41 Harnessing developmental cues for cardiomyocyte production 2 cell; IGF1, insulin-like growth factor; Isl1, ISL LIM homeobox 1; MAPK, mitogen-activated protein kinase; Meis2, meis homeobox 2; p38i, p38 inhibitor; pHH3, phosphohistone H3; PI3K, phosphoinositide 3 kinases; Rb1, retinoblastoma; S1P/LPA, sphingosine-1-phosphate/ lysophosphatidic acid; SAG, smoothened agonist; SB, SB431542; (si)RNA, small interfering RNA; SMAD, mothers against decapentaplegic; TGFβ, transforming growth factor β. However, insulin does not rescue cell cycle arrest, likely due to altered substrate use and fatty acid metabolism in the treated cardiomyocytes, so this effect is short-lived (Mills et al., 2017). During development and hiPSC differentiation, the GSK-3β/ Wnt/β-catenin signaling cascade exerts multiphasic effects on cardiac differentiation and proliferation. The Wnt/β-catenin pathway is essential for cardiac repair and has been implicated in cardiac diseases (Titmarsh et al., 2016). Multiple studies in 2D and 3D in vitro cell models have described a significant proliferative effect of small molecules that inhibit GSK-3β and activate Wnt/β-catenin signaling, such as CHIR99021 (Buikema et al., 2013; Titmarsh et al., 2016; Sharma et al., 2018b; Uosaki et al., 2013) (Table 2). Attempts have also been made to increase cardiomyocyte yield by first expanding the production of hiPSCs and then inducing cardiac differentiation (Le and Hasegawa, 2019). Conventional methods for this approach include the culture of hiPSCs in a 2D monolayer at larger surface areas (using T175 flasks, CompacT SelecT, roller bottles or CellCube) (Tohyama et al., 2017; Soares et al., 2014). Although these 2D culture monolayers can increase hiPSC cell number efficiently, hiPSC medium is very costly, and the differentiation efficiency is highly variable due to lack of control of important culture parameters such as the confluency of the hiPSCs and the timing of compound addition (Tohyama et al., 2017). More recently, advanced 3D culture strategies such as microcarriers, 3D aggregation and bioreactors have attracted interest and have proven suitable for large-scale culturing of hiPSCs (Borys et al., 2020; Abecasis et al., 2017). This 3D culture approach improves the efficiency of hiPSC-CM production and reduces variability in differentiation outcomes (Hofer and Lutolf, 2021). However, the cost of the medium and the need for dissociation during harvesting represent hurdles for subsequent differentiation applications (Le and Hasegawa, 2019). Recently, we discovered that cell-cell contact promotes terminal differentiation and maturation, rather than proliferation, in densely cultured hiPSC-CMs (Buikema et al., 2020). Concomitant activation of Wnt signaling by CHIR99021 and cell-cell contact inhibition by low cell density serial passaging resulted in a massive proliferative response in the hiPSC-CMs (Buikema et al., 2020; Maas et al., 2021) (Figure 4). These hiPSC-CMs exhibited immature functional properties, such as reduced contractility and underdeveloped sarcomeres; however, upon withdrawal of CHIR99021, cells quickly exited the cell cycle and terminally differentiated (Buikema et al., 2020). It appears that insulin is required for long-term hiPSC-CM culture recapitulating cardiac development (Lian et al., 2013;
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