Figure 5.

Ultrastructural Organization and Proteomics of Later-Stage hiPSC-CMs on Nanopatterned Surfaces Reveal Immature Sarcomeric Composition

Single cells in sparse cultures on nanopatterned surfaces were investigated for sarcomeric ultrastructures.

(A) Transmission electron microscopy (TEM) images of early-stage (control₁), later-stage (control₁), and FCM-hiPSC-CMs (FCM-CMs) at low magnification (12,000×) show the presence of aligned myofibrils. Early stages look quite immature with contractile machinery forming but early occurrence of myofibril alignment. Later stages (both control and FCM-CMs) were more mature showing bundling of Z bands (arrowheads).

(B) TEM images acquired at higher magnification (40,000×) confirm a clear alignment of Z disks (arrowheads), but variable-spaced thick filaments remarkably visible in FCM-hiPSC-CMs with evidence of myofibril disarray in some cases. Both later-stage cell lines expressed I-Z-I bands with no clear evidence of H zones. In FCM-CMs, the sarcomeric ultrastructure was highly variable with sparse expression of M lines (stars).

(C) Proteomics of later-stage hiPSC-CMs (100 days p.d.) were compared with fetal left ventricular (LV) samples (130 gestational days). β-myosin was predominant at this stage of maturation indicating α to β isoform switch has occurred. CTNI expression indicated a higher grade of maturation. However, SSTNI was still expressed, confirming immature phenotype as seen for fetal LV. Representative western blot images were cropped. Scale bars represent 1 μm for the overview and 500 nm for the detailed view.