The Fragile Site Landscape of Induced Pluripotent Stem Cells: Hierarchy, Variability, Tissue Specificity, and Links to Culture-Acquired Rearrangements.

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.