Depletion of TP53 in Human Pluripotent Stem Cells Triggers Malignant-Like Behavior

Joaquin Montilla-Rojo; Thomas F. Eleveld; Marnix van Soest; Sanne Hillenius; Dennis M. Timmerman; Ad J.M. Gillis; Bernard A.J. Roelen; Christine L. Mummery; Leendert H.J. Looijenga; Daniela C.F. Salvatori

doi:10.1002/adbi.202400538

Depletion of TP53 in Human Pluripotent Stem Cells Triggers Malignant-Like Behavior

Joaquin Montilla-Rojo, Thomas F. Eleveld, Marnix van Soest, Sanne Hillenius, Dennis M. Timmerman, Ad J.M. Gillis, Bernard A.J. Roelen, Christine L. Mummery, Leendert H.J. Looijenga, Daniela C.F. Salvatori^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Human pluripotent stem cells (hPSCs) tend to acquire genetic aberrations upon culture in vitro. Common aberrations are mutations in the tumor suppressor TP53, suspected to confer a growth-advantage to the mutant cells. However, their full impact in the development of malignant features and safety of hPSCs for downstream applications is yet to be elucidated. Here, TP53 is knocked out in hPSCs using CRISPR-Cas9 and compared them with isogenic wild-type hPSCs and human germ cell tumor lines as models of malignancy. While no major changes in proliferation, pluripotency, and transcriptomic profiles are found, mutant lines display aberrations in some of the main chromosomal hotspots for genetic abnormalities in hPSCs. Additionally, enhanced clonogenic and anchorage-free growth, alongside resistance to chemotherapeutic compounds is observed. The results indicate that common TP53-depleting mutations in hPSCs, although potentially overlooked by standard analyses, can impact their behavior and safety in a clinical setting.

Original language	English
Article number	2400538
Journal	Advanced Biology
Issue number	4
DOIs	https://doi.org/10.1002/adbi.202400538
Publication status	E-pub ahead of print - 6 Jan 2025

Bibliographical note

Publisher Copyright:
© 2025 The Author(s). Advanced Biology published by Wiley-VCH GmbH.

Funding

The authors acknowledge the Utrecht Sequencing Facility (USEQ) for providing sequencing service and data. USEQ is subsidized by the University Medical Center Utrecht and The Netherlands X-omics Initiative (NWO project 184.034.019). The LU07 (LUMC007iCTRL01) and H9 (WA09) cells were kindly provided by Christian Freund, LUMC, Leiden, The Netherlands. This work was supported by Kinderen Kankervrij (KiKa) foundation (L.L., T.E.) and the Novo Nordisk Foundation Center for Stem Cell Medicine reNEW (NNF21CC0073729) (S.H., L.H.J.L).

Funders	Funder number
University Medical Center Utrecht
Netherlands X-omics Initiative (NWO)	184.034.019
Kinderen Kankervrij (KiKa) foundation
Novo Nordisk Foundation Center for Stem Cell Medicine reNEW	NNF21CC0073729

Keywords

genetic aberration
human germ cell tumor
human pluripotent stem cell
malignancy
pluripotency
TP53

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Montilla-Rojo, J., Eleveld, T. F., van Soest, M., Hillenius, S., Timmerman, D. M., Gillis, A. J. M., Roelen, B. A. J., Mummery, C. L., Looijenga, L. H. J., & Salvatori, D. C. F. (2025). Depletion of TP53 in Human Pluripotent Stem Cells Triggers Malignant-Like Behavior. Advanced Biology, (4), Article 2400538. Advance online publication. https://doi.org/10.1002/adbi.202400538

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abstract = "Human pluripotent stem cells (hPSCs) tend to acquire genetic aberrations upon culture in vitro. Common aberrations are mutations in the tumor suppressor TP53, suspected to confer a growth-advantage to the mutant cells. However, their full impact in the development of malignant features and safety of hPSCs for downstream applications is yet to be elucidated. Here, TP53 is knocked out in hPSCs using CRISPR-Cas9 and compared them with isogenic wild-type hPSCs and human germ cell tumor lines as models of malignancy. While no major changes in proliferation, pluripotency, and transcriptomic profiles are found, mutant lines display aberrations in some of the main chromosomal hotspots for genetic abnormalities in hPSCs. Additionally, enhanced clonogenic and anchorage-free growth, alongside resistance to chemotherapeutic compounds is observed. The results indicate that common TP53-depleting mutations in hPSCs, although potentially overlooked by standard analyses, can impact their behavior and safety in a clinical setting.",

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AB - Human pluripotent stem cells (hPSCs) tend to acquire genetic aberrations upon culture in vitro. Common aberrations are mutations in the tumor suppressor TP53, suspected to confer a growth-advantage to the mutant cells. However, their full impact in the development of malignant features and safety of hPSCs for downstream applications is yet to be elucidated. Here, TP53 is knocked out in hPSCs using CRISPR-Cas9 and compared them with isogenic wild-type hPSCs and human germ cell tumor lines as models of malignancy. While no major changes in proliferation, pluripotency, and transcriptomic profiles are found, mutant lines display aberrations in some of the main chromosomal hotspots for genetic abnormalities in hPSCs. Additionally, enhanced clonogenic and anchorage-free growth, alongside resistance to chemotherapeutic compounds is observed. The results indicate that common TP53-depleting mutations in hPSCs, although potentially overlooked by standard analyses, can impact their behavior and safety in a clinical setting.

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Depletion of TP53 in Human Pluripotent Stem Cells Triggers Malignant-Like Behavior

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