Figure 1.

Prolonged Notch Activation in 3D Gels Is Dose-Dependent and Promotes hESC-CM Proliferation

(A) Time course analysis of Notch-mediated luciferase expression in U2OS CSLluc/ren Notch reporter cell line indicates a prolonged Notch signal response in 3D culture conditions compared to the 2D Delta-1 platform. Luciferase signal is expressed normalized to IgG controls (2D or 3D) and plotted against days in culture. IgG controls were normalized to 1 (dashed line). (B) Dose-dependent Notch activation. Culturing the U2OS CSLluc/ren Notch reporter cell line in the 3D Delta-1 gel results in Delta-1 dose-dependent activation of Notch signaling, as indicated by fold luciferase expression compared to IgG control gels. (C) hESC-CMs in 3D-engineered cardiac tissues proliferate in response to Delta-1 (Delta). Cardiomyocyte proliferation was measured by histology as double-positive βMHC⁺/BrdU⁺ cells, which results in a significant increase in response to Delta with day 15 cells and day 30 cells over IgG controls (control). Note that although proliferation slows over time, significant augmentation by Delta is still possible at day 30. For (B), p-values were calculated using a one-way ANOVA with all samples compared to 0 μg/mL followed by Dunnett’s multiple comparison test. For (C), p-values were calculated using a multiple unpaired t-test without assuming a consistent SD. ∗p < 0.05, ∗∗p < 0.005. Error bars denote SEM. See also Figures S1 and S2.