Induced pluripotent stem cells in Hirschsprung disease: Modeling, mechanisms, and translational challenges

Abstract

Hirschsprung disease (HSCR) is a congenital disorder of the enteric nervous system (ENS) caused by impaired migration, proliferation, survival, and differentiation of enteric neural crest cells (ENCCs) during intestinal development. Although many HSCR-associated genes and regulatory variants have been identified, most cases are oligogenic or multifactorial, and mechanistic studies are limited by scarce human embryonic enteric tissues. Patient-derived induced pluripotent stem cells (iPSCs) preserve individual genetic backgrounds and can be differentiated into ENCCs, enteric neurons, and intestinal or colonic organoids, providing human platforms for modeling early ENS development and disease-relevant phenotypes. This review summarizes iPSC platform establishment, ENCC differentiation, two-dimensional ENCC models, recombined or co-culture organoids, emerging bioengineered gut systems, drug screening, and regenerative strategies. We discuss how genetic complexity, isogenic controls, multi-omics integration, electrophysiological maturation, and ENCC-microenvironment interactions influence model interpretation. Current applications remain limited by developmental immaturity, inter-line and inter-batch variability, incomplete functional validation, safety concerns, manufacturing requirements, and pediatric regulatory considerations. At present, iPSC-based HSCR models are best viewed as complementary tools for mechanistic investigation and preclinical development rather than direct routes to clinical therapy.

Key Words: Hirschsprung disease; Induced pluripotent stem cells; Disease modeling; Drug screening; Cell therapy

Core Tip: Patient-derived induced pluripotent stem cell (iPSC) platforms provide human models for linking Hirschsprung disease (HSCR)-associated genetic backgrounds with enteric neural crest cell (ENCC) developmental phenotypes and tissue-level interactions. This review summarizes iPSC platform establishment, two-dimensional ENCC models, recombined and bioengineered gut systems, drug screening, and cell-based regenerative strategies. We emphasize genetic complexity, reproducibility, functional maturation, electrophysiological validation, and translational feasibility. Current iPSC-based HSCR models are most useful for mechanistic investigation and preclinical development, whereas drug screening and cell therapy remain exploratory.

INTRODUCTION

Hirschsprung disease (HSCR) is a congenital disorder of the enteric nervous system (ENS) and a leading cause of functional intestinal obstruction in neonates[1]. It occurs in approximately 1 in 5000 live births, with a male predominance of 2.8:1 to 4.0:1. The disease is caused by defects in the migration, proliferation, and differentiation of enteric neural crest cells (ENCCs) during intestinal development. This developmental defect results in aganglionosis of the distal bowel, persistent contraction of the affected segment, functional obstruction, and secondary dilatation of the proximal ganglionated intestine[2]. Most affected infants are diagnosed during the neonatal period, typically presenting with delayed meconium passage, abdominal distension, and bilious vomiting. Hirschsprung-associated enterocolitis (HAEC) is the most severe complication, affecting 10%-30% of patients either pre- or post-surgery, and is a major contributor to HSCR-related mortality[3].

HSCR is clinically classified by the length of the aganglionic segment into short-segment HSCR, long-segment HSCR, and total colonic aganglionosis, which account for approximately 72%, 15%, and 8% of cases, respectively[4,5]. Although this classification reflects the extent of the disease and clinical severity, it does not fully capture the genetic heterogeneity underlying HSCR[6]. High-penetrance monogenic variants, primarily involving RET proto-oncogene (RET), endothelin receptor type B (EDNRB), zinc finger E-box-binding homeobox 2, SRY-box transcription factor 10 (SOX10), paired-like homeobox 2B (PHOX2B), neuregulin 1, and a few other genes[7-10], are responsible for only a small proportion of cases and are more often linked to familial forms and severe phenotypes (Figure 1). In contrast, most sporadic short-segment cases are thought to result from oligogenic or multifactorial inheritance, involving rare coding variants, non-coding regulatory variants, and incomplete penetrance[11,12]. Even for RET[13,14], the most well-established HSCR susceptibility gene, identified variants account for only part of the disease’s heritability.

Figure 1 Major Hirschsprung disease-associated genes and signaling pathways regulating enteric neural crest cell development. This schematic summarizes representative genetic and microenvironmental regulators involved in enteric neural crest cell (ENCC) proliferation, migration, differentiation, and survival during enteric nervous system (ENS) formation. Glial cell line-derived neurotrophic factor-RET proto-oncogene signaling, endothelin 3-endothelin receptor type B signaling, and neuregulin 1-erb-b2 receptor tyrosine kinase 2 signaling are shown as major pathways influencing ENCC behavior. Key transcriptional regulators, including SRY-box transcription factor 10, paired-like homeobox 2B, and zinc finger E-box-binding homeobox 2, are illustrated within the ENCC regulatory network. Extracellular matrix cues are also depicted as part of the intestinal microenvironment that modulates ENCC colonization. Coordinated ENCC proliferation, migration, and differentiation support normal ENS formation, whereas impaired pathway activity or disrupted regulatory interactions can lead to failed distal bowel colonization and aganglionosis in Hirschsprung disease (HSCR). This figure highlights representative mechanisms rather than all genetic or regulatory factors involved in HSCR pathogenesis. GDNF: Glial cell line-derived neurotrophic factor; RET: RET proto-oncogene; EDN3: Endothelin 3; EDNRB: Endothelin receptor type B; NRG1: Neuregulin 1; ERBB2: Erb-b2 receptor tyrosine kinase 2; ENCC: Enteric neural crest cell; ZEB2: Zinc finger E-box-binding homeobox 2; SOX10: SRY-box transcription factor 10; PHOX2B: Paired-like homeobox 2B; ENS: Enteric nervous system.

Animal models, particularly those involving mice and zebrafish, have significantly advanced our understanding of ENS development and facilitated the identification of numerous HSCR-associated genes[15-17]. However, these models have limitations when applied to studying the genetic architecture of human HSCR. Many animal models rely on complete gene knockout, while most patients with HSCR, especially those with sporadic short-segment disease, exhibit complex genetic risk factor combinations[18,19]. Additionally, some human disease-associated non-coding variants reside in regulatory regions that are not well conserved across species, limiting their functional evaluation in traditional animal models[20]. The availability of primary human ENCCs from relevant embryonic stages is also severely restricted due to ethical and logistical challenges. Together, these limitations make it difficult to link patient-specific genetic variants with human ENS developmental phenotypes.

Surgical resection of the aganglionic bowel, followed by pull-through procedures, remains the standard treatment for HSCR and has significantly improved patient survival[21]. However, this approach does not restore the missing enteric neural circuitry. Consequently, many patients experience long-term complications such as constipation, fecal incontinence, recurrent obstructive symptoms, and postoperative HAEC[22,23]. These persistent issues suggest that anatomical correction alone is insufficient for some patients, highlighting the need for experimental models that can better elucidate disease mechanisms and guide the development of novel therapeutic strategies[24,25].

Patient-derived induced pluripotent stem cells (iPSCs) offer a human cellular model for studying HSCR in a disease-relevant genetic context[26-28]. Unlike animal models or immortalized cell lines, iPSCs retain patient-specific coding and non-coding genetic variations and can be differentiated into ENCCs, enteric neurons, and intestinal organoids[29-31]. Previous reviews, including the comprehensive review by Lui and Ngan[32], have outlined two-dimensional and three-dimensional pluripotent stem cell-based models for HSCR and helped establish a basic framework for the field. This review focuses on how iPSC-based HSCR models should be interpreted and applied. We discuss the impact of oligogenic and multifactorial inheritance on patient-derived iPSC modeling, differences among model platforms in disease relevance and functional readouts, and the need for electrophysiological and tissue-level validation beyond lineage marker expression.

LITERATURE SEARCH STRATEGY AND SELECTION CRITERIA

A narrative literature search was conducted to identify studies relevant to iPSC-based modeling and translational research in HSCR. PubMed was searched from inception to June 2026. Reference lists of relevant articles were also screened manually to identify additional studies. The search used combinations of the following terms: “Hirschsprung disease”, “HSCR”, “induced pluripotent stem cells”, “iPSC”, “human pluripotent stem cells”, “enteric neural crest cells”, “ENCCs”, “enteric nervous system”, “enteric neurons”, “intestinal organoids”, “colonic organoids”, “gut-on-chip”, “organ-on-chip”, “CRISPR”, “gene editing”, “electrophysiology”, “calcium imaging”, “multi-electrode array”, “spatial transcriptomics”, “oxidative phosphorylation”, “oxidative stress”, “cell therapy”, and “transplantation”.

Original research articles, reviews, and methodological articles were considered if they were relevant to HSCR genetics, ENS development, iPSC generation, ENCC differentiation, organoid or bioengineered gut models, functional validation, drug screening, or translational cell therapy. Priority was given to studies directly involving HSCR, patient-derived iPSCs, ENCCs, enteric neurons, intestinal or colonic organoids, and ENS-related regenerative approaches. Foundational studies in pluripotent stem cell biology, genome editing, electrophysiology, organ-on-chip systems, and cell therapy regulation were also included when they provided important methodological or conceptual context.

Articles were excluded if they were not written in English, were not relevant to HSCR, ENS development, iPSC-based modeling, or stem-cell-based translational research, or were available only as conference abstracts without sufficient methodological detail. Duplicate records and studies outside the scope of this review were also excluded. Studies were selected according to their relevance to the review topic, methodological importance, recency, and contribution to mechanistic or translational understanding. Because this article is a narrative review rather than a systematic review or meta-analysis, no formal risk-of-bias assessment or quantitative evidence synthesis was performed.

IPSC PLATFORMS FOR HSCR RESEARCH

Since Takahashi and Yamanaka’s groundbreaking generation of mouse and human iPSCs[28,33], this technology has become a cornerstone for studying human diseases, developmental biology, and regenerative medicine[34]. In the context of HSCR research, the utility of patient-derived iPSCs hinges not only on successful reprogramming and maintenance of pluripotency but also on the ability to differentiate into enteric lineage cells relevant to disease pathogenesis[35]. Given that HSCR results from abnormalities in ENS development, iPSC-based models must be evaluated based on their capacity for reproducible differentiation into ENCCs and enteric neurons. Therefore, developing reliable iPSC platforms for HSCR necessitates careful donor cell selection, standardized reprogramming protocols, and stringent lineage-specific quality control[36-38].

Previous reviews on iPSC-based HSCR models have given limited attention to the construction of the iPSC platform itself[32]. However, as the field progresses from proof-of-concept studies using a small number of lines to larger cohorts and, ultimately, translational applications, these platform-level decisions play an increasingly critical role in determining the scope of research questions that can be addressed and the confidence with which results can be interpreted[39,40]. The requirements for a research-grade iPSC line to study single-gene defects differ substantially from those necessary for lines intended for cell therapy development[41,42]. In practice, donor cell source, reprogramming method, and quality control standards determine not only the robustness of the iPSC platform but also the types of biological and translational questions it can support[43,44]. Accordingly, this review examines donor cell selection, reprogramming strategies, and lineage-specific quality control as key steps in building iPSC platforms for HSCR research (Figure 2).

Figure 2 General workflow for establishing patient-derived induced pluripotent stem cell platforms for Hirschsprung disease research. Somatic cells can be obtained from patients with Hirschsprung disease using several sources, including skin fibroblasts, peripheral blood mononuclear cells, urinary epithelial cells, and hair keratinocytes. These donor cells are reprogrammed into patient-derived induced pluripotent stem cells (iPSCs) using either integrative or non-integrative strategies. Integrative approaches may achieve reprogramming but carry potential risks related to transgene insertion and mutagenesis. Non-integrative approaches, such as Sendai virus-based reprogramming, messenger RNA-based reprogramming, or episomal plasmid-based reprogramming, can generate transgene-free iPSC lines and are generally more suitable for downstream translational applications. Platform-level quality control should include confirmation of general iPSC identity, genomic integrity, and clearance of reprogramming vectors when applicable. After quality control, patient-derived iPSCs can be differentiated and applied to disease modeling, drug screening, and preclinical evaluation of cell-based therapeutic strategies. The figure emphasizes that donor cell source, reprogramming strategy, and quality control standards directly influence reproducibility, scalability, and translational suitability. PBMCs: Peripheral blood mononuclear cells; iPSC: Induced pluripotent stem cell.

Selection of somatic cell sources for patient-derived iPSCs

The selection of donor somatic cells impacts reprogramming efficiency, sample availability, donor recruitment, and potential residual epigenetic effects, all of which influence the feasibility and interpretation of HSCR iPSC studies[45-48]. Given that many patients with HSCR are neonates or young children, minimally invasive sampling methods are particularly important[49]. Dermal fibroblasts have been widely used for iPSC generation and remain a well-characterized cell source; however, the need for skin biopsies can limit their use in pediatric cohorts and large-scale recruitment efforts[48,50].

Peripheral blood mononuclear cells (PBMCs) are currently one of the most practical sources for HSCR iPSC generation. These cells can be easily obtained via routine venous blood collection, allowing for repeated sampling and cohort expansion[51]. Although the reprogramming efficiency of PBMCs may be lower than that of fibroblasts, their accessibility makes them ideal for studies involving diverse HSCR subtypes and genetically heterogeneous patient populations[42,52,53]. Alternative non-invasive sources, such as urine-derived epithelial cells and hair follicle keratinocytes[54-58], offer potential options, but their use in infants may be limited by inconsistent cell yield, culture stability, and reprogramming variability[59]. Overall, PBMCs strike a practical balance between accessibility, scalability, and reprogramming efficiency for patient-derived HSCR iPSC studies.

Reprogramming strategies for HSCR-related iPSC studies

The selection of a reprogramming strategy should align with the intended application of the iPSC line[36]. For mechanistic studies, key factors include efficiency, reproducibility, and compatibility with diverse donor samples. In translational or preclinical contexts, non-integrating methods, vector clearance, genomic stability, and adherence to good manufacturing practices are also critical. Sendai virus-based reprogramming[60,61], commonly used in HSCR-related iPSC studies, offers high efficiency, even with low-input pediatric samples, and utilizes a non-integrating cytoplasmic RNA virus. These features make Sendai virus-based reprogramming well suited for generating research-grade iPSC lines and disease-specific cohorts. However, for therapeutic development, thorough verification of viral component clearance is essential. Synthetic mRNA-based reprogramming avoids both integration and viral persistence, though it is technically challenging and may exhibit reduced efficiency in certain pediatric samples[62]. Episomal plasmid-based systems are simple, cost-effective, and non-integrating, but they typically show lower efficiency than Sendai virus-based methods, particularly when starting material is limited[63,64]. Small-molecule reprogramming bypasses the need for exogenous genetic material but remains an exploratory approach in HSCR research, requiring further refinement[65-67]. Currently, the selection of reprogramming methods depends on the specific research goals: Sendai virus-based approaches are ideal for efficiently generating research-grade lines, mRNA-based methods are suited for translational applications, and episomal systems may be appropriate for biobanking when lower efficiency is acceptable.

Improving reprogramming efficiency and ensuring line quality

Reprogramming efficiency remains a key challenge in HSCR studies, particularly when limited pediatric samples are available. Several strategies, including chromatin-modifying agents, small-molecule combinations, and regulators like Pramef12 and Gadd45a[68,69], have been reported to enhance reprogramming efficiency or kinetics. However, in the context of HSCR modeling, an increase in colony number alone is insufficient. Protocols aimed at improving reprogramming efficiency must also preserve genomic integrity, pluripotency, and stable differentiation potential.

Standard quality control for newly generated iPSC lines typically includes the assessment of pluripotency markers such as OCT4, SOX2, NANOG, SSEA4, and TRA-1-60, along with karyotyping, copy number variation analysis, and verification of reprogramming vector clearance[37,38,70]. While these assays are essential for confirming pluripotent stem cell identity, they do not ensure that a line can consistently generate enteric neural crest derivatives. This distinction is particularly critical in HSCR, as disease-relevant phenotypes may arise from subtle differences in ENCC migration, proliferation, or neuronal differentiation.

Thus, the characterization of HSCR iPSC lines should extend beyond general pluripotency assessment. In addition to confirming pluripotency, genomic stability, and vector clearance, investigators should verify that each line retains a stable capacity for enteric lineage differentiation across independent batches. This platform-level quality control is important for reducing variation introduced by donor cell source, reprogramming method, clone selection, or early culture expansion[71,72]. More detailed criteria for defining iPSC-derived ENCC identity and function are discussed in the following model sections.

IPSC-BASED MODELS RELEVANT TO HSCR RESEARCH

Patient-derived iPSCs offer a human cellular system for studying developmental processes directly relevant to HSCR, such as ENCC migration, proliferation, neuronal differentiation, and interactions with the intestinal microenvironment. However, available iPSC-based models differ in their disease relevance, experimental readouts, and intended applications. Some have been directly used to study HSCR patient-derived cells or HSCR-associated variants, whereas others were developed mainly in normal pluripotent stem cell systems and remain relevant as emerging experimental platforms. In this review, these models are discussed in three groups: Two-dimensional iPSC-derived ENCC and enteric neuron models, three-dimensional recombined or co-culture organoid models, and emerging integrated or bioengineered gut platforms with potential relevance to HSCR. This organization helps clarify which models are most suitable for studying cell-intrinsic defects, tissue-level interactions, or future functional validation, while also indicating where HSCR-specific evidence remains limited[32] (Figure 3).

Figure 3 Induced pluripotent stem cell-based modeling platforms relevant to Hirschsprung disease research. The figure summarizes three major categories of human induced pluripotent stem cell (iPSC)-based and emerging gut models used to study Hirschsprung disease (HSCR). In two-dimensional models, HSCR patient-derived iPSCs are directed toward enteric neural crest cells (ENCCs) through neural crest induction using developmental signaling modulation, such as bone morphogenetic protein inhibition, transforming growth factor-beta inhibition, and WNT pathway activation. ENCCs can then be differentiated into enteric neurons and assessed using cellular phenotypes such as migration and neuronal differentiation. In three-dimensional recombined organoid models, iPSC-derived gut epithelial spheroids or intestinal and colonic organoids are combined with ENCCs to generate innervated organoid systems. These models allow assessment of ENCC colonization, neuroepithelial interaction, in vivo maturation after transplantation, and tissue-level readouts such as contractile responses. Emerging integrated and bioengineered gut platforms include de novo innervated gut tissues and intestine-enteric nervous system microphysiological systems. These platforms may provide additional opportunities to study epithelial-neural communication, mechanical stimulation, barrier function, and dynamic multicellular interactions. Overall, these model systems are complementary rather than hierarchical, and each should be selected according to the biological question and required functional endpoint. 2D: Two-dimensional; 3D: Three-dimensional; HSCR: Hirschsprung disease; iPSC: Induced pluripotent stem cell; BMP: Bone morphogenetic protein; TGF: Transforming growth factor; ENCC: Enteric neural crest cell; GDNF: Glial cell line-derived neurotrophic factor; L-AA: L-ascorbic acid; EGF: Epidermal growth factor; FGF4: Fibroblast growth factor 4; RSPO1: R-spondin 1; HIOs: Human intestinal organoids; HCOs: Human colonic organoids.

Two-dimensional iPSC-derived ENCC and enteric neuron models

Two-dimensional cultures of iPSC-derived ENCCs remain the most widely used and experimentally feasible system for investigating cell-autonomous defects in HSCR[73]. Directed differentiation protocols typically mimic key developmental stages of neural crest induction, vagal patterning, and enteric lineage specification through the staged modulation of bone morphogenetic protein (BMP), WNT, transforming growth factor-beta, and retinoic acid signaling[29,74]. A rapid differentiation protocol developed by Gogolou et al[75] generates vagal neural crest-like cells within six days, with these cells expressing early neural crest markers, including SOX10, PAX3, PAX7, and p75NTR. Subsequent exposure to retinoic acid induces the expression of vagal HOX genes (HOXB4, HOXB5) and ENS progenitor markers such as ASCL1 and PHOX2B[76]. These stepwise strategies have since been applied in HSCR-related research[77].

For HSCR modeling, reproducible ENCC differentiation and standardized identity criteria are essential. The identity of iPSC-derived ENCCs should be defined not only by lineage marker expression, but also by disease-relevant cellular behavior. These criteria may include neural crest and enteric lineage markers, differentiation efficiency, migratory capacity, proliferative behavior, neuronal differentiation potential, and neurite outgrowth[78]. Because HSCR-associated phenotypes are often quantitative rather than binary, modest differences in differentiation efficiency among iPSC lines, clones, or batches may affect phenotypic interpretation. Standardized protocols and quantitative quality control are therefore needed to distinguish genotype-associated effects from technical variation.

Several iPSC-based studies have demonstrated the value of ENCC models in mechanistic research. For instance, studies from the University of Hong Kong revealed that RET dosage influences ENCC behavior. Heterozygous or homozygous RET deletion in control iPSCs impaired ENCC migration and neuronal differentiation, whereas correction of RET-associated defects in patient-derived iPSCs improved these phenotypes[26]. Similar approaches have been employed to investigate other candidate genes, such as VCL[79], which is implicated in ENCC development, and BACE2, whose disruption is linked to abnormal ENCC differentiation via a proposed BACE1-APP-BACE2 axis[80]. CRISPR/Cas9-mediated correction of RET and VCL variants in patient-derived iPSCs has provided direct functional evidence linking candidate variants to impaired ENCC migration and neuronal differentiation. These studies also illustrate how genome editing can generate isogenic controls and help separate variant-specific effects from broader genetic-background effects[26]. More recently, Li et al[27] utilized single-cell transcriptomic analysis of patient-derived ENCCs, identifying shared transcriptional changes involving HDAC1-associated regulatory programs and oxidative phosphorylation.

These findings suggest that HSCR-related ENCC dysfunction may reflect the combined effects of genetic susceptibility, transcriptional and epigenetic regulation, and metabolic disturbance, rather than isolated gene defects alone. RET, EDNRB, SOX10, PHOX2B, and related developmental regulators influence ENCC identity, survival, migration, and responsiveness to intestinal trophic cues, while HDAC-associated transcriptional regulation may shape ENCC responses to these developmental signals. In parallel, oxidative phosphorylation and mitochondrial stress responses may influence the energy-dependent processes required for ENCC migration, proliferation, and differentiation[81]. Broader work on oxidative stress biology may help interpret this metabolic vulnerability[82,83], although direct causal links between oxidative stress and ENCC developmental failure in human HSCR remain insufficiently defined.

Overall, two-dimensional iPSC-derived ENCC models are useful for identifying cell-intrinsic defects, testing candidate genes, generating isogenic controls, and evaluating early developmental phenotypes. However, improvements in migration or differentiation in vitro do not necessarily indicate restoration of tissue-level ENS function. Most HSCR cases are not caused by a single pathogenic variant, and the effect of a given variant may depend on the broader genetic background. These models are therefore best suited for mechanistic analysis and candidate gene validation, rather than for fully recapitulating the complexity of HSCR pathogenesis.

Genetic complexity and implications for iPSC-based HSCR modeling

HSCR should not be viewed as a uniform monogenic disorder[11,12]. Although pathogenic variants in genes such as RET, EDNRB, SOX10, PHOX2B, EDN3, and other developmental regulators have provided important mechanistic insights[7,9,14], most sporadic HSCR cases are considered oligogenic or multifactorial. For iPSC-based disease modeling, this means that a phenotype observed in a patient-derived iPSC line may not reflect the effect of a single candidate variant alone. Modifier genes, non-coding regulatory variants, epigenetic background, and interactions among susceptibility loci may all contribute to the observed ENCC phenotype[14,18,84,85]. This complexity may help explain why patients carrying similar pathogenic variants can differ in disease severity, aganglionic segment length, or associated clinical manifestations.

Patient-derived iPSCs are valuable for HSCR modeling because they preserve the donor-specific genetic background, including coding and non-coding variants. This allows ENCC development to be examined in a more patient-relevant context, but it also complicates causal interpretation. Impaired ENCC migration, proliferation, or neuronal differentiation in a patient-derived iPSC line may be driven by the variant under study, additional modifier variants, polygenic burden, epigenetic differences, or clonal changes acquired during reprogramming and culture. Single-patient iPSC models and single-gene perturbation systems should therefore be interpreted as mechanistic tools, rather than as models that fully represent the genetic heterogeneity of HSCR.

To address this genetic complexity, future studies should combine isogenic gene-edited controls with larger panels of patient-derived iPSC lines. Isogenic controls are useful for testing the effect of individual variants under a matched genetic background, whereas patient-derived cohorts are needed to capture inter-patient variability, incomplete penetrance, and polygenic risk. Genetic analysis alone, however, is unlikely to explain all disease-relevant phenotypes. Integrating single-cell transcriptomics, chromatin accessibility profiling, DNA methylation analysis, spatial transcriptomics, and developmental gene regulatory network reconstruction may help clarify how genetic susceptibility, epigenetic state, ENCC identity, metabolic activity, and tissue microenvironmental cues converge during ENS development[20,86,87]. Such systems-level approaches are particularly important for oligogenic HSCR, in which disease phenotypes may arise from the cumulative effects of multiple modest-risk variants and background-dependent regulatory changes.

Functional maturation and electrophysiological validation

A major limitation of current iPSC-based HSCR models is that marker-defined differentiation does not necessarily indicate functional maturation. Neural crest and neuronal markers, such as SOX10, PHOX2B, p75NTR, RET, TUJ1, and HuC/D, are useful for tracing lineage progression, but these markers alone cannot demonstrate that derived cells have acquired mature enteric neuronal properties. Recent studies on iPSC-derived enteric neurons further support the need to evaluate electrophysiological properties, neuronal subtype specification, synaptic connectivity, and network-level behavior rather than relying only on lineage marker expression. Mature ENS function also depends on neurotransmitter responsiveness and coordinated interaction with smooth muscle and epithelial compartments.

Electrophysiological assays therefore represent an important component of functional validation. Patch-clamp recording can directly evaluate resting membrane potential, action potential firing, ion-channel activity, and synaptic currents[88,89]. Multi-electrode array platforms may further provide information about network-level activity and maturation[90]. Calcium imaging is widely used because it allows dynamic assessment of neuronal activity in living cultures and organoids. However, calcium transients mainly reflect intracellular calcium dynamics and cellular responsiveness and should not be interpreted as a substitute for direct electrophysiological evidence of action potentials, synaptic transmission, or neuronal subtype-specific function[91].

A more reliable assessment of HSCR models will require integrating marker-based assays with electrophysiological and tissue-level functional readouts. In addition to migration assays and lineage markers, iPSC-derived ENCCs and enteric neurons should be evaluated using neuronal subtype markers, synaptic protein expression, neurotransmitter responsiveness, patch-clamp or multi-electrode array recordings, calcium imaging, neuromuscular co-culture systems, and contraction-based assays in organoids or engineered intestinal tissues. Combining these readouts would help determine whether improved ENCC migration or neuronal differentiation reflects functionally meaningful restoration of enteric neural activity.

Three-dimensional recombined or co-culture organoid models

ENS development occurs within the intestinal microenvironment, shaped by interactions among epithelial, mesenchymal, smooth muscle, immune, and extracellular matrix components[92,93]. Compared with monolayer cultures, three-dimensional intestinal organoids provide a more suitable setting for studying these tissue interactions. In recombined or co-culture models, intestinal or colonic organoids derived from pluripotent stem cells are combined with separately generated neural crest cells or ENCCs. This strategy allows investigators to examine ENCC colonization, neuroepithelial interaction, neuromuscular patterning, and tissue-level maturation in a more physiologically relevant setting[94,95].

iPSC-derived intestinal organoids are typically generated through sequential differentiation into definitive endoderm, hindgut, and intestinal lineages[30]. When combined with iPSC-derived ENCCs, these organoids form innervated intestinal tissues displaying features such as epithelial organization, smooth muscle development, and neuron-dependent contractile activity, particularly after in vivo maturation following transplantation[96-98]. Given that HSCR predominantly affects the distal bowel, human colonic organoids may provide a more relevant tissue context than intestinal organoids. Transient BMP activation has been employed to guide pluripotent stem cells toward a colonic identity[31]. Incorporating HSCR patient-derived ENCCs into colonic organoids could help explore region-specific defects in ENCC colonization, neuromuscular development, and epithelial-mesenchymal signaling[99].

Recombined organoid models are particularly valuable for studying non-cell-autonomous mechanisms in HSCR. Workman et al[96] combined wild-type or PHOX2B-mutant ENCCs with wild-type intestinal organoids and showed that PHOX2B perturbation affected both ENCC development and mesenchymal differentiation in the surrounding tissue. This finding supports the view that HSCR pathogenesis cannot be explained by ENCC-intrinsic defects alone. Altered developmental regulators may also disrupt reciprocal signaling between ENCCs and surrounding epithelial, mesenchymal, extracellular matrix, or neuromuscular compartments during intestinal colonization.

Organoid models also enable tissue-level readouts that are difficult to capture in monolayer cultures, including tissue organization, ENCC colonization, neuromuscular patterning, calcium responsiveness, and contractile responses. These readouts, however, should be interpreted with caution. Contractile activity or calcium responsiveness alone does not necessarily demonstrate mature ENS function and should ideally be supported by evidence of neuronal subtype specification, synaptic connectivity, electrophysiological or network-level activity, and coordinated neuromuscular transmission. In addition, current organoid systems remain limited by developmental immaturity, batch-to-batch variability, and incomplete standardization across laboratories. Accordingly, three-dimensional recombined or co-culture organoid models are best viewed as platforms for tissue-level mechanistic studies and functional validation, rather than as fully mature models of HSCR pathophysiology or reliable predictors of therapeutic efficacy.

Emerging integrated and bioengineered gut platforms with potential relevance to HSCR

In addition to recombined organoids, newer human gut models aim to generate or organize multiple intestinal lineages within more integrated systems. These include de novo innervated gut tissues, in which enteric neuroglial populations arise during coordinated differentiation or tissue engineering, as well as organ-on-chip and intestine-ENS microphysiological systems that incorporate spatial organization, fluid flow, mechanical stimulation, and multicellular interactions[100]. Compared with conventional recombined organoids, these approaches may better capture tissue scale, regional organization, neuromuscular maturation, and dynamic epithelial-neural communication.

Recent differentiation systems have generated complex human intestinal organoids containing epithelial, mesenchymal, enteric neuroglial, endothelial, and organized smooth muscle components within a single differentiation framework. Transient spheroid confinement-based approaches have also been used to generate large-scale, elongated, and functional human small intestinal, colonic, and gastric tissues with de novo ENS development. These systems may reduce the need for post hoc assembly of neural and non-neural components and may improve tissue size, organization, and maturation. In parallel, gut-on-chip and intestine-ENS chip platforms offer controlled settings for studying neuroepithelial communication, barrier function, mechanical stimulation, fluid flow, and multicellular responses to environmental cues. Recent microfluidic systems have enabled compartmentalized co-culture of enteric neurons and intestinal epithelial cells, providing a useful approach for studying gut neuroepithelial communication under defined spatial conditions.

These emerging systems need to be interpreted cautiously in HSCR research. Most de novo innervated gut tissues and organ-on-chip models have been developed using normal pluripotent stem cells or non-HSCR experimental settings, and their ability to reproduce patient-specific HSCR phenotypes remains unclear. It is not yet known whether they can reliably model ENCC migration failure, oligogenic risk, variable penetrance, distal bowel aganglionosis, or long-term neuromuscular dysfunction. They are also technically demanding, costly, relatively low-throughput, and require further standardization of cell composition, maturation state, and functional readouts. At this stage, these systems are better viewed as experimental tools for extending functional studies, rather than established patient-specific HSCR disease models.

Taken together, the current models are best viewed as complementary tools rather than as a linear hierarchy of increasing sophistication. Two-dimensional ENCC cultures remain the most practical option for analyzing cell-intrinsic defects, testing candidate genes, and conducting early compound screens. Recombined or co-culture organoids add a tissue context and are better suited for studying ENCC colonization, neuroepithelial communication, and neuromuscular patterning, although their results depend on the timing, efficiency, and proportion of ENCC incorporation. Integrated organoids and bioengineered gut systems may further expand tissue-level and dynamic functional readouts, but most have not yet been validated with patient-specific HSCR cells or variants. Model choice should therefore be guided by the biological question and the required endpoint, while taking into account reproducibility, physiological relevance, throughput, cost, and translational feasibility (Table 1).

Table 1 Comparison of induced pluripotent stem cell-based and emerging gut modeling platforms relevant to Hirschsprung disease research.

Comparison domain	2D iPSC-derived ENCC models	3D recombined/co-culture organoid models	Emerging integrated/bioengineered platforms
Model design	ENCCs or enteric neurons in monolayer culture	Gut organoids combined with ENCCs	Integrated organoids, de novo innervated tissues, or gut-on-chip systems
HSCR-specific validation	Relatively established	Moderate	Limited
Main use	Cell-autonomous defects and candidate gene testing	ENCC colonization and tissue-level interaction	Dynamic multicellular interaction and future translational modeling
Reproducibility/scalability	Relatively high; scalable	Moderate to low; less scalable	Variable; technically demanding
Physiological relevance	Low to moderate	Moderate to high	Potentially high, but not yet validated in HSCR
Functional/drug-screening value	Migration, proliferation, differentiation, early compound testing	Neuromuscular patterning, calcium activity, contractile response	Barrier function, flow, mechanical cues, exploratory testing
Major limitations	Lacks tissue context and mature ENS function	Batch variability and incomplete maturation	High cost, low throughput, limited HSCR-specific evidence

2D: Two-dimensional; iPSC: Induced pluripotent stem cell; ENCC: Enteric neural crest cell; 3D: Three-dimensional; HSCR: Hirschsprung disease; ENS: Enteric nervous system.

DRUG SCREENING AND TARGET EVALUATION USING IPSC-BASED HSCR MODELS

Pharmacological treatment is not currently a primary therapeutic strategy for HSCR. The hallmark pathological feature of HSCR is the absence of enteric ganglia in the distal bowel, resulting from abnormal ENS development during embryogenesis[1]. Once aganglionosis is established, it cannot be reversed by available postnatal drug therapies.

In this context, iPSC-based models are primarily used to evaluate whether disease-associated developmental defects can be modulated in vitro. Patient-derived ENCCs offer a human cellular system for studying key processes in HSCR pathogenesis, including ENCC migration, proliferation, and neuronal differentiation. Such studies can help identify disease-relevant pathways and nominate candidate compounds for further validation, rather than directly defining clinically applicable drug treatments[101].

Several studies have utilized iPSC-derived ENCCs to evaluate candidate compounds in disease-relevant genetic contexts[73]. Research from the University of Hong Kong demonstrated that ENCCs derived from iPSCs could be used to assess whether pharmacological treatments improve migration or neuronal differentiation[27]. These findings suggest that some HSCR-associated cellular defects are responsive to pharmacological modulation in vitro. However, the number of compounds tested in iPSC-based HSCR models remains limited, and most studies have focused on early developmental phenotypes rather than mature intestinal function.

A major limitation of current drug screening studies is the absence of validated functional endpoints. Commonly used readouts, such as migration distance, migration velocity, neurite outgrowth, and expression of neuronal or ENCC markers, are valuable for detecting cellular phenotypes. However, their correlation with clinically meaningful outcomes, such as bowel motility, neuromuscular coordination, or postoperative functional recovery, remains unclear. Statistically significant improvements in these assays may not necessarily reflect therapeutic benefits in vivo.

Future drug-related applications of iPSC-based HSCR models will require more predictive and standardized assays. Two-dimensional ENCC models may be useful for early-stage compound screening because they are relatively scalable and compatible with quantitative cellular readouts. Three-dimensional organoid or engineered gut systems may serve as secondary validation platforms for assessing tissue-level responses, including ENCC colonization, neuromuscular patterning, calcium activity, and contractile behavior. However, these readouts still require further standardization and functional correlation with in vivo outcomes. At present, iPSC-based drug screening in HSCR should be viewed as an exploratory approach for evaluating mechanism-based targets, rather than a direct path to clinical pharmacotherapy.

IPSC-BASED CELL THERAPY AND TRANSLATIONAL PERSPECTIVES IN HSCR

HSCR is characterized by the absence of enteric ganglia in the distal bowel, making enteric neural cell replacement an attractive experimental concept[24,102]. Over the past two decades, the transplantation of enteric neural crest-derived cells into aganglionic bowel has been explored in experimental models[24,103]. iPSC technology may provide a renewable source of patient-specific or donor-derived ENCCs, enabling these cells to be generated, expanded, characterized, and potentially modified under controlled conditions[88].

At present, no iPSC-derived ENCC therapy has entered clinical trials for HSCR. Current evidence comes from in vitro systems, ex vivo human tissue studies, and animal models. These studies support the biological feasibility of ENCC replacement, but they do not establish clinical efficacy or long-term safety. Durable engraftment, directional migration, subtype-appropriate neuronal differentiation, synaptic integration, and coordinated interactions with smooth muscle and epithelial compartments remain insufficiently validated. For this reason, iPSC-derived ENCC transplantation should be viewed as an experimental approach for studying ENS repair rather than a therapy ready for clinical use.

The therapeutic target may extend beyond the aganglionic segment, as studies have revealed abnormalities in the proximal ganglionated bowel of HSCR patients, such as altered neuronal subtypes, neurochemical coding, and contractile properties[104-106]. These findings suggest that future cell-based therapies may need not only to replace missing enteric neurons but also to modulate the function of the remaining intestine. Before any target product profile can be defined, several questions remain unresolved, including disease pathology, cell identity, delivery route, tissue integration, and long-term safety[40,107-110]. The following sections discuss the main strategies under investigation and the barriers that currently limit their clinical application (Figure 4).

Figure 4 Translational strategies and major barriers for induced pluripotent stem cell-based cell therapy in Hirschsprung disease. This schematic illustrates two major preclinical strategies for induced pluripotent stem cell (iPSC)-based enteric neural cell replacement. In autologous cell therapy, somatic cells are obtained from an individual patient, reprogrammed into patient-derived iPSCs, genetically modified when appropriate through gene correction or chimeric antigen receptor (CAR) transduction, differentiated into enteric neural crest cells (ENCCs), subjected to quality control, and then considered for transplantation back into the patient. This approach offers potential advantages in genetic matching and reduced immune rejection, but it remains individualized, time-consuming, and difficult to scale. In allogeneic or universal donor strategies, donor-derived iPSC lines may undergo genetic engineering, including gene correction or CAR transduction, before differentiation into ENCC products that may be preserved and used as off-the-shelf cell sources. Regardless of the source, iPSC-derived ENCC products require rigorous preclinical evaluation before clinical application. Key translational barriers include cell identity, purity, and potency; genomic stability and absence of residual pluripotent cells; tumorigenicity and ectopic differentiation; and durable engraftment, migration, and neuromuscular integration within the host bowel. These requirements are particularly important in the pediatric setting, where long-term safety, intestinal growth, and tissue remodeling must be considered before clinical translation can be attempted. iPSC: Induced pluripotent stem cell; ENCC: Enteric neural crest cell; CAR: Chimeric antigen receptor.

Autologous gene-corrected iPSC-ENCC transplantation

Autologous gene-corrected transplantation is a personalized approach in which iPSCs are derived from a patient, genetically corrected via genome editing, differentiated into ENCCs, and transplanted back into the affected bowel. This strategy offers potential advantages, including genetic matching and a reduced risk of immune rejection. It may also enable the correction of pathogenic variants before transplantation in patients with defined monogenic defects. Lai et al[26] demonstrated the feasibility of this approach by correcting RET-associated defects in patient-derived iPSCs, resulting in improved ENCC migration and neuronal differentiation in vitro.

Despite this proof-of-concept evidence, autologous gene-corrected iPSC-ENCC transplantation remains far from clinical use. For each patient, the workflow would involve reprogramming, genome editing, clonal selection, directed differentiation, and extensive quality control, making the process costly and difficult to scale. The use of gene editing also raises safety concerns, including off-target effects, chromosomal alterations, and clonal instability[108]. Beyond genetic correction, the final ENCC product would need to show stable lineage identity, sufficient purity, controlled proliferative capacity, and the ability to engraft, migrate, differentiate, and functionally integrate within the host bowel. Patient selection represents another major constraint. This approach is most relevant to patients carrying clearly defined pathogenic variants in single genes, whereas most HSCR cases, especially sporadic short-segment disease, are likely oligogenic or multifactorial. In such cases, the causative defects may not be fully identified, which makes targeted correction difficult. Therefore, autologous gene-corrected iPSC-ENCC transplantation is best viewed as a proof-of-concept platform for studying gene repair and cell replacement, rather than as a broadly applicable clinical strategy for HSCR at present[111].

Allogeneic or universal iPSC-based cell sources

Allogeneic iPSC-derived ENCCs may offer a more scalable alternative to patient-specific autologous products[112]. Potential approaches include HLA-matched iPSC banks, HLA-edited universal donor lines, and hypoimmunogenic iPSC lines[41,113,114]. In theory, these approaches could provide a more readily available source of ENCCs for HSCR research and future preclinical development.

However, immune compatibility alone is insufficient to define a safe and clinically applicable product. Donor-derived or engineered iPSC lines would still need to demonstrate genomic stability, reproducible ENCC differentiation, absence of residual undifferentiated cells, and stable enteric lineage identity after expansion. After transplantation, the derived ENCCs would also need to survive, migrate, avoid ectopic differentiation or abnormal growth, and integrate with host neuromuscular circuits in the developing intestine.

The pediatric setting adds another layer of complexity. Most patients with HSCR undergo surgery early in life, and the risk-benefit balance of allogeneic cell therapy, especially if immunosuppression is required, may differ from that of adult regenerative medicine applications. Further preclinical studies are therefore needed to determine whether allogeneic iPSC-derived ENCCs can provide durable functional benefit with an acceptable long-term safety profile.

Tissue engineering and organoid-based regenerative approaches

Tissue engineering represents another potential strategy for HSCR treatment[115-119]. Instead of delivering dissociated or clustered ENCCs into the aganglionic bowel, this approach aims to generate innervated intestinal tissue that could replace or support the affected bowel segment. iPSC-derived intestinal or colonic organoids may supply epithelial and mesenchymal components, while iPSC-derived ENCCs provide the neural component necessary for ENS formation.

Several studies have demonstrated that iPSC-derived intestinal tissues, combined with ENCCs, can form multilayered structures containing polarized epithelium, smooth muscle, and enteric neurons[99]. More recent differentiation systems have generated epithelial, mesenchymal, ENS, endothelial, and smooth muscle lineages within a single iPSC-derived organoid platform[120]. These advancements suggest that iPSC-based organoid systems can model selected aspects of intestinal neuromuscular development and may provide experimental platforms for evaluating future regenerative concepts. Nevertheless, they should not be interpreted as evidence that organoid-based intestinal replacement is close to clinical application.

Several practical barriers include vascularization, tissue scale-up, mechanical strength, luminal organization, peristaltic coordination, and surgical integration with the host bowel. Functional connection between the engineered tissue and the host enteric and extrinsic nervous systems remains underdeveloped. Additionally, current organoid systems are developmentally immature and exhibit batch-to-batch variability. Consequently, organoid-based regenerative therapy for HSCR should currently be regarded as a preclinical research avenue rather than a near-term clinical option.

Preclinical evidence, safety, and translational barriers

Preclinical studies have demonstrated that transplanted ENCCs can survive, migrate, and differentiate within aganglionic bowel in animal models[121-125]. Some reports also suggest partial improvement in intestinal motility or contractile responses[77]. While these findings support the feasibility of ENCC transplantation, they do not yet establish clinical efficacy. Most evidence still comes from small-animal models, which cannot fully address delivery to longer bowel segments, cell distribution within the intestinal wall, survival under luminal and peristaltic stress, integration with host neuromuscular circuits, or procedure-related complications[126]. Large-animal studies will therefore be needed before these approaches can be evaluated in a clinically relevant setting.

Safety remains a central concern. iPSC-derived products may contain residual undifferentiated cells or highly proliferative progenitors, and genomic alterations may arise during reprogramming, expansion, gene editing, or differentiation. Long-term studies are needed to assess tumorigenicity, ectopic differentiation, inappropriate lineage commitment, and stability of the enteric phenotype after transplantation. The recipient bowel environment may further influence transplanted cell behavior. Local inflammation, fibrosis, microbial cues, resident immune cells, and postoperative remodeling could affect cell survival, migration, differentiation, and integration.

Cellular heterogeneity adds another layer of uncertainty. Although evidence from cancer biology and broader translational studies cannot be directly extrapolated to HSCR, it illustrates that cell behavior may vary with genetic background, tissue context, intercellular communication, and disease state[127,128]. For iPSC-derived ENCC or organoid-based products, differences in cell composition, maturation state, proliferative capacity, and lineage stability may influence engraftment, immune recognition, ectopic growth, and long-term function. Preclinical studies should therefore assess not only engraftment and neuronal marker expression, but also lineage stability, proliferative behavior, immune compatibility, tissue-context-dependent responses, patient-specific variability, and durable functional recovery.

Delivery and integration also remain unresolved. A clinically useful ENCC product would need to survive delivery, migrate over an appropriate distance, distribute within the target bowel wall, differentiate into appropriate enteric neuronal and glial populations, and functionally integrate with host smooth muscle, epithelial compartments, and neural circuits. These requirements are particularly important in the developing pediatric intestine, where growth and tissue remodeling may alter long-term safety and function.

Regulatory, manufacturing, and pediatric considerations

Biological proof of concept is only one part of the translational pathway. Before clinical testing could be considered, any iPSC-derived ENCC product for HSCR would need a clearly defined manufacturing and regulatory strategy. Clinical-grade production would require Good Manufacturing Practice-compatible and preferably xeno-free culture conditions, validated differentiation and expansion protocols, and predefined release criteria. These criteria should include cell identity, purity, potency, viability, sterility, genomic stability, absence of residual pluripotent cells, controlled proliferative capacity, and evidence supporting enteric lineage maturation. For gene-edited products, additional testing would be needed to assess off-target changes, chromosomal rearrangements, clonal selection effects, and post-editing stability.

The pediatric context creates further practical constraints. Many patients with HSCR undergo surgery early in life, which may limit the feasibility of individualized autologous products that require reprogramming, gene correction, clonal selection, expansion, differentiation, and release testing. Universal donor or HLA-edited iPSC lines may improve availability and scalability, but they also raise questions regarding immune evasion, immune surveillance, genomic stability, and long-term safety. Future development would therefore require standardized potency assays, batch-release criteria, product traceability, and post-transplant surveillance plans.

Ethical considerations are closely linked to the pediatric setting. Genome editing of patient-derived iPSCs raises questions about the acceptable extent of genetic manipulation, uncertainty around off-target effects, and responsibility for long-term follow-up. Even when transplanted cells are intended for somatic repair rather than germline modification, extended monitoring would be needed to evaluate tumorigenicity, ectopic differentiation, immune responses, and durable neuromuscular integration during intestinal growth and development.

Economic feasibility represents another important consideration. Autologous gene-corrected iPSC products are individualized, time-consuming, and costly, whereas universal donor strategies may reduce production time but introduce additional regulatory and safety uncertainties. Thus, the clinical translation of iPSC-based therapy for HSCR will depend not only on improved disease modeling and cell biology, but also on realistic manufacturing, regulatory, ethical, and economic planning. At present, iPSC-derived ENCC transplantation should remain framed as a research and preclinical development platform until standardized manufacturing, robust functional assays, long-term integration, and stringent safety criteria are established.

CURRENT CHALLENGES AND FUTURE DIRECTIONS

Although iPSC-based approaches have advanced HSCR research, several limitations hinder their broader application. A major challenge is the developmental immaturity of current iPSC-derived ENCCs and intestinal organoids. Most models resemble embryonic or fetal stages of ENS and intestinal development, while many clinically relevant outcomes, such as bowel motility, postoperative obstruction, constipation, and enterocolitis, are observed in the postnatal intestine. Improved maturation strategies are necessary to generate models that better reflect human intestinal neuromuscular function.

Another limitation is the incomplete representation of HSCR’s genetic complexity. Many existing studies focus on single-gene perturbations, selected monogenic cases, or small numbers of patient-derived iPSC lines. While these approaches are valuable for mechanistic analysis, they may not fully capture the oligogenic and multifactorial inheritance typical of most sporadic short-segment HSCR cases. Larger and genetically diverse iPSC cohorts, together with isogenic gene-edited controls, will be needed to distinguish variant-specific effects from broader genetic-background effects. Multi-omics, chromatin accessibility analysis, DNA methylation profiling, spatial transcriptomics, and developmental regulatory network analysis may further help connect genotype, epigenetic state, ENCC cell state, tissue context, and functional phenotype.

Reproducibility and functional validation also remain major challenges. Variability may arise from donor background, somatic cell source, reprogramming method, iPSC clone, passage number, differentiation batch, culture matrix, growth factor activity, and laboratory-specific protocols. Standardized criteria for iPSC-derived ENCC identity should include not only neural crest and enteric lineage markers, but also migratory capacity, proliferative behavior, neuronal and glial differentiation potential, and reproducibility across independent batches. Functional validation should move beyond surrogate readouts such as marker expression, migration distance, neurite outgrowth, calcium activity, or contraction-related responses. Future studies should combine these assays with patch-clamp recording, multi-electrode array analysis, neuronal subtype profiling, synaptic marker assessment, neurotransmitter responsiveness, neuromuscular co-culture systems, and tissue-level motility assays.

For translational applications, safety and manufacturing barriers remain unresolved. iPSC-derived ENCC products would require stable genomic integrity, high purity, reproducible potency, minimal risk of residual undifferentiated cells, and durable integration with host intestinal circuits. Clinical translation should therefore proceed cautiously and only after large-animal validation, Good Manufacturing Practice-compatible production, clear regulatory pathways, long-term pediatric safety monitoring, and economically feasible manufacturing strategies have been established.

CONCLUSION

Patient-derived iPSC platforms provide a valuable human system for studying early ENCC development and ENS formation in HSCR. By preserving patient-specific genetic backgrounds and enabling differentiation into ENCCs, enteric neurons, and intestinal organoid systems, these models help connect genetic findings with disease-relevant cellular defects, including altered ENCC migration and differentiation, gene-regulatory programs, metabolic vulnerability, and ENCC-microenvironment interactions.

At present, the main value of iPSC-based HSCR models lies in mechanistic investigation and preclinical model development. Their use in drug screening and regenerative medicine remains exploratory. Important limitations include developmental immaturity, inter-line and inter-batch variability, incomplete modeling of oligogenic and background-dependent disease architecture, limited functional validation, and unresolved safety and manufacturing requirements. Addressing these limitations will require more mature and standardized models, genetically diverse patient-derived cohorts, and rigorous long-term preclinical validation. Rather than being viewed as a direct route to clinical application, iPSC-based platforms should be used as complementary systems for linking patient genetic background, ENCC developmental phenotypes, tissue-level interactions, and realistic translational barriers.