Figure 5: Ablating regions of high cell-adhesion tension inhibits, whereas mechanical stretching promotes mesoderm specification.

(A) Cartoon of eyebrow knife experiment.

(B) Representative brightfield images and traction stress maps before and after eyebrow knife ablation.

(C) Representative brightfield and time-lapse images of T-mNeonGreen expression following BMP4 stimulation for triangle hESC colonies with one corner ablated using the eyebrow knife prior to BMP4 addition. Arrow indicates site of eyebrow knife ablation.

(D) Plot of time to T-mNeonGreen expression in the corners of triangle hESC colonies with and without eyebrow knife ablation. Line and bars represent mean ± 95% CI for n = 10 (2, 5, 3) colonies.

(E) Representative traction stress map prior to BMP4 addition and corresponding time-lapse images of T-mNeonGreen expression following BMP4 stimulation for Pac-Man hESC colonies. Arrow indicates concave edge (“mouth”) of Pac-Man.

(F) Map of average traction stresses measured for Pac-Man hESC colonies. n = 20 (3, 10, 4, 3) colonies.

(G) Normalized average intensity map of T-mNeonGreen expression within Pac-Man hESC colonies at 30 h BMP4. n = 20 (3, 10, 4, 3) colonies.

(H) Cartoon of mechanical stretching experiment and representative brightfield image of an hESC colony cultured on the stretching device.

(I) Representative images of T-mNeonGreen expression for hESC colonies with and without mechanical stretching during 48 h BMP4.

(J) Normalized average intensity maps of T-mNeonGreen expression for hESC colonies with and without mechanical stretching during 48 h BMP4. n = 27 (12, 8, 7) stretched colonies and n = 39 (21, 10, 8) control colonies.

For (A)-(G): Scale bars = 250 μm. For (H)-(J): Scale bars = 100 μm. Pa = Pascals. Avg = average. a.u. = arbitrary units. *p < 0.05.