70 Chapter 3 Figure 3: Phenotypic assessment of hiPSC-CMs following GSK-3β inhibition and removal of cell-cell contact inhibition (A-B) Confocal microscopy images of P3 (D35) hiPSC-CMs treated previous with either DMSO (1:5000) (CTR) or CHIR (2.0 μM) since P0 and (for CHIR-treated cells) subsequently treated for 6 days with either continuation of CHIR (2.0 μM) or replacement of CHIR with C59 (2.0 μM) (CHIR>C59) and immunostained on micropatterned surfaces for the expression of troponin T (TnT) and alpha-s actinin (α-SA) or phospho Histone H3 (pHH3). Treated hiPSC-CMs were micropatterned as single cells to adopt rectangular morphology with 7:1 aspect ratio. (C-D) Representative images of distribution of sarcomere fiber alignment. Color codes represent angle of α-SA with respect to cellular long axis (e.g. Z-disc-registered α-SA angle = 90o) (n=10) (E) Contractility measurements in cells treated in (B). (F) Ca2+ transients (Fluo-4AM) fluorescence expressed relative to baseline [F/F0] in hiPSC-CMs at day (D) 42 in hiPSC-CMs treated with DMSO (CTR), CHIR (CHIR) or CHIR followed by C59 (CHIR>C59). Plots displaying the Ca2+ transient (G) Frequency, (H) Amplitude and Decau Tau for each group. Fold increases in the expression of sarcomere (I), electrophysiological (J), and metabolic genes (K) at the indicated days in hiPSC-CMs treated with CHIR from D12 to D28 followed by either continued treatment with CHIR, CHIR withdraw (CHIR>CTR) or replacement of CHIR with C59 (CHIR>C59). (L) UMAP plots of day 12 hiPSC-CMs treated with DMSO (1:2500) (grey), CHIR (4.0 μM) (yellow), or C59 (4.0 μM) (black) for 24 hrs. (M) UMAP plots for indicated treatments and genes of single-cell sequencing data. (N) Average normalized (over CTR) expression of the selected mature cardiac genes for indicated treatments. (O) UMAP plots for cell cycle analysis, S-phase (S)(blue), G2/M-phase (G2M)(green) and G1-phase (red) for the indicated treatments. (P) Bar graph displaying the percentage of cells from (L) in S, G2M, or G1 phase for indicated treatments. Scale bars represent 100μm. Dot plots represent biological replicates and average. Bar charts represent mean. Graphs represent average±SD. *p<0.05 and **p<0.005 by unpaired t-test. NS=not significant. Supplementary Table 1 specifies the replicates per experiment. YAP activity is uncoupled from cell density-dependent CHIR-induced hiPSC-CM proliferation. The Hippo signaling pathway has been shown to regulate heart growth and Wnt signalingmediated cardiomyocyte proliferation (Heallen et al., 2011). Hippo pathway consists of a core kinase cascade that modulates activity of transcriptional co-activator, yes-associated protein (YAP), which is one of the major nuclear effectors of Hippo pathway. Previous studies have
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