Genetic instability in human pluripotent stem cells (hPSCs) is a well-documented challenge. Recurrent cytogenetic abnormalities arise during culture and confer undesirable or unwanted phenotypes. We previously showed that routine single-cell passaging of hPSCs can result in a high incidence of de novo genetic abnormalities, and that eTeSR™, a novel hPSC maintenance medium optimized for single-cell passaging, can significantly reduce the appearance of these recurrent abnormalities.

To further demonstrate the genomic stability of routinely single-cell passaged hPSCs in eTeSR™, 135 clonal sublines (derived from H1, H9, and SCTi003-A hPSCs) were independently passaged for 20 weeks in eTeSR™ while 136 clonal sublines were also independently passaged as single-cells in two other commercially-available hPSC media.

Single nucleotide polymorphism microarray analysis revealed that, after 20 weeks, 70% of clonal sublines maintained in control media acquired at least one de novo abnormality compared to only 25% eTeSR™-maintained sublines. This can be attributed to fewer small (< 10,000 kb) structural variants detected in cells maintained in eTeSR™ (3%) compared to control media (69%). Notably, 51% of control sublines displayed a gain in chromosome 20q11, a well-characterized, recurrent copy number variant which conveys a selective advantage. Conversely, the 20q11 abnormality was not detected in any of the eTeSR™ samples. This study underscores the role of innovative media formulations in mitigating culture-acquired genetic aberrations in hPSCs, addressing a critical challenge in the field.