Figure 3. Wnt and Notch signaling affects blast colony formation from the hemangioblast.
(A) Role of Wnt signaling in blast colony development. 4×104 Flk-1+ cells isolated from the day 2.75 EBs generated from either sb-cat or Notch1-IC ES cells were cultured in M10 serum-free hemangioblast media in the presence or absence of doxycyclin (Dox; 2 μg/ml) and DKK1(DKK; 300ng/ml) as indicated. Dox was added at 0 hr of culture, while DKK1 was added at 6 hr of culture. Colonies were washed with media at 24 hr to deplete DKK1 and Dox, and replated back into fresh M10. Blast and core colonies were scored following 4 days of culture. (B) Effect of DKK1 treatment on blast and core colony development. (C) Hematopoietic potential of DKK1 treated colonies. 1600 colonies of each group were picked at day 3 of culture, pooled, dissociated and replated into hematopoietic methylcellulose cultures. Ep: primitive erythroid colonies; Def: combined macrophage, bipotential macrophage/erythroid, definitive erythroid and multipotential myeloid/erythroid colonies. (D) Role of Numb in blast colony development. gSI γ-secretase inhibitor X (2.5 μM) (E) Role of Notch signaling in blast colony development. (A-E) Error bars represent standard deviations of the mean of number of colonies from n independent experiments (A, n=3; B, n=3; C, n=3; D, n=4; E, n=4). (**P-value<0.01; ***P-value<0.001)