116 Chapter 5 Figure 3. Selected gene expression profiles for developmental growth and cell cycle regulators in serially passaged immature hiPSC-CMs and metabolic matured hiPSC-CMs. (A) Representative immunofluorescence for Hoechst (blue), Ki67 (red), and a-actinin (green) in hiPSC-CMs treated with CHIR99021 at passage 1 (P1) and 4 (p > 3), versus metabolic matured hiPSC-CMs at day 77. Graph displays the percentage of Ki67 positive nuclei per condition (P1, >P3 or day 77) (n = 3 differentiations with 17, 32 and 24 quantified images). (B) Heatmap showing the expression patterns of 40 selected genes involved in the proliferation and/or development of hiPSC-CMs between expanding hiPSC-CMs (passage 1–3), metabolic matured hiPSC-CMs (day 77, 84 and 97) and adult human heart tissue. (C) Heatmap showing the expression patterns of cell cycle phase-related genes between expanding hiPSC-CMs (passages 1–3), metabolic matured hiPSC-CMs (day 77, 84 and 97) and adult human heart tissue. Red indicates high expression and blue indicates low expression of represented genes. Heatmaps are clustered and show the RPKMs for the selected genes. GSK3β Inhibition Increases Non-Viral Vector Incorporation Efficiency in hiPSC-CMs The efficiency of non-viral vector incorporation in low-proliferative hiPSC-CMs is usually below 20%, which highly challenges its usefulness in, e.g., molecular knockdown experiments.26 We reasoned that GSK3β inhibition in hiPSC-CMs, promoting a high-proliferative status, would also potentially enhance the incorporation and expression of introduced plasmid DNA. To this end, we used day 30 high-proliferative hiPSC-CMs treated with CHIR99021 and lowproliferative non-GSK3β inhibition treated age-matched controls and transfected these for 72 h with a PF256-H2B-mCherry plasmid (mCherry expression controlled by the histone cluster 1 promotor in the presence of Lipofectamine or ViaFect transfection reagent.27 Non-viral vector incorporation efficiency was measured by flowcytometry for mCherry and Hoechst and fluorescence-based immunohistochemistry for α-actinin and expression of mCherry 72 h after the start of transfection (Supplementary Figure 3A–C). We observed that in the presence of CHIR99021 30.5 ± 4.9% of day 30, high-proliferative hiPSC-CMs expressed mCherry versus 10.8 ± 5.4% (p = 0.03) of low-proliferative hiPSC-CMs cultured without a GSK3β inhibitor (Figure 4A). To investigate the cell cycle-independent effects of the GSK3β inhibition of non-viral vector incorporation efficacy, we also transfected day ~80 metabolic matured
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