Human pluripotent stem cells (hPSCs) are pivotal in regenerative medicine, yet their in vitro expansion often leads to genetic abnormalities, raising concerns about their safety in clinical applications. This study analyzed ten human embryonic stem cell lines across multiple passages to elucidate the dynamics of chromosomal abnormalities and single-nucleotide variants (SNVs) in 380 cancer-related genes. Prolonged in vitro culture resulted in 80% of the lines acquiring gains of chromosome 20q or 1q, both known for conferring an in vitro growth advantage. 70% of lines also acquired other copy number variants (CNVs) outside the recurrent set. Additionally, we detected 122 SNVs in 88 genes, with all lines acquiring at least one de novo SNV during culture. Our findings showed higher loads of both CNVs and SNVs at later passages, which were due to the cumulative acquisition of mutations over a longer time in culture, and not to an increased rate of mutagenesis over time. Importantly, we observed that SNVs and rare CNVs followed the acquisition of chromosomal gains in 1q and 20q, while most of the low-passage and genetically balanced samples were devoid of cancer-associated mutations. This suggests that recurrent chromosomal abnormalities are potential drivers for the acquisition of other mutations.

Human pluripotent stem cells (hPSC) are increasingly used in clinical trials, with retinal pigment epithelium (RPE) being among the most transplanted cell types. However, hPSC frequently acquire chromosomal abnormalities, whose impact on differentiation and transplant safety is incompletely understood. In this work, we investigate how aneuploidy influences the progression of hPSC through undirected RPE differentiation. Large-scale omics analysis of genetically normal hPSC cultures reveals pervasive low-grade mosaicism, with 3–6% of cells carrying different aneuploidies. During undirected differentiation, all aneuploid cells are lost except those with a gain of chromosome arm 1q. These cells differentiate efficiently only when co-cultured with wild-type cells, which promote neural differentiation through paracrine signals that 1q-gain cells are uniquely receptive to. This interaction enables 1q-gain cells not only to persist but to eventually dominate the culture, owing to their competitive advantage.

Background: Gains of chromosome 20q11.21 are among the most common culture-acquired abnormalities in human pluripotent stem cells (hPSC), conferring a well-defined survival advantage while altering differentiation capacity. However, it remains unclear whether this advantage persists during differentiation, how the aneuploidy alters ectodermal and retinal pigment epithelium (RPE) lineage specification, and which genes within the minimal amplicon drive these effects. Methods: We used three isogenic human embryonic stem cell line pairs (wild-type and 20q11.21 gain) and assessed their behaviour in two neuroectoderm differentiation systems: directed neuroectoderm induction (dual SMAD inhibition) and long-term spontaneous RPE differentiation. Competitive dynamics were measured in mixed cultures, and lineage outcomes were analysed using immunostaining, gene expression profiling and single-cell RNA sequencing. To identify driver genes, we generated BCL2L1 and ID1 overexpression lines and tested their effects under both directed and spontaneous differentiation conditions. Results: Across all lines and conditions, 20q cells expanded from a minor fraction to dominate mixed cultures, indicating that their competitive advantage persists beyond the undifferentiated state. Despite this dominance, pure 20q cells failed to specify to neuroectoderm or RPE. Single-cell transcriptomics revealed consistent diversion toward non-neural ectodermal and extraembryonic fates. Mechanistically, overexpression of BCL2L1 and ID1 alone or in combination impaired neuroectoderm specification, while synergistic effect of both genes promoted non-neural ectodermal outcomes under directed differentiation conditions. In spontaneous differentiation, both genes could disrupt differentiation. Conclusions: The 20q11.21 gain couples a persistent survival advantage with a disruption of neural and RPE lineage competence, redirecting cells toward alternative ectodermal and extraembryonic fates. These effects arise from the combined action of two dosage-sensitive genes BCL2L1 and ID1 within the amplicon, illustrating how regional gene dosage can reshape developmental signalling responses in hPSC.

C-C motif chemokine receptor-like 2 (CCRL2) is an atypical chemokine receptor (ACKR) that binds chemerin with high affinity but lacks classical G protein-coupled signaling. Instead, it functions as a non-signaling presenter of chemerin to CMKLR1-expressing cells, modulating antitumor immunity. CCRL2 is highly expressed in the tumor microenvironment and various human cancers, and its expression has been linked to delayed tumor growth in mouse models, primarily through the chemerin/CMKLR1 axis. While CCRL2’s role in immune surveillance is well established, its tumor cell-intrinsic functions remain less clear. Here, we investigated the impact of CCRL2 overexpression and knockout on tumor cell behavior in vitro. Although CCRL2 did not affect proliferation, migration, or clonogenicity in B16F0 melanoma and LLC cells, it significantly influenced spheroid morphology in B16F0 cells. Transcriptomic analysis revealed that CCRL2 modulates innate immune signaling pathways, including TLR4 and IFN-γ/STAT1, with context-dependent downstream effects. These findings suggest that CCRL2 shapes tumor architecture by rewiring inflammatory signaling networks in a cell-intrinsic manner. Further studies in other cancer types and cell models are needed to determine whether CCRL2’s regulatory role is broadly conserved and to explore its potential as a therapeutic target in solid tumors.