Panel A shows bright field microscopy images of human induced pluripotent stem cells during CHIR treatment with or without inhibitor, illustrating cell retraction over time. Panel B shows confocal orthogonal images of cells, x-axis represents spatial depth and y-axis represents cell structure with DNA and filamentous actin staining. Panel C shows maximum intensity projection microscopy images comparing nuclear and actin organization at different time points. Panel D shows violin plot, x-axis shows time after CHIR treatment and y-axis shows monolayer height. Panel E shows fluorescence microscopy images of nuclei during treatment. Panel F shows violin plot, x-axis shows treatment conditions and y-axis shows nuclear area. Panel G shows scatter plot, x-axis shows cell count and y-axis shows nuclear area with correlation trends. Panel H shows fluorescence microscopy images of phosphorylated myosin light chain distribution over time. Panel I shows violin plot, x-axis shows time after treatment and y-axis shows percentage of phosphorylated myosin light chain positive area. Panel J shows violin plot, x-axis shows treatment conditions and time points and y-axis shows retardance measurements.
hiPSC differentiation along the cardiac mesoderm increases actomyosin-driven contractility. (A) Bright-field imaging of WT hiPSCs treated with CHIR in the presence or absence of a pan-caspase inhibitor Q-VD-OPH. The retraction area is highlighted in yellow. Time is presented as hh:mm. Scale bar = 20 µm (related to Video 1). (B and C) Confocal XZ orthogonal views (B) and MaxIPs (C) of hiPSCs at the basal state (0-h CHIR) or after CHIR treatment (24- and 48-h CHIR), stained for DNA (cyan) and F-actin (gray). Scale bar = 10 µm. (D) Quantification of the monolayer height at 0 h, 24, and 48 h after CHIR treatment from B. Violin plots represent individual measurements (small dots) averaged for each biological repeat (large dots). Median (plain red line) and quartiles (dotted black lines) are displayed. n = 32 technical repeats across N = 7 independent biological repeats. A Kruskal–Wallis test with Dunn’s multiple comparisons posttest was performed on biological repeats. (E–G) Representative MaxIP immunofluorescence of WT hiPSCs during CHIR treatment. Fixed cells were stained for DNA marker (Hoechst). Scale bar = 20 µm (E). Average nuclear size from n = 15 fields of view across N = 3 independent biological repeats. Median (plain red line) and quartiles (dotted black lines) are displayed. A two-tailed unpaired t test was performed (F). Correlation of nuclear area over cell number for each field of view during CHIR treatment. Biological repeats are color-coded. Pearson’s correlation coefficient is displayed for each treatment (G). (H) Representative MaxIP immunofluorescence of hiPSCs at the basal state (0 h) or after CHIR treatment (24 and 48 h) stained for ppMLC2. Pixels are color-coded by intensity using the fire LUT. Scale bar = 50 μm. (I) Percentage of ppMLC2-positive area at 0, 24, and 48 h after CHIR is reported as violin plots. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 15 technical repeats across N = 3 independent biological repeats. A Kruskal–Wallis test with Dunn’s multiple comparisons posttest was performed on the technical repeats. (J) Retardance measurements obtained from hiPSCs at the basal state (0 h), after CHIR treatment (24, 48, 72 h), or treated with 10 μM Y-27632 at 0 h (Y-27) using QPOL imaging. Median (plain red line) and quartiles (dotted black lines) are displayed. n = 240 technical repeats for 0, 24, 48, and 72 h and n = 120 technical repeats for Y-27 across N = 3 independent biological repeats. A one-way ANOVA with Šidák’s multiple comparisons posttest was performed on the biological repeats. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.