As pluripotent cells, they can differentiate into any cell type in the body, including specialized cells such as neurons, blood cells, liver cells, and gametes, while also possessing unlimited self-renewal capabilities.

Since their inception, iPSCs have been commonly used to understand human development and disease modeling, perform drug screening, and facilitate the development of cell therapies. In fact, their ability to differentiate into any cell type makes them valuable for regenerative medicine, personalized treatments, and understanding complex diseases like cancer, neurodegenerative diseases, and genetic disorders.

Characteristics of induced pluripotent stem cells

The characteristics of iPSCs closely resemble those of ESCs, making them an ideal tool for advancing research and drug development while addressing ethical concerns.

Plasticity and versatility

Induced pluripotent cells offer remarkable plasticity and versatility, enabling unlimited expansion, genetic engineering, and differentiation into various somatic cell types for a range of applications in:

• Drug screening and toxicity studies
• Cell therapies
• Disease modeling
• Regenerative medicine (under research and pre-clinical stage)

Comparison to embryonic stem cells (ESCs)

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are both pluripotent stem cells (PSCs). However, ESCs are derived from the inner mass of the blastocysts of an embryo, raising ethical concerns. In contrast, iPSCs are produced in vitro by reprogramming adult somatic cells, offering a more ethical and efficient approach to personalized treatments.

How are induced pluripotent stem cells created?

Induced pluripotent stem cells are produced by introducing reprogramming factors into somatic/adult stem cells, often via retroviral or lentiviral transduction.

Non-integrative methods like episomal plasmids and reprogramming mRNAs or proteins and peptides are also used to address safety concerns, such as integration of the viral genome into the cell genome and epigenetic modifications, potentially activating oncogenes and forming tumors. These methods enable the generation of pluripotent cells even in species where deriving embryonic stem cells has been challenging.

Overview of the reprogramming steps

The iPSC reprogramming process involves introducing specific transcription factors into somatic/ adult stem cells, which induces epigenetic changes and reprograms them into a pluripotent state capable of differentiating into various cell types.

Key factors in reprogramming (Yamanaka factors)

Takahashi and Yamanaka demonstrated that overexpression of four transcription factors can reprogram the somatic cells to a pluripotent state by rearranging their epigenetic landscape, proving cellular identity is epigenetically regulated. Reprogramming of iPSCs causes downregulation of the somatic cell signatures and induction of the pluripotent markers.

These four transcriptional factors include Oct4, Sox2, Klf4 and c-Myc (OSKM). These are collectively called Yamanaka factors.

Role of essential genes: Oct4, Sox2, and others involved in inducing pluripotency

Somatic cells like pancreatic cells, hepatocytes, human fibroblasts, neural cells, etc, are reprogrammed into iPSCs using core factors like:

• Oct4, Sox2, Klf4 and c-Myc in retroviral system
• Oct4, Sox2, Nanog and Lin28 in lentiviral system

These cocktails of transcription factors regulate signaling pathways, epigenetic modifications, and microRNAs to establish pluripotency.

The factors also interact to stabilize stemness, enhance reprogramming efficiency, and maintain self-renewal. Reprogramming facilitates epigenetic changes, chromosomal modification, and various cell functions such as metabolism, cell signaling, intracellular transport, and proteostasis.

Reprogramming techniques

Reprogramming techniques for iPSC production involve inserting genes into the genome using viral vectors, which poses risks like oncogene activation and tumor formation. Other non-viral techniques avoid genomic integration, offering safer, transgene-free iPSCs suitable for clinical applications.

Viral and non-viral methods

The generation of iPS cells can be achieved through:

• Viral-based methods: These involve integrating exogenous genes into the somatic cells. This technique includes the incorporation of transcriptional factors derived from a retrovirus or lentivirus. However, they may cause immune responses, have cargo limitations, and risk genomic integration, leading to tumorigenicity. Additionally, the quality of the iPSCs may be compromised due to the failure to turn on the expression of the endogenous genes associated with pluripotency.
• Non-viral methods: They help avoid the integration of genes and are preferred for clinical applications due to safety concerns and reduced risk of unwanted gene reactivation.

Examples of virus-based method

• The most common viral-based methods for generating iPSCs include the use of retroviruses and lentiviruses, which can efficiently deliver the necessary reprogramming genes (such as Oct4, Sox2, Klf4, and Myc) into somatic cells to convert them into pluripotent iPSCs. These viral vectors have high transduction efficiency, which early iPSC studies relied on.
• Derived from HIV-1, Lentiviruses (single-stranded RNA viruses) can transduce dividing as well as non-dividing cells, allowing for highly efficient reprogramming. These viruses raise concerns about insertional mutagenesis as they integrate into the genome.
• Other less commonly used viral vectors explored for the generation of iPSC are Sendai virus (an RNA virus), Adenoviruses, and Epstein-Barr Virus (double-stranded DNA viruses), which enable efficient iPSC generation through their non-integrating characteristic, lowering the risk of insertional mutation. These viruses may be safer for clinical use as they are extrachromosomally replicated and are lost eventually through cell divisions. Nonetheless, their reprogramming efficiency is lower compared to that of retrovirus- or lentivirus-based methods.

Examples of non-viral methods

• Elastin-like polypeptides (ELP) based transfection generates virus-free iPSCs from mouse fibroblasts, demonstrating a safe method for reprogramming without the risks of viral genome integration.
• Episomal iPSCs are reprogrammed using non-integrative plasmid methods, offering a safer alternative to viral approaches. This shows great potential in clinical applications and functional restoration.
• Small-molecule-mediated reprogramming is a technique that includes small molecules and growth factors used for drug screening, disease modeling, and cell therapy while addressing the challenges of viral DNA integration and oncogene risks associated with iPSCs. Owing to the simpler structure, low cost, availability, cell permeation, and stability, they are advantageous. Some reported small molecule compounds used for iPSC reprogramming are DNA methyltransferase and histone deacetylase (HDAC) inhibitors, especially VPA, glycogen synthase kinase 3 (GSK-3) inhibitor, CHIR99021¹.

Cell sources for reprogramming

The source of somatic cells used for iPSC generation significantly influences the cells' epigenetic roles, heterogeneity, differentiation potential, and mutational burden.

After the induction of reprogramming, the culture conditions need to be optimized during the transition from somatic cells to iPSCs. Traditionally, iPSCs are typically cultured on feeder layers that are mitotically inactive, such as mouse embryonic fibroblasts. Today, advanced feeder-free systems are developed using extracellular matrices like hydrogel, Matrigel, vitronectin, etc., the use of increased research momentum due to the ease of use, consistency in output, and ease of availability.

The choice of media selected for the expansion of iPSCs also contributes significantly to the expansion of iPSCs. Certain formulations like Essential 8 or mTeSR1 etc, are commonly used along with serum-free media options to maintain cell viability as well as pluripotency in an in vitro set-up.

To preserve cell purity during the reprogramming process, cell morphology must be closely observed. The cell population is diverse in the early phases of culture, exhibiting a range of colony subtypes. These colonies are moved to the new culture dish for additional growth after being chosen by hand or by selective enzymatic digestion. The success of this technique is largely dependent upon handling and experience.

Characterization and validation of the cells are also essential to maintain pluripotency and stemness of the iPSCs without further differentiation during long-term culturing. This is typically achieved through advanced genomic techniques like CRISPR-cas9 via the detection and quantification of pluripotency markers like OSKM. Additionally, certain functional assays, like embryoid body formation, differentiation assays, karyotyping analysis, etc, are further suggested to maintain cellular integrity and long-term stability in culture.

Types of adult cells used

Adult cells commonly used for iPSC generation include skin fibroblasts, liver, neurons, astrocytes, peripheral blood mononuclear cells (PBMCs), and keratinocytes. These cells are preferred for their accessibility, ease of collection, and reprogramming efficiency.

Applications of induced pluripotent stem cells in research and medicine

Induced pluripotent technology has transformed in vitro research, with iPSC-derived models used for drug screening, disease studies, and developing cell therapies for conditions like neurological disorders, COVID-19, and cancer.

Regenerative medicine and cell therapy

Induced pluripotent cells offer a groundbreaking approach to treating diseases. This technique holds promise for addressing previously untreatable conditions in regenerative medicine. In regenerative medicine, impaired or degenerative tissues are repaired by the generation of the functional tissues obtained by iPSC, followed by transplantation at the site of injury.

Studies are underway on the use of this method for treating several types of injury and diseases, such as musculoskeletal injury², spinal cord injury³, muscular dystrophies⁴, and immunodeficiencies⁵. iPSCs enable the creation of engineered immune cells⁶ with enhanced cancer-killing abilities. Advanced genome editing technologies also offer novel possibilities for more effective immunotherapies⁷.

iPSCs also offer a promising anti-aging strategy⁸ through reprogramming-induced rejuvenation, where transient expression of Yamanaka factors can reduce biological age by leveraging epigenetic rejuvenation without complete dedifferentiation.

Disease modeling

iPSCs enable disease modeling by replicating patient-specific cellular conditions, allowing researchers to study disease mechanisms and test potential therapies in vitro. For example, human prostrate and urinary tract cells were used to generate iPSCs that were further studied to understand the differentiation process of prostrate and urinary tract cells.

Some diseases such as adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III are studied by using iPSCs. Other diseases that can be studied using iPSCs to establish disease models are neurodegenerative diseases, chromosomal disorders, type 1 diabetes mellitus, etc.

Development of disease-specific iPSCs

Human iPSCs hold significant potential for treating diseases with no current cures, such as neurodegenerative diseases, cardiac infarctions, and diabetes, with the advantage of patient-specificity for autologous transplantation.

Recent research into iPSC-derived neural cells for neuroscience and iPSC transplantation studies offers potential breakthroughs in treating neurodegenerative diseases. Patient-derived iPSCs are grown into dopaminergic neurons to evaluate Parkinson's disease-specific cellular dysfunctions, for example, α-synuclein buildup and mitochondrial abnormalities⁹.

High-throughput pharmacological screening for potential neuroprotective treatments is another benefit of these models. Likewise, researchers can develop individualized treatments for amyotrophic lateral sclerosis through the use of iPSC-derived motor neurons for enhanced understanding of neurodegenerative pathways such as RNA dysregulation and axonal transport abnormalities¹⁰.

iPSCs can be developed into pancreatic beta cells, facilitating diabetes pathophysiology assessment. In type 1 diabetes, iPSC-derived beta cells are employed to assess immunomodulatory treatments and autoimmune-associated damage. Studies are underway regarding transplanting iPSC-derived beta cells into patients as a cell replacement therapy to restore insulin production¹¹.

iPSC-derived cardiomyocytes (iPSC-CMs) are often employed to imitate cardiac arrhythmias and heart failure. In hypertrophic cardiomyopathy, iPSC-CMs from patients show aberrant sarcomere structure and calcium handling, providing insight into disease progression¹². In addition, iPSC-CMs are utilized to screen for drug toxicity, predict unwanted cardiac effects of novel therapies, and evaluate cardioprotective treatments¹³.

iPSC-derived retinal pigment epithelial cells are being used to investigate age-related macular degeneration. These models can help us understand RPE dysfunction, oxidative stress responses, and inflammation. Some clinical trials are looking into the transplantation of iPSC-derived RPE cells to restore eyesight in patients with macular degeneration¹³.

iPSCs are utilized to create tumor organoids to study cancer progression and the response to treatments. In leukemia, iPSC-derived hematopoietic stem cells are used to examine clonal evolution and treatment resistance mechanisms¹⁴. Furthermore, patient-specific iPSC models offer individualized cancer treatment by enhancing drug selection based on unique genetic profiles¹⁵.

Human iPSC-derived cellular models have also been used to study SARS-CoV-2 infection, revealing human-specific disease phenotypes, tissue susceptibilities, and long-term effects, such as neurological damage and organ-specific replication of the virus.

Drug discovery and toxicity testing

Non-integrative methods are preferred for generating safer iPSCs with minimal risk of secondary disease-causing mutations, making them more suitable for therapeutic applications. Using iPSCs is potentially better than conventional tests of toxicology and drug testing as they mimic the microenvironment of the human physiological systems. For example, iPSCs have been used for cytotoxicity-cardiotoxicity, hepatotoxicity, and embryotoxicity testing¹⁶.

Screening potential drugs

iPSCs provide scalable, patient-derived models that help in studying disease pathophysiology, enabling phenotype-based drug screening, biomarker identification, and therapeutic target discovery for neurological disorders.

iPSC-derived models hold promise for advancing precision medicine and improving central nervous system (CNS) drug discovery and toxicity studies.

Advantages of induced pluripotent stem cells

Induced pluripotent cells allow for high-throughput drug screening and complex phenotypic assays, facilitating the discovery of therapeutic candidates through innovative techniques such as machine learning and high-content imaging.

Ethical considerations

ESCs are obtained from the developmental stages of the embryo that lead to embryo destruction. Moreover, the use of ESCs requires informed consent due to health and safety concerns. However, iPSCs generated from somatic cells are a promising alternative to embryonic stem cell therapy, offering patient specificity and bypassing ethical concerns.

Patient-specific cells and reduced immune rejection

Patient-specific iPSCs, particularly from monogenic disease patients, are valuable for creating in vitro models to study disease mechanisms and screen for better drugs. They offer significant advantages in drug discovery, regenerative medicine, and toxicology, providing a solution free from immune rejection and ethical concerns.

Flexibility in cell source

iPSC-derived cellular models offer flexibility in drug development by enabling the use of various somatic cell types, including those that are difficult to access from primary sources, to test drug efficacy and toxicity in disease-specific genetic contexts.

Challenges and limitations of iPSCs

Despite having numerous advantages over traditional approaches, iPSCs have their limitations and face various ethical and technical challenges.

Technical challenges

iPSCs face technical challenges such as incomplete reprogramming, low efficiency, genetic instability, and difficulty in scaling production for therapeutic use.

Reprogramming efficiency and reproducibility

Reprogramming somatic cells into iPSCs is often complex and inefficient, with only a small fraction successfully becoming pluripotent, leading to variability in the quality of the resulting cells.

Additionally, reproducibility is a challenge, as differences in reprogramming methods and conditions can cause inconsistencies in iPSC properties, impacting their reliability for research and therapy.

Issues and implications

Ensuring the quality, consistency, and scalability of iPSCs is important for their reliable use in clinical and research settings, as variability can affect both experimental outcomes and the safety of iPSC-based therapies.

Long-term culture of iPSCs potentially causes genomic instability and epigenetic alterations that affect their suitability as cancer models.

Genetic and epigenetic abnormalities

A major challenge in iPSC-based cancer therapy is the technical difficulty of generating high-quality iPSCs.

iPSCs may acquire genetic mutations and retain epigenetic memory, leading to genomic instability and variability in differentiation. Additionally, the potential for tumorigenicity due to undifferentiated cells in iPSC-derived products raises significant safety concerns.

Ethical considerations surrounding iPSC research

Genetic modifications in iPSC research present significant ethical concerns, with other key factors including obtaining informed consent from donors and ensuring the responsible use of patient-specific cells in clinical applications.

Regulatory guidelines

• As per global regulations, iPSCs are subject to varying policies across different regions (USA, EU, Japan, etc.) with strict guidelines for clinical and research applications.
• Ethical guidelines emphasize obtaining informed consent from donors and ensuring donor privacy.
• Countries like Japan have set clear protocols for clinical trials, including patient safety, long-term follow-up, and reporting.
• Policies often prohibit deriving stem cell lines and iPSCs from embryos, emphasizing alternative ethical approaches.
• Regulatory bodies such as the Food and Drug Administration (FDA) and European Medicines Agency (EMA) authorize pre-clinical studies, including safety and efficacy evaluations before clinical application.
• Gene modification tools like clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), and zinc finger nuclease (ZFN) require rigorous quality checks due to potential off-target effects.
• Patent laws surrounding iPSC technologies ensure the protection of innovations while promoting public health advancements.

Current solutions and ongoing research and future perspectives in iPSC research

Advances in genetic engineering, like CRISPR/Cas9, are enhancing the safety and functionality of iPSCs by correcting genetic abnormalities.

Moreover, developing hypoimmunogenic iPSCs and implementing stringent quality control measures may improve the reliability and safety of iPSC-based therapies.

Future perspectives in iPSC research focus on improving reprogramming efficiency, enhancing the clinical applications of iPSC-derived therapies, and overcoming challenges such as safety, immune rejection, and tumorigenicity.

Innovations in reprogramming techniques

Human induced pluripotent stem cells (hiPSC) can be used to generate bone marrow-like organoids (BMOs) that mimic the structure and function of the human bone marrow niche, including hematopoietic stem/progenitor cells and a vascular network^{16, 17}.

These BMOs provide a physiologically relevant in vitro model for studying hematopoietic development, bone marrow diseases, and inborn errors.

Certain cell lines are generated from hiPSCs using cellular reprogramming technology.

CRISPR and genome editing

Genome editing technology may be used in specific cases like genetic disease correction.

CRISPR/Cas9-based genome editing in iPSCs has been developed to overcome limitations like low efficiency and off-target effects, with a new electroporation-mediated plasmid delivery approach enabling rapid editing within two weeks.

This method avoids long-term side effects by minimizing Cas9 gene integration and preserves the pluripotency and differentiation ability of iPSCs, enhancing genome editing in research.

FAQs

What are induced pluripotent stem cells?

Induced pluripotent stem cells (iPSCs) are unspecialized and artificially reprogrammed somatic cells that revert to a pluripotent state, allowing them to differentiate into any cell type. They are created by introducing specific genes that reset the cell’s developmental potential.

How are human induced pluripotent stem cells created?

Human induced pluripotent stem cells (iPSCs) are created by introducing a set of specific genes, typically using viruses or other methods, into human somatic cells like skin or blood cells. These genes reprogram the cells back to a pluripotent state, allowing them to differentiate into various cell types.

What are the benefits of iPSCs?

iPSCs offer significant benefits, including the ability to generate patient-specific cells for personalized medicine and disease modeling without the ethical concerns associated with embryonic stem cells. They also hold potential for regenerative therapies, as they can be differentiated into various cell types for tissue repair and organ regeneration.

What is the difference between pluripotent and induced pluripotent stem cells?

Pluripotent stem cells (PSCs) are naturally occurring cells, such as embryonic stem cells, that can differentiate into almost any cell type in the body. Induced pluripotent stem cells (iPSCs) are adult somatic cells that have been reprogrammed through genetic manipulation to acquire pluripotency, mimicking the properties of embryonic stem cells.

References:

1. Kim, Y., Jeong, J., Choi, D. Small-molecule-mediated reprogramming: a silver lining for regenerative medicine. Experimental and molecular medicine. 52, 213–226 (2020).
2. Sanjurjo-Rodríguez, C., Castro-Viñuelas, R., Piñeiro-Ramil M., et al. Versatility of induced pluripotent stem cells (iPSCs) for improving the knowledge on musculoskeletal diseases. International journal of molecular sciences. 21, 6124 (2020).
3. Sugai, K., Sumida, M., Shofuda, T., et al. First-in-human clinical trial of transplantation of iPSC-derived NS/PCs in subacute complete spinal cord injury: Study protocol. Regenerative therapy. 18, 321–333 (2021).
4. Piga, D., Salani, S., Magri, F., et al. Human induced pluripotent stem cell models for the study and treatment of Duchenne and Becker muscular dystrophies. Therapeutic advances in neurological disorders. 12, (2019).
5. Karagiannis, P., Yamanaka, S., Saito, M.K. Application of induced pluripotent stem cells to primary immunodeficiency diseases. Experimental hematology. 71, 43–50 (2019).
6. Zhou, Y., Li, M., Zhou, K., et al. Engineering induced pluripotent stem cells for cancer immunotherapy. Cancers (Basel). 14, 2266 (2022).
7. Fang, M., Allen, A., Luo, C., et al. Unlocking the potential of iPSC-derived immune cells: engineering iNK and iT cells for cutting-edge immunotherapy. Frontiers in immunology. 15, 1457629 (2024).
8. Jothi, D., Kulka, L.A.M. Strategies for modeling aging and age-related diseases. Nature partner journal – aging. 10, 32 (2024).
9. Avazzadeh, S., Baena, J.M., Keighron, C., et al. Modelling Parkinson's disease: iPSCs towards better understanding of human pathology. Brain science. 11, 373 (2021).
10. Lisha, Y., Katarina, S.D., Adria, S., et al. Sporadic ALS hiPSC-derived motor neurons show axonal defects linked to altered axon guidance pathways. Neurobiology of disease 206, 106815 (2025)
11. Maxwell, K.G., Millman, J.R. Applications of iPSC-derived beta cells from patients with diabetes. Cell rep medicine. 2, 100238 (2021).
12. Wu, H., Yang, H., Rhee, J.W., et al. Modelling diastolic dysfunction in induced pluripotent stem cell-derived cardiomyocytes from hypertrophic cardiomyopathy patients. European heart journal. 40, 3685–3695 (2019).
13. Dehghan, S., Mirshahi, R., Shoae-Hassani, A., et al. Human-induced pluripotent stem cells-derived retinal pigmented epithelium, a new horizon for cell-based therapies for age-related macular degeneration. Stem cell research and therapy. 13, 217 (2022).
14. Chehelgerdi, M., Behdarvand Dehkordi, F., Chehelgerdi, M., et al. Exploring the promising potential of induced pluripotent stem cells in cancer research and therapy. Molecular cancer. 22, 189 (2023).
15. Hao, M., Cao, Z., Wang, Z., et al. Patient-derived organoid model in the prediction of chemotherapeutic drug response in colorectal cancer. American Chemical Society biomaterials science & engineering. 8, 3515–3525 (2022).
16. Singh, V.K., Kalsan, M., Kumar, N., et al. Induced pluripotent stem cells: applications in regenerative medicine, disease modeling, and drug discovery. Frontiers in cell and developmental biology. 3, 2 (2015).
17. Frenz-Wiessner, S., Fairley, S.D., Buser M., et al. Generation of complex bone marrow organoids from human induced pluripotent stem cells. Nature methods. 21, 868–881 (2024).