The risk of tumorigenicity in induced pluripotent stem cells (iPSCs) is substantial and represents a major barrier to their clinical translation. Indeed, the intrinsic properties of self-renewal and pluripotency that render these cells therapeutically promising also confer a fundamentally significant tumorigenic potential (Lee et al., 2013).
What causes tumors in hPSCs?
iPSCs are exposed to numerous factors that promote oncogenic transformation, including genomic insertion of reprogramming vectors, overexpression of oncogenic transcription factors, and global hypomethylation resembling that observed in cancers (Lee et al., 2013). iPSC tumorigenicity also appears to originate from epigenomic instability, primarily attributable to aberrant DNA methylation (Iida et al., 2017). Additionally, there is the well-documented risk of incomplete differentiation of iPSCs prior to transplantation, which can leave residual undifferentiated cells that may proliferate uncontrollably and subsequently develop into tumors (Keller & Spits, 2021; Lee et al., 2013; Movahed et al., 2025).
Tumors promoted by genetic abnormalities
The genetic instability of iPSCs poses a major concern in this respect because chromosomal abnormalities and sub-karyotypic alterations acquired during reprogramming, in vitro maintenance, or differentiation could increase the tumorigenic potential of the cells. Such genetic aberrations can activate oncogenes, including MYC, and inactivate tumor suppressor genes, such as TP53, thereby elevating the risk of tumorigenicity.
It is therefore critical for cell manufacturers and clinicians to rigorously monitor the genomic stability of their iPSC cultures and identify potential cancer-associated variants early in their workflow.
The complexity of early genomic abnormality detection
The study by Hirayama et al. illustrates the complexity of early genomic abnormality detection. This clinical trial for the treatment of bullous keratopathy revealed a de novo mutation in the EP300 gene at a late stage, after the treatment had already been administered.
This occurred despite rigorous testing and adherence to the notification requirements set by the Japanese Ministry of Health, Labor and Welfare and compliance with the Food and Drug Administration (FDA) draft guideline. The team performed two whole-genome sequencing analyses that showed no abnormalities in the cell line or the differentiated cells at the preclinical stage. This was further confirmed by whole-genome sequencing analysis of the cell bank lot designated for this clinical study. The abnormality in the EP300 gene was only detected when a final whole-genome sequencing analysis was performed on the transplanted cells.
The EP300 gene is implicated in chromatin remodeling and cell proliferation and is listed in the Cancer Gene Census. The mutation was attributed to expansion in culture rather than differentiation.
Monitoring continues, and the patients did not experience treatment-related adverse effects during the one-year follow-up period (Hirayama et al., 2025). This case, however, demonstrates the challenge of translating iPSCs safely to the clinic.
A proposed method to mitigate genomic abnormalities in iPSCs
As sequencing costs have declined rapidly, NGS assays are becoming more broadly adopted in iPSC research and clinical workflows. Lezmi & Benvenisty even describe sequencing assays as the only suitable methodology to fully capture nucleotide-level variation in hPSCs thanks to their sensitivity (Lezmi & Benvenisty, 2021).
However, most NGS-based panel assays for identifying oncogenic variants available on the market are designed for general clinical diagnostics. They are used by default by stem cell researchers but have several limitations:
- The genes selected are not always representative of recurrent variants found in hPSC lines, such as TP53 (Lezmi & Benvenisty, 2021; Merkle et al., 2017) or BCOR (Rouhani et al., 2022), and/or the gene panels are too small to capture the abundance of potential mutations in hPSCs.
- The result interpretation is not optimized for PSC researchers, who can become overwhelmed by the extensive data generated, complicating the decision-making process.
The Stem-Seq™ range: NGS-based assays designed for scientists working on PSCs
This NGS-based range of genomic detection assays has been specifically designed to address the needs of scientists working on hPSCs. It combines comprehensive coverage and high sensitivity for the timely detection of tumor-associated abnormalities in hPSCs.
The Stem-Seq™ Panel:
- A custom-designed, targeted panel comprising 361 genes selected for their relevance to stem cell research.
- Detects single-nucleotide variants (SNVs) associated with cancers (including TP53, BCOR, EP300 etc.) as well as selected variants specific to pluripotent stem cells and their impact on cellular development in culture.
The Stem-Seq™ Plus:
- The Stem-Seq™ Plus includes the Stem-Seq™ Panel for SNV detection and provides the additional capability to detect copy number variations (CNVs) across the entire genome. This comprehensive assay enables the detection of CNVs in regions that may not have been previously considered or that could harbor unexpected variations. This unbiased, genome-wide view provides significant additional information on the genomic stability of your cells.
Both assays include a fully interpreted report by our team of experts.
For more information about the Stem-Seq™ range and how you could integrate it into your workflow, click here.
Hirayama, M., Hatou, S., Nomura, M., Hokama, R., Hirayama, O. I., Inagaki, E., Aso, K., Sayano, T., Dohi, H., Hanatani, T., Takasu, N., Okano, H., Negishi, K., & Shimmura, S. (2025). A first-in-human clinical study of an allogenic iPSC-derived corneal endothelial cell substitute transplantation for bullous keratopathy. Cell Reports Medicine, 6(1). https://doi.org/10.1016/j.xcrm.2024.101847
Keller, A., & Spits, C. (2021). The impact of acquired genetic abnormalities on the clinical translation of human pluripotent stem cells. In Cells (Vol. 10, Number 11). MDPI. https://doi.org/10.3390/cells10113246
Lee, A. S., Tang, C., Rao, M. S., Weissman, I. L., & Wu, J. C. (2013). Tumorigenicity as a clinical hurdle for pluripotent stem cell therapies. In Nature Medicine (Vol. 19, Number 8, pp. 998–1004). https://doi.org/10.1038/nm.3267
Lezmi, E., & Benvenisty, N. (2021). Identification of cancer-related mutations in human pluripotent stem cells using RNA-seq analysis. In Nature Protocols (Vol. 16, Number 9, pp. 4522–4537). Nature Research. https://doi.org/10.1038/s41596-021-00591-5
Merkle, F. T., Ghosh, S., Kamitaki, N., Mitchell, J., Avior, Y., Mello, C., Kashin, S., Mekhoubad, S., Ilic, D., Charlton, M., Saphier, G., Handsaker, R. E., Genovese, G., Bar, S., Benvenisty, N., McCarroll, S. A., & Eggan, K. (2017). Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature, 545(7653), 229–233. https://doi.org/10.1038/nature22312
Movahed, A. Y., Bagheri, R., Savatier, P., Šarić, T., & Moradi, S. (2025). Elimination of tumorigenic pluripotent stem cells from their differentiated cell therapy products: An important step toward ensuring safe cell therapy. In Stem Cell Reports (Vol. 20, Number 7). Cell Press. https://doi.org/10.1016/j.stemcr.2025.102543
Rouhani, F. J., Zou, X., Danecek, P., Badja, C., Amarante, T. D., Koh, G., Wu, Q., Memari, Y., Durbin, R., Martincorena, I., Bassett, A. R., Gaffney, D., & Nik-Zainal, S. (2022). Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells. Nature Genetics, 54(9), 1406–1416. https://doi.org/10.1038/s41588-022-01147-3
