Figure 5

Effect of Wnt signaling inhibition on Ca²⁺ oscillation of CMs differentiated from CPCs in 3D vs 2D culture. Human iPSC-CPCs were plated in 3D aligned nanofiber and 2D plates with the addition of 53AH, XAV939 and DMSO control from day 0 to day 3, followed by culture in assay medium until day 7 or day 14 of differentiation. The cells were then loaded with FLIPR Calcium 5 dye and intracellular Ca²⁺ oscillation was recorded on a FLIPR Tetra system. (a–d) Representative recordings of intracellular Ca²⁺ oscillation from cells treated with 1.1 µM 53AH (blue tracers), 10 µM XAV939 (red tracers) or DMSO control (black tracers), and cultured in 2D plates for 7 days (a), 14 days (b) and in 3D aligned nanofiber plates for 7 days (c) and 14 days (d) with arrows indicating the time point of adding isoproterenol. (e,f) The peak frequency quantification of Ca²⁺ oscillation from cells differentiated with 53AH in 3D aligned nanofiber culture under the basal and isoproterenol stimulated conditions (e), and the peak amplitude of Ca²⁺ oscillation in the basal condition in cells treated with 53AH or XAV939 (f). Quantification results are presented as scatter dot plots with mean ± SEM. n = 3–4. *p < 0.05 indicates significant difference between Day 7_3D and Day 14_3D cultures. ⁺p < 0.05 indicates significant difference between the basal and isoproterenol stimulated conditions.