74 Chapter 3 Figure 5: CHIR and low-density plating activate β-catenin and AKT signaling to enhance hiPSC-CM proliferation via Cyclin D2-dependent kinases and prevent maturation via repression of sarcomere gene expression. (A) TOPFlash (TCF/LEF) luciferase analysis of hiPSC-CMs treated with CTR or CHIR in the presence/ absence of PNU74654 for 24 hrs. (B) Fold increase in TnT+ cell number after DMSO (CTR) or CHIR (2.0 μM) treatment in the presence/absence of PNU74654 (32 μM). (C) Normalized gene expression of Wnt target genes and maturation markers after treatment with CTR or the indicated compounds. Note the complete recovery of maturation gene expression when canonical Wnt signaling is abolished by PNU74654 treatment. (D) A mini-screen of 43 kinase targets after CHIR treatment demonstrated an increase in phosphorylation (p) levels at AKT, HSP27, and others. (E) Confirmation of AKT T308 phosphorylation by Western Blotting analysis. (F) Quantification of pAKT protein expression level after CHIR treatment. (G) Immunofluorescence analysis for pAKT T308 expression in TnT+ (green) day 20 hiPSC-CMs cultured for 6 days with the indicated treatment. Quantification of the number of TnT+ (H) and pAKT T308+ (I) cells for each treatment with the indicated compounds. (J) TOPFlash luciferase analysis of hiPSCCMs after treatment of CHIR for 24 hrs in the presence/absence of MK2206 (1.0 μM). (K) Expression of Wnt target genes and maturation markers after treatment with CTR or the indicated compounds. Note that AKT signaling is not changing the Wnt dependent maturation related gene expression. (L) A schematic diagram of the inhibitory relationship between GSK3 and downstream canonical Wnt signaling and their de-repression with CHIR. The role of PNU74654 to inhibit β-catenin-TCF/LEF activity is highlighted. Dashed lines indicate a correlation between AKT-CCND2. (M) Immunofluorescence analysis for Cyclin D2 (CCND2) (pink) expression in TnT+ (green) day 20 hiPSC-CMs cultured for 6 days with the indicated compounds. Quantification of the number of CCND2+/TnT+ cells per treatment group (N). (O) Immunofluorescence analysis for phospho Histone H3 (pHH3) (pink) and TnT (green) expression at day 20 hiPSC-CMs cultured for 6 days with the indicated compounds. Quantification of the number of pHH3+/TnT+ cells per treatment group (P). Scale bars represent 100μm, dot plots represent biological replicates and average. Bar charts represent mean±SD. *p<0.05 and **p<0.005 by unpaired t-test. Supplementary Table 1 specifies the replicates per experiment.
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