Fig. 4.

Overview of Wnt modulation during hiPSC-CM differentiation and expansion. (A) Schematic overview of human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation. Created with BioRender.com. (B) Brightfield images of hiPSC-CMs over multiple passages including expansion illustrating the process of cell-cell contact removal via sparse passaging (∼2.5×10⁴ cells/cm²) with concomitant CHIR99021 administration in B27+insulin/RPMI media to facilitate massive expansion. (C) Brightfield images of hiPSC-CMs in expansion from days 15-20. (D) Immunofluorescence images of hiPSC-CMs stained for α-actinin (green), proliferation marker Ki67 (red) and nuclei (blue) as indicated. (E) Quantification of Ki67⁺ cells indicates that a 37% increase in hiPSC-CM proliferation can be promoted by administering 2 μM CHIR99021. (F) Quantification of hiPSC-CM number from P1 to P5 by sequentially expanding the hiPSC-CMs using CHIR99021 (CHIR). D, differentiation day; P, passage; RPMI, Roswell Park Memorial Institute medium. Figure is adapted from Maas et al. (2021) and is available to view on Figshare alongside detailed Materials and Methods: 10.6084/m9.figshare.23607282. Scale bars: 200 µM (B,C); 100 µM (D).