Transformative Potential of Induced Pluripotent Stem Cells in Congenital Heart Disease Research and Treatment

Abstract

Congenital heart disease (CHD), the most common congenital anomaly, remains a significant lifelong burden despite advancements in medical and surgical interventions. Induced pluripotent stem cells (iPSCs) have emerged as a groundbreaking platform in CHD research, offering patient-specific models to investigate the genetic, epigenetic, and molecular mechanisms driving the disease. Utilizing technologies such as CRISPR/Cas9 gene editing, cardiac organoids, and high-throughput screening, iPSCs enable innovative strategies in disease modeling, precision drug discovery, and regenerative therapies. However, clinical translation faces challenges related to immaturity, differentiation variability, large-scale feasibility, and tumorigenicity. Addressing these barriers will require standardized protocols, bioengineering solutions, and interdisciplinary collaboration. This review examines the critical role of iPSCs in advancing CHD research and care, demonstrating their potential to revolutionize treatment through patient-specific, regenerative approaches. By addressing current limitations and advancing iPSC technology, the field is positioned to pave the way for precision-based CHD therapies for this lifelong condition.

1. Introduction

Congenital heart disease (CHD) represents a significant challenge to global healthcare, as the most common congenital anomaly [1] and as a leading contributor to childhood morbidity and mortality [2,3]. Despite substantial progress in surgical and medical interventions, CHD often necessitates lifelong care, leaving survivors vulnerable to complications that persist into adulthood. This burden emphasizes the need for transformative approaches that address the underlying causes of CHD rather than solely mitigating its structural and symptomatic effects [4].

The discovery of induced pluripotent stem cells (iPSCs) has revolutionized the landscape of biomedical research, providing an innovative platform to investigate and address complex conditions such as CHD [5,6]. First introduced in 2006 by Takahashi and Yamanaka, iPSCs have revolutionized the field [7]. These cells are generated by reprogramming mature somatic cells into a pluripotent state via key transcription factors (OCT4, SOX2, KLF4, c-MYC) [7,8]. This groundbreaking approach overcomes many ethical and practical limitations associated with embryonic stem cells [9,10], while retaining the remarkable ability to generate diverse cell types. From cardiomyocytes to endothelial and smooth muscle cells, iPSCs offer an unparalleled tool for replicating the complexity of human biology, firmly establishing themselves as a key innovation in disease modeling, drug discovery, and regenerative medicine [11]. Figure 1 illustrates the process of iPSC generation and its applications in CHD research.

A critical advantage of iPSCs lies in their ability to create patient-specific models that capture the unique genetic and phenotypic characteristics of individuals [12,13]. This capability is particularly relevant for CHD, which arises from a complex interplay of genetic, epigenetic, and environmental factors [14,15,16]. By creating controlled environments to study genetic mutations, signaling pathways, and cellular interactions, iPSC-based models provide crucial insights into disease mechanisms and support the development of targeted therapies [17,18,19,20,21,22,23].

Moreover, iPSCs facilitate precision medicine by enabling personalized drug testing and cell replacement therapies without the need for immunosuppression [24]. iPSC-derived cardiomyocytes (iPSC-CMs) have facilitated high-throughput drug screening to assess both therapeutic efficacy and cardiotoxicity in CHD research [25,26]. Advances in tissue engineering using iPSCs, such as the development of cardiac patches and valves, offer new opportunities for regenerative therapies to repair structural heart defects [27].

Despite these promising advancements, several challenges remain, including the immaturity of iPSC-derived cells, variability in differentiation, and large-scale feasibility, all of which hinder their clinical application. However, innovations in bioengineering, 3D culture systems, and co-culture techniques are steadily addressing these barriers. This review explores how iPSCs are bridging critical gaps in disease modeling, drug discovery, and regenerative therapies for CHD. It emphasizes the importance of translational applications and interdisciplinary collaboration in accelerating the development of precision-based treatments.

2. iPSC-Based Modeling of CHD

2.1. Advances in iPSC-Based Disease Modeling

Traditionally, CHD research has relied on animal models, including genetically engineered mice, to study cardiac development and disease mechanisms. While these models have provided essential insights, they often fail to accurately replicate human CHD phenotypes due to differences in genomic content, physiology, and disease progression [28]. For instance, orthologous genetic variants in mice may not produce the same clinical manifestations observed in humans. These limitations underscore the need for human-based platforms that better reflect the complexities of CHD [28].

iPSCs offer an advanced solution by providing human-derived cells capable of replicating key aspects of cardiac development and disease progression (Figure 1). Unlike animal models, iPSCs can be differentiated into patient-specific cardiomyocytes, enabling researchers to study both genetic and epigenetic factors that influence CHD. For example, studies have shown that epigenetic memory can enhance the efficiency of cardiac differentiation, as demonstrated by differences in promoter methylation of transcription factors such as NKX2-5 and GATA4 between fibroblast-derived and cardiac progenitor cell (CPC)-derived iPSCs [29]. Such findings illustrate how iPSCs can uncover critical regulatory mechanisms and support the development of targeted interventions.

By overcoming the limitations of animal models, iPSCs provide a robust foundation for translational research, enabling scientists to better evaluate therapeutic strategies in human-relevant contexts (Figure 2).

2.2. Patient-Specific iPSC Models for CHD

iPSCs derived from CHD patients enable the modeling of specific subtypes, including hypoplastic left heart syndrome (HLHS) [22,30,31,32], Tetralogy of Fallot (TOF) [17,33], pulmonary atresia with intact ventricular septum (PAIVS) [34,35], bicuspid aortic valve (BAV) and calcific aortic valve disease (CAVD) [36], supravalvular aortic stenosis (SVAS) [37], cardiac septal defects [38], and Barth syndrome (BTHS) [39] (Table 1). iPSC models have revealed how defects in key pathways, such as NOTCH signaling, contribute to abnormal cardiac morphogenesis in HLHS [22,23,31,32,40]. For example, an HLHS patient-specific iPSC line carrying a heterozygous

NOTCH1 mutation has been established and characterized, offering a valuable platform for dissecting disease mechanisms and testing therapeutics [32]. Similarly, iPSC-CMs from TOF patients have shown altered expression of genes implicated in collagen pathways, such as BICC1 and MYH11, which are linked to disease pathology [17]. These patient-specific models allow researchers to analyze disease mechanisms on an individualized basis, supporting the development of precision therapies tailored to specific genetic mutations and phenotypes (Figure 1).

2.3. iPSC-Derived Cardiac Organoids

Organoids are three-dimensional (3D) self-organizing structures [41] that replicate mature tissue architecture [42] and are often described as models that closely resemble the composition of adult tissues [43,44]. In cardiac systems, however, organoids typically represent embryonic and immature stages derived through the recapitulation of developmental programs [45,46,47]. Despite this immaturity, cardiac organoids derived from iPSCs mark a significant advancement in disease modeling by replicating the architecture and functionality of native cardiac tissue, offering a more physiologically relevant model than traditional two-dimensional cultures [48]. iPSC-derived organoids enable the study of complex disease phenotypes, such as structural abnormalities and functional defects in CHD [49].

For example, studies using TBX5 knockout cardiac organoids have demonstrated that this transcription factor plays a critical role in first heart field development and chamber formation [50]. These models revealed defects ranging from delayed morphogenesis to the absence of chamber structures [51,52]. These defects are accompanied by disruptions in electrophysiological and contractile functions, closely mimicking congenital heart defects such as atrial and ventricular septal defects. Similarly, Tbx1 knockout organoids have demonstrated significant disruption in second heart field development, resulting in impaired anterior heart tube elongation [53] and outflow tract (OFT) malformations [54,55,56], as seen in conditions like DiGeorge syndrome.

Additionally, organoid models have been employed to investigate the effects of environmental factors, such as pharmaceutical agents, on cardiac development. Research has shown that drugs like thalidomide and doxorubicin can disrupt myocardial development [57] and electrophysiological activity [47,58]. Thalidomide downregulates critical cardiac genes like Nppa and Vegf, leading to abnormal morphogenesis [59], while doxorubicin induces apoptosis and impairs contractile function [58], mimicking tissue damage observed in clinical cases of congenital defects.

2.4. Single-Cell Technologies in iPSC-Based CHD Research

Single-cell technologies, such as single-cell RNA sequencing (scRNA-seq), have significantly advanced the understanding of iPSC-derived cardiac cells [60,61] by revealing their heterogeneity [62] and offering insights into cellular mechanisms underlying CHD [63]. For example, scRNA-seq analysis of iPSC-CMs from patients with PAIVS identified distinct subpopulations with downregulated contractile and maturation genes alongside upregulated immature isoforms. These findings provide crucial insights into disrupted signaling pathways and transcription factors like HOPX and PDLIM3, which contribute to right ventricular hypoplasia and contractile dysfunction [64].

Similarly, transcriptomic profiling of iPSC-CMs derived from CHD patients, including those with single ventricle defects (SVD) and TOF, has uncovered significant gene expression differences compared to non-CHD controls. For example, 919 differentially expressed genes (DEGs) in SVD-iPSC-CMs were linked to developmental processes, while TOF-iPSC-CMs exhibited 250 DEGs with enrichment in developmental pathways [18]. These findings emphasize the importance of single-cell analysis in revealing how genetic mutations impact cardiac cell differentiation and function. Integrating single-cell and spatial transcriptomics with patient-specific iPSC models and organoid technologies provides a robust framework for mapping cellular heterogeneity and gene expression gradients during cardiac development [65,66]. This approach is invaluable for uncovering molecular mechanisms that drive CHD progression and guiding the development of targeted therapies [67,68]. Building upon advancements in modeling, iPSCs also offer unparalleled insights into the genetic and molecular underpinnings of CHD, enabling targeted therapeutic development.

Table 1. Induced pluripotent stem cell-based models in congenital heart disease.

CHD Subtype	iPSC-Based Model	Key Findings	Genetic Pathways
HLHS	iPSC-CMs, cardiac organoids, endothelial cells	Altered calcium handling, mitochondrial dysfunction [69,70], dysregulated histone acetylation patterns that impaired differentiation [71,72,73], endothelial-mesenchymal transition defects [23]	NKX2-5, NOTCH1 [22,23,40], Myh6 [19]
TOF	Patient-derived iPSC-CMs, CRISPR-engineered TOF models	Dysregulated collagen expression [17], contractile defects, abnormal right ventricular development, disrupted metabolic pathways (butanoate metabolism) [74,75].	GATA4, TBX1, JAG1 [17]
BAV	iPSC-derived endothelial and smooth muscle cells	Abnormal valvulogenesis, endothelial dysfunction, early calcification	GATA4 [76,77], NOTCH1 [36]
SVAS	iPSC-derived vascular smooth muscle cells	Elastin deficiency leading to vascular abnormalities and stenosis [37]	ELN [37]
Cardiac Septal Defects ASD, VSD, AVSD	Patient-specific iPSC-CMs, 3D cardiac tissue models	Defective septal development, impaired myocardial proliferation, disrupted signaling pathways [38]	TBX5, GATA4 [78], NKX2-5 [38,78,79,80]
BTHS	iPSC-CMs with TAZ mutations	Mitochondrial dysfunction, impaired cardiolipin remodeling, excessive ROS generation [39]	TAZ, PPAR pathways [39]
LQTS	iPSC-CMs carrying patient-specific KCNQ1, KCNH2 mutations	Prolonged action potential duration, abnormal ion channel activity	KCNQ1, KCNH2, SCN5A [81,82,83]
LVNC	iPSC-CMs, Fibroblasts	Decreased ventricular development, deep trabeculae, metabolic maturation defects [84]	Mkl2, Myh7, Nkx2-5 [84]
HOS	iPSC-CMs	Epigenetic alterations affecting cardiac developmental genes [85]	TBX5 [86]
OFT malformations	iPSC-CMs, organoid [54,55,56]	Decreased transcription levels in cardiomyocytes	GATA6 [87]

Abbreviations: ASD, atrial septal defect; AVSD, atrioventricular septal defect; BAV, bicuspid aortic valve; BTHS, Barth syndrome; CHD, congenital heart disease; CRISPR, clustered regularly interspaced short palindromic repeats; HLHS, hypoplastic left heart syndrome; HOS, Holt–Oram syndrome; iPSC, induced pluripotent stem cell; iPSC-CMs, iPSC-derived cardiomyocytes; LQTS, long QT syndrome; LVNC, left ventricular non-compaction; OFT, outflow tract; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species; SVAS, supravalvular aortic stenosis; TAZ, Tafazzin; TOF, tetralogy of Fallot; VSD, ventricular septal defect.

Table 1. Induced pluripotent stem cell-based models in congenital heart disease.

CHD Subtype	iPSC-Based Model	Key Findings	Genetic Pathways
HLHS	iPSC-CMs, cardiac organoids, endothelial cells	Altered calcium handling, mitochondrial dysfunction [69,70], dysregulated histone acetylation patterns that impaired differentiation [71,72,73], endothelial-mesenchymal transition defects [23]	NKX2-5, NOTCH1 [22,23,40], Myh6 [19]
TOF	Patient-derived iPSC-CMs, CRISPR-engineered TOF models	Dysregulated collagen expression [17], contractile defects, abnormal right ventricular development, disrupted metabolic pathways (butanoate metabolism) [74,75].	GATA4, TBX1, JAG1 [17]
BAV	iPSC-derived endothelial and smooth muscle cells	Abnormal valvulogenesis, endothelial dysfunction, early calcification	GATA4 [76,77], NOTCH1 [36]
SVAS	iPSC-derived vascular smooth muscle cells	Elastin deficiency leading to vascular abnormalities and stenosis [37]	ELN [37]
Cardiac Septal Defects ASD, VSD, AVSD	Patient-specific iPSC-CMs, 3D cardiac tissue models	Defective septal development, impaired myocardial proliferation, disrupted signaling pathways [38]	TBX5, GATA4 [78], NKX2-5 [38,78,79,80]
BTHS	iPSC-CMs with TAZ mutations	Mitochondrial dysfunction, impaired cardiolipin remodeling, excessive ROS generation [39]	TAZ, PPAR pathways [39]
LQTS	iPSC-CMs carrying patient-specific KCNQ1, KCNH2 mutations	Prolonged action potential duration, abnormal ion channel activity	KCNQ1, KCNH2, SCN5A [81,82,83]
LVNC	iPSC-CMs, Fibroblasts	Decreased ventricular development, deep trabeculae, metabolic maturation defects [84]	Mkl2, Myh7, Nkx2-5 [84]
HOS	iPSC-CMs	Epigenetic alterations affecting cardiac developmental genes [85]	TBX5 [86]
OFT malformations	iPSC-CMs, organoid [54,55,56]	Decreased transcription levels in cardiomyocytes	GATA6 [87]

Abbreviations: ASD, atrial septal defect; AVSD, atrioventricular septal defect; BAV, bicuspid aortic valve; BTHS, Barth syndrome; CHD, congenital heart disease; CRISPR, clustered regularly interspaced short palindromic repeats; HLHS, hypoplastic left heart syndrome; HOS, Holt–Oram syndrome; iPSC, induced pluripotent stem cell; iPSC-CMs, iPSC-derived cardiomyocytes; LQTS, long QT syndrome; LVNC, left ventricular non-compaction; OFT, outflow tract; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species; SVAS, supravalvular aortic stenosis; TAZ, Tafazzin; TOF, tetralogy of Fallot; VSD, ventricular septal defect.

3. Molecular Insights and Precision Medicine Using iPSCs

3.1. Genetic and Epigenetic Contributions in iPSC Models

iPSC models have significantly advanced the understanding of CHD by elucidating the roles of genetic mutations in cardiac development. Mutations in key transcription factors such as GATA4, NKX2-5, and TBX5, which regulate cardiac development, have been linked to defects such as atrial and ventricular septal defects and TOF [88,89]. One notable study demonstrated that GATA4 mutations associated with atrial septal defects (ASDs) disrupt transcriptional networks responsible for atrial specification, impairing key signaling pathways such as Sonic Hedgehog and altering TBX5 recruitment at cardiac enhancers [38,79]. Likewise, iPSCs from TOF patients with TBX1 mutations highlighted defects in CPC proliferation, offering critical insights into OFT malformations [17]. Additionally, mutations in GATA6, a master regulator of heart formation, have also been linked to diverse congenital heart defects, including both cardiac malformations and pancreatic agenesis [90], underscoring its pivotal role during embryogenesis [91,92].

Recent research has also highlighted the role of de novo mutations in chromatin regulation genes, revealing genetic overlap between CHD and neurodevelopmental disorders [93,94,95]. In parallel, iPSC models have proven instrumental in exploring genotype–phenotype discordance and incomplete penetrance in inherited cardiac disorders. For example, a study using iPSC-CMs from individuals within the same LQTS Type 2 (LQT2) family revealed striking differences in cellular electrophysiology despite identical KCNH2 mutations. This approach led to the identification of genetic modifiers, such as REM2 and KCNK17, that explained disease variability and underscored the power of iPSC modeling in dissecting complex inheritance patterns [96].

In addition to genetic alterations, epigenetic mechanisms, including histone modifications, DNA methylation, and non-coding RNAs, play critical roles in regulating cardiac gene expression [97]. While genetic mutations provide foundational insights, epigenetic factors add complexity, shaping gene expression and cellular behavior. iPSC models have provided a platform for studying how dysregulated epigenetic states contribute to CHD [98]. For example, studies of HLHS-derived iPSCs revealed dysregulated histone acetylation patterns that impaired cardiomyocyte differentiation [71,72,73]. Furthermore, non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs, also play crucial roles in cardiac development [99]. iPSC-CMs have identified 96 miRNAs that promote cardiomyocyte proliferation, many of which act on the Hippo signaling pathway by regulating components such as LATS2, TEAD1, and YAP, thereby highlighting their critical roles in cardiac biology and development and contributing to our understanding of CHD pathogenesis [100].

Transcriptomic analyses of iPSC-CMs have also uncovered distinct RNA profiles, shedding light on disrupted pathways regulating cardiomyocyte function and development. These findings highlight the significance of RNA-based regulatory mechanisms and their contribution to CHD pathogenesis, promoting further exploration of epigenetic influences [18]. Continued advancements in next-generation sequencing technologies are uncovering additional genetic variations, such as single-nucleotide polymorphisms and copy number variants, which further expand the potential for developing targeted therapies [101].

3.2. Patient-Specific Therapies Enabled by iPSCs

3.2.1. Drug Testing and Screening with iPSC-CMs

iPSC-CMs have become invaluable tools for drug discovery and personalized treatment development. Large-scale studies, such as the Comprehensive In Vitro Proarrhythmia Assessment initiative, have demonstrated the ability of iPSC-CMs in predicting drug-induced arrhythmias across multiple laboratories [102,103]. These studies validate the utility of iPSC-CMs for assessing arrhythmogenic potential and modeling patient-specific responses. For instance, iPSC-CMs from patients with trastuzumab-induced cardiotoxicity have revealed underlying metabolic disruptions, enabling the identification of potential therapeutic targets [104,105]. Similarly, studies using iPSCs derived from various cardiovascular and neurological conditions have demonstrated their capacity to restore calcium signaling and contractile function through targeted drug interventions [106].

In the context of CHD and inherited cardiac disorders, iPSC-CMs have been instrumental in testing drugs for conditions such as long QT syndrome (LQTS) and catecholaminergic polymorphic ventricular tachycardia, successfully recapitulating disease phenotypes to evaluate antiarrhythmic therapies [81,82,83]. Patient-specific iPSC models of LQTS have provided key insights into the electrophysiological abnormalities associated with various LQTS subtypes and have facilitated precision medicine approaches by enabling high-throughput drug testing and genotype-specific treatment strategies [107]. Furthermore, research using HLHS-derived iPSCs has uncovered significant cellular defects, including altered calcium handling and mitochondrial dysfunction. Metabolic interventions targeting these defects have demonstrated improved contractility, suggesting potential therapeutic avenues [69,70]. iPSC-based studies on TOF have identified disrupted metabolic pathways, such as butanoate metabolism, further emphasizing the potential of metabolic modulators as therapeutic strategies [74,75].

Importantly, because CHD primarily affects pediatric populations, there is growing interest in utilizing iPSC-based platforms to assess pediatric-specific drug responses, developmental toxicity, and long-term safety profiles in children [21]. These models are especially valuable for younger patients with complex CHD subtypes who require tailored pharmacological strategies. Cardiomyocytes derived from pediatric patient-specific iPSCs offer unique advantages by capturing age-dependent electrophysiological and metabolic characteristics, enabling developmentally relevant modeling of drug efficacy and toxicity [108]. For example, iPSC-CMs from patients with HLHS have demonstrated mitochondrial and contractile dysfunctions, provided mechanistic insights, and revealed potential therapeutic targets [21]. Pediatric-focused iPSC models thus hold great promise for optimizing treatment outcomes and minimizing adverse effects in vulnerable pediatric populations, aligning with the goals of precision medicine in CHD care [108].

3.2.2. Gene Therapy and Genome Editing Using iPSC Models

The combination of iPSC technology with CRISPR/Cas9 gene editing holds great promise for correcting disease-causing mutations [109], as shown earlier in Figure 1. One example involved the correction of a GATA4 mutation in iPSCs derived from ASD patients, resulting in restored cardiomyocyte differentiation and function [78]. This study exemplifies the potential of gene-edited iPSCs for personalized regenerative therapies aimed at repairing damaged cardiac tissue. Furthermore, preclinical studies have demonstrated that transplantation of gene-edited iPSC-CMs can improve cardiac function in animal models of heart disease [110,111]. For example, a meta-analysis highlighted functional recovery in ischemic heart disease models following iPSC-CM transplantation [111]. Research involving large animal models has further confirmed the feasibility of using iPSC-CMs for myocardial repair, paving the way for clinical applications [110].

In addition to therapeutic applications, CRISPR/Cas9 has advanced functional genomics research by enabling precise genetic modifications in iPSC models. This allows researchers to investigate the role of specific mutations in cardiac development [112,113]. For example, TBX5 knockout studies have elucidated the gene’s role in Holt–Oram syndrome (HOS) [114], while the correction of TBX5 mutations restored normal gene function and enhanced understanding of regulatory pathways [86]. Moreover, CRISPR technology facilitates the exploration of complex genetic contributions to CHD, including oligogenic inheritance [84]. By introducing or correcting combinations of genetic variants in iPSCs, researchers can study how multiple genetic factors interact to influence disease progression [115,116]. This capability enhances our understanding of CHD’s genetic complexity and supports the development of targeted therapies [117].

3.2.3. Clinical Trials and Regenerative Applications of iPSC-Based Therapies

The first-in-human phase I/II clinical trial (LAPiS) demonstrated the feasibility of using iPSC-derived heart muscle cells injected directly into a patient’s myocardium during coronary bypass surgery. This trial marked a significant milestone in regenerative medicine for heart failure treatment [118]. Building on these advancements, a clinical trial by the Mayo Clinic investigates the potential of autologous iPSC-derived cardiac cells to strengthen heart function in CHD patients. In this study, a patient’s own skin cells are reprogrammed into iPSCs, differentiated into cardiac cells, and then delivered into the heart muscle to assess their therapeutic potential. This pioneering initiative exemplifies how iPSC-based therapies are transitioning from laboratory research to clinical practice [119].

Successful clinical translation of iPSC-based therapies will require alignment with regulatory frameworks, such as those outlined by the U.S. Food and Drug Administration (FDA) for cell-based products [120]. This includes preclinical safety evaluation, standardization of manufacturing under current good manufacturing practices (cGMP), and phase I/II clinical trial designs to assess safety, dosing, and preliminary efficacy [121]. Furthermore, partnerships with industry collaborators, including biotechnology companies and translational research institutes, are critical to scaling production, navigating regulatory approval, and advancing therapies toward clinical implementation [122].

4. iPSC-Based Drug Discovery and Testing for CHD

iPSC-derived models have revolutionized the drug discovery process, providing platforms for testing therapeutic efficacy and cardiotoxicity in patient-specific contexts. Recent advancements have enabled high-throughput screening to accelerate drug development for CHD.

4.1. High-Throughput Screening Platforms Using iPSC-CMs

iPSC-CMs serve as powerful tools for high-throughput drug screening, allowing researchers to evaluate compounds for their effects on contractility, calcium signaling, and electrophysiology (Figure 3). Studies using iPSC-CMs have identified drugs with both therapeutic potential and cardiotoxic risk [123].

To further enhance drug discovery efforts, advanced systems for generating and analyzing iPSC-CMs have significantly improved drug safety testing and disease modeling. Automated patch clamp (APC) systems have become a cornerstone of modern electrophysiological drug screening, offering high-throughput and highly precise measurements of ion channel activity and action potentials. When integrated with traditional techniques such as microelectrode arrays (MEAs), calcium imaging, and optical mapping, these platforms enable comprehensive, high-capacity assessments of cardiac electrophysiology and drug-induced effects [124]. High-throughput functional analysis of iPSC-CMs enables rapid and efficient screening of drug responses, accelerating the identification of effective treatments for CHD [125]. Complementing these efforts, innovations in automated physiological recording techniques, such as video motion tracking and optical mapping, have enabled precise, quantitative assessments of physiological defects in iPSC-derived cardiac cells carrying CHD-associated mutations. These technologies have contributed to the creation of scalable, high-throughput platforms that are instrumental in advancing drug discovery and personalized therapies [70,126].

Adding to these advancements, cardiac microphysiological systems (MPS) using hiPSC-CMs represent a cutting-edge approach to human-specific cardiac modeling. By creating 3D-aligned cardiac tissues with physiologically relevant properties, MPS platforms enable robust cardiotoxicity testing and high-throughput drug screening. This innovative technology bridges the gap between in vitro and in vivo models, offering more accurate predictions of human cardiac responses to therapeutic compounds [127]. These high-throughput platforms not only accelerate the identification of effective treatments but also provide a critical foundation for functional restoration studies, where compounds are tested for their ability to rescue impaired cardiac function in patient-specific models.

4.2. Functional Restoration Studies in iPSC Models

Phenotypic screening using iPSC-CMs allows for the identification of compounds that restore normal cardiac function in patient-specific models (Figure 3). This approach is particularly useful for investigating drugs that target impaired contractility and calcium handling, which are often disrupted in CHD. Among the tools used in this context, APC systems are especially valuable for quantifying how candidate compounds reverse abnormal electrophysiological properties in patient-specific iPSC-CMs. This level of precision facilitates the identification of drugs that restore normal cardiac function by correcting mutation-induced defects in ion channel behavior [124,128,129]. For instance, iPSC-CMs with CHD-associated mutations have helped identify small molecules capable of rescuing these functional defects, paving the way for targeted therapeutic interventions [5,6]. By focusing on patient-derived cells, researchers gain insights into the impact of specific genetic mutations on drug responses, enabling more effective and personalized treatments [108,130].

4.3. Technical Advances in iPSC-CM Differentiation and Scale-Up

To enable disease modeling and drug screening applications, several well-established protocols have been developed to convert iPSCs into iPSC-CMs. A widely adopted approach involves temporal modulation of Wnt/β-catenin signaling using small molecules: activation during mesoderm induction (e.g., CHIR99021), followed by inhibition (e.g., IWP2 or Wnt-C59) to promote cardiac specification. This monolayer-based protocol enables efficient and reproducible differentiation and has been adapted for multiple disease models [131]. For scalable production, bioengineering strategies such as suspension culture in spinner flasks, microcarrier-based expansion, and 3D cardiac organoids have been implemented to increase yield and enhance structural and functional maturation of iPSC-CMs [132,133,134]. Moreover, automation platforms and cGMP-compliant protocols are being developed to support high-throughput screening and therapeutic applications [133]. These technical innovations are critical for advancing iPSC-based CHD models toward translational and clinical utility.

4.4. Drug Repurposing Using iPSC-Derived Models

iPSC-based models have provided unique opportunities to repurpose existing drugs for CHD treatment. For example, statins, traditionally used for lipid regulation, have shown potential benefits in modulating cardiac remodeling in iPSC-derived CHD models. This strategy accelerates the translation of therapies into clinical practice, providing timely interventions for CHD [135].

5. Limitations and Challenges of iPSCs in CHD Research

Despite the transformative potential of iPSC technology, several limitations hinder its clinical application. These challenges necessitate innovative solutions and ongoing research to improve the reliability of iPSC-derived models.

5.1. Immaturity of iPSC-CMs

Human iPSC-CMs exhibit fetal-like characteristics, with disorganized myofibril structures and a lack of T-tubules, which contrast with the mature architecture of adult cardiomyocytes [136]. Efforts to promote maturation, such as the use of hormones [137], physical and electrical conditioning [138], and co-culture systems [139], have shown promise but remain limited in replicating adult cardiac phenotypes. Additionally, while 3D cardiac organoids provide a more physiologically relevant model [140], their ability to fully capture CHD pathogenesis remains uncertain.

5.2. Large-Scale Feasibility and Reproducibility

The large-scale production of iPSC-derived cells with consistent quality is a significant obstacle. Variability in reprogramming techniques, differentiation protocols, and genetic backgrounds often leads to inconsistent outcomes [141,142]. Addressing this requires standardized protocols, bioengineering innovations, and automation platforms, which can improve reproducibility [143,144]. Techniques discussed earlier, such as advanced high-throughput screening systems, are paving the way toward more efficient large-scale applications [125].

Several major collaborative initiatives are actively working to overcome variability and enhance reproducibility in iPSC research. For example, the National Institutes of Health (NIH)’s iPSC initiatives aim to develop standardized protocols for cell generation and differentiation to promote consistency across institutions [145]. The International Stem Cell Initiative (ISCI) provides a global framework for assessing the genetic stability, pluripotency, and differentiation capacity of iPSC lines under harmonized conditions [146]. In the context of drug safety, the Comprehensive in vitro Proarrhythmia Assay (CiPA) incorporates iPSC-CMs into multi-center studies to benchmark electrophysiological performance and evaluate proarrhythmic risk [147]. Together, these efforts help establish quality control benchmarks, reduce inter-laboratory variability, and expand the clinical and research utility of iPSC-based models.

5.3. Tumorigenicity and Genomic Instability

The pluripotent nature of iPSCs introduces potential risks of tumorigenesis, stemming from residual undifferentiated cells, sustained reprogramming activity, and genomic instability acquired during culture [148]. Advanced purification techniques [149], non-integrating reprogramming techniques [150,151,152], and ongoing assessments of genomic stability are critical to mitigating these risks and ensuring safety for clinical applications.

5.4. Immunogenicity and Compatibility Issues

Although autologous iPSCs are expected to minimize immune rejection, practical limitations in generating autologous therapies have led to the exploration of allogeneic approaches. These approaches carry the risk of immune responses due to genetic and epigenetic differences. Future research must focus on refining protocols to reduce immunogenicity while maintaining clinical feasibility [153].

5.5. Cell Engraftment and Integration Challenges

Achieving stable engraftment and functional integration of iPSC-derived cells into the host myocardium remains a major challenge. Poor survival, limited electromechanical coupling, and incomplete syncytium integration have hindered therapeutic success [154,155,156]. Approaches such as tissue-engineered scaffolds, improved delivery techniques, and the identification of paracrine mediators offer promising pathways to enhance therapeutic outcomes [157].

5.6. Cost, Expertise, and Ethical Considerations

The generation, maintenance, and differentiation of iPSCs require substantial financial resources and specialized expertise, which can limit accessibility. Streamlining processes through innovations in automation and artificial intelligence (AI)-driven platforms may reduce costs and improve large-scale feasibility, making iPSC-based solutions more feasible on a broader scale. Further advancements in AI-integrated protocols could optimize cell differentiation and quality control, accelerating the development process.

Additionally, while iPSCs bypass many ethical concerns associated with embryonic stem cells, challenges remain regarding donor cell sourcing, clinical-grade production, and regulatory compliance. Initiatives such as Japan’s iPSC library [158] highlight potential solutions, but logistical and financial barriers persist.

Emerging ethical concerns related to iPSC use in CHD research and therapy also warrant attention. These include the responsible sourcing and consent for human-derived cells [159], equitable access to iPSC-based therapies [160], and the implications of gene editing technologies in pediatric populations [159,161]. As clinical applications advance, it is essential to establish transparent ethical frameworks that balance innovation with patient safety, informed consent, and long-term monitoring of interventions [162].

Moreover, the integration of clinically applicable imaging modalities, such as speckle tracking echocardiography (STE), can enhance the translational impact of iPSC-based studies. As an angle-independent technique increasingly used in both fetal and pediatric cardiology [163,164], STE enables detailed and reproducible assessment of global and regional myocardial function. In preclinical models, STE has also been used to evaluate the functional effects of iPSC-derived cell therapies on myocardial strain and remodeling [165]. Further studies are needed to explore its role in assessing the impact of iPSC-based interventions in congenital heart disease, particularly in evaluating regional and global myocardial responses.

6. Conclusions

Induced pluripotent stem cells have emerged as a transformative platform in CHD research and treatment. These technologies offer unparalleled opportunities to address critical gaps in understanding disease mechanisms, enabling innovative approaches to drug discovery, disease modeling, and regenerative therapies. Patient-specific iPSC models replicate the genetic and phenotypic heterogeneity of CHD, providing a foundation for precision medicine and targeted therapeutic interventions. Despite significant advancements, challenges such as the immaturity of iPSC-derived cells, variability in differentiation protocols, large-scale feasibility, and concerns about tumorigenicity remain barriers to clinical translation. To overcome these challenges, the development of standardized protocols, advanced bioengineering techniques, and rigorous quality control measures is crucial. Moreover, efforts to streamline costs, improve accessibility, and ensure regulatory compliance will be vital for widespread clinical adoption of iPSC-based therapies.

Looking ahead, the integration of cutting-edge technologies promises to further accelerate advancements in iPSC research. AI-driven approaches, such as predictive modeling and machine learning algorithms, are increasingly being applied to analyze large-scale iPSC data. These tools can enhance our understanding of how complex genetic and environmental factors influence CHD phenotypes, leading to more accurate disease prediction and personalized treatment strategies. Additionally, combining AI with high-throughput screening platforms may significantly optimize drug discovery, accelerating the identification of effective compounds tailored to patient-specific needs.

To fully realize the translational potential of iPSC-based technologies in CHD, strategic interdisciplinary collaboration is essential. Fields such as bioinformatics and computational biology are critical for analyzing genomic, transcriptomic, and electrophysiological data generated from iPSC-derived cardiac models. Materials science and tissue engineering contribute to the development of biomimetic scaffolds, 3D cardiac tissues, and organoids that better mimic physiological conditions. In parallel, clinical pharmacology and regulatory science support the optimization of dosing strategies and ensure safety and compliance throughout the translational process. By integrating expertise from these diverse disciplines, the field can accelerate discovery, improve model accuracy, and overcome key translational barriers in iPSC-based CHD research.

The continued evolution of iPSC research holds the promise of reshaping CHD care and advancing regenerative medicine. By fostering interdisciplinary collaboration among scientists, clinicians, and industry experts, the field can drive innovation toward patient-centered healthcare solutions. With sustained investment in research and a commitment to addressing existing challenges, iPSC technology is set to play a significant role in next-generation healthcare, offering renewed hope to patients and families affected by CHD worldwide.