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Human Pluripotent Stem Cell-Derived Cardiovascular Cells: From Developmental Biology to Therapeutic Applications

2019; Elsevier BV; Volume: 25; Issue: 3 Linguagem: Inglês

10.1016/j.stem.2019.07.010

1934-5909

Stephanie Protze, Jee Hoon Lee, Gordon Keller,

Congenital heart defects research

Advances in our understanding of cardiovascular development have provided a roadmap for the directed differentiation of human pluripotent stem cells (hPSCs) to the major cell types found in the heart. In this Perspective, we review the state of the field in generating and maturing cardiovascular cells from hPSCs based on our fundamental understanding of heart development. We then highlight their applications for studying human heart development, modeling disease-performing drug screening, and cell replacement therapy. With the advancements highlighted here, the promise that hPSCs will deliver new treatments for degenerative and debilitating diseases may soon be fulfilled. Advances in our understanding of cardiovascular development have provided a roadmap for the directed differentiation of human pluripotent stem cells (hPSCs) to the major cell types found in the heart. In this Perspective, we review the state of the field in generating and maturing cardiovascular cells from hPSCs based on our fundamental understanding of heart development. We then highlight their applications for studying human heart development, modeling disease-performing drug screening, and cell replacement therapy. With the advancements highlighted here, the promise that hPSCs will deliver new treatments for degenerative and debilitating diseases may soon be fulfilled. Human embryonic and induced pluripotent stem cells (human pluripotent stem cells [hPSCs]) (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (11349) Google Scholar, Thomson et al., 1998Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Google Scholar) have the potential to transform our understanding of human cardiovascular diseases and radically change our approaches to treating them. hPSCs provide, for the first time, the ability to establish models of human cardiovascular development and diseases in vitro and the opportunity to design human cardiomyocyte-based platforms for drug discovery and predictive toxicology. Additionally, they represent an unlimited source of human cardiac cells for cell-based therapies to treat patients with the most debilitating forms of heart disease. The translation of this remarkable potential into practice is dependent on our ability to direct the differentiation of hPSCs to the desired cell type(s). As hPSCs represent an early, pre-gastrulation stage of development, the differentiation of functional cardiovascular cells involves transitions through developmental stages comparable to those that lead to the formation of heart tissue in the embryo. Given this, the design of differentiation protocols is often guided by our understanding of cardiovascular development in model organisms. The initial hPSC differentiation protocols yielded mixed populations of immature cardiomyocytes composed of ventricular, atrial, and pacemaker cells. These mixed populations are not well suited for modeling diseases and testing drugs that target specific myocyte subtypes in the adult heart. Similarly, cell-based therapies aimed at remuscularizing the damaged ventricle following a myocardial infarction (MI) or the development of a biological pacemaker will require access to highly enriched populations of specific cardiomyocyte subtypes, such as left ventricular cardiomyocytes or pacemaker cells. To overcome these limitations, more refined protocols have been developed that enable the generation of populations enriched in the major cardiomyocyte subtypes including atrial, ventricular, and sinoatrial pacemaker cells, as well as non-myocyte cell types including epicardial cells and derivative fibroblasts and vascular smooth muscle cells. In addition, recent studies focused on engineering cardiac tissue and on mimicking the metabolic and hormonal changes occurring at birth have made progress in promoting the maturation of the hPSC-derived cardiomyocytes in vitro. In this perspective, we review the remarkable progress made in generating cardiovascular cells from hPSCs over the past two decades and the advances in applying this new biology to the study and treatment of human heart diseases. We also highlight the important role that developmental biology has played in this success. To be able to generate the different cardiac cell types from hPSCs, it is essential to understand how they develop in the early embryo. Lineage tracing and gene targeting studies over the past two decades have provided important insights into the origin of different regions of the heart and showed that many derive from distinct progenitor populations that are specified early in development (Kelly, 2012Kelly R.G. The second heart field.Curr. Top. Dev. Biol. 2012; 100: 33-65Crossref PubMed Scopus (103) Google Scholar, Meilhac and Buckingham, 2018Meilhac S.M. Buckingham M.E. The deployment of cell lineages that form the mammalian heart.Nat. Rev. Cardiol. 2018; 15: 705-724Crossref PubMed Scopus (7) Google Scholar, Moorman et al., 2007Moorman A.F. Christoffels V.M. Anderson R.H. van den Hoff M.J. The heart-forming fields: one or multiple?.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2007; 362: 1257-1265Crossref PubMed Scopus (0) Google Scholar, Rosenthal and Harvey, 2010Rosenthal N. Harvey R.P. Heart development and regeneration. Elsevier/Academic Press, 2010Google Scholar). Undoubtedly, the most-studied and best-characterized progenitors are those that develop from distinct subpopulations of cardiovascular mesoderm referred to as the first heart field (FHF) and second heart field (SHF) (Meilhac and Buckingham, 2018Meilhac S.M. Buckingham M.E. The deployment of cell lineages that form the mammalian heart.Nat. Rev. Cardiol. 2018; 15: 705-724Crossref PubMed Scopus (7) Google Scholar). These progenitors can be distinguished based on characteristic marker expression and their location in the developing embryo. The FHF progenitors, identified by the expression of HCN4 and TBX5 and lack of expression of ISL1, form the ventral portion of the cardiac crescent and give rise to the primitive heart tube. These progenitors generate the majority of the cells in the left ventricle (Bruneau et al., 1999Bruneau B.G. Logan M. Davis N. Levi T. Tabin C.J. Seidman J.G. Seidman C.E. 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Cell Biol. 2013; 15: 1098-1106Crossref PubMed Scopus (92) Google Scholar). The SHF progenitors, by contrast, express ISL1, but not HCN4 or TBX5, and are positioned dorsomedial in the crescent. They contribute to the anterior and posterior poles of the heart tube and ultimately give rise to the majority of the cardiomyocytes found in the right ventricle and the outflow tract. The FHF/SHF contribution to the atrial chambers is less clear as different studies have reported that both progenitor populations can give rise to atrial cardiomyocytes (Buckingham et al., 2005Buckingham M. Meilhac S. Zaffran S. Building the mammalian heart from two sources of myocardial cells.Nat. Rev. Genet. 2005; 6: 826-835Crossref PubMed Scopus (747) Google Scholar, Laugwitz et al., 2008Laugwitz K.L. Moretti A. Caron L. Nakano A. Chien K.R. Islet1 cardiovascular progenitors: a single source for heart lineages?.Development. 2008; 135: 193-205Crossref PubMed Scopus (179) Google Scholar, Meilhac et al., 2004Meilhac S.M. Esner M. Kelly R.G. Nicolas J.F. Buckingham M.E. The clonal origin of myocardial cells in different regions of the embryonic mouse heart.Dev. Cell. 2004; 6: 685-698Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Within the SHF, lineage tracing studies have provided evidence that the atrial cells derive from progenitors specified within the posterior region, whereas the right ventricular and outflow tract cardiomyocytes are generated from progenitors that develop in the anterior region (Galli et al., 2008Galli D. Domínguez J.N. Zaffran S. Munk A. Brown N.A. Buckingham M.E. Atrial myocardium derives from the posterior region of the second heart field, which acquires left-right identity as Pitx2c is expressed.Development. 2008; 135: 1157-1167Crossref PubMed Scopus (0) Google Scholar, Verzi et al., 2005Verzi M.P. McCulley D.J. De Val S. Dodou E. Black B.L. The right ventricle, outflow tract, and ventricular septum comprise a restricted expression domain within the secondary/anterior heart field.Dev. Biol. 2005; 287: 134-145Crossref PubMed Scopus (297) Google Scholar). To map the embryonic origins of these different progenitors, several groups have used lineage tracing approaches to characterize the developmental potential of early emerging Mesp1+ mesodermal cells. Devine et al., 2014Devine W.P. Wythe J.D. George M. Koshiba-Takeuchi K. Bruneau B.G. Early patterning and specification of cardiac progenitors in gastrulating mesoderm.eLife. 2014; 3 (Published online October 8, 2014)https://doi.org/10.7554/eLife.03848Crossref PubMed Scopus (77) Google Scholar used a Mesp1-Cre driver mouse line together with a MADM Cre-reporter to clonally label cardiogenic mesoderm and showed that Mesp1-derived clones contribute to distinct anatomical locations within the heart, including the atria, the left ventricle, and the right ventricle. Clones contributing to multiple regions were rare, supporting the interpretation that progenitors with distinct potential are specified early, at the stage of mesoderm induction. To establish the temporal allocation of progenitor fates, Lescroart et al., 2014Lescroart F. Chabab S. Lin X. Rulands S. Paulissen C. Rodolosse A. Auer H. Achouri Y. Dubois C. Bondue A. et al.Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development.Nat. Cell Biol. 2014; 16: 829-840Crossref PubMed Scopus (92) Google Scholar used a doxycycline inducible Mesp1 rtTA/TetO-Cre driver line in combination with a confetti Cre-reporter to label Mesp1+ mesoderm at different developmental stages. With this approach, they were able to show that cells labeled at E6.25 preferentially generated left ventricular cardiomyocytes, whereas those labeled at E7.25 gave rise to clones of cardiomyocytes found in the atria, the right ventricle, or the outflow tract. Somewhat different patterns of lineage development were recently reported by Bardot et al., 2017Bardot E. Calderon D. Santoriello F. Han S. Cheung K. Jadhav B. Burtscher I. Artap S. Jain R. Epstein J. et al.Foxa2 identifies a cardiac progenitor population with ventricular differentiation potential.Nat. Commun. 2017; 8: 14428Crossref PubMed Scopus (11) Google Scholar, who traced Foxa2+ cells in early embryos and identified a population specified by E6.5 that gave rise to right and left ventricular cardiomyocytes, but not to atrial cells. These findings indicate that both ventricular progenitors share the expression of Foxa2 and that at least some of the right ventricular cells are specified early. This interpretation is not inconsistent with the above study as Lescroart et al., 2014Lescroart F. Chabab S. Lin X. Rulands S. Paulissen C. Rodolosse A. Auer H. Achouri Y. Dubois C. Bondue A. et al.Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development.Nat. Cell Biol. 2014; 16: 829-840Crossref PubMed Scopus (92) Google Scholar also found that a small number of clones labeled at E6.25 can contribute to the right ventricle. The Lescroart et al., 2014Lescroart F. Chabab S. Lin X. Rulands S. Paulissen C. Rodolosse A. Auer H. Achouri Y. Dubois C. Bondue A. et al.Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development.Nat. Cell Biol. 2014; 16: 829-840Crossref PubMed Scopus (92) Google Scholar study also identified clones consisting of only endothelial cells or epicardial cells, suggesting that progenitors restricted to these lineages are also specified early. These observations are consistent with previous studies that suggest the cardiomyocyte and endothelial lineages segregate early in development (Wei and Mikawa, 2000Wei Y. Mikawa T. Fate diversity of primitive streak cells during heart field formation in ovo.Dev. Dyn. 2000; 219: 505-513Crossref PubMed Scopus (46) Google Scholar). Notably, some of the clones marked at the later stage contained both cardiomyocytes and endothelial cells or cardiomyocytes and smooth muscle cells, indicating that a subset of these cells in the heart develop from a bipotential progenitor. This finding is in line with studies using the pluripotent stem cell model that identified bi- and tri-potent cardiovascular progenitors that are able to generate cardiomyocytes and smooth muscle cells, or cardiomyocytes and endothelial and smooth muscle cells (Kattman et al., 2006Kattman S.J. Huber T.L. Keller G.M. Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages.Dev. Cell. 2006; 11: 723-732Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, Moretti et al., 2006Moretti A. Caron L. Nakano A. Lam J.T. Bernshausen A. Chen Y. Qyang Y. Bu L. Sasaki M. Martin-Puig S. et al.Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification.Cell. 2006; 127: 1151-1165Abstract Full Text Full Text PDF PubMed Scopus (720) Google Scholar, Wu et al., 2006Wu S.M. Fujiwara Y. Cibulsky S.M. Clapham D.E. Lien C.L. Schultheiss T.M. Orkin S.H. Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart.Cell. 2006; 127: 1137-1150Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, Yang et al., 2008Yang L. Soonpaa M.H. Adler E.D. Roepke T.K. Kattman S.J. Kennedy M. Henckaerts E. Bonham K. Abbott G.W. Linden R.M. et al.Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.Nature. 2008; 453: 524-528Crossref PubMed Scopus (973) Google Scholar). In a more recent study, this same group (Lescroart et al., 2018Lescroart F. Wang X. Lin X. Swedlund B. Gargouri S. Sànchez-Dànes A. Moignard V. Dubois C. Paulissen C. Kinston S. et al.Defining the earliest step of cardiovascular lineage segregation by single-cell RNA-seq.Science. 2018; 359: 1177-1181Crossref PubMed Scopus (25) Google Scholar) profiled the E6.75 and E7.25 Mesp1+ cells by single-cell RNA sequencing and identified distinct clusters representative of endothelial cell, FHF cardiomyocyte, and anterior and posterior SHF fates, confirming the heterogeneity of these mesodermal populations. Collectively, these lineage tracing and molecular profiling studies provide compelling evidence that cardiovascular progenitors with distinct developmental potential are specified during gastrulation. These findings support the general concept of the FHF/SHF model in showing that progenitors that give rise to the left ventricle are distinct from those that contribute to the right ventricle and the outflow tract. In addition, they provide strong evidence that the cardiovascular progenitor pool displays a high degree of heterogeneity and essentially represents a continuum of cells with distinct developmental potential that are specified at different times during gastrulation. The temporal analyses of Lescroart et al., 2014Lescroart F. Chabab S. Lin X. Rulands S. Paulissen C. Rodolosse A. Auer H. Achouri Y. Dubois C. Bondue A. et al.Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development.Nat. Cell Biol. 2014; 16: 829-840Crossref PubMed Scopus (92) Google Scholar indicate that the atrial progenitors appear at the same time as the SHF progenitors that contribute to the right ventricle and the outflow track, following the emergence of the FHF progenitors. These observations suggest that the FHF may be more restricted to the left ventricle than previously thought and that atrial potential is largely skewed toward the SHF. If progenitors with different potential are specified during gastrulation as the lineage tracing studies suggest, the signaling pathways that regulate these early developmental decisions, including Nodal, Wnt, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF), are likely to play a role in establishing these cardiac fates. Support for this interpretation comes from studies using the hPSC model that showed that different levels of activinA/Nodal and BMP signaling can induce distinct atrial and ventricular mesoderm populations in vitro (Lee et al., 2017Lee J.H. Protze S.I. Laksman Z. Backx P.H. Keller G.M. Human Pluripotent Stem Cell-Derived Atrial and Ventricular Cardiomyocytes Develop from Distinct Mesoderm Populations.Cell Stem Cell. 2017; 21: 179-194Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). In addition to these pathways, studies in the chick and mouse have shown that retinoic acid (RA) signaling plays a pivotal role in early cardiac development. This pathway was found to be important for development of the inflow structures of the heart, including the atria, as inhibition of RA signaling resulted in atrial malformation, whereas excessive signaling led to the development of enlarged atria at the expense of ventricular chamber formation (Heine et al., 1985Heine U.I. Roberts A.B. Munoz E.F. Roche N.S. Sporn M.B. Effects of retinoid deficiency on the development of the heart and vascular system of the quail embryo.Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1985; 50: 135-152Crossref PubMed Scopus (97) Google Scholar, Yutzey et al., 1994Yutzey K.E. Rhee J.T. Bader D. Expression of the atrial-specific myosin heavy chain AMHC1 and the establishment of anteroposterior polarity in the developing chicken heart.Development. 1994; 120: 871-883Crossref PubMed Google Scholar). These effects of RA were restricted to a narrow window of embryonic development, specifically Hamburger-Hamilton stages (HHs)4–7 in the chick and E7.5–8.5 in the mouse, a time corresponding to the formation of the cardiac crescent (Hochgreb et al., 2003Hochgreb T. Linhares V.L. Menezes D.C. Sampaio A.C. Yan C.Y. Cardoso W.V. Rosenthal N. Xavier-Neto J. A caudorostral wave of RALDH2 conveys anteroposterior information to the cardiac field.Development. 2003; 130: 5363-5374Crossref PubMed Scopus (109) Google Scholar, Xavier-Neto et al., 1999Xavier-Neto J. Neville C.M. Shapiro M.D. Houghton L. Wang G.F. Nikovits Jr., W. Stockdale F.E. Rosenthal N. A retinoic acid-inducible transgenic marker of sino-atrial development in the mouse heart.Development. 1999; 126: 2677-2687PubMed Google Scholar). A role for this pathway at this early developmental stage was also supported by studies that identified a gradient of RA signaling spanning from the emerging mesoderm population to the posterior region of the cardiac crescent (Moss et al., 1998Moss J.B. Xavier-Neto J. Shapiro M.D. Nayeem S.M. McCaffery P. Dräger U.C. Rosenthal N. Dynamic patterns of retinoic acid synthesis and response in the developing mammalian heart.Dev. Biol. 1998; 199: 55-71Crossref PubMed Scopus (153) Google Scholar). This pattern completely overlapped with expression of the RA synthesizing enzyme RALDH2, suggesting that these cells can both synthesize and respond to RA. The most straightforward interpretation of these observations is that the RALDH2+ cells represent the sub-population of cardiovascular mesoderm that contributes specifically to the posterior regions of the cardiac crescent and gives rise to the atria and sinus venosus derivatives. Given their positioning within the crescent, the FHF and SHF progenitors are exposed to different signaling environments that regulate their proliferation and differentiation to cardiovascular fates (Evans et al., 2010Evans S.M. Yelon D. Conlon F.L. Kirby M.L. Myocardial lineage development.Circ. Res. 2010; 107: 1428-1444Crossref PubMed Scopus (143) Google Scholar, Kelly, 2012Kelly R.G. The second heart field.Curr. Top. Dev. Biol. 2012; 100: 33-65Crossref PubMed Scopus (103) Google Scholar, Vincent and Buckingham, 2010Vincent S.D. Buckingham M.E. How to make a heart: the origin and regulation of cardiac progenitor cells.Curr. Top. Dev. Biol. 2010; 90: 1-41Crossref PubMed Scopus (252) Google Scholar). BMP and FGF secreted by the hepatogenic mesoderm together with Dkk1 produced by the anterior visceral endoderm specify a cardiac fate in the FHF progenitors. The SHF progenitors by contrast are exposed to Wnt, FGF and Hedgehog pathway agonists secreted from the pharyngeal mesoderm. These signaling pathways promote proliferation and expansion of the SHF ISL1+ progenitor pool while delaying their differentiation (Cohen et al., 2008Cohen E.D. Tian Y. Morrisey E.E. Wnt signaling: an essential regulator of cardiovascular differentiation, morphogenesis and progenitor self-renewal.Development. 2008; 135: 789-798Crossref PubMed Scopus (183) Google Scholar, Dyer and Kirby, 2009Dyer L.A. Kirby M.L. Sonic hedgehog maintains proliferation in secondary heart field progenitors and is required for normal arterial pole formation.Dev. Biol. 2009; 330: 305-317Crossref PubMed Scopus (70) Google Scholar, Tzahor, 2007Tzahor E. Wnt/beta-catenin signaling and cardiogenesis: timing does matter.Dev. Cell. 2007; 13: 10-13Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). As the SHF progenitors migrate ventrally toward the heart tube they encounter a region of BMP and non-canonical Wnt signaling that specifies cardiogenic differentiation (Schleiffarth et al., 2007Schleiffarth J.R. Person A.D. Martinsen B.J. Sukovich D.J. Neumann A. Baker C.V. Lohr J.L. Cornfield D.N. Ekker S.C. Petryk A. Wnt5a is required for cardiac outflow tract septation in mice.Pediatr. Res. 2007; 61: 386-391Crossref PubMed Scopus (0) Google Scholar, Tirosh-Finkel et al., 2010Tirosh-Finkel L. Zeisel A. Brodt-Ivenshitz M. Shamai A. Yao Z. Seger R. Domany E. Tzahor E. BMP-mediated inhibition of FGF signaling promotes cardiomyocyte differentiation of anterior heart field progenitors.Development. 2010; 137: 2989-3000Crossref PubMed Scopus (80) Google Scholar). To integrate the findings of the different studies discussed in this section, we propose a scheme of cardiovascular development building on an earlier model put forward by Rosenthal and Xavier-Neto that defined progenitors based on dependency on RA signaling (Rosenthal and Xavier-Neto, 2000Rosenthal N. Xavier-Neto J. From the bottom of the heart: anteroposterior decisions in cardiac muscle differentiation.Curr. Opin. Cell Biol. 2000; 12: 742-746Crossref PubMed Scopus (40) Google Scholar). In this model, cardiovascular development is initiated by E6.25 with specification of the left ventricular progenitors that form the most anterior region of the crescent and generate the left ventricular cardiomyocyte population (Figure 1). These progenitors, which can be considered as FHF progenitors, develop in the absence of RA signaling. The right ventricular progenitors are specified next and migrate to a position posterior to the left ventricular progenitors within the anterior region of the crescent. The generation of the right ventricular progenitors is also independent of RA signaling. Following this, the RALDH2+ atrial and sinoatrial (SAN) progenitors are specified and contribute to the posterior ends of the crescent, establishing an anterior-posterior RA gradient. Their differentiation to atrial and SAN derivatives is dependent on RA signaling. In this model, the right ventricular and atrial progenitors would represent the anterior and posterior portion of the SHF, respectively. This model is supported by molecular analyses carried out in the Lescroart et al. study (Lescroart et al., 2014Lescroart F. Chabab S. Lin X. Rulands S. Paulissen C. Rodolosse A. Auer H. Achouri Y. Dubois C. Bondue A. et al.Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development.Nat. Cell Biol. 2014; 16: 829-840Crossref PubMed Scopus (92) Google Scholar) that show significantly higher levels of RALDH2 expression in the late emerging progenitors (E7.25) than in those specified at the early stage (E6.25). Based on this model, one would predict that the early stages of human cardiovascular development are also characterized by the specification of RALDH+ and RALDH– progenitors. The concept of modeling human cardiovascular development using hPSCs was supported by studies with mouse pluripotent stem cells (mPSCs) that showed that it was possible to generate cardiovascular cells in vitro, even in poorly defined cultures that contained fetal bovine serum (FBS) (Kattman et al., 2006Kattman S.J. Huber T.L. Keller G.M. 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The initial studies demonstrating cardiomyocyte development from hPSCs were also carried out in cultures that included FBS as a source of cytokines to promote differentiation (Itskovitz-Eldor et al., 2000Itskovitz-Eldor J. Schuldiner M. Karsenti D. Eden A. Yanuka O. Amit M. Soreq H. Benvenisty N. Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.Mol. Med. 2000; 6: 88-95Crossref PubMed Google Scholar, Kehat et al., 2001Kehat I. Kenyagin-Karsenti D. Snir M. Segev H. Amit M. Gepstein A. Livne E. Binah O. Itskovitz-Eldor J. Gepstein L. Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes.J. Clin. Invest. 2001; 108: 407-414Crossref PubMed Scopus (1383) Google Scholar). While this approach successfully documented the cardiac potential of the hPSCs, the efficiency of differentiation was exceptionally low. Therefore, most groups moved to defined serum-free media formulations and the use of recombinant pathway agonists and antagonists that allow for the manipulation of developmentally relevant signaling pathways. Studies using this approach demonstrated that the pathways known to regulate primitive streak (PS) formation and mesoderm induction, including activinA/Nodal, Wnt, and BMP (Tam and Behringer, 1997Tam P.P. Behringer R.R. Mouse gastrulation: the formation of a mammalian body plan.Mech. Dev. 1997; 68: 3-25Crossref PubMed Scopus (340) Google Scholar) promoted efficient development of cardiac populations from hPSCs (Kattman et al., 2011Kattman S.J. Witty A.D. Gagliardi M. Dubois N.C. Niapour M. Hotta A. Ellis J. Keller G. 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