Comprehensive Assessment of the Genomic Stability of Human Induced Pluripotent Stem Cells for Clinical Applications

Background Human induced pluripotent stem cells (hiPSCs) acquire genomic instability during reprogramming and culture, which poses significant risks for clinical applications. Current detection methods, such as karyotyping analysis, often fail to identify critical submicroscopic variations. This highlights an urgent need for comprehensive genomic surveillance strategies. Methods Three human iPSC lines were continually cultured in vitro for 50 passages, with genome stability evaluated every 10 passages. The evaluation methods included karyotyping to detect chromosomal abnormalities, optical genome mapping (OGM) to identify copy number variations (CNVs) and structural variants (SVs), whole-exome sequencing (WES) to detect coding mutations, and RNA sequencing (RNA-seq) to detect the changes of gene expression. Results We detected accumulating chromosomal abnormalities (e.g., trisomy 12), SVs, CNVs, and sequence mutations in three hiPSC lines during extended culture. OGM effectively identified SVs and CNVs below karyotyping resolution, particularly recurrent genome abnormalities such as gains on chr17q, chr12p and chr20q. WES revealed coding mutations, including germline short variants and newly acquired somatic mutations, some of which were associated with tumors or diseases, such as CDH1 , BCOR . Transcriptional changes correlated with genomic alterations, including dysregulation of oncogenes such as BCL2L1 , KRAS and MDM2 . Results demonstrate that each method had unique detection capabilities and limitations, and only integrative approaches can comprehensively identify genomic abnormalities. Conclusions This study established a comprehensive strategy for evaluating the genetic stability of hiPSCs by integrating karyotyping, OGM, WES, and RNA-seq. This comprehensive strategy can be applied to scenarios such as hiPSC clone screening, establishment of cell bank passages, and quality control of hiPSC-derived products. It provides a reliable genetic stability evaluation protocol to support the safe clinical application of hiPSC-related products.