Toward improved understanding of cardiac development and congenital heart disease: The advent of cardiac organoids

Abstract

Central MessageHuman cardiac organoid systems hold significant promise for mechanistic studies of early heart morphogenesis and an improved understanding of congenital cardiac disease.This Invited Expert Opinion provides a perspective on the following papers: Cell. 2021 Jun 10;184(12):3299-3317.e22. https://doi.org/10.1016/j.cell.2021.04.034. and N Engl J Med. 2021 Aug 26;385(9):847-849. https://doi.org/10.1056/NEJMcibr2108627.Feature Editor's Introduction—During the past decade, we have witnessed remarkable progress in genome editing technology, stem cell research, and bioengineering. The fundamental basic research discoveries accelerate rapidly into clinical translation, paving the way for myocardial regeneration, better understanding of the structural heart disease, and bioengineering of heart structures and even entire hearts. The new horizon is vast and diverse, ranging from creating universal stem cell biobanking to genome edited heart xenotransplantation. Herein, a group of experts from Duke University discuss the state of the art and the possible influence of cardiac organoids on our understanding of structural heart disease. It may not be immediately clear now in what practical ways this technology will be translated into our daily work, yet the current progress in bioengineering will likely have a very significant influence on our surgical practice.Igor E. Konstantinov, MD, PhD, FRACS Cardiac birth defects remain a significant health burden and cause of death in the United States and worldwide, with congenital heart malformations affecting 1% to 2% of live births.1van der Linde D. Konings E.E. Slager M.A. Witsenburg M. Helbing W.A. Takkenberg J.J.M. et al.Birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis.J Am Coll Cardiol. 2011; 58: 2241-2247Crossref PubMed Scopus (1771) Google Scholar Surgical advances have promoted longer, healthier lives in those with severe genetic anomalies, but a more complete mechanistic understanding of early cardiac development and congenital structural disease could open doors to minimally invasive or nonsurgical interventions. To achieve such a lofty goal, the fields of developmental cardiology and congenital heart surgery require new models to provide critical insights into biological processes underlying early human cardiac development and disease. The in vitro self-organizing human cardiac organoid (CO) systems, such as those recently reported by Hofbauer and colleagues,2Hofbauer P. Jahnel S.M. Papai N. Giesshammer M. Deyett A. Schmidt C. et al.Cardioids reveal self-organizing principles of human cardiogenesis.Cell. 2021; 184: 3299-3317.e3222Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar represent powerful platforms to study early cardiac morphogenic events and related abnormalities, offering the promise to eventually improve the current standard of care for patients with congenital heart disease via numerous applications (Figure 1). Organoids are 3-dimensional (3D), self-assembled, cellular structures that can recapitulate important spatial and functional relationships in developing and adult mouse and human tissues.3Kim J. Koo B.K. Knoblich J.A. Human organoids: model systems for human biology and medicine.Nat Rev Mol Cell Biol. 2020; 21: 571-584Crossref PubMed Scopus (502) Google Scholar As such, organoids have been increasingly used to model early organ development, physiology, disease, and drug responses in vitro.3Kim J. Koo B.K. Knoblich J.A. Human organoids: model systems for human biology and medicine.Nat Rev Mol Cell Biol. 2020; 21: 571-584Crossref PubMed Scopus (502) Google Scholar Compared with traditional animal models, organoids have the primary advantage of using human cells and having improved ability to interrogate cellular processes, higher experimental throughput, and control over 3D cellular and matrix composition and structural organization. On the other hand, they have a small size due to the absence of perfusable vasculature, possess simplified architecture, and lack complex chemical and physical cues present in vivo. The typical protocols for organoid formation (Figure 2), involve culturing stem cells on a plate, reconstituting them into a single cell suspension, and embedding them in an extracellular matrix analog (eg, Matrigel [Corning Life Sciences], a solubilized basement membrane matrix secreted by mouse sarcoma cells). Over several days, the embedded cells self-aggregate into 3D spheroid structures, followed by differentiation, migration, and formation of solid or luminal structures, depending on the cell types used and modulation of specific signaling pathways.3Kim J. Koo B.K. Knoblich J.A. Human organoids: model systems for human biology and medicine.Nat Rev Mol Cell Biol. 2020; 21: 571-584Crossref PubMed Scopus (502) Google Scholar,4Cakir B. Xiang Y. Tanaka Y. Kural M.H. Parent M. Kang Y.-J. et al.Engineering of human brain organoids with a functional vascular-like system.Nat Methods. 2019; 16: 1169-1175Crossref PubMed Scopus (307) Google Scholar Organoids may be constructed out of terminally differentiated cells; however, the use of pluripotent stem cells (PSCs) or their multipotent derivatives uniquely allows studies of early lineage specification and tissue morphogenesis in a controlled, in vitro setting. Recent advances in organoid development have resulted in the successful generation of in vitro models of noncardiac tissues such as the intestines5Silva A.C. Matthys O.B. Joy D.A. Kauss M.A. Natarajan V. Lai M.H. et al.Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids.Cell Stem Cell. 2021; 28: 2137-2152.e2136Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and brain.6Velasco S. Kedaigle A.J. Simmons S.K. Nash A. Rocha M. Quadrato G. et al.Individual brain organoids reproducibly form cell diversity of the human cerebral cortex.Nature. 2019; 570: 523-527Crossref PubMed Scopus (376) Google Scholar Self-organized COs mimicking early heart development5Silva A.C. Matthys O.B. Joy D.A. Kauss M.A. Natarajan V. Lai M.H. et al.Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids.Cell Stem Cell. 2021; 28: 2137-2152.e2136Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,7Moris N. Anlas K. van den Brink S.C. Alemany A. Schroder J. Ghimire S. et al.An in vitro model of early anteroposterior organization during human development.Nature. 2020; 582: 410-415Crossref PubMed Scopus (150) Google Scholar, 8Drakhlis L. Biswanath S. Farr C.M. Lupanow V. Teske J. Ritzenhoff K. et al.Human heart-forming organoids recapitulate early heart and foregut development.Nat Biotechnol. 2021; 39: 737-746Crossref PubMed Scopus (94) Google Scholar, 9Rossi G. Broguiere N. Miyamoto M. Boni A. Guiet R. Girgin M. et al.Capturing cardiogenesis in gastruloids.Cell Stem Cell. 2021; 28: 230-240.e236Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar and chamberogenesis2Hofbauer P. Jahnel S.M. Papai N. Giesshammer M. Deyett A. Schmidt C. et al.Cardioids reveal self-organizing principles of human cardiogenesis.Cell. 2021; 184: 3299-3317.e3222Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar,10Lee J. Sutani A. Kaneko R. Takeuchi J. Sasano T. Khoda T. et al.In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.Nat Commun. 2020; 11: 4283Crossref PubMed Scopus (39) Google Scholar,11Lewis-Israeli Y.R. Wasserman A.H. Gabalski M.A. Volmert B.D. Ming Y. Ball K.A. et al.Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease.Nat Commun. 2021; 12: 5142Crossref PubMed Scopus (54) Google Scholar are some of the latest additions to this rapidly evolving field. In the original and increasingly used formulation of COs, multiple terminally differentiated cardiac cell types are coaxed together to form spheroid-shaped microtissues. For example, the COs made of human induced PSC-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells can permit relatively high-throughput studies of drug responses,3Kim J. Koo B.K. Knoblich J.A. Human organoids: model systems for human biology and medicine.Nat Rev Mol Cell Biol. 2020; 21: 571-584Crossref PubMed Scopus (502) Google Scholar hypoxic insults,12Richards D.J. Li Y. Kerr C.M. Yao J. Beeson G.C. Coyle R.C. et al.Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity.Nat Biomed Eng. 2020; 4: 446-462Crossref PubMed Scopus (127) Google Scholar or heterocellular interactions underlying heart diseases.13Giacomelli E. Meraviglia V. Campostrini G. Cochrane A. Cao X. van Helden R.W.J. et al.Human-iPSC-derived cardiac stromal cells enhance maturation in 3D cardiac microtissues and reveal non-cardiomyocyte contributions to heart disease.Cell Stem Cell. 2020; 26: 862-879.e811Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar More complex structures made from multiple spheroid COs have been generated using 3D bioprinting to position and fuse organoids in predefined spatial patterns and study cardiac scarring.14Daly A.C. Davidson M.D. Burdick J.A. 3D bioprinting of high cell-density heterogeneous tissue models through spheroid fusion within self-healing hydrogels.Nat Commun. 2021; 12: 753Crossref PubMed Scopus (114) Google Scholar Although these methods do not replicate developmental organogenesis, they can recreate the realistic macroscopic architecture of an adult heart and its components, including ventricles, atria, valves, and coronary vasculature.15Kupfer M.E. Lin W.H. Ravikumar V. Qiu K. Wang L. Gao L. et al.In situ expansion, differentiation, and electromechanical coupling of human cardiac muscle in a 3D bioprinted, chambered organoid.Circ Res. 2020; 127: 207-224Crossref PubMed Scopus (103) Google Scholar Overall, whereas 2-dimensional micropatterning16Ma Z. Wang J. Loskill P. Huebsch N. Koo S. Svedlund F.L. et al.Self-organizing human cardiac microchambers mediated by geometric confinement.Nat Commun. 2015; 6: 7413Crossref PubMed Scopus (123) Google Scholar and 3D bioprinting17Lee A. Hudson A.R. Shiwarski D.J. Tashman J.W. Hinton T.J. Yerneni S. et al.3D bioprinting of collagen to rebuild components of the human heart.Science. 2019; 365: 482-487Crossref PubMed Scopus (718) Google Scholar technologies can be used to preform complex organoid structures, they do not recreate dynamic spatiotemporal signals and self-organizing processes that drive early cardiac development and morphogenesis. Recently, several studies have demonstrated generation of submilimeter to millimeter-sized COs where cells are initially mixed with Matrigel, followed by a staged addition of small molecules and growth factors to guide early cardiac differentiation, lineage specification, and formation of stratified ventricular walls and chamber-like lumens. Although the initial efforts to mimic early cardiac development in organoids have been limited to specification and rudimentary patterning of first and second heart fields with mouse PSCs,18Andersen P. Tampakakis E. Jimenez D.V. Kannan S. Miyamoto M. Shin H.K. et al.Precardiac organoids form two heart fields via Bmp/Wnt signaling.Nat Commun. 2018; 9: 3140Crossref PubMed Scopus (68) Google Scholar recent studies that utilize glycogen synthase kinase-3 inhibition to activate wingless and Int-1 (Wnt) signaling have demonstrated the formation of multilineage COs containing spatially distinct cardiac mesoderm and endodermal, gut-like regions.5Silva A.C. Matthys O.B. Joy D.A. Kauss M.A. Natarajan V. Lai M.H. et al.Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids.Cell Stem Cell. 2021; 28: 2137-2152.e2136Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,7Moris N. Anlas K. van den Brink S.C. Alemany A. Schroder J. Ghimire S. et al.An in vitro model of early anteroposterior organization during human development.Nature. 2020; 582: 410-415Crossref PubMed Scopus (150) Google Scholar,9Rossi G. Broguiere N. Miyamoto M. Boni A. Guiet R. Girgin M. et al.Capturing cardiogenesis in gastruloids.Cell Stem Cell. 2021; 28: 230-240.e236Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Under exogenously supplied growth factors (ie, ascorbic acid, basic fibroblast growth factor, and vascular endothelial growth factor) and endogenous heterocellular cues, cardiac lineage cells within these COs undergo early differentiation and morphogenic events, eventually forming linear heart tube-like structures, but not proceeding to generate cardiac chamber.19Baar K. Birla R. Boluyt M.O. Borschel G.H. Arruda E.M. Dennis R.G. Self-organization of rat cardiac cells into contractile 3-D cardiac tissue.FASEB J. 2005; 19: 275-277Crossref PubMed Scopus (113) Google Scholar A more accurate recapitulation of the interactions between human cardiac mesoderm and foregut endoderm8Drakhlis L. Biswanath S. Farr C.M. Lupanow V. Teske J. Ritzenhoff K. et al.Human heart-forming organoids recapitulate early heart and foregut development.Nat Biotechnol. 2021; 39: 737-746Crossref PubMed Scopus (94) Google Scholar resulted in multilineage organoids containing an outer layer of epicardial cells, cardiomyocytes, smooth muscle cells, and gut epithelial cells. Although this system could allow studies of early cardiac patterning events, including those involving aberrant NKX2 Homeobox 5 (NKX2.5), signaling, it did not result in cardiac chamberogenesis. Only recently have 2 studies with human2Hofbauer P. Jahnel S.M. Papai N. Giesshammer M. Deyett A. Schmidt C. et al.Cardioids reveal self-organizing principles of human cardiogenesis.Cell. 2021; 184: 3299-3317.e3222Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar,11Lewis-Israeli Y.R. Wasserman A.H. Gabalski M.A. Volmert B.D. Ming Y. Ball K.A. et al.Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease.Nat Commun. 2021; 12: 5142Crossref PubMed Scopus (54) Google Scholar and 1 with mouse10Lee J. Sutani A. Kaneko R. Takeuchi J. Sasano T. Khoda T. et al.In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.Nat Commun. 2020; 11: 4283Crossref PubMed Scopus (39) Google Scholar cells been able to create in vitro conditions for the spontaneous generation of chamber-like cavities within COs (Table 1). The cavity formation was induced by manipulating Wnt and/or retinoic acid signaling and did not require recreation of all developmental stages such as formation of the cardiac crescent, linear heart tube, or cardiac looping. Similar to other CO studies, the use of mouse PSCs yielded more advanced cardiac morphogenesis compared with use of human PSCs, with manipulation of extracellular matrix proteins and fibroblast growth factor 4 signaling resulting in the formation of primitive atrial and ventricular chambers.10Lee J. Sutani A. Kaneko R. Takeuchi J. Sasano T. Khoda T. et al.In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.Nat Commun. 2020; 11: 4283Crossref PubMed Scopus (39) Google Scholar A more rigorous structural and functional characterization of these COs and improved methods to decrease their variability in shape and cellular composition will be highly instructive for the future generation of developmentally mimetic COs made from human PSCs.Table 1Overview of cardiac organoid systemsSpeciesArchitectureModulated pathwayFindingsAdvantages; DisadvantagesReferenceOrganoids with single-cell-type composition HumanSpheroid with a microchamberWnt/β-cateninBiophysical cues dictate cell specification in cardiac organoidsStudying mechanical forces in cardiac development;Simple structure with 1 microchamber16Ma Z. Wang J. Loskill P. Huebsch N. Koo S. Svedlund F.L. et al.Self-organizing human cardiac microchambers mediated by geometric confinement.Nat Commun. 2015; 6: 7413Crossref PubMed Scopus (123) Google Scholar HumanSpheroid with a microchamberWntNoradrenaline and oxygen gradients can be used to study ischemiaMature cardiomyocytes relevant for adult disease;Simple structure with 1 microchamber12Richards D.J. Li Y. Kerr C.M. Yao J. Beeson G.C. Coyle R.C. et al.Human cardiac organoids for the modelling of myocardial infarction and drug cardiotoxicity.Nat Biomed Eng. 2020; 4: 446-462Crossref PubMed Scopus (127) Google ScholarMultilineage organoid systems MousePrimitive, multilineage organoid without chambersBMP/WntBMP and Wnt signaling govern first and second heart field specificationCo-existence of both heart fields in single organoids; Use of murine instead of human cells18Andersen P. Tampakakis E. Jimenez D.V. Kannan S. Miyamoto M. Shin H.K. et al.Precardiac organoids form two heart fields via Bmp/Wnt signaling.Nat Commun. 2018; 9: 3140Crossref PubMed Scopus (68) Google Scholar MouseTube-like structure with cardiac and vascular progenitor cellsWntMouse ESCs can recapitulate key cardiac morphogenic events in vitroHigh fidelity recreation of cardiac crescent and heart tube; Use of murine instead of human cells9Rossi G. Broguiere N. Miyamoto M. Boni A. Guiet R. Girgin M. et al.Capturing cardiogenesis in gastruloids.Cell Stem Cell. 2021; 28: 230-240.e236Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar HumanMulticell type spheroid (ECs, cardiomyocytes, fibroblasts)cAMPIntercellular crosstalk via cAMP signaling improves cardiomyocyte maturationIncreased cardiomyocyte maturation;Use of differentiated cells not akin to development13Giacomelli E. Meraviglia V. Campostrini G. Cochrane A. Cao X. van Helden R.W.J. et al.Human-iPSC-derived cardiac stromal cells enhance maturation in 3D cardiac microtissues and reveal non-cardiomyocyte contributions to heart disease.Cell Stem Cell. 2020; 26: 862-879.e811Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar HumanMultilineage spheroid: cardiac, foregut endoderm, and vascularWntNKX2.5 knockout disrupts formation of compact myocardiumMimicking early cardiac development;No clear cardiac chamber formation8Drakhlis L. Biswanath S. Farr C.M. Lupanow V. Teske J. Ritzenhoff K. et al.Human heart-forming organoids recapitulate early heart and foregut development.Nat Biotechnol. 2021; 39: 737-746Crossref PubMed Scopus (94) Google ScholarOrganoids with spontaneous formation of multiple chambers MouseMultilineage organoid with atrial and ventricular mimetic chambersBMP/Wnt, FGF4FGF4 supplementation gives rise to conduction cells, cardiomyocytes, fibroblasts, and ECsCardiac chamberogenesis with multiple resident cells;Use of murine but not human cells10Lee J. Sutani A. Kaneko R. Takeuchi J. Sasano T. Khoda T. et al.In vitro generation of functional murine heart organoids via FGF4 and extracellular matrix.Nat Commun. 2020; 11: 4283Crossref PubMed Scopus (39) Google Scholar HumanMultiple primitive chambersWntModeling effects of gestational diabetes on heart developmentMultiple cardiac cell types and vascularization; rudimentary chambers11Lewis-Israeli Y.R. Wasserman A.H. Gabalski M.A. Volmert B.D. Ming Y. Ball K.A. et al.Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease.Nat Commun. 2021; 12: 5142Crossref PubMed Scopus (54) Google Scholar HumanChambered cardiac organoidWnt/BMP/HAND1Requisite need for HAND1 in the cardiac chamber formationFormation of ventricular-like chambers;No regeneration despite fetal cardiomyocytes2Hofbauer P. Jahnel S.M. Papai N. Giesshammer M. Deyett A. Schmidt C. et al.Cardioids reveal self-organizing principles of human cardiogenesis.Cell. 2021; 184: 3299-3317.e3222Abstract Full Text Full Text PDF PubMed Scopus (89) Google ScholarWnt, Wingless and Int-1; BMP, bone morphogenic protein; ESCs, embryonic stem cells; cAMP, cyclic adenosine monophosphate; FGF4, fibroblast growth factor 4; HAND1, heart and neural crest derivatives expressed 1; ECs, endothelial cells. Open table in a new tab Wnt, Wingless and Int-1; BMP, bone morphogenic protein; ESCs, embryonic stem cells; cAMP, cyclic adenosine monophosphate; FGF4, fibroblast growth factor 4; HAND1, heart and neural crest derivatives expressed 1; ECs, endothelial cells. The most advanced method for forming a cavity resembling a primitive left ventricle in human COs has been reported by Hofbauer and colleagues.2Hofbauer P. Jahnel S.M. Papai N. Giesshammer M. Deyett A. Schmidt C. et al.Cardioids reveal self-organizing principles of human cardiogenesis.Cell. 2021; 184: 3299-3317.e3222Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar The authors named these COs “cardioids,” using the term Baar and colleagues19Baar K. Birla R. Boluyt M.O. Borschel G.H. Arruda E.M. Dennis R.G. Self-organization of rat cardiac cells into contractile 3-D cardiac tissue.FASEB J. 2005; 19: 275-277Crossref PubMed Scopus (113) Google Scholar originally coined to describe self-assembled aligned cylindrical cardiac tissues made from neonatal rat heart cells. Approximately 1 to 2 mm in size, each human cardioid was made starting from 5000 to 7500 PSCs, which, for improved reproducibility, were cultured within individual wells of a commercially available AggreWell (Stem Cell Technologies) dish. Upon induction of cardiac mesoderm, chamber-like cavities rapidly formed in the presence of Wnt, activin, and retinoic acid signals and were maintained in a stable fashion during subsequent cardiomyocyte specification and maturation for up to 3 months. Although exogenously induced and endogenous changes in vascular endothelial growth factor signaling led to partial lining of cavities with endothelial cells, neither endoderm nor endothelium was required for chamber formation. Instead, chambers were formed solely by self-organization of cardiac mesoderm, through a process regulated via the Wnt-bone morphogenic protein-heart and neural crest derivatives expressed 1 (HAND1) signaling axis. Specifically, and similar to cardiac development in vivo, chamber formation was critically dependent on the activity of HAND1, the genetic deletion of which resulted in small, poorly formed COs with rudimentary chambers. These experiments further revealed that cavity formation and cardiomyocyte specification in cardioids occur in parallel but are distinctly regulated. Interestingly, addition of PSC-derived epicardial cell aggregates to pre-formed cardioids generated stable structures in which epicardial cells both spread on the CO surface and, upon migration into the organoid, upregulated endothelial cells and fibroblast markers, thus yielding a more in vivo-like ventricle cell composition. The critical difference between fetal and adult mammalian hearts is the lack of proliferative capacity in adult cardiomyocytes, representing the primary barrier to robust regenerative response following myocardial injury.20Karra R. Poss K.D. Redirecting cardiac growth mechanisms for therapeutic regeneration.J Clin Invest. 2017; 127: 427-436Crossref PubMed Scopus (39) Google Scholar In contrast, developing fetal hearts consist of cardiomyocytes with significant proliferative and regenerative ability.21Sturzu A.C. Rajarajan K. Passer D. Plonowska K. Riley A. Tan T.C. et al.Fetal mammalian heart generates a robust compensatory response to cell loss.Circulation. 2015; 132: 109-121Crossref PubMed Scopus (54) Google Scholar The epicardium-supplemented cardioids by Hofbauer and colleagues2Hofbauer P. Jahnel S.M. Papai N. Giesshammer M. Deyett A. Schmidt C. et al.Cardioids reveal self-organizing principles of human cardiogenesis.Cell. 2021; 184: 3299-3317.e3222Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar did not regenerate after a localized cryoinjury and instead mounted a fibrotic response characterized by fibroblast, fibronectin, and collagen I accumulation at the injury site and no cardiomyocyte proliferation. As in mature hearts, this fibrogenic outcome in cardioids was aided by epicardial cells,22Quijada P. Trembley M.A. Small E.M. The role of the epicardium during heart development and repair.Circ Res. 2020; 126: 377-394Crossref PubMed Scopus (50) Google Scholar yet the lack of injury-induced proliferation of evidently immature cardiomyocytes is puzzling and suggests either a nonphysiological nature of cryoinjury or requisite roles for additional resident nonmyocytes or systemic influences (eg, immune system) in mounting a robust regenerative response in a fetal heart. The modular nature of cardioids can be used to gain further mechanistic insights into these clinically important phenomena. Although essential roles of various transcription factors and their interactions have been extensively studied in cardiac specification, morphogenesis, function, and disease,23Luna-Zurita L. Stirnimann C.U. Glatt S. Kaynak B.L. Thomas S. Baudin F. et al.Complex interdependence regulates heterotypic transcription factor distribution and coordinates cardiogenesis.Cell. 2016; 164: 999-1014Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar genetic manipulations in cardioid systems have revealed new, stage-specific changes in transcriptional hierarchy during early cardiogenesis. Specifically, by generating organoids from human PSCs and induced PSCs with HAND1 or NKX2.5 knockout, the authors determined that, in the cardiac mesoderm stage, HAND1 functions upstream of NKX2.5, whereas in the cardiomyocyte stage it functions downstream of NKX2.5. Because HAND1 and NKX2.5 mutations are known pathogenic drivers of ventricular anomalies, including hypoplastic left heart syndrome, it is conceivable that additional genetic manipulations of relevant transcription factors in cardioids (eg, GATA4, TBX5, MEF2, and SRF) could enhance our understanding of early cardiac morphogenetic defects.8Drakhlis L. Biswanath S. Farr C.M. Lupanow V. Teske J. Ritzenhoff K. et al.Human heart-forming organoids recapitulate early heart and foregut development.Nat Biotechnol. 2021; 39: 737-746Crossref PubMed Scopus (94) Google Scholar,11Lewis-Israeli Y.R. Wasserman A.H. Gabalski M.A. Volmert B.D. Ming Y. Ball K.A. et al.Self-assembling human heart organoids for the modeling of cardiac development and congenital heart disease.Nat Commun. 2021; 12: 5142Crossref PubMed Scopus (54) Google Scholar On the other hand, cardioids are primarily made of first heart field derivatives and do not include second heart field or neural crest cells, which play important roles in outflow tract septation, valvulogenesis, and development of the cardiac conduction system.24Keyte A. Hutson M.R. The neural crest in cardiac congenital anomalies.Differentiation. 2012; 84: 25-40Crossref PubMed Scopus (168) Google Scholar As such, cardioids do not model coemergence of a variety of intra- and extracardiac cell lineages that contribute the formation of linear heart tube, cardiac looping, and patterning of later, more complex cardiac structures (eg, right ventricle, atria, cardiac cushions, valves, Purkinje fibers, inflow/outflow tract, vasculature, and lymphatics25Meilhac S.M. Buckingham M.E. The deployment of cell lineages that form the mammalian heart.Nat Rev Cardiol. 2018; 15: 705-724Crossref PubMed Scopus (98) Google Scholar). Because most congenital malformations arise during these later stages of heart development, cardioids will require significant improvements to allow modeling of the common cardiac birth defects. Specifically, improving methods for human PSC differentiation and cardiomyocyte maturation26Shadrin I.Y. Allen B.W. Qian Y. Jackman C.P. Carlson A.L. Juhas M.E. et al.Cardiopatch platform enables maturation and scale-up of human pluripotent stem cell-derived engineered heart tissues.Nat Commun. 2017; 8: 1825Crossref PubMed Scopus (232) Google Scholar,27Funakoshi S. Fernandes I. Mastikhina O. Wilkinson D. Tran T. Dhahri W. et al.Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells.Nat Commun. 2021; 12: 3155Crossref PubMed Scopus (37) Google Scholar and integration of advanced bioengineering methods28Pomeroy J.E. Helfer A. Bursac N. Biomaterializing the promise of cardiac tissue engineering.Biotechnol Adv. 2020; 42: 107353Crossref PubMed Scopus (37) Google Scholar for 3D cell culture will be important first steps toward realizing this goal. Nevertheless, in their state-of-the-art form, COs can currently permit systematic studies of early cardiac self-patterning, including dissecting and simultaneously investigating cardiac morphogenetic and specification events. Self-organizing COs derived from human PSCs represent important emerging platforms for studies of early heart development. Despite their structural simplicity, these in vitro systems may hold the key to improved understanding of early cardiac morphogenesis and mechanistic underpinnings of congenital heart disorders, eventually leading to improved patient care.