Figure 5.
Impact of chronic optical pacing (C-OP) on structural maturation of h-iPSC ECTs: (a) Representative, linear and cylindrical h-iPSC ECT constrained at the long axis ends by mesh nylon anchors. (b–c) Representative central paraffin sections of a (b) D14 control ECT showing 54% CM and (c) D14 C-OP ECT showing 52% CM and 13% ChIEF-transfected cells. Nuclei were stained with DAPI (blue), CM with cTnT (green) and ChIEF-transfected cells with tdTomato (red). CM were predominantly located at the outer surface and the majority of transfected cells were CM. Inset in (c) shows higher magnification of double-positive cTnT and tdTomato cells. (d) Whole mount confocal image of a control D14 ECT. (e) Mean CM fraction and (f) mean alignment concentration (κ) were similar in D14 control (n = 9) and C-OP (n = 10) h-iPSC ECTs. Error bars show standard deviations. (g–j) Representative local CM orientations within D14 (g) control and (i) C-OP h-iPSC ECTs. Color-coded lines indicate local CM orientation magnitudes in the circumferential (green), radial (red), and axial (blue) directions. For clarity, only a subset of the orientations is shown. Insets are 3X magnification of regions enclosed by the yellow dashed lines. (h and j) Spherical histograms of local CM orientations. CM in both control and C-OP aligned parallel to the ECT long axis. The volume of each ray represents the relative count in each direction and the mean CM orientation is shown by the thick red line. κ for the control sample (h) was 3.89 while the C-OP sample (j) was 3.46. Scale bars: 5 mm (a), 250 μm (b, c, d, g, i), and 100 μm (c inset). Orientation axes also serve as scale bars in (d, g, i).