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REVIEWS

Navigating The Labyrinth of Cardiac RegenerationErin Lambers* and Tsutomu Kume*

Feinberg Cardiovascular Research Institute, Feinberg School of Medicine, Northwestern University, Chicago, Illinois

Heart disease is the number one cause of morbidity and mortality in the world and is a major health and economic burden,costing the United States Health Care System more than $200 billion annually. A major cause of heart disease is the massiveloss or dysfunction of cardiomyocytes caused by myocardial infarctions and hypertension. Due to the limited regenerativecapacity of the heart, much research has focused on better understanding the process of differentiation toward cardiomyo-cytes. This review will highlight what is currently known about cardiac cell specification during mammalian development,areas of controversy, cellular sources of cardiomyocytes, and current and potential uses of stem cell derived cardiomyocytesfor cardiac therapies. Developmental Dynamics 000:000–000, 2016. VC 2016 Wiley Periodicals, Inc.

Key words: heart development; cardiomyogenesis; directed differentiation; direct lineage conversion; adult stem cells;

reprogramming

Submitted 6 November 2015; First Decision 26 January 2016; Accepted 10 February 2016; Published online 0 Month 2014

Health and Economic Burdens ofCardiovascular Disease

Despite medical advances in modern medicine, heart failurecaused by ischemic heart disease (IHD) is the leading cause ofdeath worldwide, with myocardial infarction (MI) being the mostcommon presentation (Finegold et al., 2013). In the United States,MI prevalence has reached a staggering 8.5 million cases (Lloyd-Jones et al., 2010). The Global Burdon of Disease Study estimatedthat 17.3 million deaths worldwide are attributed to cardiovascu-lar disease which is a 41% increase since 1990 (GBD 2013 Mor-tality and Causes of Death Collaborators, 2015). In addition to thehigh prevalence, patients with heart failure are faced with thepoor prognosis of a 5-year mortality rate equaling nearly 50%(Stewart et al., 2001). From an economic perspective, there is anenormous financial burden associated with cardiovascular dis-ease costing the United States healthcare system approximately$204.4 billion annually (Mozaffarian et al., 2015). Currently,heart transplantation is an established therapy for heart failure,but it is inundated with major problems including limited donorhearts, chronic immunosuppressant therapy, and graft vasculop-athy (Stehlik et al., 2011). Therefore, to avoid these major compli-cations, there has been focused attention on new strategies toregenerate damaged heart tissue.

An Overview of Heart Development

Much of what the medical scientific community has learnedabout cardiac development and regeneration originates fromstudies of model organisms (Gould et al., 2013) such as Drosoph-ila (Seyres et al., 2012), zebrafish (Liu and Stainier, 2012), Xeno-pus (Kaltenbrun et al., 2011), chick (Martinsen, 2005), and mouse(de Boer et al., 2012). We have learned that cardiac developmentis governed by a complex circuitry involving signaling molecules(Fig. 1). During vertebrate embryogenesis the heart is the firstorgan to form (Moorman et al., 2003). During gastrulation, theprocess in which the three germ layers of an embryo are formed,a subset of epiblast cells moves as a sheet to the primitive streakand undergoes an epithelial-to-mesenchymal transition (EMT)(Gould et al., 2013). This sheet of epiblast cells then ingresses totransiently form the mesendoderm, a time-restricted rather thanspatially defined intermediate germ layer, which later becomesthe endoderm and the mesoderm (Gould et al., 2013).

Studies in embryos and ESCs have shed light on this process (vanVliet et al., 2012). This process is controlled through families of sig-naling molecules that include wingless integrated (Wnt), fibroblastgrowth factor (FGF), and the transforming growth factor-beta (TGF-beta) superfamily. Nodal and bone morphogenetic protein-2 (BMP2)(Schlange et al., 2000), members of the TGF-beta family, worktogether with the Wnt/Beta-Catenin (Sumi et al., 2008) and FGFpathways (Willems and Leyns, 2008) by sending activating/inhibi-tory signals in a time and space dependent manner, patterning theembryo and allowing the successive formation of the mesendoderm,mesoderm, and cardiac mesoderm (Tada et al., 2005) (Fig. 1B).

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Grant sponsor: National Institutes of Health; Grant numbers:EY019484, HL126920; Grant sponsor: American Heart Association;Grant number: 15PRE25080006.*Correspondence to: Erin Lambers, Feinberg Cardiovascular ResearchInstitute, Department of Medicine, Northwestern University School ofMedicine, 303 E Chicago Avenue, Chicago, IL 60611. E-mail: e-lambers@northwestern.edu and Tsutomu Kume, Ph.D., Feinberg CardiovascularResearch Institute, Department of Medicine, Northwestern UniversitySchool of Medicine, 303 E Chicago Ave. Chicago IL 60611. E-mail:t-kume@northwestern.edu

Article is online at: http://onlinelibrary.wiley.com/doi/10.1002/dvdy.24397/abstractVC 2016 Wiley Periodicals, Inc.

DEVELOPMENTAL DYNAMICS 00:00–00, 2016DOI: 10.1002/DVDY.24397

1

Cardiac progenitors can be first found in mouse embryo atembryonic day (E) 7.0 at the cardiac crescent stage (Vincent andBuckingham, 2010). The cardiac mesoderm is derived from atleast two mesodermal progenitor populations: The first heart field(FHF) is located in the anterior mesoderm forming the cardiaccrescent, and the second heart field (SHF) is located medial andanterior to the cardiac crescent (Buckingham et al., 2005) (Fig. 1).Genetic tracing, explant experiments, and fluorescent dye label-ing of cells using embryos have shown that FHF and SHF progen-itors transiently express a distinct set of gene expressionpatterns, which subsequently contribute to distinct regions of theadult heart (Meilhac et al., 2015). Nkx2.5, Gata4, and Mef2c aretranscription factors transiently expressed in both FHF and SHFprogenitors (Heikinheimo et al., 1994; Dodou et al., 2004; Zhanget al., 2014). While Tbx5 and Hcn4 are transiently and predomi-nantly expressed in FHF progenitors, Tbx1 and Isl1 are transi-

ently and predominantly expressed in SHF progenitors (Sp€ateret al., 2013, 2014; Xin et al., 2013; Rana et al., 2014).

Although genetic fate mapping of Isl1 expressing cells hasshown contribution to both heart fields (Ma et al., 2008), deletionof Isl1 in mouse models produce a phenotype that corresponds todefects of the SHF lineage and is considered a marker of the SHF(Cai et al., 2003) (Fig. 1C). Cardiac looping occurs at E9.0, duringwhich time cells undergo uneven growth and remodeling. FromE10 to E15, septation formation in the atria and ventricles formthe four chambers of the heart. Meanwhile, the myocardiumundergoes a process called compaction during which time loosenetworks of trabeculae, which are thought to allow adequateoxygenation and growth, diminish and cardiomyocytes formtighter connections and cell–cell junctions (Dellefave et al.,2009). Ultimately the FHF lineage predominantly gives rise to theleft ventricle and the SHF lineage predominantly contributes to

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Fig. 1. Comparison of embryonic stem cell (ESC) differentiation into cardiomyocytes and early heart development. A: Time course of the embry-onic stages of cardiac development in the mouse embryo. During gastrulation the cells of the inner cell mass (ICM) form three distinct germ layers:endoderm, ectoderm, and mesoderm. The heart is derived from the mesoderm. The first cardiac progenitors are found at the cardiac crescentstage forming the first heart field (FHF, red) and the second heart field (SHF, blue). Following looping, during which time cells undergo unevenmigrations and proliferation to form the heart tube, the FHF lineage predominantly give rise to the left ventricle and the SHF lineage predominantlycontributes to the right ventricle and the outflow tract (OFT) and portions of the atria. B: An analogous time course of pluripotent stem cell (PSC)differentiation into cardiomyocyte lineages recapitulating heart development. Key signaling pathways involved at different stages of cardiac devel-opment are shown: FHF (red), SHF (blue), atrial cardiomyocytes (orange), and ventricular cardiomyocytes (green). C: Key transcription factorsinvolved at different stages of cardiac development. Transcription factors transiently and predominantly expressed in FHF progenitors (red), SHFprogenitors (blue), and both progenitor populations (purple). Markers of lineage committed atrial cardiomyocytes (orange), ventricular cardiomyo-cytes (green), and both atrial and ventricular markers (gray).

2 LAMBERS AND KUME

the right ventricle, the outflow tract (OFT), and portions of theinflow tract and atria (Fig. 1A). A recent study published inNature exploited levels of tbx5 and pitx2 to control FHF and SHFcontributions to the zebrafish heart. They found that alterationsof the FHF and SHF contributions disrupt cardiac function andspeculate that FHF and SHF progenitors may give rise to cardio-myocytes with distinctive physiological properties (Mosimannet al., 2015).

Additionally, cells that form the coronary vessels and the car-diac fibrous matrix are derived from the epicardium and arerequired for proper cardiac function (Gittenberger-de Groot et al.,1998). The genesis of epicardial derived cells (EPDCs) throughEMT and lineage determination is also regulated by a complexnetwork of transcription factors, including Wt1, Tbx18, Nfatc1,and basic helix–loop–helix (bHLH) transcription factors (Kirsch-ner et al., 2006; von Gise et al., 2011; Combs et al., 2011;Acharya et al., 2012). Specifically, Tcf21, a bHLH factor, has beenshown to be a key regulator of cell lineage development in EPDCs(Braitsch et al., 2012; Acharya et al., 2012; Tandon et al., 2013).Additionally, a recent study demonstrated that cardiac regenera-tion in zebrafish is guided by the outflow tract and hedgehog sig-naling which may have implications for mobilizing epicardialcells for cardiac repair (J Wang et al., 2015). Detailed develop-mental stages and regulation of cardiogenesis is further reviewedin Xin et al. (2013).

Neonatal Regeneration

Because the neonatal mammalian heart can substantially regen-erate after injury through cardiomyocyte proliferation until post-natal day 7 (Porrello et al., 2011), it makes an excellent model tostudy important factors involved in mammalian regeneration.Meis1, for example, was shown to be a critical regulator of thecardiomyocyte cell cycle in neonatal mice. Meis1 deletion wassufficient to increase the postnatal proliferative window of cardi-omyocytes, reactivate entry into mitosis, with no harmful effecton cardiomyocyte function (Mahmoud et al., 2013). Others usedthe regenerating neonatal mouse heart to identify a regulator ofcell cycle entry, interleukin 13, in the regenerating mouse heart(O’Meara et al., 2015).

Neonatal models have also been useful in identifying otherprocesses aside from cardiomyocyte renewal, which contribute toregeneration. Recently, Mahmoud et al. demonstrated that phar-macological cholinergic nerve inhibition impairs neonatal miceheart regeneration and could be rescued by administration ofneuregulin1 and nerve growth factor (Mahmoud et al., 2015).Using a genetic approach to label neural crest lineages, Whiteet al. similarly demonstrated that sympathetic denervation with6-OHDA inhibits neonatal heart regeneration (White et al., 2015).

Adult Cardiac Regeneration and Areas ofControversy

If and how the adult heart regenerates after injury has beenwidely debated. Ventricular dilation and wall thinning are funda-mental structural aspects of heart failure due to cardiomyopathiesand ischemic heart disease (Sutton and Sharpe, 2000). Theremodeling of the heart’s architecture effects ventricular perform-ance and characterizes myocardial disease (Sutton and Sharpe,2000). Infarction causes massive loss of cardiomyocytes, which

are replaced by scar tissue (Sutton and Sharpe, 2000). Histori-cally, remodeling and plasticity of the heart was thought to bedue principally to cellular processes of cardiomyocyte hypertro-phy and death, with no existence of cardiomyocyte renewal (Zak,1973). However, in the past decade, studies have shown evidenceof significant cardiomyocyte turnover in the heart (Mollova et al.,2013).

Seminal studies that have contributed to the paradigm shiftinclude studies using retrospective [14C] birth dating of cardiomy-ocytes (Bergmann et al., 2009) and cardiomyocyte genetic fatemapping (Hsieh et al., 2007), showing that cardiomyocyte turn-over in the heart does exist at approximately 1% per year (Berg-mann et al., 2009). Additionally, many studies have reportedcardiomyocyte progenitor populations in the heart as sources ofregeneration such as side population (Pfister et al., 2008),Sca1þ(Matsuura et al., 2004), cardiosphere derived progenitorcells (Smith et al., 2007), and c-kitþ multipotent resident stemcells (Beltrami et al., 2003) also providing evidence for adult car-diac regeneration.c-kitþ cells, in particular, have been hotlydebated as a source of regeneration in the adult heart. C-kitþprogenitor cells were originally reported to be found in nicheswithin the heart (Urbanek et al., 2006). These niches were thoughtto be important for physiological turnover of cardiac cells, governthe migration and commitment of c-kitþ (Urbanek et al., 2006;Hosoda et al., 2009;), and significantly repair infarcted adulthearts after transplantation thereby attenuating left ventricularremodeling and improving left ventricular function (Beltramiet al., 2003; Dawn et al., 2005; Bolli et al., 2006; Bearzi et al.,2007; Rota et al., 2008; Fischer et al., 2009; Tang et al., 2010).However, many independent groups through much effort couldnot replicate the findings demonstrating that c-kitþ were impor-tant for significant physiological turnover of cardiac cells in theadult heart (Murry et al., 2004; Nygren et al., 2004; Jesty et al.,2012). Van Berlo et al. (2014) and Sultana et al. (2015) publishedtwo important studies concluding that c-kitþ cells are an irrele-vant source of cardiomyocytes in the heart. The results of thestudy by Van Berlo et al. revealed that it was possible for c-kitþcells to make new myocytes during development, with aging, andafter injury, but at an extremely low level (van Berlo et al., 2014).Similarly, with five independently derived lineage tracing mousemodels, Sultana et al. found that c-kitþ cells have the capacity togenerate less than 0.05% cardiomyocytes during development,aging, or after infarct and are instead a subpopulation of endo-thelial cells (Sultana et al., 2015).

Current and Preclinical Cellular Therapiesfor Cardiac Regeneration

Adult Progenitor Stem Cells

Due to the limited regenerative capacity of the heart, stem celltherapies have emerged as strategies to promote cardiac regenera-tion. Clinical trials have tested the safety and efficacy of severaldifferent adult progenitor stem cell populations for myocardialrepair. The cell types used in clinical trials include bone marrow-derived mononuclear cells (BMMNCs), bone marrow or peripheralblood derived endothelial progenitor cells (EPCs), adipose or bonemarrow derived mesenchymal stromal cells (MSCs), and cardiacderived cardiac progenitor cells (CPCs) (Table 1).

Approximately 2,000 patients have been treated withBMMNCs, representing the majority of patients receiving cell

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4 LAMBERS AND KUME

therapy compared with all other cell types combined. Meta-analysis of BMMNC clinical trials suggest a very modest improve-ment in heart function due to improved infarct size and improvedremodeling (Tian et al., 2014). Implanted cells survive only brieflyafter implantation and, therefore, the modest changes in left ven-tricular ejection fraction (LVEF) and remodeling are thought to bedue to paracrine effects, rather than by contribution of cells them-selves (Alfaro and Young, 2012). The most pronounced improve-ment in LVEF was seen in the recent SCIPIO RCT using c-kitþcells (Bolli et al., 2011). However, the trial has been under investi-gation for concerns about the integrity of data (The LancetEditors, 2014). Overall, the benefits of adult progenitor stem celltherapy have not produced the functional outcomes that wereonce anticipated. Thus, new avenues of generating cardiomyo-cytes for cellular transplantation therapies are being explored.

Directed Differentiation

Human embryonic stem cells (ESCs) were first derived in 1998(Thomson et al., 1998) and since then have been acclaimed as apromising source of cellular material to be used in the treatmentof many diseases such as heart failure. In 2006, Dr. Shinya Yama-naka discovered that forced expression of four transcription fac-tors (Oct4, Sox2, Klf4, and c-Myc) in mature fibroblasts wasenough to reprogram fully committed somatic cells back into apluripotent state (Takahashi and Yamanaka, 2006). This Nobelprize winning discovery generating induced pluripotent stemcells (iPSCs) has allowed stem cell researchers to bypass ethicalcontroversies surrounding the use of human ESCs to more effec-tively study methods to generate cells such as cardiomyocytes foruse in drug discovery (Sinnecker et al., 2014), modeling cardiacdiseases (Itzhaki et al., 2011), and cardiac regeneration (LiyingZhang et al., 2015).

The first efforts to differentiate human pluripotent stem cells(PSCs) into cardiomyocytes used basic methods of embryoid body(EB) formation in which the cells would differentiate spontane-ously into different cellular lineages, yielding a low percentage ofcardiomyocytes. Further studies have revealed that many of thesignaling pathways that control cardiac development in vivo arecapable of more efficiently directing the differentiation of PSCsinto cardiomyocytes in vitro. Induction and inhibition of Wnt-B-Catenin signaling pathways and induction of Activin/Nodal/TGFb, BMP, FGF, and noncanonical Wnt signaling pathways atvarious stages of cardiac differentiation can increase cardiomyo-cyte production up to 50% (Laflamme et al., 2007; Yang et al.,2008; Paige et al., 2010; Kattman et al., 2011; Noseda et al.,2011; Lian et al., 2012) (Fig. 2B).

Purification methods yielding up to 98% purity have also beeninitiated isolating cardiomyocytes from other cell types based oncardiomyocyte specific traits. These include the expression of thesignal regulatory protein alpha (SIRPA) (Dubois et al., 2011), dif-ferences in cardiomyocyte glucose metabolism versus lactatemetabolism in noncardiomyocytes (Tohyama et al., 2013), andhigher content of mitochondria found in cardiomyocytes usingmitochondrial specific dyes (Hattori et al., 2010). A recent studyhas shown that human ESC-derived cardiomyocytes can bepropagated on clinical scale, transplanted, and integrate func-tionally into nonhuman primate recipient hearts (Chong et al.,2014). Although this demonstrates ESC-derived cardiomyocytesare getting closer to human clinical applications, safety concernsinvolving cardiac arrhythmias are still a major hurdle to over-

come. Regarding cell transplantation therapies, additional atten-tion will need to be placed on methods that can further directPSC differentiation into specific cardiomyocytes of the heart suchas ventricular cardiomyocytes, atrial cardiomyocytes, and cardiacconduction cells. These will allow tailored cell transplantationtherapies to repair specific regions of damaged heart tissue andlower risks of complications such as cardiac arrhythmias.

Direct Lineage Conversion

In 1987, a seminal discovery was made showing that ectopicexpression of a single skeletal muscle gene, MYOD1, could con-vert fibroblasts into skeletal muscle cells (Davis et al., 1987). Thiswas the first example of what we now call direct reprogrammingin which a terminally differentiated adult cell type can becomeanother adult cell type without ever passing through a pluripo-tent state. Inspired by this seminal discovery, a group recentlyintroduced a cocktail of three transcription factors known to reg-ulate normal cardiac development (Gata4, Mef2c, and Tbx5;together known as GMT) into mouse hearts with myocardialinfarction. They showed that these factors were able to reprogramcardiac fibroblasts, cells comprising scar tissue, into functionalcardiomyocytes capable of electrical coupling and showed areduction in scar tissue (Qian et al., 2013). It was demonstratedthat the introduction of Hand2 to the cocktail (GHMT) was ableincrease direct reprogramming efficiency from approximately 7%to 20% (Song et al., 2012).

To translate this direct reprogramming system from mouse tohuman, it was discovered that Myocd was additionally required(Nam et al., 2013). Recently, a group used polycistronic vectors toexpress different ratios of the GMT factors and found that, ifMef2c expression is increased compared with Gata4 and Tbx5,then reprogramming efficiency of fibroblasts into cardiomyocytesincreases three- to five-fold (L. Wang et al., 2015). This studydemonstrated that the stoichiometry of reprogramming factors iscritical for reprogramming efficiency and may explain why otherreports using the same factors demonstrate different reprogram-ming efficiencies (Muraoka and Ieda, 2015).

Epigenetic factors have also recently been found to be impor-tant for cardiac reprogramming. Four miRNAs, miR-1, miR-133,miR-208, and miR-499, were found to be sufficient to reprogramfibroblasts into cardiomyocytes in vitro and in vivo and this effi-ciency was enhanced by the inhibition of JAK1, janus kinase 1(Jayawardena et al., 2012) (Fig. 2B). Another epigenetic factor, anATP dependent chromatin remodeling factor, has also been iden-tified as an important regulator of the cardiac transcriptional pro-gram, BRG1-Associated factor 60c (BAF60c). The overexpressionBAF60c together with Gata4 and Tbx5 have been shown totrans-differentiate multipotent mesodermal cells into beating car-diomyocytes (Takeuchi and Bruneau, 2009).

Recently, there have been exciting advancements in the fieldof direct reprogramming demonstrating the ability to reprogramcardiomyocytes into specific subtypes of cardiac cells includingPurkinje cells and sinoatrial-like conduction cells (Fig. 2C). Acti-vation of Notch signaling has resulted in the conversion of cardi-omyocytes into Purkinje like cells (Rentschler et al., 2012). Forcedexpression of transcription factor Tbx3 or Tbx18 in cardiomyo-cytes was shown to induce pacemaker like cells in vitro and invivo (Bakker et al., 2012; Kapoor et al., 2013). In a follow-uplarge animal study, transcription factor Tbx18 was injected intothe hearts of pigs with complete heart block using minimally

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CARDIAC REGENERATION 5

invasive methods to successfully create new sinus nodes as analternative to pacemakers (Hu et al., 2014).

Short Perspectives

Clinical trials using bone marrow derived cells, mesenchymalstem cells and cardiac progenitor cells have demonstrated safety

of catheter based cell delivery, however, all with very modestimprovement in heart function. Overall, poor engraftment andlow survival have greatly limited the potential of adult progenitorcells to differentiate into cardiomyocytes to repair damaged myo-cardium. Thus, new avenues for cell transplantation are beingexplored. Insights that we have gained from the normal develop-ing embryonic heart, including key signaling molecules,

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Fig. 2. Cellular sources for cardiac transplantation. A: Adult progenitor stem cells that have been tested in human clinical trials. The major celltypes used for cardiac therapies include: (1) bone marrow mononuclear cells (BMMNCs, purple) and endothelial progenitor cells (EPCs, purple)derived from the bone marrow, (2) mesenchymal stem cells (MSCs, green) derived from adipose tissue, and (3) cardiac progenitor cells (CPCs,orange) derived from the heart. B: Pluripotent stem cell (PSC) derived cardiomyocytes have been generated from embryonic stem cells (ESCs)and induced-PSCs (iPSCs) (blue) induced from somatic cells (green). Differentiation efficiency into cardiac progenitors (red and blue) and cardio-myocytes (pink) depends on the introduction of signaling molecules involved during development in a finely tuned time and dose dependent man-ner. C: Generation of cardiomyocytes from direct lineage conversion bypasses the pluripotent state by reprogramming terminally differentiatedadult cells directly into another type of fully committed adult cell. This has been achieved in human fibroblasts (green) with the forced expressionof four developmental transcription factors: Gata4, Mef2c, Tbx5, and Hand2. It has also been accomplished in fibroblasts using four miRNAs mir-1, mir-133, mir-208, and mir499 along with the inhibition of Jak1 signaling. Direct lineage conversion has also been performed allowing the conver-sion of cardiomyocytes (pink) into Purkinje-like and pacemaker-like cells using Notch activation and Tbx18, respectively.

6 LAMBERS AND KUME

transcription factors, and epigenetic factors, have allowed thediscovery of innovative methods to direct the differentiation ofboth ESCs and iPSCs, pluripotent stem cells (PSCs), into cardio-myocytes and to reprogram adult cell types into cardiomyocytes.

There still exist many limitations and barriers associated withmethods of reprogrammed cardiomyocytes that need to be over-come before their use in clinical cellular transplantation therapiescan be realized. Although PSC and direct conversion derived car-diomyocytes beat spontaneously, express many cardiomyocytespecific genes, and recapitulate some electrophysiological fea-tures of adult cardiomyocytes, they still possess immature fea-tures of embryonic cardiomyocytes. These features include higherproliferation rates (Snir et al., 2003; McDevitt et al., 2005; Berg-mann et al., 2009), smaller and more rounded morphologies lack-ing t-tubules and bi-nucleation (Smolich, 1995; Lieu et al., 2009),and potassium currents considered to be responsible for arrhyth-mias (Abdul Kadir et al., 2009; Lahti et al., 2012). Although timein culture creates a more mature cardiomyocyte phenotype, it isclear more work needs to be done to drive the maturation of PSCderived cardiomyocytes (Robertson et al., 2013).

Additionally strategies used to create reprogrammed cardiomy-ocytes commonly use methods such as lentiviral delivery bywhich defined factors are randomly integrated into the genome,thereby creating genomic instability and increase risk of tumorformation. To bypass this shortcoming, new strategies going for-ward should be improved using nonintegrating methods such asepisomal delivery (Caxaria et al., 2016).

Directed differentiation and direct conversion methods of car-diomyocyte generation need to be further refined. The maincauses of death due to cardiovascular diseases include myocardialinfarction and heart failure, which primarily affect the ventriclesof the heart. Therefore, future strategies will need to bias differen-tiation and reprogramming strategies toward ventricular cardio-myocytes. Ventricular cardiomyocytes are electrically silent anddo not beat spontaneously, but only in response to electricalstimulation. Therefore, future studies will need to propagateand use ventricular cardiomyocytes to avoid transplantationcomplications such as arrhythmias caused by spontaneous beat-ing or inefficient coupling of endogenous and exogenouscardiomyocytes.

The key to understanding how to effectively repair the heartlies in elucidating the intricate pathways that construct the heartduring normal development. As we are overcoming barriers andobstacles in our path toward understanding cardiac development,the closer we are getting to the center labyrinth toward cardiacregeneration. Continued research in preclinical and clinical cellu-lar therapies is needed to fully realize our public health goals ofsignificantly decreasing the high cardiovascular mortality ratesthat plague the world.

AcknowledgmentsWe thank the members of the Kume Lab and of the Feinberg Cardi-ovascular Institute for their valuable discussion over many topicsof the review article. We also thank Dr. Margaret Buckingham ofthe Pasteur Institute for her intellectual contributions regardingcardiac lineage specification in FHF and SHF progenitor popula-tions. T.K. was funded by the NIH, and E.L. was funded by theAmerican Heart Association.

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