Identification and functional relevance of cancer-related mutations in human pluripotent stem cells and their differentiated progenies

Human pluripotent stem cells (hPSCs) are known to acquire chromosomal aberrations in culture, affecting their growth as well as their tumorigenic potential. We have recently shown using whole exome sequencing and RNA-sequencing, that hPSCs acquire cancer-related point-mutation in the tumor suppressor gene TP53, suggesting the presence of cancer-related mutations also in other genes. Using our established methodology for the identification of cancer-related point-mutations using RNA-sequencing, we analyzed published data of the prevalent hPSC lines; WA01 and WA09, to investigate whether hPSCs acquire additional cancer related mutations in culture. By comparing 172 RNA-sequencing samples, originating from 44 studies of 40 different research groups, to early passage WES of the same cell lines, we identified cancer-related mutations in 33% of the samples. While TP53 remains the most mutated gene in hPSCs, we have identified less frequent events also in other genes. Our most recent analysis identified cancer-related point-mutations not only in undifferentiated hPSCs, but also in their differentiated progenies. Importantly, we highlight the persistent expression of dominant-negative mutant alleles in differentiated hPSCs-derived cells, as well as the functional implications of such mutations in both the undifferentiated and differentiated states. Thus, we show the acquisition of pathogenic mutations characteristic to human tumors, during the culturing of hPSCs, and present a bioinformatic pipeline for the identification of such mutations using RNA-sequencing.

Part of this work was recently been published in:

Avior, Y., Lezmi, E., Eggan, K. & Benvenisty, N. Cell Stem Cell (2021)

Lezmi, E. & Benvenisty, N. Nature Protocols (2021)