In a study published in Stem Cell Reports on June 18, a team led by Prof. ZHENG Ping from the Kunming Institute of Zoology of the Chinese Academy of Sciences, and collaborators from Zhejiang University School of Medicine, constructed the first high-resolution map of early replication fragile sites (ERFSs) in human embryonic stem cells (hESCs), and addressed why copy number variations (CNVs) and single nucleotide variants (SNVs) recurrently accumulate at specific genomic regions during prolonged hESC culture—a key concern for regenerative medicine.

The researchers developed a two-step cell cycle synchronization strategy combining low-dose drug treatment with hydroxyurea-induced replication stress, which overcomes hESC sensitivity to conventional synchronization agents. Using EdU labeling and CUT&Tag profiling of replication stress-associated proteins (RPA, BRCA1, SMC5) and the DNA damage marker γH2AX, they identified 2,404 high-confidence ERFSs in H9 cells and 1,438 in TJ-1# cells.

The researchers found that ERFSs were enriched in GC content, short interspersed nuclear elements (SINEs), and exhibited high chromatin accessibility, with ~70% located in gene promoters and ~30% overlapping validated enhancers. Associated genes were predominantly involved in pluripotency, proliferation, and DNA damage response.

Critically, the researchers revealed that ERFS hotspots significantly overlapped with CNV gain events (including the recurrent 20q11.21 locus) and showed elevated SNV frequencies with mutational signatures resembling cancer genomes. Unlike common fragile sites, ERFS formation correlated with open chromatin and transcriptional activity rather than gene size.

This ERFS map provides a structural framework for understanding selective genetic variant accumulation in hESC culture, and may help to optimize culture conditions to reduce genomic instability at its source.