37 Harnessing developmental cues for cardiomyocyte production 2 areas in ~35% of the culture surface (Mummery et al., 2003). Due to the addition of insulin and serum, however, only 2-3% of the cells were cardiomyocytes. The number of beating colonies increased 10-fold with the administration of serum- and insulin-free media, whereafter each beating colony contained ~25% cardiomyocytes. Moreover, in situ hybridization demonstrated that the streak ectoderm displays the highest level of mRNA for both Wnt3a and Wnt8c (Marvin et al., 2001). In retrospect, the expression of Wnt inhibitors in the ectoderm may be the underlying mechanism for the increased cardiomyocyte differentiation efficiency (Piccolo et al., 1999). Overall, it appears that the timing and relative expression of different growth factor combinations induce then pattern the cardiogenic mesoderm. Subsequent studies identified important roles for the activin A, insulin, Wnt and BMP pathways in the establishment of cardiovascular cells (Klaus et al., 2007; Schneider and Mercola, 2001; Liu et al., 1999). Activin A and transforming growth factor β1 (TGFβ1) stimulation promotes cardiac mesoderm formation in mouse embryonic stem cells (Moretti et al., 2006; Kattman et al., 2 006) and addition of activin A and BMP4 induces endogenous Wnt signaling and mesoderm-like cells from human embryonic stem cell sources (Paige et al., 2010). Remarkably small changes in BMP4 and activin A concentrations can drive the specification of the FHF, the anterior SHF and the posterior SHF (Yang et al., 2022) from distinct stem cell-derived mesoderm populations (Kattman et al., 2011), ultimately affecting the relative numbers of atrial versus ventricular cardiomyocytes (Lee et al., 2017). Although cardiomyocytes produced in this way may be high quality, scaling up these protocols is challenging due to the stability and cost of growth factors involved, which must be precisely applied to achieve this delicate balance. The ability to reprogram patient somatic cells into induced pluripotent stem cells (hiPSCs) represented a key turning point for the field (Takahashi et al., 2007). The potential to differentiate these patient-specific cells into many cell types, including functional cardiomyocytes (hiPSC-CMs) fueled a search for signaling molecules to replace the growth factors. In vitro, molecular inhibition of GSK-3β using small molecules such as CHIR99021 leads to nuclear accumulation of β-catenin and thus the transcription of Wnt target genes (Lian et al., 2012). Activation of Wnt signaling in monolayer-based hESC and hiPSC cultures results in efficient mesoderm-like cell formation, and subsequent inhibition of the Wnt signaling pathway promotes the terminal differentiation of these mesoderm-like cells into cardiomyocytes (Paige et al., 2010; Lian et al., 2012). The current standard in monolayer-based directed differentiation protocols uses this two-step Wnt pathway modulation to generate >80% hiPSC-CMs within 7 days of culture (Paige et al., 2010; Lian et al., 2012) (Figure 3). Altogether, the development of small molecule-based directed differentiation protocols has facilitated reliable and cost-effective production of both atrial and ventricular hiPSCCMs. However, cell type heterogeneity presents a challenge, as homogeneous populations of subtype-specific cardiomyocytes are essential for drug discovery, cardiovascular disease modeling and cardiotoxicity screens (Zhang et al., 2009).
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