Senior Scientist STEMCELL Technologies Inc. Vancouver, British Columbia, Canada
Abstract: Genetic instability in human pluripotent stem cells (hPSCs) is a well-documented challenge. Recurrent cytogenetic abnormalities arise during culture and confer a selective advantage to genetically variant cells through mechanisms including resistance to cell death, increased cell proliferation and reduced differentiation capacity. 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 hPSC sublines (derived from H1, H9, and SCTi003-A hPSCs) were independently expanded for 20 weeks in eTeSR™ while 136 clonal sublines were independently expanded 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 and is often undetectable by G-band karyotyping. Conversely, the 20q11 abnormality was not detected in any of the eTeSR™ samples. To investigate the reduced incidence of 20q11 observed in eTeSR™-maintained sublines, we performed a competitive mixing assay in which hPSCs with a gain of 20q11.21 were mixed with fluorescently labelled wild-type cells (1:20 ratio) and cultured for 5 passages. We observed a 4-fold reduction in the takeover rate of 20q11.21 when cells were cultured in eTeSR™ (14 ± 8%) compared to 62 ± 7% in an alternative commercial single-cell workflow (mean ± STDEV; n = 4). This study underscores the role of innovative media formulations in mitigating culture-acquired genetic aberrations in hPSCs, addressing a critical challenge in the field.
