42 Chapter 2 Figure 4. Overview of Wnt modulation during hiPSC-CM differentiation and expansion. (A) Schematic overview of human induced pluripotent stem cellderived 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 hiPSCCM 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). Govindsamy et al., 2018), so we also chose to optimize our protocol for proliferation induction with B-27 medium containing insulin. In summary, Wnt activation together with removal of cell-cell contacts transiently increases the window for massive expansion of immature hiPSC-CMs. Massively expanded hiPSC-CMs are fully functional and could form a powerful source for tissue engineering applications and/or myocardial biopatches (Miyagawa et al., 2022; Gao et al., 2018). These protocols are still limited to producing a 100- to 250-fold increase
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