Sequential genetic and epigenetic alterations in human pluripotent stem cells for recurrent 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 ‘culture-adapted phenotype’ acquisitions of 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.’ Notably, hPSCs with a copy number gain of 20q11.21 during early passage did not present ‘culture-adapted phenotypes’ nor BCL2L1 induction. Single-cell RNAseq and ATACseq analyses revealed active transcriptional regulation at 20q11.21 loci at late-passaged hESCs with the induced BCL2L1 and TPX2 to trigger ‘culture-adapted phenotypes’ was associated with epigenetic changes facilitating TEA domain (TEAD) binding. These results suggest that copy number 20q11.21 gain and additional epigenetic changes are necessary for expressing ‘culture-adapted phenotypes’ by activating gene transcription at this specific locus.