Longitudinal analysis of genetic and epigenetic changes in human pluripotent stem cells in the landscape of culture-induced abnormality

Human embryonic stem cells (hESCs) are naturally equipped to maintain genome integrity to minimize genetic mutations during early embryo development. However, genetic aberration risks and subsequent cellular changes in hESCs during in vitro culture pose a significant threat to stem cell therapy. While a few studies have reported specific somatic mutations and copy number variations (CNVs), the molecular mechanisms underlying the acquisition of 'culture-adapted phenotypes' by hESCs are largely unknown. Therefore, we conducted comprehensive genomic, single-cell transcriptomic, and single-cell ATAC-seq analyses of an isogenic hESC model displaying definitive 'culture-adapted phenotypes'. We found that hESCs lacking TP53, in which loss-of-function mutations were identified in human pluripotent stem cells (hPSCs), presented a surge in somatic mutations. Notably, hPSCs with a copy number gain of 20q11.21 during early passage did not present 'culture-adapted phenotypes' or BCL2L1 induction. Single-cell RNA-seq and ATAC-seq analyses revealed active transcriptional regulation at the 20q11.21 locus. Furthermore, the induction of BCL2L1 and TPX2 to trigger 'culture-adapted phenotypes' was associated with epigenetic changes facilitating TEA domain (TEAD) binding. These results suggest that 20q11.21 copy number gain and additional epigenetic changes are necessary for expressing 'culture-adapted phenotypes' by activating gene transcription at this specific locus. Human pluripotent stem cells (hPSCs) hold significant potential in regenerative medicine due to their ability to produce all types of cells. This study examines how genetic changes in hPSCs impact their stability and safety. Researchers used a set of identical human embryonic stem cells, grown for up to six years, and analyzed them using whole genome sequencing and single-cell sequencing techniques. The study found that certain genetic aberrations, especially mutations in the TP53 gene and a recurrent gain at 20q11.21, become more frequent as hPSCs are grown longer. These findings emphasize that TP53 mutations and 20q11.21 gains can alter the biological characteristics of hPSCs, affecting their safety more than the number of times the cells have been replicated. Understanding these changes is important for establishing guidelines to ensure the safety of stem cell therapy.This summary was initially drafted using artificial intelligence, then revised and fact-checked by the author.