Figure 2.

Characterization of Human HE along Human EB Differentiation

(A) Flow cytometry analysis of CD31⁺CD144⁺ populations at different times of EB differentiation (representative plots from four different experiments). Circled gates indicate the HE-enriched isolated populations along with the percentage of CD43⁺ cells found within these gated populations or without gating. CD31⁺CD144⁺CD43⁺ cells were excluded during the sort.

(B) Kinetic of endothelial (CD31⁺CD144⁺) and hematopoietic (CD43⁺) profiles obtained after re-plating in hematopoietic condition the purified CD31⁺CD144⁺ populations obtained at days 6, 8, and 10 of EB differentiation. Data are representative of three independent experiments after 4 and 7 days in culture on gelatin-coated plates.

(C) Quantification and statistical analysis of the total number of cells and hematopoietic-committed cells (CD43⁺) generated at days 4 and 7 of culture. Error bars indicate the SEM of data from three independent experiments. The significance of the difference between samples was confirmed using two-way ANOVA; p value: adjusted p value using SIDAK multiple comparison: ^∗p < 0.05, ^∗∗p < 0.001, ^∗∗∗p = 0.0002, ^∗∗∗∗p < 0.0001.

(D) Representative pictures of CD31⁺CD144⁺ populations isolated at the indicated times of EB differentiation after 4 and 7 days of culture on gelatin-coated plates in hematopoietic-inducing condition. Red arrows indicate endothelial clusters with hemogenic ability. Red arrowheads indicate emerging blood cells and clusters of blood cells. Black arrowheads indicate endothelial cell clusters with no hemogenic potential.

(E) Quantification of colony-forming unit (CFU) potential of 10⁴ cells harvested after 4 days of culture from the CD31⁺CD144⁺ populations isolated at days 6, 8, and 10 of EB differentiation. Error bars indicate the SEM of data from three independent experiments.

(F) Pictures of representative colonies and cytospin of cells stained with O-dianisidine MGG. Scale bars, 100 μm. M, macrophages; N, neutrophils; E, eosinophils; Ba, basophils; Mo, monocytes; Ery, erythrocytes. Brown staining is indicative of hemoglobinization of the erythrocytes. Scale bars, 20 μm.