66 Chapter 3 Figure 1: GSK-3β inhibition promotes hiPSC-CM proliferation in a cell-density dependent manner. (A) Schematic of Wnt-modulated directed cardiac differentiation and subsequent expansion. Day 12, hiPSC-CMs were plated densely (~100,000 cells/cm2), sparsely (~10,000 cells/cm2) for serial passaging or cultured without passaging (~500,000-1M cells/cm2) in the presence of CHIR99021 (CHIR) (2.0M) (B) Graph displaying growth of hiPSC-CMs represented as fold increase over day 12 for each passage (P) number. Data are means ± SEM. (C) Immunohistochemistry for proliferation marker Ki67 (green), cardiac troponin T (TnT) (cyan) and nuclei (red) in hiPSC-CMs for the indicated culture conditions. (D) Quantification of the Ki67+ hiPSC-CMs mentioned in C. Immunofluorescence (E) and quantification (F) of the mitotic phospho-histone H3 (pHH3) (green), TnT (cyan) and nuclei (red) in hiPSC-CMs cultured in varying cell densities. Data (B-F) are in means ± SEM. *p<0.05, ****p<0.001, n.s. p>0.05 by One-way ANOVA with Tukey’s post hoc multiple comparisons test. (G) Illustration of conditionedmedia of densely cultured cells, subsequent factor concentration and application to sparsely cultured hiPSCCMs. (H) Immunofluorescence images for the indicated antibodies in sparsely passaged hiPSC-CMs cultured with varying concentrations of conditioned medium from densely cultured hiPSC-CMs. (I) Quantification of the percentages of Ki67+/TnT+ cells in H. Data are means ± SEM. n.s. p>0.05 by One-way ANOVA Dunnett’s post hoc multiple comparisons to control “no C/M” group (blue open circle). (J) Schematic of culturing fixed hiPSC-CMs cell numbers with (dense) or without (sparse) cell-cell contact. Immunofluorescence (K) and quantification (L) of the percent proliferative (Ki67+/TnT+) cells with (dense) and without sparsely cell-cell contact. Same for mitotic (pHH3+/TnT+) cells (M) and (N). Data are means ± SEM. *p<0.05, **p<0.01 by unpaired t-test. Supplementary Table 1 specifies the replicates per experiment.
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