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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., (© 2025 The Author(s). Advanced Biology published by Wiley‐VCH GmbH.)

The development of human induced pluripotent stem cell (iPSC)-based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN's prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter's versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.