Human pluripotent stem cells (hPSCs) have become indispensable model systems because of their ability to self-renew indefinitely and differentiate into virtually any cell type in the body. These properties have led to widespread adoption of hPSCs for disease modeling, drug discovery, and the development of advanced therapy medicinal products (ATMPs).
hPSCs can harbor genetic alterations originating from the donor cell population as well as acquire de novo changes during reprogramming and in vitro expansion1-3. These include chromosomal abnormalities and smaller sequence-level changes such as single-nucleotide variants and insertions/deletions (indels). Many of these alterations confer a selective growth advantage, allowing abnormal subclones to quickly outcompete their normal counterparts and dominate the culture.
Studies have identified a number of variants in cancer-associated genes including TP53, EGFR, CDK12, and APC in cultured hPSC lines3-5. These acquired oncogenic variants can impact genomic integrity, differentiation potential, and introduce product safety risks for clinical use. Puigdevall et al. found that somatic mutations in the BCOR gene in iPSC models of neurodevelopment were strongly associated with impaired dopaminergic neuron differentiation2. Because many mutations occur at the sequence level, they are not detectable by conventional karyotyping, and other genome wide methods of characterization.
Next-generation sequencing (NGS) enables high-resolution detection of both chromosomal and sequence-level variants, providing sensitivity beyond traditional cytogenetic approaches. While karyotyping remains useful for identifying large-scale chromosomal changes, targeted NGS-based methods offer a complementary strategy for detecting pathogenic variants and assessing the genomic integrity of hPSC lines.
WiCell’s Oncopanel Assay
The WiCell Oncopanel Assay was developed to enable detection of pathogenic and likely pathogenic genetic variants in hPSCs that can influence cell behavior, differentiation outcomes, and translational suitability. Available as either a whole genome sequencing (WGS) or whole exome sequencing (WES) assay, it provides high-resolution assessment of cancer-associated genes relevant to stem cell quality and genomic integrity.
The curated 65-gene panel was designed to identify inherited and acquired variants in high-priority oncogenes and tumor suppressor genes that may compromise hPSC function, even when present at low levels of mosaicism (Figure 1).
| APC | DDX3X | H3-3A | MUC16 | PTEN |
| ASXL1 | DNMT3A | IDH1 | MYD88 | PTPN11 |
| BAP1 | EGFR | IDH2 | MYOD1 | RB1 |
| BCOR | EIF1AX | IKZF1 | NF1 | SETD2 |
| BCORL1 | FAS | JAK2 | NOTCH1 | SF3B1 |
| BRAF | FAT4 | KIT | NPM1 | SMAD4 |
| CALR | FBXW7 | KMT2D | NRAS | SMARCB1 |
| CDKN2A | FGFR4 | KRAS | NSD2 | STAT3 |
| CDC | FLT3 | LRP1B | PAX5 | TET2 |
| CREBBP | GNA11 | MAP2K1 | PBRM1 | TP53 |
| CSMD3 | GNAQ | MAP3K1 | PHF6 | U2AF1 |
| CTNNB1 | GNAS | MET | PIK3CA | VHL |
| CYSLTR2 | GRIN2A | MTOR | PIM1 | WT1 |
Figure 1. Genes covered in WiCell’s Oncopanel Assay.
Gene selection was guided by publicly available cancer genomics resources, including the Genomic Data Commons (GDC) and the Catalogue of Somatic Mutations in Cancer (COSMIC), as well as large-scale analyses of somatic mutation frequencies in human cancers6. Selection criteria prioritized genes that are frequently mutated and have established roles in cancer development, with particular emphasis on genes associated with both iPSC donor cell types and differentiated target cell populations, especially hematopoietic lineages.
In addition to cancer genomics datasets, the panel incorporates genes known to acquire recurrent mutations during prolonged hPSC culture1,3. For example, Merkle et al. identified recurrent dominant-negative mutations in TP53 across a large cohort of hPSCs3. A subsequent analyzing 146 hPSC lines found 64% had TP53 mutations, which conferred a pronounced selective advantage, perturbed target gene expression and altered cellular differentiation4.
This data-driven approach ensures coverage of genetic variants most relevant to stem cell maintenance, differentiation outcomes, and translational safety considerations.
Ensuring Success from Research to Clinical Translation
As hPSC-based research and development of ATMPs continue to advance, a monitoring strategy to detect cancer-associated variants is becoming an increasingly important component of genomic quality assessment. The WiCell Oncopanel Assay provides insight into the genomic integrity of hPSC lines and helps mitigate risks that may impact downstream research and clinical development.
Learn more about the assay, including sample requirements and additional details: https://www.wicell.org/test-cells/oncopanel-assay/
References
- Rouhani FJ, Zou X, Danecek P, et al. Substantial somatic genomic variation and selection for BCOR mutations in human induced pluripotent stem cells. Nat Genet. 2022;54(9):1406-1416. doi:10.1038/s41588-022-01147-3
- Puigdevall P, Jerber J, Danecek P, Castellano S, Kilpinen H. Somatic mutations alter the differentiation outcomes of iPSC-derived neurons. Cell Genom. 2023;3(4):100280. Published 2023 Mar 23. doi:10.1016/j.xgen.2023.100280
- Merkle FT, Ghosh S, Kamitaki N, et al. Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations. Nature. 2017;545(7653):229-233. doi:10.1038/nature22312
- Lezmi E, Jung J, Benvenisty N. High prevalence of acquired cancer-related mutations in 146 human pluripotent stem cell lines and their differentiated derivatives. Nat Biotechnol. 2024;42(11):1667-1671. doi:10.1038/s41587-023-02090-2
- Al Delbany D, Ghosh MS, Krivec N, et al. De Novo Cancer Mutations Frequently Associate with Recurrent Chromosomal Abnormalities during Long-Term Human Pluripotent Stem Cell Culture. Cells. 2024;13(16):1395. Published 2024 Aug 21. doi:10.3390/cells13161395
- Mendiratta G, Ke E, Aziz M, Liarakos D, Tong M, Stites EC. Cancer gene mutation frequencies for the U.S. population. Nat Commun. 2021;12(1):5961. Published 2021 Oct 13. doi:10.1038/s41467-021-26213-y