38 Chapter 2 Developmental shortcuts for human pluripotent stem cell-derived cardiomyocyte expansion Robust generation of large quantities of hiPSC-CMs from patient donors remains a significant hurdle. Conventional approaches to tackle this problem use large numbers of hiPSCs, which is very costly, and the differentiation efficiency is highly variable due to lack of control of important culture parameters (Laco et al., 2018). Moreover, the subsequent expansion of hiPSC-CM cultures is generally modest (<10-fold) (Table 2). In vitro studies have identified multiple pathways controlling the proliferation of hiPSC-CMs. Computational approaches and chemical screens for cardiomyocyte proliferation regulators found 67 miRNAs targeting different components of the Hippo pathway (Diez-Cuñado et al., 2018) (Table 2). Hippo signaling appears to be upregulated in hiPSC-CMs cultured on plastic, but altering substrate stiffness has no effect on the fraction of proliferative cardiomyocytes, and none of the small molecule Hippo pathway inhibitors has been reported to enhance cardiomyocyte proliferation in vitro (Buikema et al., 2020). The TGFβ pathway regulates the cell cycle in hiPSC-CMs, where it directly boosts the production of p27 in a Smad-dependent manner, allowing p27 to inhibit the activity of G1 cyclins (Toyoshima and Hunter, 1994; Kodo et al., 2016). The combined upregulation of CDK1, CCNB, CDK4 and CCND successfully upregulates cardiomyocyte proliferation markers in hiPSCCMs (Mohamed et al., 2018), and inhibiting Wee1 and TGFβ promotes CDK1 phosphorylation, thus promoting G2/M phase entry (Mohamed et al., 2018) (Table 2). Multiple hormones are implicated in the regulation of fetal growth, and many of these take on considerably different roles during early postnatal life. In utero, the human heart relies predominantly on high concentrations of carbohydrates, whereas the postnatal myocardium also uses fatty acid-rich substrates (Kolwicz et al., 2013). Insulin, prolactin, IGF1, IGF2 and thyroid-associated hormones are involved in anabolism during heart development (Díaz Del Moral et al., 2021). The growth factor IGF1 was shown to enhance the proliferation of hESCderived cardiomyocytes via activation of the PI3K/Akt signaling pathway (McDevitt et al., 2005). Thyroid hormone or 3,5,3′-triiodothyronine (T3) inhibits proliferation and drives the maturation of cardiomyocytes before birth (Chattergoon et al., 2019). In parallel to IGF1, insulin increases proliferation of hiPSC-CMs and cardiac organoids (Mills et al., 2017) (Table 2). Aurora B, aurora kinase B; bFGF, basic fibroblast growth factor; BrdU, 5-bromo-2′-deoxyuridine; CCNB, cyclin B1; CCND, cyclin D2; CDK1, cyclin-dependent kinase 1; CDK4, cyclin-dependent kinase 4; DOX, doxycycline; EdU, 5-ethynyl-2′-deoxyuridine; ERK, extracellular signal-regulated kinase; FGF1, fibroblast growth factor 1; FOXO, forkhead box transcription factors; G2/M, inter/ mitosis and cytokinesis phase; GLI1, GLI family zinc finger 1; hESC-CM, human embryonic stem cell-derived cardiomyocyte; hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocyte; hiPSC-CPC, human induced pluripotent stem cellderived cardiac progenitor
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