From retinal repair to diabetes treatment, induced pluripotent stem cell (iPSC)-derived cell therapies are entering clinical trials with increasing numbers over the last few years since their first generation and characterization 18 years ago. One of the most overlooked perils in mammalian cultures in general and in iPSC-derived products in particular is the accumulation of culture-induced genetic variants—mutations that can silently compromise safety, efficacy, and subsequent regulatory approval.
The stem cell community is aware of the need for a harmonized and effective way to assess the genetic integrity of stem cell-based therapy programs. But what is expected of a cell therapy developer to ensure compliance to meet regulators’ expectations across regions?
What Are Culture-Acquired Genetic Variants in iPSCs?
During in vitro expansion, iPSCs, like all mammalian cell lines, can acquire and accumulate spontaneous chromosomal abnormalities, oncogenic variants, and epigenetic alterations. This includes the most common and recurrent iPSC abnormalities such as whole or partial gains on chromosomes 1, 12, 17, 20, and X 1 which often escape detection by standard karyotyping assays. As Vales and Barbaric (2024) explain 2, these mutations persist because they offer a growth advantage, allowing abnormal cells to dominate the culture over time.
This genetic drift can have serious consequences (Figure 1). One of the most significant safety concerns in cell therapy is the risk that engrafted cells will form tumors in patients. In a widely cited case, Han et al. reported the development of immature teratomas in a patient post iPSC-derived β cell transplantation caused by a rare mosaic mutation that went undetected during early screening 3.
Furthermore, these genomic changes can alter the cells’ behavior, leading to variability in their capacity to differentiate into specific lineages, growth dynamics, functional output and maturation, ultimately affecting downstream differentiation outcomes. Research by Kilpinen et al. (2017) revealed that both inherited genomic background and mutations acquired during reprogramming or expansion contribute to this heterogeneity 4. This compromises experimental reproducibility and poses significant challenges for clinical translation, where consistent and predictable differentiation is paramount.
Figure 1: Genomic changes in iPSCs can cause multiple downstream consequences.
Key Factors in iPSC Clone Risk Profiling: Reprogramming Method, Donor Characteristics, Passage Number, Genetic Test Results
Regulatory agencies recommend a risk-based, stage-appropriate testing strategy. A practical way to implement this is by applying a risk stratification model early in the development pipeline to evaluate iPSC clones before advancing them to clinical manufacturing. Factors such as the method of reprogramming, donor characteristics, passage number, and results from genetic testing all contribute to the risk profile of a clone. By assessing clones based on these criteria, developers can take targeted actions—from banking and advancing low-risk lines to excluding high-risk ones from clinical pipelines (Figure 2).
Figure 2: Genomic changes in iPSCs require the development of a risk stratification strategy.
Why does Genetic Screening throughout iPSC-based therapy development matter?
Early detection of culture-induced genetic variants is essential to ensure the safety and consistency of iPSC-derived therapies. According to Benvenisty et al. 5, the field must adopt systematic approaches to identify and interpret genetic variants, including functional annotation and database-driven analysis. Without this, even low-frequency mutations can derail clinical outcomes.
As Kirkeby et al. 6 note, since 2020, the stem cell field has seen a surge in first-in-human clinical trials using iPSC-derived cells for retinal, corneal, pancreatic, and neural applications. In this rapidly evolving landscape, genetic integrity is no longer optional—it is mission-critical.
Regulatory Guidance on Genetic Integrity in iPSC Therapy
In regulatory guidance documents issued by authorities such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), Japan Pharmaceuticals and Medical Devices Agency (PMDA) and agencies in and other parts of Asia, there is a clear emphasis on maintaining genetic integrity throughout the manufacturing and clinical development of iPSC-based therapies.
These agencies require developers to adopt a risk-based, stage-appropriate framework for genetic stability testing, extending from donor selection and raw materials to final drug product release 7, 8, 9. Such guidance specifies that the extent and frequency of genetic testing should be commensurate with the risk profile of the differentiated product. Factors influencing this profile include the degree of cell manipulation, the intended route of administration, duration of persistence in the body, the final differentiated drug product, and the target patient population. To ensure genetic fidelity and minimize the risk of tumorigenicity or unexpected differentiation, regulatory bodies recommend or require the application of validated analytical methods. These typically include G-banding karyotyping and next-generation sequencing (NGS) approaches to detect subtle mutations or chromosomal rearrangements.
In summary, the preservation of genetic integrity across the iPSC product lifecycle is a central pillar of regulatory compliance and clinical viability, demanding robust, risk-stratified quality control strategies and full traceability across manufacturing stages.
REPROCELL’s Solution: NGS-Based Oncogenetic Profiling for iPSCs
At REPROCELL, we believe that when it comes to assessing genetic safety in iPSCs, a focused approach is both practical and informative. Rather than scanning the entire genome and generating long lists of variants of uncertain significance, it is more meaningful to concentrate on a select group of well-established driver genes linked to cancer or abnormal cell proliferation — such as TP53, KRAS or BCOR. Using high-sensitivity techniques such as WGS, developers can reliably detect or rule out critical mutations. Demonstrating the absence of key oncogenic mutations with precision offers far greater value than producing an exhaustive whole-genome sequencing report that may include low-confidence or clinically irrelevant findings (Figure 3).
Every StemRNA Clinical iPSC Seed Clone, its corresponding donor material, and any subsequent GMP MCB batch are assessed for genetic integrity by performing a combined approach of two complementary techniques:
Each report includes a review of:
- ClinVar annotations for clinical relevance
- Functional impact scoring to assess risk
This allows researchers to confidently select the right iPSC starting clones for their specific cell therapy product —minimizing risk and maximizing differentiation fidelity.
Figure 3. WGS of StemRNA Clinical Seed iPSC Clones and the corresponding Donor Fibroblasts
As a cell line provider, REPROCELL is looking to demonstrate the absence of genetic variants associated with tumorigenicity risk utilizing targeted panels to support safety testing. The collected raw WGS data can be re-analyzed for disease-specific gene panels, such as cardiac, neurological, or other condition-related panels. Reports can be tailored to your individual needs, whether that is a focused gene panel or comprehensive genomic insight (Figure 3).
Culture-acquired mutations are a silent threat to the success of iPSC-based therapies. Robust data and clear documentation, aligned with FDA, EMA, and PMDA guidance, are now essential for regulatory submission and safe clinical translation. REPROCELL can help with this by integrating early, comprehensive genetic screening.
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Contact us today to learn how our oncogenetic profiling can support your research or clinical pipeline.
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References
- Baker, D., Hirst, A.J., Gokhale, P.J., Juarez, M.A., Williams, S., Wheeler, M., Bean, K., Allison, T.F., Moore, H.D., Andrews, P.W., and Barbaric, I. Detecting Genetic Mosaicism in Cultures of Human Pluripotent Stem Cells. Stem Cell Reports. 7(5):998-1012. (2016).
- Vales, J. P. and Barbaric, I. Culture acquired genetic variation in human pluripotent stem cells: Twenty years on. BioEssays, 46(12), e2400062. (2024).
- Han, L., Shi, Y., Zhang, Y., Li, T., Liu, B., Chen, J. and Zhang, J. Distinctive clinical and pathologic features of immature teratomas after iPSC derived β cell therapy in a diabetes patient. Stem Cells and Development. 31(5-6). (2022).
- Kilpinen, H., Goncalves, A., Leha, A., Afzal, V., Alasoo, K., Ashford, S., Bala, S., Bensaddek, D., Casale, F. P., Culley, O. J., Danecek, P., Faulconbridge, A., Harrison, P. W., Kathuria, A., McCarthy, D. J., McCarthy, S. A., Meleckyte, R., Memari, Y., Moens, N., Soares, F., Mann, A., Streeter, I., Agu, C. A., Alderton, A., Nelson, R., Harper, S., Patel, M., White, A., Patel, S. R., Clarke, L., Halai, R., Kirton, C. M., Kolb Kokocinski, A., Beales, P. L., Birney, E., Danovi, D., Lamond, A. I., Ouwehand, W. H., Vallier, L., Watt, F. M., Durbin, R., Stegle, O., and Gaffney, D. J. Common genetic variation drives molecular heterogeneity in human iPSCs. Nature, 546(7658), 370–375. (2017).
- Benvenisty, N., Draper, J. S., Gokhale, P. J., Healy, L., Hewitt, Z., Hursh, D., Hodgson, A., Ludwig, T. E., Mah, N., McClelland, S. E., Mennecozzi, M., Merkle, F. T., Mountford, J. C., Pera, M. P., Prigione, A., Rodriguez, T. A., Rossi, A., Rouhani, F. J., Saeb Parsy, K., Selfa Aspiroz, L., Shakiba, N., Spits, C., Tonge, P. D. and Barbaric, I. A call to action for deciphering genetic variants in human pluripotent stem cells for cell therapy. Cell Stem Cell, 32(4), 508–512. (2025).
- Kirkeby, A., Main, H. and Carpenter, M. Pluripotent stem cell derived therapies in clinical trial: A 2025 update. Cell Stem Cell, 32(1), 10–37. (2025).
- EMA’s “Guideline on Human Cell‑Based Medicinal Products” (2008).
- FDA’s Draft Guidance for Industry “Safety Testing of Human Allogeneic Cells Expanded for Use in Cell‑Based Medical Products” (2024).
- FDA’s draft guidance: Considerations for the Use of Human- and Animal-Derived Materials in the Manufacture of Cellular and Gene Therapy and Tissue-Engineered Medical Products. (2024).
