Probing Single Ventricle Heart Defects with Patient-Derived Induced Pluripotent Stem Cells and Emerging Technologies

Abstract

Single ventricle heart defects (SVHDs) are a severe type of congenital heart disease with poorly understood pathogenic mechanisms. New research using patient-specific induced pluripotent stem cells (iPSCs) as a cellular model is beginning to uncover genetic and cellular etiologies of SVHDs. Hypoplastic left heart syndrome (HLHS) is a type of SVHD that is characterized by an underdeveloped left ventricle and other malformations in the left side of the heart. Hypoplastic right heart syndrome (HRHS), the second type of SVHD, is characterized by an underdeveloped right heart, including malformed tricuspid and pulmonary valves. Despite a noticeable lack of research on SVHD, emerging technologies offer a promising future to further probe the genetic and cellular mechanisms of these diseases. Pediatric cardiovascular research is at the dawn of a new era in terms of what can be discovered with patient-specific iPSCs in conjunction with other technologies (e.g., organoids, single-cell genomics, CRISPR/Cas9 genome editing). In this review, we present recent approaches and findings utilizing patient-specific iPSCs to identify cellular mechanisms responsible for improper cardiac organogenesis in HLHS and HRHS.

Introduction

The cardiovascular system is the first organ system to develop during fetal development (Donovan MF, 2020). During the early stages of heart development, structural malformations of the heart and its associated vessels can emerge, producing congenital heart disease (CHD) in newborns (Diz et al., 2021). Approximately one-third of infants with CHD must undergo at least one surgery within the first year of life to survive (Dittrich et al., 2003). CHD occurs in less than 1% of live births, with a higher incidence in males (Diz et al., 2021). CHD is a broad term for all structural heart defects present at or before birth. Each case of CHD is typically stratified into one of four main categories: cardiac septation defects, conotruncal and aortic arch artery anomalies, right- and/or left-sided inflow and/or outflow tract obstructiion, and left-right abnormalities (heterotaxy) (Bruneau, 2008; Garg, 2006; Lin et al., 2021). Cardiac septation defects include atrial septal defects, ventricular septal defects, and atrioventricular septal defects. Tetralogy of Fallot, truncus arteriosus, and interrupted aortic arch are typical examples of CHD that are classified as conotruncal and aortic arch defects. Right-sided outflow tract obstructive defects describe cardiac abnormalities such as pulmonary stenosis (PS) and pulmonary atresia with intact ventricular septum (PA-IVS) and in the most severe cases present as hypoplastic right heart syndrome (HRHS). Left-sided outflow tract obstructive defects include bicuspid aortic valve, aortic valve stenosis, and hypoplastic left heart syndrome (HLHS). While modern medical technology has advanced and improved the survival prognosis of CHD newborns into adulthood, the underlying mechanisms that cause defective heart formation remain unknown in the majority of cases.

In recent years, some advancements in determining the pathogenetic mechanisms underlying CHD have been made using induced pluripotent stem cells (iPSCs). In 2006, a groundbreaking study published by Takahashi and Yamanaka demonstrated that differentiated somatic cells could be reprogrammed into an embryonic-like state through the forced expression of Oct3/4, Sox2, c-Myc, and Klf4 (Takahashi & Yamanaka, 2006). Over the next 15 years, researchers have built upon Takahashi and Yamanaka’s protocol to develop more efficient methods to generate human iPSCs. These iPSCs can proliferate, be differentiated into various cell types, and retain the genetic information of affected patients. Cardiovascular researchers have utilized patient-specific iPSCs to study the heart’s genetic composition and link mutations to CHD. Recent studies have had success in generating iPSCs from a small volume of blood from a patient and subsequently be differentiated into cardiomyocytes (CMs) (Ye et al., 2021; Zhao, Ye, et al., 2020). Patient iPSCs have a promising future in cardiovascular research as they provide the means to take a disease-specific look into the genetic determinants of CHD. This review aims to describe research on two SVHDs with their clinical presentations and highlight recent progress in modeling CHD using patient-derived iPSCs to better understand the etiology of SVHD.

Hypoplastic Left Heart Syndrome

Affecting 3% of infants born with CHD, HLHS is the most complex and severe form of CHD (Grossfeld et al., 2019). Yet, with an incidence in the United States of 1 in 3,841 live births, HLHS is rare compared to most congenital cardiac disorders (Mai et al., 2019). Although only about 1,025 infants are born yearly with HLHS in the United States, this type of SVHD is responsible for 25% of cardiac-related infant deaths (Diz et al., 2021; Mai et al., 2019). HLHS is characterized by underdevelopment of the structures that constitute the left side of the heart (Figure 1) including the mitral valve, left ventricle, aortic valve, and aorta (Reamon-Buettner et al., 2008). The defect was first described by a German physician, Dr. von Bardeleben, in the mid-19^th century, who noted the significant cardiac pathologies and recognized that infants with HLHS relied upon a patent ductus arteriosus (PDA) to supply blood from the pulmonary artery to the body, bypassing the underdeveloped left heart (Gehrmann et al., 2001). After birth, with closure of the ductus arteriosus, there is rapid onset of RV failure, shock, and death without emergent treatment. Over a century later, Noonan and Nadas proposed the name hypoplastic left heart syndrome after analyzing heart structural commonalities (Noonan & Nadas, 1958). Interestingly, paleopathologists recently discovered a 6,500-year-old mummy of a young Peruvian child who most likely succumbed from complications of having an underdeveloped left ventricle (Grossfeld et al., 2019). Although HLHS has been present for thousands of years of human history, only within the past 20 years have infants been able to survive what was once a death sentence.

Figure 1. Diagrams with highlighted anatomical defects associated with single ventricle heart defects in comparison to healthy cardiac morphology.

Common single ventricle heart defects include hypoplastic left heart syndrome (HLHS), pulmonary atresia with intact ventricular septum (PA-IVS), and tricuspid atresia (TA). The images in this figure are courtesy of Centers for Disease Control and Prevention.

There are three main structural variants of HLHS, differentiated by varying degrees of stenosis or atresia of the aortic and mitral valves and chamber hypoplasia (Crucean et al., 2017). The most common structural variant presents as a severely thickened left ventricular wall, restricting the volume of blood it is able to hold (Grossfeld et al., 2019). Furthermore, the endocardium of the hypoplastic left ventricle is affected by fibroelastosis, whereas the aortic valve is either atretic or stenotic. Endocardial fibroelastosis of the left ventricle is thought to be caused by a reduction of blood flow due to ventricular hypoplasia (Friehs et al., 2013). In patients with HLHS, there are improper hemodynamic properties in the circulatory system. Based on insufficient blood flow through the left heart, many researchers theorize that improper hemodynamics may contribute to the hypoplastic left ventricle (deAlmeida et al., 2007). Even if not an etiological determinant of HLHS, altered blood flow resulting from this disease can lead to neurodevelopmental issues that negatively influence brain growth (Reich et al., 2019). In the second variant, both the mitral and aortic valves are atretic (Grossfeld et al., 2019). Because both valves associated with the left ventricle are completely closed off, blood never enters the already severely hypoplastic left ventricular cavity. An inaccessible left ventricle requires blood to shunt through the foramen ovale or an atrial septal defect to enable blood to be rerouted through the pulmonary artery and cross over to the aorta through the PDA to reach systematic circulation. Interestingly, there is no fibroelastic lining of the left ventricle in this variant, which may be due to the complete lack of blood flow. The third variant has the presence of a severe aortic coarctation (Grossfeld et al., 2019). Otherwise known as the “hypoplastic left heart complex,” this variant contributed to the majority of HLHS cases studied by Noonan and Nadas (Noonan & Nadas, 1958). The combination of hypoplastic left ventricle with an obstructed aortic arch increases disease severity and adds another degree of complexity for cardiothoracic surgeons when planning surgical intervention.

Patients usually undergo a series of corrective surgeries designed to redirect blood directly into the lungs. By doing so, surgeons are able to buy time for patients by increasing the amount of oxygenated blood that is pumped away from the heart into the rest of the body. The first palliative intervention is the Norwood procedure in which a Blalock-Taussig shunt tube is placed to allow oxygen-rich and oxygen-poor blood to be mixed and systemically circulated. This procedure usually occurs when SVHD patients are days old, and about 5–15% of infants that undergo this procedure are at risk of passing away. At about 6 months old, patients then go through a Glenn procedure where the superior vena cava is disconnected from the heart and attached to the pulmonary artery. During this operation, the Blalock-Taussig shunt is also removed. Again, 5–15% of infants are at risk of death. Finally, by the time babies are about three years old, they would have completed a Fontan surgery. Similar to the Glenn procedure, deoxygenated blood is rerouted through the pulmonary artery. By the end of these series of interventional surgeries, deoxygenated blood completely bypasses the heart, and the heart perfuses oxygen-rich blood. With these advances of modern medicine and interventional procedures, infants born with SVHDs can now live well into adulthood into their 30s and 40s. Interventions have even had much success in the fetal stage of development to prolong the life expectancy of fetuses diagnosed with SVHDs. In a fetal balloon atrial septostomy, for example, surgeons insert a fine needle through the mother’s abdomen and into the fetal heart. The needle pierces the wall between the left and right atria to allow oxygenated and deoxygenated blood to mix, so the baby can survive long enough for corrective surgery (Plackett, 2021). Unfortunately, patients who survive into adulthood have a significantly higher chance of experiencing diseases such as thyroid dysfunction, kidney disease, stroke, and liver cirrhosis when compared to healthy adults, thus emphasizing the need to study the developmental etiologies of SVHDs (Garmendia Madariaga et al., 2014; Plackett, 2021).

Compared to other congenital heart disorders and congenital disorders in general, little is understood about the genetic determinants of HLHS. Studies point to mutations in the NOTCH1, a transcriptional regulator, as a determinant of HLHS, with frameshift or nonsense mutations often resulting in aortic valve anomalies (Garg et al., 2005; Kerstjens-Frederikse et al., 2016; McBride et al., 2008; Riley et al., 2011; Theis, Hrstka, et al., 2015). Other studies have garnered significant evidence that the NOTCH pathway, if mutated, disrupts normal cardiac valve development, especially with respect to the aortic valve (Iascone et al., 2012). HAND genes encode proteins that are responsible for the formation of heart chambers during cardiogenesis, and also play a role in HLHS. Specifically, dHAND/HAND2 is highly correlated with the formation of the right heart chambers, whereas eHAND/HAND1 is highly correlated with the chambers of the left heart. Due to this specificity, a mutated eHAND gene results in an underdeveloped left ventricle (Thomas et al., 1998). Furthermore, a basic helix-loop-helix (bHLH) transcription factor encoded by eHAND/HAND1 gene is responsible for the rightward looping of the heart tube, that when mutated, disrupts cardiac looping leading to a hypoplastic left ventricle and outflow tract anomalies in mouse models. In a case study, 24 of 31 hypoplastic human hearts were found to carry mutations in the bHLH domain of HAND1 (Reamon-Buettner et al., 2008). This study used old hearts that were formalin preserved for decades, and is inconsistent with Hand1 frameshift mutation in transgenic mice (Firulli et al., 2017). The data likely represents sequencing artifact, as other studies have not been able to replicate these findings using fresh cardiac tissue. Additional research found that specific genes such as FOXC2/FOXL1 (transcription factors within the Forkhead family) (Stankiewicz et al., 2009), IRX4 (a member of the Iroquois homeobox family) (Hu et al., 2018), MYH6 (part of the myosin protein) (Theis, Zimmermann, et al., 2015), and NKX2–5 (a transcription factor essential for cardiac morphogenesis) (Elliott et al., 2003) have active roles in heart development and, if mutated, may contribute to HLHS (Pierpont et al., 2018).

Animal models have been invaluable for studying the morphological and genetic abnormalities of HLHS. Studying the hemodynamic implications of a physically altered mitral valve, Hahn et al. found that chicken embryos developed various abnormalities including hypoplastic left atrium and ventricle, mitral atresia, and aortic stenosis (Harh et al., 1973). Subsequently, Fishman et al. showed the effects of hemodynamics on cardiogenesis by altering either the left ventricular inflow or outflow tract in lamb embryos (Fishman et al., 1978). Left ventricular inflow obstruction decreased left ventricular output 30%, decreased the left to right ventricle weight ratio 70%, and reduced the mean chamber volume >50%. In comparison, left ventricular outflow obstruction reduced left ventricular output >60%, doubled left ventricular wall thickness, and decreased mean left ventricular volume >50%. Collectively, these early studies using animal models to study HLHS demonstrate that hemodynamics play an integral role in heart development and that pathological hemodynamics could contribute to the etiology of HLHS. Moreover, using a genome-edited mouse model of HLHS, Liu et al. identified novel variants in Sap130 and Pcdha9 could cause ventricular hypoplasia and aortic valve abnormalities in HLHS (Liu et al., 2017). However, this study is controversial as another study has claimed that double-outlet right ventricle is not HLHS in the abovementioned transgenic mouse heart (Chaudhry et al., 2019).

The 21^st century has seen the birth and growth of a novel field of study: induced pluripotent stem cells (iPSCs). iPSC technology is advancing cardiovascular research into new realms as scientists can utilize patient-specific iPSCs to examine developmental processes. During and after the differentiation process of transforming iPSCs into cardiomyocytes (iPSC-CMs), specific genes, proteins, and regulation of early human cardiogenesis have advanced our understanding of the etiology of HLHS and other forms of CHD. HLHS patient-derived iPSCs (HLHS-iPSC) were shown to have a multitude of functional differences when compared to normal iPSCs derived from a healthy patient. Morphologically, HLHS-iPSCs produced fewer beating clusters in comparison to healthy control iPSCs and showed variation in ventricular/atrial cardiomyocyte commitment (Jiang et al., 2014). A study examining mechanisms to reverse the effects of HLHS in a developing heart found that HLHS-iPSC-derived cardiomyocytes exposed to hypoxic conditions during the differentiation phase rescued the cardiac cells from fibrosis through TGF-β1 inhibition (Gaber et al., 2013). More recently, HLHS-iPSC research has indicated that sarcomere disorganization of cardiac tissue is not the driving force of impaired contractility (a phenomenon that arises in an HLHS heart) whereas mitochondrial dysfunction may be the cause (Paige et al., 2020). In addition, dysfunctional NOTCH signaling pathway and NOTCH1-dependent nitric oxide signaling deficiency are observed in HLHS iPSC-derived cardiomyocytes, highlighting the contribution of NOTCH1 variants to the impaired cardiogenesis in HLHS (Hrstka et al., 2017; Yang et al., 2017). Similarly, studies using HLHS iPSC-derived endothelial cells reveal an important role for endocardium in the cardiac pathogenesis of HLHS, as endocardial defects lead to abnormal endothelial-to-mesenchymal transition, reduced cardiomyocyte proliferation, and disrupted fibronectin-integrin signaling (Miao et al., 2020). Single-cell transcriptomics and 3D modeling with HLHS iPSC-derived cardiac cells also uncover intrinsic defects in cell cycle regulation and unfolded protein response, leading to sequential defects in cardiac lineage commitment and cardiomyocyte maturation in HLHS (Krane et al., 2021).

Hypoplastic Right Heart Syndrome

Structural abnormalities of the right side of the heart that form during fetal development cause HRHS. Unlike HLHS, there is a large degree of anatomic variability in the types of congenital heart defects that can fall under the term “HRHS”. Pulmonary atresia with intact ventricular septum, tricuspid atresia, and tricuspid stenosis are cardiac malformations that fall under the realm of HRHS (Jacobstein et al., 1985). A hypoplastic right ventricle and underdeveloped pulmonary and tricuspid valves are defining characteristics among HRHS defects. Due to lack of blood flow through pulmonary circulation, HRHS is a cyanotic CHD where deoxygenated blood enters systemic circulation. Survival of an infant born with HRHS is dependent upon the pulmonary blood supply and an atrial septal defect, which shunts blood from the right to the left atrium (Mohan et al., 2016). Hypoxemia occurs in HRHS because blood skips pulmonic circulation and enters the left atria without proper (or any) oxygenation. There are varying degrees of HRHS depending on the severity of valve and ventricle hypoplasia. For most cases, surgical intervention is essential, as the infant will not survive without treatment (Daubeney et al., 2005). Much like HLHS, these infants undergo a similar three-stage surgery that ultimately results in deoxygenated blood bypassing the right side of the heart, into the lungs, and then returning to the left heart to be pumped through systemic circulation (Mohan et al., 2016).

First described in 1783, PA-IVS is an extremely rare type of CHD, yet the third most common form of cyanotic CHD (Gorla & Singh, 2021). The incidence of PA-IVS ranges from 4–8 per 100,000 live births based upon studies performed in the United States and United Kingdom. PA-IVS is characterized by pulmonary valve atresia or the lack of pulmonary valve formation and a normal ventricular septum. Clinically, the size and morphology of the RV varies in PA-IVS (LaPar & Bacha, 2019). The RV may be severely hypoplastic with a small cavity and marked muscular hypertrophy or it may be large, dilated, and thin-walled. The majority (60–80%) of PA-IVS patients have well-formed tripartite RVs including an inlet, a trabecular body, and an infundibular outlet (Daubeney et al., 2002). Roughly, 15–30% of PA-IVS patients have bipartite RVs comprising just an inlet and trabecular body with a loss of functional chamber size. In rare cases (2–10%), PA-IVS patients show a unipartite RV with a severely hypoplastic ventricle composed of only an inlet. PA-IVS patients present with a spectrum of RV hypoplasia and they have long-term outcomes of single ventricle palliation (1v), 1 ½ ventricle palliation (1.5v), or bi-ventricular repair (2v) after surgical or catheter-based intervention to increase pulmonary blood flow (Gorla & Singh, 2021). Additionally, hypoplasia or dysplasia may affect the tricuspid valve in PA-IVS.

Tricuspid atresia in HRHS results in the complete lack of tricuspid valve development in the right heart. Multiple types of tricuspid atresia can be seen dependent on the great artery relationship and size of the ventricular septal defect (VSD). The great arteries may be normally related, transposed, or double outlet from the RV. Either the pulmonary artery or aorta may be undeveloped. The anatomic makeup dictates the surgical pathway. Type I tricuspid atresia has normally related great arteries and may or may not have an accompanying VSD, which when present allows blood to bypass the lungs. A PDA allows for blood flow to the pulmonary arteries and is essential for survival. Type II tricuspid atresia differs from Type I in that there is D-transposition of the great arteries along with a VSD. The pulmonary artery stems from the left ventricle and is involved in systemic circulation, while the aorta is directly connected to the hypoplastic right ventricle. Type III tricuspid atresia includes other complex defects including malposition of the great arteries and double outlet right ventricle (DORV). Characteristic of all types is left ventricular overload (sole pump for both systemic and pulmonic circulation).

Genetic etiologies of HRHS are not well understood. Very few studies have researched the genetic and molecular mechanisms responsible for the anatomical deformities associated with HRHS. However, a recent study described copy number variants (CNV) within genes responsible for cardiac development (Giannakou et al., 2018). For example, CNVs detected in ERBB4, encoding a neuregulin receptor, may affect cardiomyocyte differentiation and cell fate determination of human pluripotent stem cells. ERBB4 is linked to the Wnt signaling pathway which is an important mechanism during cardiogenesis (Fu et al., 2019). CNVs were also identified in SALL1, NKD1, TRABD2B, and JADE1, which are related to the Wnt pathway. SALL1 defines the fate of cardiac progenitor cells and is expressed in the mesoderm pre-outflow tract formation (Morita et al., 2016). NKD1 antagonizes a normal Wnt pathway and can stabilize a dysfunctional Wnt pathway (Angonin & Van Raay, 2013). TRABD2B and JADE1 are general regulators of the Wnt pathway (Borgal et al., 2014; Thienpont et al., 2010). Further biomolecular research must be conducted to paint a better picture of the genetic etiologies of HRHS.

Most infants born with HRHS must undergo a series of surgical interventions during the first few years of life to establish proper hemodynamics. There are a few options depending on the type and severity of HRHS, each with benefits and risks. A pulmonary artery band procedure (PABP) is useful in patients with Type I tricuspid atresia, where there is excessive blood in pulmonic circulation due to the ventricular septal defect. A PABP will, as the name implies, place a band around the pulmonary artery. The goal of this procedure is twofold: limit high blood pressure exposure in pulmonary vasculature and restore sufficient blood volume in systemic circulation to prevent severe hypoperfusion. A procedure that creates a Blalock-Taussig shunt is useful in HRHS patients with limited blood flow and cyanosis, but is a temporary procedure to connect systemic circulation to pulmonic circulation. A shunt connects the innominate artery to the right pulmonary artery and serves as an artificial PDA. More commonly, a Glenn procedure is performed when the patient is approximately 4–6 months old to establish a systemic-pulmonic connection via an artificial anastomosis between the superior vena cava and pulmonary artery. Finally, a Fontan procedure, the most complex option, allows for passive circulation of low-pressure pulmonary blood after surgically connecting the inferior vena cava and superior vena cava to the pulmonary artery.

There have been no in-depth studies using animal models to understand HRHS. However, a few studies examined abnormal valvular phenotypes and their implications in mouse models. A spectrum of cardiac malformations (e.g. ventricular septal defects, tetralogy of Fallot, and tricuspid artresia) are expressed in Hey2 mutant mice (Donovan et al., 2002). Deletion of the Zfpm2 gene also results in tricuspid atresia in mice. Heterozygous Zfpm2 mice exhibit normal cardiogenesis, while homozygous mice with Zfpm2 knockout show a variety of deformities including pulmonary stenosis, ventricular septal defect, and atrial septal defect, in addition to tricuspid atresia (Svensson et al., 2000). Genetically edited pigs carrying Sap130 mutation present tricuspid dysplasia, with tricuspid atresia leading to early embryonic lethality, suggesting that large animal models could provide new opportunities for illustrating developmental mechanisms and conducting preclinical studies in HRHS (Gabriel et al., 2021). To better understand HRHS, future research must use functional animal hearts to model the valvular and ventricular abnormalities unique to HRHS.

There are rare published studies on the use of iPSCs to study HRHS. Of the current research using iPSC-CMs to study the development of PA-IVS cardiac tissue, Lam et al. found that compared to healthy controls, PA-IVS iPSC-CMs have reduced contractility and show diminished expression of genes responsible for cardiac contractility (Lam et al., 2020). Recently, several groups have developed protocols to generate cardiac valve endothelial-like cells and pre-valvular endocardial cells from human iPSCs (Cheng et al., 2021; Neri et al., 2019). Interestingly, patient iPSC-derived valvular interstitial cells (VICs) harboring a DCHS1 mutation recapitulate features of mitral valve prolapse that are similar to primary VICs derived from the explanted mitral valve (Neri et al., 2019). However, there are few publications that focus on the pulmonary or tricuspid valves using patient-specific iPSCs. The field of iPSC technology and its use as a model to study HRHS is still in its infancy, but offers promising genetic and molecular biological discoveries yet to be found. Important genes linked to SVHDs are listed in Table 1.

Table 1.

Anatomical malformations and genes associated with SVHDs as studied in humans and mice.

SVHDs	Anatomical Defects	Associated Gene Mutations	References
HLHS	Hypoplastic left ventricle	NOTCH1	(Garg et al., 2005)
	Aortic stenosis	dHAND/eHAND	(Thomas et al., 1998)
	Aortic atresia	HAND1	(Reamon-Buettner et al., 2008)*
	Endocardial fibroelastosis	FOXC2	(Stankiewicz et al., 2009)
	Mitral atresia	FOXL1	(Stankiewicz et al., 2009)
	Mitral stenosis	IRX4	(Hu et al., 2018)
	Atrial septal defect	MYH6	(Theis, Zimmermann, et al., 2015)
		NKX2–5	(DeLaughter et al., 2016; Elliott et al., 2003)
HRHS	Hypoplastic right ventricle	ERBB4	(Fu et al., 2019)
	Pulmonary stenosis	SALL1	(Morita et al., 2016)
	Pulmonary atresia	NKD1	(Angonin & Van Raay, 2013)
	Tricuspid stenosis	TRABD2B	(Thienpont et al., 2010)
	Tricuspid atresia	JADE1	(Borgal et al., 2014)
	Atrial septal defect

^*Data presented in this study have not been replicated.

Emerging Technologies to Study SVHDs using iPSCs

Groundbreaking discoveries using iPSC technology are advancing our understanding of the pathophysiological mechanisms at play in CHD. An overview of the creation of patient iPSC-derived cardiac cells and their potential applications is shown in Figure 2. However, like any model there are flaws, such as those associated with standard 2D monolayer culture of iPSCs that in a simplified in vitro environment struggle to mimic the complex nature of cardiac development in a more complex in vivo setting (van Mil et al., 2018). Although patient-specific iPSC-CMs are used as a tool to model the developing embryonic heart, there is some dissonance in cells cultured in vitro compared to those within heart tissue growing in the uterine environment. Additionally, iPSC-CMs require time to generate and must be purified, which can lead to difficulties harvesting a sufficient yield of atrial-, ventricular-, and nodal-like cardiomyocytes (Ye et al., 2021; Zhao, Shao, et al., 2020). The cost to harvest these cells can also be a restrictive factor. From the collection of the patient’s blood to relatively mature day 30 iPSC-CMs (Figure 3), it may take over six months (depending on the procedure) to produce enough iPSC-CMs for experimentation. Furthermore, the cost of iPSC banking, labor, and other miscellaneous materials can add up to thousands of dollars (Huang et al., 2019). Even though iPSCs are patient specific, currently there is little to no clinical therapeutic or transplantation use of differentiated cardiomyocytes due to immunogenicity and functional barriers (Gao & Pu, 2021). Attempts to transplant a sheet of iPSC-CMs or cardiac patch into a myocardial infarction in rat, monkey and porcine model had partial success (Chong et al., 2014; Gao et al., 2018; Laflamme et al., 2007; Liu et al., 2018; Murry & MacLellan, 2020). A high yield of pure iPSC-CMs improved short term cardiac function of ischemic cardiac tissue through secretions of cytokines and paracrine effects; however, long-term analysis (8 weeks post-transplantation) showed very few surviving cardiomyocytes (Kawamura et al., 2012). Similar results have been obtained in rat (Masumoto et al., 2014) and guinea pig models (Weinberger et al., 2016). Stem cell therapy is in its infancy with improvements needed in both efficiency and effectivity. However, by combining animal models with a better understanding of biological features informed by patient-specific iPSC-CMs may allow clinical applications to be realized sooner. As of yet, there are about 13 clinical trials underway using stem cells as a therapy for HLHS listed in the ClinicalTrial.gov. In one study, HLHS patients were injected with autologous cardiosphere-derived cells to test their feasibility as a therapy to improve ventricular function. Compared to controls, cardiosphere-treated patients saw improved right ventricular ejection fraction, improved somatic growth, and reduced heart failure status (Ishigami et al., 2015). Thus, even with its respective limitations, iPSC-derived cardiac cells hold a promising future for SVHD research. New emerging technologies discussed below may help resolve questions that iPSC technology alone is unable to answer.

Figure 2. General workflow in generating iPSC-derived cardiac cells from patients.

Emerging technologies such as organoids, CRISPR/Cas9 genome editing, and single-cell genomics would enhance the advantage of using patient-specific iPSCs to study genetic and cellular etiologies of single ventricle heart defects. Illustration was created with BioRender.com.

Figure 3. Immunofluorescent staining of human iPSC-CMs shows features of cardiomyocytes: elongated rod-like morphology and sarcomeric structure with intercalated distribution of cardiac troponin T and α-actinin.

Nuclei were stained with DAPI in blue (A), α-actinin was labeled in red (B), and cardiac troponin T was marked in green (C). Yellow arrows highlight the elongated cardiomyocytes in the merged image (D) of panels A, B and C. Scale bars: 100 μm.

Organoids offer a 3D perspective to studying disease mechanisms and whole organ development. More specifically, an organoid is a pluripotent stem cell-derived 3D structure capable of self-organization that mimics aspects of organs found in vivo (Lancaster & Knoblich, 2014). Rudimentary organoids were described in the mid-to-late 20^th century, but there have been significant developments within the past decade that increase their potential to model organogenesis. Currently, there are organoids that serve as models for most organs or organ systems including specific areas in the brain, intestinal tract, respiratory tract, reproductive system, and cardiovascular system (Clevers, 2016). Recently, a novel process of manufacturing a heart organoid, termed a “cardioid,” was developed through a stepwise process utilizing the Wnt signaling pathway that is initiated through specific chemicals and growth factors. These cardioids form multi-lineage cell types, model development of embryonic heart fields, develop chambers, and are semi-functional (Drakhlis et al., 2021; Hofbauer et al., 2021; Lewis-Israeli et al., 2021). There are, however, a few setbacks to organoid technology. Depending on the target organ, organoids may lack proper innervation, blood circulation, and presence of immune cells; thus, disease processes cannot be fully modeled. Additionally, the time and cost to manufacture an organoid may exceed the benefits as the process for development is more complex than that of iPSC-CMs. Even though the shortcomings of this state-of-the-art technology may act as a deterrent to widespread adoption, organoids may be an improved model of CHD as they better mimic in vivo environments due to their 3D architecture. Alternatively, three dimensional bioprinted heart tissues can be used to construct small organ-like structures using patient-specific iPSCs (Alonzo et al., 2019; Tasnim et al., 2018). Probing the underlying cellular biomolecular physiology has also become feasible with the ability to study cell-to-cell communication with the greater organizational complexity.

Single-cell genomics can be used to obtain a more detailed understanding of the cellular processes associated with disease. Single-cell genomics enables the genetic profiling of individual cells yielding insight into molecular mechanisms (Tanay & Regev, 2017). DeLaughter et al. performed single cell RNA-sequencing of specific cells over an embryonic developmental time span of mouse cardiac tissue and found that Nkx2.5 mutations are responsible for lineage-specific developmental defects (DeLaughter et al., 2016). Using single-cell RNA sequencing, Li et al. further demonstrated that murine fetal hearts homozygous for Nkx2.5 knockout exhibit an altered transcriptional profile and that the homozygous knockout cardiac cells are unable to differentiate into ventricular cardiomyocytes (Li et al., 2016). The ability to examine a single cell and determine its transcriptional profile offers the ability to understand the bigger picture of how specific cells accumulate and affect cardiogenesis and CHD (Paik et al., 2020).

CRISPR-Cas9 enables genome editing, or, more precisely, the deletion, substitution, or insertion of gene fragments into an organism’s genome. CRISPR-Cas9 gene editing, when coupled with iPSCs, offers a targeted approach to understand the roles that specific genes play in CHD (Adli, 2018). Essentially, iPSCs can undergo genetic alteration via a CRISPR-Cas9 method to help determine phenotypic changes that arise during iPSC development toward cardiomyocytes or other heart tissues. Genes can be knocked out or mutations can be knocked in (removed or inserted, respectively) in iPSCs (Motta et al., 2017). Kim et al. used this approach to elucidate the role of the MYH6 gene in patients with HLHS. Although MYH6 variants are common in HLHS, there was vague understanding of its role in the disease. Through genome-edited iPSCs, Kim et al. discovered an intrinsic disrupted sarcomere structure and cardiomyogenic differentiation defects that recapitulate the HLHS phenotype (Kim et al., 2019). Using CRISPR-Cas9 technology, HLHS patients with MYH6 variants were found to lack sarcomere organization in atrial tissue but have normal sarcomere structure and function in ventricular tissue (Kim et al., 2020). Recent in vivo CRISPR/Cas9 genome editing significantly improved muscle function and restored dystrophin expression in mouse models of Duchenne muscular dystrophy, reflecting promising therapeutic applications (Long et al., 2016; Nelson et al., 2016). A clinical trial using CRISPR-Cas9 in vivo gene editing show positive outcomes with mild adverse event for treating hereditary transthyretin amyloidosis (Gillmore et al., 2021). Single-dose in vivo CRISPR base editing in PCSK9 in cynomolgus monkeys can reduce low-density lipoprotein cholesterol level and could be potentially translated for treating atherosclerotic cardiovascular disease (Musunuru, 2022; Musunuru et al., 2021). The advantages of using CRISPR/Cas9 is to interrogate the biochemical, developmental, and phenotypic changes caused by a specific genetic variant, which can be highly beneficial to study inherited SVHDs.

Conclusions and Perspectives

The emergence, adoption, and continuous development of iPSCs, organoids, single-cell genomics, and CRISPR/Cas9 genome editing have paved new avenues to study the etiologies of single ventricle heart diseases. Hypoplastic left heart syndrome is a rare, yet serious congenital heart defect that is poorly understood at the molecular or genetic level. Studies using iPSC-derived cardiac cells are producing ground-breaking discoveries that reveal the physiological and structural differences between HLHS and normal cardiac tissue. Hypoplastic right heart syndrome is even rarer, as demonstrated by the glaring lack of research. Although publications on single ventricle heart disease are lacking in number, there is a promising future. The continued development of iPSC-derived cardiovascular cells and “cardioids” in combination with single-cell genomic data, CRISPR/Cas9 genome editing, and use of relevant animal models, cardiovascular researchers are on the verge of discovering the underlying roots responsible for single ventricle heart disease. This knowledge can then pave the way to develop therapies and diagnostics to better serve those who suffer from such a serious and complex congenital heart disease.