72 Chapter 3 (Figure 5C). We also show that the expression of markers of hiPSC-CM maturation (MYL2, MYH7, MYOM1) were down-regulated by CHIR treatment and recovered fully upon the cotreatment with PNU746554 (Figure 5C). These results demonstrate that β-catenin-mediated canonical Wnt signaling prevents hiPSC-CM maturation and is responsible for roughly 50% of the observed CHIR-induced expansion of hiPSC-CMs. Figure 4: Density-dependent CHIR-induced hiPSC-CM proliferation is uncoupled from YAP activity. (A) Representative immunofluorescence images for yes-associated protein (YAP)(green), cardiac troponin T (TnT) (cyan) and nuclei (red) in dense or sparse culture conditions. (B) Graph displaying nuclear/cytoplasmic YAP ratios for the indicated culture condition. Data are means ± SEM. ****p<0.0001 by unpaired t test. (C) Schematic displaying potential biophysical effects of sparse and dense cell culture on cell-sensing of the substrate stiffness. (D) Immunofluorescence for Ki67 (green), TnT (cyan) and nuclei (red) in hiPSC-CMs cultured on substrates with varying stiffness (kPa) or on tissue culture plastic (TCP)(infinite stiffness). (E) Quantification of the percent Ki67+/TnT+ cells for the indicated substrate stiffness in D. (F) Representative immunofluorescence images as in A of hiPSC-CMs cultured sparsely on the substrates as in D. (G) Nuclear/cytoplasmic ratios of YAP in hiPSC-CMs in F. YAP expression (I) and (H) in hiPSC-CMs cultured in sparse or dense culture conditions with or without YAP inhibitor Verteporfin (1.0 or 10 μM). Immunofluorescence (J) and quantification (K) for ki67+ (red), TnT+ (green) cells (blue) for the same conditions as in H. Data are means ± SEM. *p<0.05. ****p<0.0001 by One-way ANOVA Dunnett’s post hoc multiple comparisons to control “Sparse-DMSO” group. Supplementary Table 1 specifies the replicates per experiment.
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