Longitudinal whole-genome analysis of human embryonic stem cells during extended expansion and differentiation

Background Human embryonic stem cells (hESCs) hold tremendous promise for regenerative medicine but require careful monitoring of genomic integrity during long-term expansion. Chemically defined media have been developed to reduce variability associated with complex culture conditions. Results Here, using longitudinal whole-genome sequencing (WGS) of established hESC lines cultured in E8 medium across 40 passages and during differentiation into dopaminergic (DA) neurons, we revealed a low-burden of detectable acquired mutations dominated by clock-like and oxidative stress-related signatures, consistent with background replication errors and reactive oxygen species (ROS) exposure. Detected variants generally showed low variant allele frequencies (VAFs) and limited recurrence across sampled passages, with no consistent VAF increases suggestive of strong positive selection or clonal expansion under the current sequencing depth and variant-calling criteria. Notably, a BCOR p.F914Wfs*2 frameshift variant was detected at passage 40 in the E8-cultured Q19 line (VAF = 0.53), although its longitudinal dynamics and biological significance remain unresolved. Similar low overall mutation burdens were observed in hESCs cultured in AIC medium across approximately 50 passages. Conclusions Collectively, these results suggest that optimized, chemically defined medium can support a low burden of detectable genomic alterations during propagation in the examined hESC lines, while larger studies across additional genetic backgrounds, together with predefined genomic quality-control criteria, and continued genomic monitoring remain essential prior to clinical application.