76 Chapter 3 Figure 6: Long-term GSK3 inhibition with low-density plating does not preclude hiPSC-CMs terminal differentiation and maturation in functional cardiac tissue. (A) Immunofluorescence image of alpha-actinin (αAct) and (B) qPCR analysis of sarcomeric gene expression in passage 4 (P4) expanded vs non-expanded hiPSC-CMs at day 42 (~D42) of differentiation. (C) Brightfield images of engineered heart tissues produced from non-expanded or P4 expanded hiPSC-CMs at ~D42 and further cultured for 25-days. (D) Brightfield image of engineered heart tissues in a 24-well plate format. (E) Immunofluorescence microscopy images of alpha-actinin (α-Act) and Troponin T (TnT) in cardiac tissue generated from previously expanded hiPSC-CMs. (F) Graphs displaying development of contraction force (mN), beating rate and time to peak (T1) in heart tissues generated from previously expanded (P4 CHIR>CTR) (yellow) or non-expanded (grey) hiPSC-CMs measured at different time points after cardiac tissue formation. (G) Plots for contraction analysis in engineered heart tissue 14 days after formation for the indicated treatment and hiPSC lines. Scale bars represent 100μm. Bar charts represent mean±SD. Dot plots represents biological replicates and mean±SEM. *p<0.05 by unpaired t-test. Supplementary Table 1 specifies the replicates per experiment.
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