Figure 2
Figure 2. Cyclopamine increases blood differentiation in hEBs and favors adult hematopoiesis. (A) Schematic representation of the hEB differentiation system and treatment with cyclopamine, Shh, or purmorphamine. (B) Activation of the Hh pathway by Shh or purmorphamine results in a strong induction of Gli1, Ptch, and Hhip expression, in contrast to the down-regulation shown after the inhibition of the pathway mediated by cyclopamine. (C) Representative flow cytometry data of the hematopoietic differentiation at day 15 of hEB differentiation after Shh or purmorphamine treatment showing inhibition of hEB-derived hematopoiesis. (D) Reduction in the percentage of mature blood cells (CD45+) and primitive blood cells (CD34+/45+), but not in hemogenic progenitors (CD45−PFV) after Shh or purmorphamine treatment. (E) Representative flow cytometry data of hematopoietic differentiation at day 15 of hEB differentiation after DMSO (control) or cyclopamine treatment showing inhibition of hEB-derived hematopoiesis. (F) Reduction in the percentage of mature blood cells (CD45+) and primitive blood cells (CD34+/45+), but not in hemogenic progenitors (CD45−PFV) after control or cyclopamine treatment. (G-H) Treatment schema (G) and percentage of mature blood cells (H) after constant, early, and late cyclopamine treatment relative to control-treated hEBs (dashed line). Early inhibition of the Hh pathway significantly increases hematopoietic differentiation compared with all other combinations. (I) Inhibition of the Hh pathway by siRNA directed against Smo with a 30% reduction of Smo expression in hEBs on day 2 after the Smo-siRNA transfection. (J) Western-blot showing a reduction in Smo protein in hEBs at day 4 after Smo-siRNA transfection. (K) Increase in the percentage of mature blood cells at day 15 after Smo-siRNA transfection. (L) Number of hemangioblast colonies is increased on cyclopamine treatment compared with control. (M) Pie charts representing the number of individual CFU types generated from control and cyclopamine-treated cells. (N) An increase in total number of CFUs is observed from equal starting numbers of control or cyclopamine-treated hemangioblasts. *P < .05; **P < .01.

Cyclopamine increases blood differentiation in hEBs and favors adult hematopoiesis. (A) Schematic representation of the hEB differentiation system and treatment with cyclopamine, Shh, or purmorphamine. (B) Activation of the Hh pathway by Shh or purmorphamine results in a strong induction of Gli1, Ptch, and Hhip expression, in contrast to the down-regulation shown after the inhibition of the pathway mediated by cyclopamine. (C) Representative flow cytometry data of the hematopoietic differentiation at day 15 of hEB differentiation after Shh or purmorphamine treatment showing inhibition of hEB-derived hematopoiesis. (D) Reduction in the percentage of mature blood cells (CD45+) and primitive blood cells (CD34+/45+), but not in hemogenic progenitors (CD45−PFV) after Shh or purmorphamine treatment. (E) Representative flow cytometry data of hematopoietic differentiation at day 15 of hEB differentiation after DMSO (control) or cyclopamine treatment showing inhibition of hEB-derived hematopoiesis. (F) Reduction in the percentage of mature blood cells (CD45+) and primitive blood cells (CD34+/45+), but not in hemogenic progenitors (CD45−PFV) after control or cyclopamine treatment. (G-H) Treatment schema (G) and percentage of mature blood cells (H) after constant, early, and late cyclopamine treatment relative to control-treated hEBs (dashed line). Early inhibition of the Hh pathway significantly increases hematopoietic differentiation compared with all other combinations. (I) Inhibition of the Hh pathway by siRNA directed against Smo with a 30% reduction of Smo expression in hEBs on day 2 after the Smo-siRNA transfection. (J) Western-blot showing a reduction in Smo protein in hEBs at day 4 after Smo-siRNA transfection. (K) Increase in the percentage of mature blood cells at day 15 after Smo-siRNA transfection. (L) Number of hemangioblast colonies is increased on cyclopamine treatment compared with control. (M) Pie charts representing the number of individual CFU types generated from control and cyclopamine-treated cells. (N) An increase in total number of CFUs is observed from equal starting numbers of control or cyclopamine-treated hemangioblasts. *P < .05; **P < .01.

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