Selection Mechanisms during Culture Adaptation in Stable Human Pluripotent Stem Cell Cultures

Human pluripotent stem cells (hPSCs) have generated a lot of interest in the scientific community based on their potential applications in tissue engineering and regenerative medicine. However, numerous research groups have continued to report a propensity for genomic alterations during hPSC culture that poses concerns for basic research and clinical applications. To realize the clinical promise of stem cells, significant research in the development of propagation systems and bioprocessing strategies that generate stable stem cells is needed. Work from our laboratory and others have demonstrated that amplification of specific chromosomal regions is correlated with increased gene expression. To date, the phenotypic association of common genomic alterations remains unclear and is a cause for concern during hPSC propagation. Determining specific genes responsible for competitive advantage of abnormal hPSCs has been elusive; and progress on our molecular understanding of why hPSC single‐cell dissociation increases aneuploid selection has been slow. In our study, we focus on a common genomic aberration and a list of candidate genes with increased gene expression to functionally validate a gene that may confer selective advantage when overexpressed. Our studies demonstrate that transduced hPSCs exhibited culture dominance in co-cultures of modified overexpression lines with non-overexpression lines. Furthermore, during low density seeding, we demonstrate increased clonality of our overexpression line against matched controls. Our results also indicate that this selective advantage could be reduced by varying the hPSC propagation and culture conditions. Our findings have important implications for hPSCs to reach their full potential in regenerative medicine, as it is critical to address the mechanisms that act as molecular switches during culture adaptation that lead to genomic instability in order to develop appropriate propagation strategies for generation of chromosomally stable hPSCs.