Induced Pluripotent Stem Cells (iPSC) and Their Use in Disease Modeling

TL;DR

This paper reviews how induced pluripotent stem cells are cultured and used to model diseases and develop personalized therapies.

Contribution

The paper provides an updated overview of iPSC methodologies and their applications in disease modeling and regenerative medicine.

Findings

iPSCs can be reprogrammed from various somatic cell types and differentiated into patient-specific cell types.

Pluripotency is induced using four transcription factors: Oct4, Sox2, Klf4, and c-Myc.

iPSCs offer opportunities for disease modeling and drug screening but face challenges like genomic instability and lack of standardized protocols.

Abstract

This paper reviews methodologies for culturing induced pluripotent stem cells (iPSCs) and highlights their applications in disease modeling and regenerative medicine. iPSCs can be reprogrammed from multiple somatic cell sources, including keratinocytes, fibroblasts, peripheral blood mononuclear cells, and urinary epithelial cells. Their ability to differentiate into patient-specific cell types provides unique opportunities to model neurodegenerative, cardiovascular, metabolic, and autoimmune disorders in vitro. Pluripotency is typically induced by the overexpression of four canonical transcription factors-Oct4, Sox2, Klf4, and c-Myc. iPSC culture is technically demanding, as the cells display genomic and epigenetic instability and require tightly controlled microenvironmental conditions to maintain viability and pluripotency. Rigorous quality control, including PCR-based assays and genomic integrity analyses, is essential. Advances in iPSC technology have enabled personalized disease modeling, mechanistic studies of pathogenesis, drug screening, and the development of precision therapies. Despite their translational promise, iPSCs remain limited by issues of genomic instability, clinical safety, and the lack of standardized culture protocols.

Tables

Table 1. The use of iPSCs in the above diseases.DMD: Duchenne muscular dystrophy; SLE: systemic lupus erythematosus; MS: multiple sclerosis

Disease		Use of iPSCs
Neurodegenerative disease	Alzheimer's disease	Studying the effect of mutations on increased tau protein phosphorylation and beta-amyloid accumulation to find effective treatments [4,44,45]
	Parkinson's disease	Studying the accumulation of alpha-synuclein in neurons to develop new therapies [3,46,47]
Heart Disease	Arrhythmias	The effect of mutations in the KCNQ1 gene on the development of congenital cardiac arrhythmias [48-50]
	Myocardial damage	Replacement transplantation of the resulting cardiomyocytes, fibroblasts, vascular smooth muscle, or endothelial cells [1,51-53]
Metabolic diseases	Cystic fibrosis	Reproduction of chloride transport defects and excessive mucus production caused by CFTR mutations and testing of new drugs, i.e., ivacaftor and lumacaftor [55-57]
	DMD	The dystrophin gene mutation and the mechanisms of muscle fiber degeneration were investigated, allowing for the development of new therapies [58-60]
	Wilson's disease	The ATP7B gene mutations were investigated, allowing for the testing of new drugs and the assessment of their effectiveness [61-63]
Autoimmune diseases	SLE	Investigation of defective B and T lymphocyte regulation and interactions with antigen-presenting cells [65]
	Rheumatoid arthritis	Obtaining fibroblast-like synoviocytes, which enabled the study of new signaling pathway inhibitors [66,67]
	Type I diabetes	Creation of functional insulin-producing cells and replication of the autoimmune attack on pancreatic islets in vitro [68,69]
	MS	Creation of oligodendrocytes and replication of remyelination and demyelination in vitro [70,71]

Full text

Introduction and background

Induced pluripotent stem cells (iPSCs) are somatic cells that have been genetically reprogrammed to acquire the defining properties of embryonic stem cells (ESCs). Reprogramming is achieved through the introduction of four transcription factors, Oct4, Sox2, Klf4, and c-Myc, which restore pluripotency and self-renewal capacity. iPSCs exhibit key stem cell characteristics, including ESC-like morphology, expression of pluripotency-associated transcriptional networks, specific surface antigen profiles, the ability to differentiate into derivatives of the three germ layers, and virtually unlimited proliferative potential. The development of iPSC technology by Shinya Yamanaka and colleagues in 2006 at Kyoto University represented a paradigm shift in regenerative medicine. By offering an ethically acceptable alternative to ESCs, iPSCs have provided an invaluable platform for basic biomedical research, disease modeling, pharmacological testing, and personalized cell-based therapies. In recognition of this groundbreaking discovery, Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medicine [1]. We reviewed papers from oldest to newest, published in the most respected medical databases, and selected those that were most frequently cited and groundbreaking.

Review

Somatic cell isolation

The initial step in generating iPSCs is the isolation of somatic cells from the donor. The choice of cell source is critical, as it directly influences the efficiency of reprogramming, the quality of the resulting iPSC lines, and their subsequent experimental or clinical applications [2]. Historically, dermal fibroblasts obtained from skin biopsies were the first cell type employed for iPSC generation. They remain a widely used starting material because, despite the invasiveness of biopsy collection, fibroblasts can be readily expanded, cryopreserved, and banked. Their high genomic stability makes them a reliable source for reprogramming [3]. An alternative, less invasive strategy is the isolation of peripheral blood mononuclear cells (PBMCs). These cells display comparable reprogramming efficiency to fibroblasts and are increasingly favored in translational studies due to the minimally invasive collection procedure [4,5]. More recently, urinary epithelial cells have emerged as an attractive source of somatic cells. They demonstrate robust reprogramming capacity while offering a completely non-invasive, reproducible, and easily repeatable method of sample acquisition, enabling the generation of multiple iPSC lines from the same donor within a short timeframe [6-8]. Keratinocytes derived from hair follicles have also been successfully utilized. Although this method yields fewer cells, the reprogramming efficiency is higher compared to fibroblasts. Other experimental sources include mesenchymal stromal cells isolated from dental pulp, synovial tissue, and hepatocytes; however, their application remains largely restricted to basic research contexts [9-11].

Pluripotency induction process

The generation of iPSCs is achieved by reprogramming somatic cells to a pluripotent state through the restoration of transcriptional and epigenetic programs characteristic of ESCs. This process reactivates the cells’ ability to differentiate into multiple lineages [12,13]. The introduction of four transcription factors, Oct4 (Pou5f1), Sox2, Klf4, and c-Myc, has been shown to be sufficient to revert fibroblasts to a pluripotent state in both murine and human cells [3]. Early approaches relied on retroviral and lentiviral vectors, which were highly efficient but carried the risk of transgene integration into the host genome, thereby increasing the likelihood of insertional mutagenesis and tumorigenesis. To mitigate these risks, integration-free methods have been developed, including episomal DNA vectors, synthetic mRNA, recombinant protein delivery, and Sendai virus-based systems. Although less efficient, these strategies significantly enhance biosafety [14,15]. Reprogramming involves two principal mechanisms: chromatin remodeling and DNA methylation resetting. Initially, the transcriptional program of the somatic cell is silenced, followed by activation of pluripotency-associated genes. Endogenous reactivation of the Oct4 promoter serves as the central stabilizing mechanism of the pluripotent state. The efficiency of this process, which typically requires several days to weeks, remains low (<0.1-several percent), depending on both technical factors (vector type, transfection method) and biological factors (donor age, cell type, epigenetic profile) [16,17]. Younger donor cells are generally reprogrammed with higher efficiency, likely due to reduced accumulation of epigenetic alterations [18-20]. A major challenge during reprogramming is the preservation of genomic stability. The forced expression of transcription factors can induce mutations and DNA damage, compromising cell function and differentiation capacity. Therefore, continuous genomic monitoring is essential throughout the reprogramming and culture process [21,22].

iPSC culture

Following successful reprogramming, the maintenance of iPSCs under in vitro culture conditions is essential to preserve their proliferative capacity and pluripotency. Suboptimal environments may trigger spontaneous differentiation or loss of stem cell properties, significantly limiting downstream applications [23-25]. Early iPSC culture protocols employed feeder layers composed of mitotically inactivated mouse embryonic fibroblasts, which secreted supportive factors. However, to enhance reproducibility and minimize xenogeneic contamination, feeder-free systems are increasingly used. These rely on extracellular matrix coatings such as Matrigel or recombinant human proteins, including laminin [26-28]. The culture medium typically consists of chemically defined formulations, such as mTeSR1 or E8, supplemented with essential growth factors (e.g., FGF2) and inhibitors of differentiation pathways (e.g., TGF-β/activin A). These media enable greater standardization and are considered more suitable for translational and clinical applications [29,30]. To sustain long-term viability, iPSCs require routine passaging, either mechanically or enzymatically (e.g., dispase, EDTA). Long-term cryopreservation is achieved using cryoprotectants such as 10% DMSO (dimethyl sulfoxide), allowing stable reconstitution of iPSC lines after thawing [31,32]. Despite advances in culture technology, challenges persist. iPSCs remain susceptible to genomic instability, particularly during extended passaging. Furthermore, small fluctuations in medium composition or environmental conditions may induce spontaneous differentiation, complicating process standardization [33,34]. Current efforts aim to establish xeno-free, chemically defined, and standardized culture conditions to support clinical translation [35].

Monitoring iPSC quality

Rigorous quality control is essential to verify the pluripotent state of iPSCs. Expression of canonical pluripotency markers, including Oct4 and Nanog, is typically assessed via PCR, immunocytochemistry, or flow cytometry. Functional pluripotency is confirmed by directed differentiation assays into all three germ layers (ectoderm, mesoderm, endoderm), generating representative cell types such as neurons, cardiomyocytes, and myocytes [36,37]. In addition, genomic integrity must be regularly evaluated, as reprogramming can introduce chromosomal abnormalities or epigenetic alterations. Such changes may compromise differentiation efficiency or predispose cells to malignant transformation [38,39].

Application of iPSCs in disease modeling

iPSCs have become an indispensable tool for modeling human diseases in vitro, enabling the generation of patient-specific cellular models that recapitulate disease pathophysiology. These models facilitate the study of molecular mechanisms, biomarker discovery, drug testing, and the development of personalized therapeutic strategies [40]. Furthermore, autologous iPSC-derived tissues reduce the risk of immune rejection in regenerative applications [41].

Neurodegenerative Diseases

iPSC-derived neuronal models have provided new insights into Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). Patient-specific neurons allow the analysis of pathogenic mechanisms and the evaluation of pharmacological interventions. For ALS, iPSCs have enabled the identification of disease biomarkers and therapeutic compounds. In AD, iPSC-derived neurons and glia reproduce hallmarks such as tau hyperphosphorylation and β-amyloid deposition, offering a platform for targeted therapeutic development [42-44]. PD models have recapitulated dopaminergic neuron degeneration in the substantia nigra and revealed the pathogenic role of α-synuclein aggregation, advancing the understanding of both sporadic and familial PD [45-47].

Cardiovascular Diseases

iPSCs differentiated into cardiomyocytes enable the study of arrhythmogenic disorders, heart failure, and myocardial injury. For example, models of congenital arrhythmias linked to KCNQ1 mutations provide a basis for precision cardiology [48-50]. In myocardial damage, iPSC-derived cardiomyocytes, fibroblasts, vascular smooth muscle cells, and endothelial cells have been explored for regenerative transplantation strategies, with promising improvements in cardiac function [1,51-53].

Metabolic Diseases

iPSCs represent a powerful platform for studying genetic and metabolic diseases, as they preserve the patient’s genotype in vitro [54]. In cystic fibrosis, iPSC-derived airway epithelial cells reproduce defective chloride transport and excessive mucus secretion caused by CFTR mutations, facilitating the evaluation of targeted drugs such as ivacaftor and lumacaftor [55-57]. In Duchenne muscular dystrophy (DMD), iPSC-derived myocytes allow mechanistic studies of muscle degeneration, and gene editing has restored dystrophin expression in vitro, highlighting therapeutic potential [58-60]. Wilson’s disease has also been modeled using iPSC-derived hepatocytes, which reproduce copper accumulation and oxidative stress, providing a platform for preclinical drug testing [61-63].

Autoimmune Diseases

Modeling autoimmune disorders has historically been challenging due to the complexity of the immune microenvironment. iPSCs provide novel opportunities to overcome these barriers [64]. In systemic lupus erythematosus (SLE), iPSC-derived B and T lymphocytes exhibit dysregulated signaling and enhanced autoantibody production, recapitulating disease-specific immune dysfunction [65]. In rheumatoid arthritis, iPSC-derived fibroblast-like synoviocytes reproduce the proinflammatory phenotype driving joint destruction, enabling the screening of targeted inhibitors [66,67]. For type 1 diabetes mellitus, iPSCs have been differentiated into insulin-producing β-like cells, which, when co-cultured with patient-derived T cells, reproduce autoimmune destruction of pancreatic islets [68,69]. In multiple sclerosis (MS), iPSC-derived oligodendrocytes replicate demyelination and remyelination processes, facilitating investigations into neuroprotective and immunomodulatory interventions [70,71].

Challenges and limitations of iPSC technology

Despite their transformative potential, iPSCs face significant technical and translational challenges that limit their widespread application in disease modeling and regenerative medicine. A major obstacle remains the low efficiency of somatic cell reprogramming, which is influenced by donor age, cell type, and the reprogramming strategy employed [72]. Furthermore, heterogeneity among iPSC lines complicates standardization and reduces reproducibility across laboratories. Genomic alterations, including chromosomal deletions, duplications, and translocations, frequently arise during long-term culture and can compromise differentiation capacity or promote neoplastic transformation [72]. Another unresolved issue is the so-called epigenetic memory of donor cells, wherein residual DNA methylation and histone modification patterns restrict full reprogramming and impair lineage-specific differentiation [73]. The risk of tumorigenesis remains one of the most serious barriers to clinical translation. Undifferentiated iPSCs within transplant populations can give rise to teratomas, while earlier viral-based reprogramming methods posed additional risks by integrating exogenous sequences into the host genome [38]. Although non-integrating systems have improved safety, the risk has not been fully eliminated. Another limitation is the incomplete maturation of iPSC-derived cells. Many exhibit a fetal-like phenotype, which restricts their utility for modeling late-onset and adult diseases. The lack of universally accepted protocols for differentiation and the absence of global quality control standards further hinder clinical progress [74]. Moreover, in vitro iPSC-derived models often fail to recapitulate the complex in vivo microenvironment, including interactions with the immune and endocrine systems. Consequently, while these models offer powerful tools to study disease mechanisms, they often require simplification that may not fully capture disease pathophysiology.

Conclusions

iPSCs represent one of the most significant innovations in experimental and translational medicine. Their ability to differentiate into nearly any cell type, combined with the possibility of generating patient-specific models, has provided unprecedented opportunities for disease modeling, mechanistic studies, and therapeutic development. iPSC-based models have already contributed to breakthroughs in understanding neurodegenerative, cardiovascular, metabolic, and autoimmune diseases. Nonetheless, iPSCs are not without limitations. Issues of genomic and epigenetic instability, risk of tumorigenesis, and difficulties in achieving complete cellular maturation remain major challenges. Standardization of culture conditions and establishment of rigorous quality control criteria are essential for their safe clinical application. Future directions include the integration of iPSC technology with genome editing tools, organoid systems, and microfluidic platforms, which may allow more physiologically relevant modeling of human tissues and accelerate the development of personalized therapies. Although numerous challenges remain, the continued refinement of iPSC methodologies holds great promise for advancing precision medicine and regenerative therapeutics.