Figure 3
Figure 3. Modulation of Notch signaling alters blood commitment of developing hEBs. (A-D) Morphologic similarity was observed between hESC (A,C) and hFib-iPSC (B,D) in undifferentiated (A-B) and day 10 developing EBs (C-D). Scale bars represent 100 μm (A-B) and 250 μm (C-D). (E) Total number of hematopoietic colonies was higher in Jag1-treated hEBs vs control (181.8 ± 20.8 vs 111.8 ± 9.9). **P < .01 (n = 4). (F-G) Knockdown HES1 using siRNA treatment reduced 81.5% of committed (F) and 82.4% of primitive blood (G) cells in hematopoietic hESC-EB development relative to siRNA controls. **P < .01 (n = 3). (H) Hematopoietic colony formation (CFU) was performed to examine the effect of HES1 knockdown on hematopoietic progenitors. siRNA-mediated knockdown of HES1 led to a reduction in the number of hematopoietic colonies (64.8 ± 9.1 vs 104.8 ± 11.5) relative to scrambled siRNA controls. *P < .05 (n = 4). (I-J) During hFib-iPSC hematopoiesis, Jag1-activated Notch1 signaling increased the number of committed (I, 199.8% increase) and primitive blood cells (J, 328.5% increase) compared with controls. *P < .05; **P < .01 (n = 3). (K) Inhibition of hFib-iPSC blood commitment by GSI. EBs treated with GSI showed 85.6% reduced blood commitment relative to controls. **P < .01 (n = 3). (L) Alterations in Notch signaling control the generation of CFU-forming hematopoietic progenitors. Jag1-induced Notch signaling increased the number of CFUs (170.25 ± 5.47), whereas GSI-treated EBs formed fewer CFUs (63.25 ± 16.58) relative to controls (101.25 ± 8.66). *P < .05; **P < .01 (n = 4). (M-N) MIEV retroviral vectors for overexpression of HES1 (M) and GFP signals in EBs transduced with control (CTRL, left panels) or HES1 (right panels) retroviral overexpression vectors (N). Scale bars represent 250 μm. (O) Transduction efficiency of both control (10.12 ± 1.7%) and HES1 (9.81 ± 0.6%) retroviruses at day 15 of EB development by FACS. (P-S) Augmentation of blood in total EBs (P-Q) or within the GFP+ cell fraction (R-S) at day 15 of EB development. Both frequency based on FACS analysis (P, 30.3 ± 1.3% vs 20.1 ± 1.5%) and total number of blood cells (Q, 10.9 ± 0.6 × 104 cells vs 6.6 ± 0.8 × 104 cells) are higher in developing hEBs transduced by HES1 overexpression vs control. Within the GFP+ cell fraction, higher frequency (R, 31.7 ± 6.5% vs 12.8 ± 1.4%) and number of blood cells (S, 11.1 ± 1.73 × 103 cells vs 4.2 ± 0.71 × 103 cells) are observed in HES1 overexpression. **P < .01 (n = 4). (T) Contribution of GFP+ cells in hematopoietic CFUs from both control and HES1 overexpression. GFP+ cells from control CFUs contributed in both hematopoietic colonies (white arrowhead) and attached endothelial colonies similarly (red arrowhead), whereas the majority of GFP+ cells are restricted to hematopoietic colonies upon HES1 overexpression. Scale bars represent 100 μm. (U) HES1 overexpression increased total number of hematopoietic colonies over the control (34.3 ± 6.4 vs 6.7 ± 1.5). **P < .01 (n = 3).

Modulation of Notch signaling alters blood commitment of developing hEBs. (A-D) Morphologic similarity was observed between hESC (A,C) and hFib-iPSC (B,D) in undifferentiated (A-B) and day 10 developing EBs (C-D). Scale bars represent 100 μm (A-B) and 250 μm (C-D). (E) Total number of hematopoietic colonies was higher in Jag1-treated hEBs vs control (181.8 ± 20.8 vs 111.8 ± 9.9). **P < .01 (n = 4). (F-G) Knockdown HES1 using siRNA treatment reduced 81.5% of committed (F) and 82.4% of primitive blood (G) cells in hematopoietic hESC-EB development relative to siRNA controls. **P < .01 (n = 3). (H) Hematopoietic colony formation (CFU) was performed to examine the effect of HES1 knockdown on hematopoietic progenitors. siRNA-mediated knockdown of HES1 led to a reduction in the number of hematopoietic colonies (64.8 ± 9.1 vs 104.8 ± 11.5) relative to scrambled siRNA controls. *P < .05 (n = 4). (I-J) During hFib-iPSC hematopoiesis, Jag1-activated Notch1 signaling increased the number of committed (I, 199.8% increase) and primitive blood cells (J, 328.5% increase) compared with controls. *P < .05; **P < .01 (n = 3). (K) Inhibition of hFib-iPSC blood commitment by GSI. EBs treated with GSI showed 85.6% reduced blood commitment relative to controls. **P < .01 (n = 3). (L) Alterations in Notch signaling control the generation of CFU-forming hematopoietic progenitors. Jag1-induced Notch signaling increased the number of CFUs (170.25 ± 5.47), whereas GSI-treated EBs formed fewer CFUs (63.25 ± 16.58) relative to controls (101.25 ± 8.66). *P < .05; **P < .01 (n = 4). (M-N) MIEV retroviral vectors for overexpression of HES1 (M) and GFP signals in EBs transduced with control (CTRL, left panels) or HES1 (right panels) retroviral overexpression vectors (N). Scale bars represent 250 μm. (O) Transduction efficiency of both control (10.12 ± 1.7%) and HES1 (9.81 ± 0.6%) retroviruses at day 15 of EB development by FACS. (P-S) Augmentation of blood in total EBs (P-Q) or within the GFP+ cell fraction (R-S) at day 15 of EB development. Both frequency based on FACS analysis (P, 30.3 ± 1.3% vs 20.1 ± 1.5%) and total number of blood cells (Q, 10.9 ± 0.6 × 104 cells vs 6.6 ± 0.8 × 104 cells) are higher in developing hEBs transduced by HES1 overexpression vs control. Within the GFP+ cell fraction, higher frequency (R, 31.7 ± 6.5% vs 12.8 ± 1.4%) and number of blood cells (S, 11.1 ± 1.73 × 103 cells vs 4.2 ± 0.71 × 103 cells) are observed in HES1 overexpression. **P < .01 (n = 4). (T) Contribution of GFP+ cells in hematopoietic CFUs from both control and HES1 overexpression. GFP+ cells from control CFUs contributed in both hematopoietic colonies (white arrowhead) and attached endothelial colonies similarly (red arrowhead), whereas the majority of GFP+ cells are restricted to hematopoietic colonies upon HES1 overexpression. Scale bars represent 100 μm. (U) HES1 overexpression increased total number of hematopoietic colonies over the control (34.3 ± 6.4 vs 6.7 ± 1.5). **P < .01 (n = 3).

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