Harnessing developmental cues for cardiomyocyte production

ABSTRACT

Developmental research has attempted to untangle the exact signals that control heart growth and size, with knockout studies in mice identifying pivotal roles for Wnt and Hippo signaling during embryonic and fetal heart growth. Despite this improved understanding, no clinically relevant therapies are yet available to compensate for the loss of functional adult myocardium and the absence of mature cardiomyocyte renewal that underlies cardiomyopathies of multiple origins. It remains of great interest to understand which mechanisms are responsible for the decline in proliferation in adult hearts and to elucidate new strategies for the stimulation of cardiac regeneration. Multiple signaling pathways have been identified that regulate the proliferation of cardiomyocytes in the embryonic heart and appear to be upregulated in postnatal injured hearts. In this Review, we highlight the interaction of signaling pathways in heart development and discuss how this knowledge has been translated into current technologies for cardiomyocyte production.

Summary: Untangling the drivers of human cardiomyocyte proliferation can stimulate cardiac regenerative approaches. This Review compares in vivo heart growth pathways with the molecular targets promoting in vitro cardiomyocyte proliferation.

Introduction

Heart failure often results from the irreversible loss of functional myocardium or malfunctioning of the individual cardiomyocytes that comprise this tissue (De Boer et al., 2003; Fox et al., 2001; Gerber et al., 2000). This loss of functional cardiomyocytes can be acute or gradual and results in adverse remodeling of the remaining healthy myocardium (Olivetti et al., 1997; Saraste et al., 1997). In turn, myocardial dysfunction results in mechanical stress and upregulation of factors including angiotensin and norepinephrine, which all act to further promote detrimental myocardial remodeling (Colucci, 1997; Adhyapak, 2022). These alterations to extracellular matrix composition, cytoskeletal architecture and cell-cell connections occur in parallel with changes to the cardiac gene profile, such as reinduction of a fetal gene program (Parker et al., 1990; Bray et al., 2008).

Although clinical therapies for heart failure have significantly improved with multiple lines of heart failure drugs and mechanical circulatory support devices (Cook et al., 2015; Mancini and Burkhoff, 2005; Shen et al., 2022; Ponikowski et al., 2016), these treatments do not repair or replace malfunctioning myocardium. A central hurdle is that the adult heart is a largely postmitotic organ, where annual turnover is between 1-2% in young adults, and less than 0.5% in older adults (Bergmann et al., 2009; 2015). Thus, it is unsurprising that the adult heart lacks regenerative capacity post injury. In contrast, before birth, expansion of fetal cardiomyocytes is crucial for proper cardiogenesis and is tightly regulated by Wnt and Hippo signaling, with varying cardiomyocyte proliferation rates depending on location and developmental stage (Drenckhahn et al., 2008; Sturzu et al., 2015; Buikema et al., 2013; Rochais et al., 2009; von Gise et al., 2012; Qyang et al., 2007).

Remarkably, the early postnatal mammalian heart possesses regenerative potential (Haubner et al., 2016), but this regenerative response is lost 1 week after birth and scar tissue is formed in response to injury instead (Porrello et al., 2011; Ye et al., 2018; Zhu et al., 2018b). Translational cardiology aims to repair or replace a broken heart with autologous material (Laflamme and Murry, 2011; Ptaszek et al., 2012). The advent of patient-specific pluripotent stem cell sources represented a major advance for the field (Burridge et al., 2012), but the generation of large numbers of functional cardiomyocytes remains challenging due to their low proliferative rates (Tani et al., 2022). Here, we review how lessons from in vivo cardiomyocyte proliferation during mammalian cardiac development have been translated into technology to generate cardiomyocytes from human pluripotent stem cell sources.

Lessons from cardiac development

Mesoderm formation

During development, most heart structures arise from the mesodermal germ layer. Mesoderm formation takes place at Carnegie stages (CS) 6-7 in humans and embryonic day (E) 6.5 in mice, at the onset of gastrulation (Zhai et al., 2022). During gastrulation, cells from the blastocyst migrate to give rise to the endoderm and mesoderm. At the end of gastrulation, the three germ layers (endoderm, mesoderm and ectoderm) are specified. At the onset of mesoderm formation, the intraembryonic mesoderm subdivides into four distinct groups: the chordamesoderm, paraxial mesoderm, intermediate mesoderm and lateral plate mesoderm (Ivanovitch et al., 2021; Chan et al., 2013). Cardiopotent cells, also called cardiac progenitor cells, are derived from the lateral plate mesoderm during early gastrulation (Yamada and Takakuwa, 2012; Moretti et al., 2006; Garry and Olson, 2006). Cardiac progenitors are prepatterned within the primitive streak, with the atrial and ventricular cells arising at different anterior-posterior positions (Chan et al., 2013). Foxa2⁺ cardiac progenitors give rise primarily to the cardiovascular cells of the ventricles and are the first cardiogenic cells that migrate to the anterior side of the embryo (Bardot et al., 2017). These cells comprise approximately half of the cardiomyocyte population (Bardot et al., 2017). The right ventricle and outflow tract progenitors are found in anterior/distal primitive streak, where cells are exposed to a higher ratio of activin A to bone morphogenetic protein 4 (BMP4) signaling, whereas atrial progenitors are specified in the proximal primitive streak, where the activin A to BMP4 ratio is low (Ivanovitch et al., 2021).

Brachyury (T) gene expression is also required for the formation of posterior mesoderm in mice and zebrafish (Schulte-Merker and Smith, 1995; Herrmann et al., 1990). Basic fibroblast growth factor (FGF) and Activin A can promote the expression of the T homolog, Xbra, in the Xenopus presumptive ectoderm (Smith et al., 1991). T-box transcription factors and T are intrinsic factors that are crucial for the initiation of mesoderm differentiation and patterning of the primitive streak and are regulated by the Wnt signaling pathway from the adjacent embryonic midline and posterior regions of the embryo, indicating the importance of Wnt signaling at this stage of development (Yamaguchi et al., 1999).

Cardiac specification

Several transcription factors for cardiac development have been identified (Olson, 2006). These include MESP1, which specifies the cardiac mesodermal population and is expressed in the primitive streak around CS6-7 or E6.5. In MESP1-null embryos, severe cardiac abnormalities are observed, leading to cardiac lethality by E10.5 (Saga et al., 1999). Furthermore, in a double knockout of MESP1 and its homolog MESP2, mesoderm progenitors do not contribute to heart development, indicating that MESP1/2 expression is essential for cardiac mesoderm formation (Kitajima et al., 2000).

By CS8 or E7.5, specific regions of the mesoderm differentiate to form cardiovascular progenitor cells, which can be divided into cells that form the first heart field (FHF) and cells that form the second heart field (SHF) (Paige et al., 2015). When Wnt inhibitors such as dickkopf 1 (DKK1) or crescent are administered to posterior lateral plate mesoderm, heart muscle development is induced and erythropoiesis is suppressed (Marvin et al., 2001, Naito et al., 2006). Meanwhile, ectopic expression of WNT8 or WNT3a in precardiac mesoderm inhibits heart muscle formation and promotes erythropoiesis (Marvin et al., 2001). In mouse embryonic stem cells, Wnt/β-catenin signaling acts biphasically; early treatment with Wnt3a stimulates mesoderm induction and cardiac differentiation, whereas late activation of β-catenin signaling impairs cardiac differentiation (Ueno et al., 2007). In combination with BMP and FGF, Wnt inhibition results in the activation of key upstream cardiac transcriptional regulatory genes NKX2-5, GATA4 and TBX5, which are required for the initiation of cardiac-like gene expression (Kelly et al., 2014).

The formation of the linear heart tube is mostly initiated by the contribution of the FHF, which eventually gives rise to the inflow tract and the majority of the left ventricle (Brade et al., 2013). FHF progenitors at this stage specifically express TBX5 and HCN4 (Später et al., 2013; Bruneau et al., 1999). The SHF develops slightly later and is less differentiated, providing cardiac progenitors that proliferate to promote the expansion of the heart tube (Kelly, 2012). The right ventricle and outflow tract are exclusively generated by the SHF (Buckingham et al., 2005). These SHF cardiomyocytes are marked with TBX1, ISL1 and HAND2 (Moretti et al., 2006; Stanley et al., 2002).

Together, these studies demonstrate that Wnt signals in different parts of the mesoderm are repressed as required for cardiac specification of these regions. Most in vitro protocols for directed cardiac differentiation of pluripotent stem cells incorporate this inhibition of Wnt signaling through the application of porcupine small-molecule inhibitors including IWP-2, IWR1 and Wnt-C59 (Lian et al., 2012; Burridge et al., 2014).

Developmental heart growth

Organ size regulation is an important aspect of cardiac development. The heart must grow large enough to generate sufficient cardiac output, and regional under- or overgrowth may result in septal wall defects, hypoplastic ventricle(s) or obstruction of the outflow tract(s) (Heallen et al., 2011). Heart growth during development is regulated by a combination of cardiomyocyte differentiation, proliferation and hypertrophy.

The human heart undergoes a dramatic proliferation period from CS9-CS16, resulting in a 600-fold increase in heart volume in just 3 weeks (de Bakker et al., 2016). First, the cardiac crescent fuses at the midline and gives rise to the FHF-derived linear heart tube, which subsequently commences beating and undergoes looping. Hereafter, the linear heart tube expands by drastic proliferation and recruitment of SHF cardiac progenitor cells that migrate from the pericardial cavity to the dorsal and caudal heart tube regions while undergoing differentiation (Kelly et al., 2014). Their rapid proliferation is regulated by canonical Wnt signaling (Günthel et al., 2018; Kwon et al., 2007). Upon the presence of the receptor-bound ligands WNT5A and WNT11, active β-catenin enters the nucleus, where it acts as a transcriptional co-activator of the T-cell factor (TCF) and leukemia enhancer factor (LEF) transcription factors to activate Wnt target genes involved in cell proliferation such as axin 2 (AXIN2), cyclin D1 (CCND1) and lymphoid enhancer binding factor 1 (LEF1) (Cadigan and Waterman, 2012; Stamos and Weis, 2013) (Fig. 1). Conversely, in the absence of Wnt ligands, a complex containing adenomatous polyposis coli (APC), casein kinase 1 (Ck1) and glycogen synthase kinase-3β (GSK-3β) mediates phosphorylation, ubiquitylation and, ultimately, degradation of β-catenin (Cadigan and Waterman, 2012; Stamos and Weis, 2013) (Fig. 1). Conditional knockout studies for β-catenin in the SHF produce outflow tract abnormalities and impaired right ventricular development (Qyang et al., 2007; Lin et al., 2007).

Fig. 1.

There is interplay between Wnt, Hippo and insulin signaling during cardiomyocyte proliferation. Activation of the Hippo, canonical Wnt or IGF1/PI3K/AKT signaling pathways causes YAP/TAZ and β-catenin to enter the nucleus and cluster with their DNA-binding partners TEAD and TCF/LEF, which activates transcription of target genes and induces cell cycle activation. Conversely, when Dsh is inactivated due to lack of Wnt proteins, activated GSK-3β can phosphorylate β-catenin or YAP/TAZ, ultimately resulting in their degradation by the proteasome. Hippo signaling induced by, for example, N-cadherin junction-mediated cell-cell contact leads to the degradation of the YAP/TAZ complex through the MST1/2-SAV1-LATS1/2-MOB1-YAP/TAZ cascade. IGF1/PI3K/AKT signaling can facilitate the entry of β-catenin into the nucleus via AKT kinase, which phosphorylates GSK-3β to inhibit its activation. AKT, RACα serine/threonine-protein kinase; APC, adenomatous polyposis coli protein; CK1α, casein kinase 1α; Dsh, disheveled; GSK-3β, glycogen synthase kinase-3; IGF1, insulin-like growth factor 1; IGF1R, IGF1 receptor; LATS1/2, large tumor suppressor homologue 1/2; LRP5/6, low-density-lipoprotein-related protein 5/6; MOB1, MOB kinase activator 1; MST1/2, mammalian STE20-like protein kinase 1/2; PI3 K, phosphoinositide 3-kinase; SAV1, protein Salvador homologue 1; TAZ, transcriptional coactivator with PDZ-binding motif; TCF/LEF, T-cell factor/lymphoid enhancer-binding factor; TEAD, TEA domain transcription factor family members; YAP, yes-associated protein; +p, phosphorylation; +u, ubiquitination.

After specification and terminal differentiation of cardiac progenitors into cardiomyocytes, the developing heart predominantly increases its size and mass via the proliferation of differentiated cardiomyocytes (Günthel et al., 2018) (Table 1). In mice, between E8.0 and E11.0, cardiomyocyte numbers increase 100-fold from ∼700 to ∼70,000 (De Boer et al., 2012) (Fig. 2A,C). During this massive growth phase, the size of the individual cardiomyocytes remains relatively constant. After E11.0, proliferation continues but at a slower rate, with cardiomyocyte numbers approaching 1,000,000 by E18.5 (de Boer et al., 2012). The ballooning ventricles exhibit the highest proliferation rates during this period (Moorman and Christoffels, 2003; Moorman et al., 2010). By contrast, the atrioventricular canal, outflow tract and inner curvature regions have lower proliferation rates and thereby preserve the slow contraction characteristics of the heart tube (De Jong et al., 1992).

Table 1.

Signaling pathways and regulators in heart development

Fig. 2.

Heart size at selected stages of normal cardiac development. (A) Brightfield images capturing murine hyperplasia over embryonic day (E)9.5 (whole embryo; dashed white lines indicate the location of the heart), E12.5, E18.5 and postnatal day (P)2. (B) Brightfield images capturing murine hypertrophy from P13 to P60. (C) Immunofluorescent images showing pHH3⁺ cells in E12.5 and Ki67⁺ in LV cells of P2 and P13 hearts. pHH3 and Ki67 are proliferation markers and TnT is a cardiomyocyte marker. Ao, aorta; H, heart; LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle; TnT, troponin T; pHH3, phosphohistone H3. Figure is adapted from Buikema et al. (2013, 2020) and is available to view on Figshare alongside detailed Materials and Methods: 10.6084/m9.figshare.23607306. Scale bars: ∼1 mm (A,C); 5 mm (B).

Proliferation rates may also vary within the same region depending on the developmental stage. Within the ventricles, proliferation rates are low during the formation of the trabecular network (Sedmera and Thompson, 2011). The trabeculae contribute to cardiac contractility, channel expression and energy metabolism, and start to develop at the luminal side of the myocardium by E9.5 or CS12 (Meyer et al., 2020; Günthel et al., 2018). Later in development, from CS12 until CS16, proliferation rates increase and the compacted ventricular chamber myocardium shows high expression of proliferative markers such as Ki67 and pHH3 (Buikema et al., 2013; Ye et al., 2015; Lin et al., 2015). These changes to ventricular proliferation rates are due to Wnt signaling. Canonical Wnt signaling is downregulated in the trabecular myocardium, which is consistent with the lower proliferation rates observed here (Buikema et al., 2013; Ye et al., 2015). By contrast, conditional knockout studies in mice have shown that epidermal growth factor receptor (EGFR) and Notch signaling are essential for trabecular development (Gassmann et al., 1995; Grego-Bessa et al., 2007). When proliferation rates in the ventricle increase, Wnt/β-catenin is essential for the exponential growth of the compacted ventricular myocardium; the increase in cardiomyocyte proliferation rates as you move from the inner trabeculae to outer compact myocardium corresponds with the graded activity of canonical Wnt signaling (Buikema et al., 2013; Ye et al., 2015). Consistent with this, β-catenin is mainly active in the compact myocardium, where it is expressed by the majority of proliferating cardiomyocytes (Buikema et al., 2013).

After CS16 and E12.5, cardiomyocyte proliferation rates decline further to maintain normal heart and embryo size ratios until birth. Postnatal heart growth is regulated via cellular hypertrophy (Günthel, Barnett, and Christoffels, 2018) (Fig. 2B,C). The insulin-like growth factor (IGF)/Akt pathway appears to promote physiological (and pathogenic) hypertrophy by activation of the ERK/MAPK and the phosphoinositide 3-kinase (PI3K)/Akt pathways in postnatal cardiomyocytes (DeBosch et al., 2006; Liu et al., 1996; Li et al., 2011).

Interplay between Wnt, Hippo and IGF signaling during cardiomyocyte expansion

The major regulator of cardiac size is the Hippo pathway. This pathway regulates growth and progenitor genes such as SOX2, SNAI2, CCND1, CDC20 and MYCL in cardiomyocytes via the MST1/2-SAV1-LATS1/2-MOB1-YAP/TAZ cascade (Heallen et al., 2011) (Fig. 1). YAP, a central protein within the Hippo signaling pathway, is activated at CS10 (Singh et al., 2016). Genetic inhibition of Hippo signaling in the embryonic heart leads to a lack of organ size control and lethal cardiomegaly shortly after birth (von Gise et al., 2012). For example, mouse models harboring various embryonic deletions of the Hippo pathway members SAV1, MST1/2 and LATS2 display overgrown hearts and thickened ventricles owing to an excess of cardiomyocytes (Del Re, 2014). Downstream analysis in Hippo mutant hearts revealed that a lack of downregulation of canonical Wnt target genes and cell cycle regulators could explain the observed hyperplasia and cardiomegaly (Heallen et al., 2011). By contrast, upregulation of Hippo signaling leads to a downregulation of YAP and Wnt/β-catenin signaling to restrain heart size (Kim et al., 2017). Cardiac-specific deletion of YAP also impedes neonatal heart regeneration, resulting in a fibrotic response that generates scar tissue (Xin et al., 2013).

Cytoplasmic YAP/TAZ can also increase cytosolic retention of β-catenin, and thereby negatively regulate the Wnt/β-catenin signaling pathway (Imajo et al., 2012). Chromatin immunoprecipitation sequencing (ChIP-seq) indicated that YAP/TAZ and β-catenin form a common regulatory complex at the SOX2 and SNAI2 gene loci (Heallen et al., 2011). Activating YAP also leads to upregulation of the IGF/Akt pathway, resulting in the inhibition of GSK-3β, stabilization of β-catenin and transcription of Wnt target genes to enhance cardiomyocyte proliferation (Xin et al., 2011) (Fig. 1). During development, IGF signaling directs cardiomyocyte proliferation between E9.5 and E12.5, and IGF knockout mice exhibit decreased ventricular proliferation (Li et al., 2011). Interestingly, knockout of IGF is non-lethal and by E14.5 the ventricular wall size is comparable with that of wild-type mice (Li et al., 2011; Díaz Del Moral et al., 2021).

The Wnt/β-catenin, IGF/Akt and Hippo/YAP pathways form a complex signaling network that regulates the balance between proliferation, differentiation and maturation of cardiomyocytes (Fig. 1). It has generally been concluded that high Wnt, high IGF and low Hippo signaling results in proliferative growth of embryonic cardiomyocytes, whereas high Hippo signaling overrules Wnt and IGF to restrain cardiomyocyte proliferation and prevent cardiomegaly.

Cardiomyocyte cell biology in vitro

Developmental shortcuts for human pluripotent stem cell-derived cardiomyocyte differentiation

Cardiomyocytes derived from pluripotent stem cells represent a promising source of cells for disease modeling, drug screens and/or cardiac regenerative medicine (Madonna et al., 2019). The untangling of cues that drive cardiac development has identified growth factors, transcriptional regulators and signaling cascades that can promote the differentiation of cardiac cells from human embryonic stem cells (hESCs). Initial protocols for embryonic stem cell differentiation required formation of embryoid bodies to generate various cell types (Rungarunlert et al., 2009), and downstream applications for cardiac cell biology were limited due to extremely low percentages of cardiomyocyte yield (Osafune et al., 2008). Previously, co-culture of hESCs with visceral endoderm-like cells induced cardiomyocyte differentiation, producing beating 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., 2006) 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) (Fig. 3). Altogether, the development of small molecule-based directed differentiation protocols has facilitated reliable and cost-effective production of both atrial and ventricular hiPSC-CMs. 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).

Fig. 3.

Summary of recent directed cardiac differentiation and expansion protocols for human pluripotent stem cell-derived cardiomyocytes. Schematics of pluripotent stem cell monolayer-based cardiomyocyte differentiation protocols. The days of each protocol are indicated by the vertical lines. Corresponding articles are shown on the left. Initial stem cell conditions, before the start of the differentiation, are shown to the left of day 0 on each timeline. Differentiation efficiency is indicated on the right side, where D indicates the day on which the differentiation efficiency was measured. The media used for differentiation are indicated by different colors below the time axis, with any additional components listed above the time axis. The red and grey boxes depicted above the time axis indicate activation or inhibition of the Wnt pathway, respectively. Key components responsible for modulating Wnt activity are indicated in bold for Wnt activation or italics for Wnt inhibition. Abbreviations: AA, L-ascorbic acid 2-phosphate; bFGF/FGF2, basic fibroblast growth factor; BMP4, bone morphogenetic protein 4; BSA, bovine serum albumin; CHIR, CHIR99021; DMEM, Dulbecco's Modified Eagle Medium; FBS, fetal bovine serum; hESCs, human embryonic stem cells; hiPSC, human induced pluripotent stem cells; IWP, inhibitor of Wnt production; KO-DMEM, knockout DMEM; LPA, lysophosphatidic acid; MEFs, mouse embryonic fibroblasts; RPMI, Roswell Park Memorial Institute medium; S1P, sphingosine-1-phosphate; VNT, vitronectin. E8, FDTA and mTeSR1 are stem cell media. C59 and IWR1 are inhibitors of the Wnt pathway. B27 is a commercially available cell culture supplement. Created with BioRender.com.

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).

Table 2.

Molecular targets for expansion of human pluripotent stem cell-derived cardiomyocytes

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 hiPSC-CMs (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 hESC-derived 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). 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) (Fig. 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; 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.

Fig. 4.

Overview of Wnt modulation during hiPSC-CM differentiation and expansion. (A) Schematic overview of human induced pluripotent stem cell-derived 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 hiPSC-CM 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).

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 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 hiPSC-CMs 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, Figs 3 and 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 lack of proliferative capacity in terminally differentiated cardiomyocytes is somewhat in line with developmental studies illustrating that Hippo signaling controls cell-cycle arrest in adult cardiomyocytes (Xin et al., 2011; Wang et al., 2014). Genetic modification of the Hippo/YAP signaling pathway results in dedifferentiation of adult myocardium with a subsequent regenerative response to ischemic injury (Leach et al., 2017; Monroe et al., 2019). Combined with novel DNA, RNA or nanoparticle local delivery technologies, signaling pathways hold promise as druggable targets in cardiovascular medicine (Braga et al., 2021). Future hiPSC-CM studies should focus on further simplification of the exact cues required for cardiomyocyte production, and should incorporate other myocyte subtypes required for regional repair of the heart, as well as more specific cardiac tissue generation.