43 Harnessing developmental cues for cardiomyocyte production 2 in cell number (Buikema et al., 2020; Maas et al., 2021) (Table 2), but they are pushing the production process towards clinically relevant cardiomyocyte numbers. For example, one batch of massively expanded hiPSC-CMs can be used to generate large numbers of 3D engineered heart tissues (EHTs) or to conduct large 2D screens (Hansen et al., 2010; Calpe and Kovacs, 2020). Moreover, hiPSC-CM-derived tissues or cell injections can form large grafts after transplantation into injured animal hearts (Shiba et al., 2016). Upon injection, previous studies have shown electrical coupling of hiPSCCMs to host myocardium, but also arrhythmic events (Hirt et al., 2012; Shiba et al., 2012). Recent work has shown that these arrhythmic events can be overcome via maturation-guided editing of hiPSC-CM ion channels, thereby improving electrophysiological function (Marchiano et al. 2023; Ottaviani et al., 2023). These approaches require 10-750 million cells for restoration of contractile function after ischemic injury in macaque monkeys (Anderson et al., 2014; Zhu et al., 2018a). Unfortunately, despite the recent methods to improve cardiomyocyte maturity, the overall maturation status of hiPSC-CMs with or without expansion remains low. Therefore, hiPSC-CM grafts retain different electrical properties compared with the host tissue, which could lead to increased arrhythmia risk (Fassina et al., 2022). DISCUSSION The further untangling of the cues that drive cardiomyocyte differentiation, proliferation and maturation will lead to increased understanding and improved drug screening options and treatments for cardiac diseases. This Review compares the pathways regulating heart growth in vivo with the molecular targets that promote in vitro cardiomyocyte production. The massive expansion of hiPSC-CMs that can be achieved via Wnt/β-catenin signaling modulation (Table 2, Figure 3 and Figure 4) provides a framework for advanced basic and translational cell biology applications in cardiovascular medicine, and several preclinical trials are making use of these hiPSC-CMs (Sharma et al., 2018a; Hnatiuk et al., 2021). The current knowledge from cardiac development has resulted in relatively easy methods for hiPSC-CM generation. Further understanding of exact cues for long-term proliferation and terminal differentiation, however, are required to produce unlimited numbers of mature cardiomyocytes. The recent discovery of partly-immortalized atrial cardiomyocytes represents a powerful approach for atrial disease modeling in vitro (Harlaar et al., 2022). However, their in vivo use may provoke safety concerns. Nevertheless, these approaches for expansion of cardiomyocytes illustrate that functional and beating cardiomyocytes can self-replicate before terminally differentiating upon growth stimuli withdrawal. Interestingly, direct activation of Wnt/ β-catenin with CHIR99021 produces cardioprotective effects upon myocardial infarction in both small and large mammals (Fan et al., 2020). However, there is little evidence for induction of terminally differentiated cardiomyocyte proliferation after administration of CHIR99021 upon ischemic injury (Fan et al., 2020). This
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