Victoria O. Pozhitnova

Cystic fibrosis is a life-threatening inherited disease caused by pathogenic variants in the CFTR gene that impair chloride and bicarbonate transport across airway epithelia. Gene editing and cell replacement strategies hold promise for restoring CFTR function, yet they inevitably generate heterogeneous cell populations in which only a fraction of cells carry a corrected allele, raising the question of how large this fraction must be for functional rescue to be detectable and whether the forskolin-induced swelling (FIS) assay is sensitive enough to capture partial correction. To address this, we generated chimeric iPSC-derived lung organoids by co-culturing GFP-labeled wild-type airway basal cells with unlabeled F508del-homozygous cystic fibrosis basal cells at seven predefined ratios (0–100% wild-type), verifying the actual wild-type fraction by flow cytometry and quantifying 24-h forskolin-induced swelling as the area ratio. Chimeric organoids showed a dose-dependent increase in swelling with rising wild-type fractions, with a statistically significant FIS response first resolved at approximately 6% wild-type cells in the pooled analysis. Separately, ROC analysis of well-level data identified two area-ratio cutoffs for classifying organoid populations—1.16 (80% sensitivity, 80% specificity) and 1.21 (98% specificity, 70% sensitivity)—which the fitted mean response reaches at approximately 14% and 34% wild-type cells, respectively. The minimum statistically detectable fraction and the classification cutoffs are distinct quantities derived from different analyses and are not interchangeable; both describe the performance of the FIS assay in heterogeneous organoids rather than thresholds for therapeutic benefit and provide a practical reference framework for designing and interpreting gene editing and cell therapy studies in cystic fibrosis.

Induced pluripotent stem cells (iPSCs) are prone to genomic instability during prolonged culture, with recurrent chromosomal aberrations conferring selective advantages. Replication stress is a major driver of this instability, yet the repertoire of replication stress-sensitive loci in iPSCs remains largely unexplored. Here, we mapped aphidicolin-sensitive fragile sites (asFS) in three independent iPSC lines using classical cytogenetic break analysis combined with Monte Carlo simulation and MiDAS mapping directly on banded metaphase chromosomes. We identified 28 asFS, which segregated into a highly active Major cluster (8 sites, accounting for 59% of breaks among asFS) and a less active Minor cluster (20 sites). Five universal asFS (9p21, 6q25-26, 20p11-12, 10q22, Xq25) were present in all three lines, representing a fragility signature associated with the pluripotent state, with Xq25 shifting into the Major cluster after correction for X chromosome dosage. Minor asFS showed preferential co-localization with physical breakpoints or minimal overlapping regions of recurrent culture-acquired aberrations, including 20q11.21 (BCL2L1), 1q32 (MDM4), 8q24 (MYC), 17q21 (WNT3-WNT9B), and 18q21 (DCC/FRA18B). MiDAS mapping validated most asFS and revealed additional replication stress-sensitive loci in pericentromeric and subtelomeric regions that are difficult to score by conventional G-banding. Comparison with fragile site maps from other cell types revealed that the iPSC asFS repertoire is distinct in rank order and relative activity, characteristic of the pluripotent state. Collectively, our findings indicate that the asFS repertoire in iPSCs is hierarchically organized into a stable universal core and a variable peripheral component, and suggest that Minor asFS may contribute to, or be associated with, the genesis of culture-acquired rearrangements. This work provides a framework for understanding how replication stress and clonal selection shape the mutational landscape of pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) are prone to genomic instability during prolonged culture, with recurrent chromosomal aberrations conferring selective advantages. Replication stress is a major driver of this instability, yet the repertoire of replication stress‑sensitive loci in iPSCs remains largely unexplored. Here, we mapped aphidico-lin‑sensitive fragile sites (asFS) in three independent iPSC lines using classical cytogenetic break analysis combined with Monte Carlo simulation and MiDAS mapping directly on banded metaphase chromosomes. We identified 28 asFS, which segregated into a highly active Major cluster (8 sites, accounting for 59% of breaks among asFS) and a less active Minor cluster (20 sites). Five universal asFS (9p21, 6q25-26, 20p11-12, 10q22, Xq25) were present in all three lines, representing a tissue‑specific fragility signature of pluripotent stem cells, with Xq25 shifting into the Major cluster after correction for X chromosome dosage. Minor asFS showed preferential co‑localization with physical breakpoints or minimal overlapping regions of recurrent culture‑acquired aberrations, including 20q11.21 (BCL2L1), 1q32 (MDM4), 8q24 (MYC), 17q21 (WNT3‐WNT9B), and 18q21 (DCC/FRA18B). MiDAS mapping validated most asFS and revealed additional replication stress‑sensitive loci in pericentromeric and subtelomeric regions that are difficult to score by conventional G‑banding. Comparison with fragile site maps from other cell types con-firmed tissue specificity, showing that the iPSC asFS repertoire is distinct in rank order and relative activity. Collectively, our findings indicate that the asFS repertoire in iPSCs is hierarchically organized into a stable universal core and a variable peripheral component, and suggest that Minor asFS may serve as initial substrates for adaptive culture‑acquired rearrangements. This work provides a framework for understanding how replication stress and clonal selection shape the mutational landscape of pluripotent stem cells

Xeroderma Pigmentosum group C (XP-C) is an autosomal recessive disorder caused by mutations in the XPC gene, leading to defective nucleotide excision repair. This defect leads to genomic instability and a profound cancer predisposition. To model this disease, we generated induced pluripotent stem cells (iPSCs) from an XP-C patient carrying a novel homozygous nonsense mutation in the XPC gene (c.1830C>A). The resulting iPSCs demonstrated typical pluripotent characteristics, including expression of key markers and trilineage differentiation capability. However, genomic assessment revealed progressive karyotypic instability during extended culture. While initial whole-genome sequencing detected no major chromosomal abnormalities, subsequent G-banding analysis identified acquired trisomy 12 in two lines (CL12 and CL27) and a derivative X chromosome in a third line (CL30). These abnormalities were absent in early-passage analyses, indicating that they were acquired and selected for during extended culture. The acquisition of a derivative X chromosome in CL30, alongside recurrent trisomy 12, represents a unique cytogenetic signature likely attributable to the underlying XPC defect. We hypothesize that the loss of GG-NER creates a permissive genomic environment, accelerating the accumulation of DNA damage and chromosomal missegregation under replicative stress. This temporal divergence in genomic integrity highlights how culture pressures drive chromosomal evolution in XP-C iPSCs independently of initial reprogramming. Our findings emphasize that XP-C iPSCs require continuous genomic surveillance and provide a model for investigating how DNA repair deficiencies interact with in vitro culture stress.

Genome instability in induced pluripotent stem cells (IPSC) poses a significant challenge for their use in research and medicine. Cataloging and precisely describing all the identified aberrations that arise during cell reprogramming, expansion, and differentiation is essential for improving approaches to instability prevention and ensuring genetic quality control. We report the karyotypic analysis of 65 cell lines derived from skin fibroblasts, urinal sediment, and peripheral blood mononuclear cells of 33 individuals, 82% of whom suffer from monogenic genetic disorders not associated with genetic instability. Trisomy of chromosomes 20 and 8 was revealed recurrently, while the 1q arm was the most frequently affected region involved in interstitial duplications and unbalanced translocations with chromosomes 15 and 18. The localization of rearrangement breakpoints identified by SNP arrays within the large DCC gene and histone gene clusters links genetic instability in IPSCs to replication-stress-induced chromosome breakage at common and early replicating fragile sites.

Substantial background level of replication stress is a feature of embryonic and induced pluripotent stem cells (iPSCs), which can predispose to numerical and structural chromosomal instability, including recurrent aberrations of chromosome 12. In differentiated cells, replication stress-sensitive genomic regions, including common fragile sites, are widely mapped through mitotic chromosome break induction by mild aphidicolin treatment, an inhibitor of replicative polymerases. IPSCs exhibit lower apoptotic threshold and higher repair capacity hindering fragile site mapping. Caffeine potentiates genotoxic effects and abrogates G2/M checkpoint delay induced by chemical and physical mutagens. Using 5-ethynyl-2′-deoxyuridine (EdU) for replication labeling, we characterized the mitotic entry dynamics of asynchronous iPSCs exposed to aphidicolin and/or caffeine. Under the adjusted timing of replication stress exposure accounting revealed cell cycle delay, higher metaphase chromosome breakage rate was observed in iPSCs compared to primary lymphocytes. Using differential chromosome staining and subsequent locus-specific fluorescent in situ hybridization, we mapped the FRA12L fragile site spanning the large neuronal ANKS1B gene at 12q23.1, which may contribute to recurrent chromosome 12 missegregation and rearrangements in iPSCs. Publicly available data on the ANKS1B genetic alterations and their possible functional impact are reviewed. Our study provides the first evidence of common fragile site induction in iPSCs and reveals potential somatic instability of a clinically relevant gene during early human development and in vitro cell expansion.