Fig. 3.

NOTCH activation at HE stage increases definitive hematopoiesis. a D4 HE were cultured with DAPT or in the presence of DLL1-Fc (see Fig. 1b schematic diagram). Cells were collected after 4 days of differentiation (D4 + 4) and used to determine frequencies of hematopoietic progenitors in CFU assay. Increase in multipotential GEMM and GM colonies in the DLL1-Fc culture condition suggests that NOTCH activation supports expansion of the most immature HPs. Results are mean ± s.e.m. for three independent experiments; two-way ANOVA Bonferroni post-hoc test, ***p < 0.001 b Flow cytometric analysis of Runx1 + 23-eGFP transgene expression in D4 HEPs cultured with DAPT or on DLL1-Fc. Runx1 + 23 enhancer activity increases in the cultures plated on DLL1-Fc and decreases in the DAPT-treated cultures. Representative contour plots from three independent experiments are shown. c T-cell potential of HP collected after 4 days of culture D4 HEs in presence of DAPT or DLL1-Fc. Bars show mean ± s.e.m. for three independent experiments; one-way ANOVA Bonferroni post-hoc test, **p < 0.01, ***p < 0.001. d Ratio of α/ζ, β/γ, and β/ε globin chain expression in erythroid cultures generated from D4 HE in presence of DAPT or DLL1-Fc. Results are mean ± s.e.m. for three independent experiments; one-way ANOVA Bonferroni post-hoc test, ***p < 0.001