} cell lines (C2^–/– and C3^–/–). Graphs show the total number of T cells generated from 1 × 10⁴ D8 floating HPs and the percentages of CD5⁺CD7⁺ and CD4⁺CD8⁺ cells (means ± SDs, n = 6 experiments). ∗∗∗∗P < .0001, 1-way ANOVA, Tukey multiple comparisons test (total number of graphs) and ∗∗∗∗P < .0001, 1-way ANOVA, Dunnett multiple comparisons test (percentages of graphs). (C-D) Flow cytometric analysis of NK-cell differentiation from WT and 2 NOTCH4^{Δin28−/Δin28−} cell lines (C2^–/– and C3^–/–). Graphs show the total number of CD56⁺ cells generated from 1 × 10⁴ D8 floating HPs and the percentages of CD56 by NK cells (means ± SDs, n = 4 experiments). ∗∗∗∗P < .0001, 1-way ANOVA, Dunnett multiple comparisons test. (E) Representative dot plots show T-cell differentiation from iSOX17 hPSCs with intact (iSOX17) and deleted NOTCH4in28 (NOTCH4^{Δin28−/Δin28−}; iSOX17C18^–/– and iSOX17C48^–/– cells). (F) Graphs show the total number of T cells generated from 1 × 10⁴ D8 floating HPs and the percentages of CD5⁺CD7⁺ and CD4⁺CD8⁺ cells (means ± SDs, n = 4 experiments). ∗∗P < .01 and ∗∗∗∗P < .0001, 2-way ANOVA, Sidak multiple comparisons test. (G) Representative dot plots show NK-cell differentiation from iSOX17 hPSC with intact (iSOX17) and deleted NOTCH4in28 (NOTCH4^{Δin28−/Δin28−}; iSOX17C18^–/– and iSOX17C48^–/– cells). (H) Graphs show the percentages and total number of CD56⁺ cells generated from 1 × 10⁴ D8 floating HPs (means ± SDs, n = 4 experiments). ∗∗∗∗P < .0001, 1-way ANOVA, Sidak multiple comparisons test. (I) Growth curve of CD56⁺ cells in NK-cell differentiation cultures initiated from 1 × 10⁴ of D8 CD43⁺ cells. D8 CD43⁺ cells were generated from DOX-treated iSOX17 hPSCs with intact and deleted NOTCH4in28 and cultured in NK-cell conditions for 3 weeks (means ± SDs, n = 4 experiments). ∗∗∗∗P < .0001, 2-way ANOVA, Tukey multiple comparisons test. ns, not significant.