Fig. 5.

Arterial-type HE undergoes EHT under high NOTCH activation and produce definitive-type HPs. a Schematic diagram of subsequent experiments. D5 CD144⁺CD43⁻CD73⁻ were sorted based on DLL4 expression (D5 HE:DLL4^+/−) using FACS and cultured on either OP9 or OP9-DLL4 for 4 days (D5 + 4). b, c Flow cytometric analysis of CD43⁺ hematopoietic and CD144⁺ endothelial cells following culture of D5 HE:DLL4⁺ and D5 HE:DLL4⁻ on either OP9 or OP9-DLL4. Bars in c are mean ± s.e.m. for at least three independent experiments. Two-way ANOVA Bonferroni post-hoc test, *p < 0.05. d The effect of NOTCH inhibition with DAPT on blood production from D5 DLL4⁺ and DLL4⁻ HE. No significant differences were found when HE:DLL4⁻ cells were treated with DAPT. Results are mean ± s.e.m. for three independent experiments. Two-way ANOVA Bonferroni post-hoc test, ***p < 0.001. e CFC potential of hematopoietic cells generated from D5 DLL4⁺ and DLL4⁻ HE following 5 days culture on OP9-DLL4. Results are mean ± s.e.m. for at least three independent experiments; two-way ANOVA Bonferroni post-hoc test, *p < 0.05, **p < 0.01. CFC-GEMMs are significantly increased in DLL4⁺ cultures on OP9-DLL4. f Ratio of α/ζ, β/γ, and β/ε globin chain expression in erythroid cultures generated form hematopoietic cells collected from D5 DLL4⁺ and DLL4⁻ HE cultured on OP9-DLL4 (D5 + 4 cells). Results are mean ± s.e.m. for three independent experiments. one-way ANOVA Bonferroni post-hoc test, **p < 0.01, ***p < 0.001. g Limiting dilution assay to determine the frequency of T-cell progenitors within the D5 + 5 HPs generated from HE:DLL4⁻ on OP9, HE:DLL4⁻ on OP9-DLL4, and HE:DLL4⁺ on OP9-DLL4