Force generation of individual day 50 Neo^r, Puro^r-selected cardiomyocytes.

CSs were isolated at day 12–13.5 and cultured until day 48 when they were dispersed and deposited onto gelatin-functionalized surfaces of polyacrylamide cast with fluorescent beads and analyzed for force generation at day 50. (A) Diagram of the apparatus containing the polyacrylamide. Platinum electrodes were used to electrically pace the cardiomyocytes at 0.5 Hz with 0.8 ms pulses of 50 volts. Inset shows micrograph of fluorescent beads. Scale bar represents 10 µm. (B) α-actinin immunostaining of hESC-derived cardiomyocytes on the functionalized surface shows characteristic striations. Scale bar represents 10 µm. (C) Bead displacements near individual cardiomyocytes (Supplemental Figures S2A,B and Movie S4) were tracked using a cross-correlation-based optical flow algorithm in order to map deformations (red arrows) or stresses (blue arrows) across the face of the gel corresponding to individual cardiomyocytes [29]. Red arrows mark local bead displacement length is as per red scale bar (1 µm), which is expanded 20× relative to that of the image (white bar represents 10 µm) to permit visualization. Blue arrows indicate force magnitude as per blue scale bar [1 nN/µm2 (equivalent to 1 kPa)]. (D) Sample plot of total, transverse and longitudinal force versus time for a cardiomyocyte as in (C).