Human pluripotent stem cells recurrently acquire and expand dominant negative P53 mutations

Abstract

Human pluripotent stem cells (hPSCs) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with acquisition of large copy number variants (CNVs) that provide mutant cells with a growth advantage in culture1–3. However, the nature, extent, and functional impact of other acquired genome sequence mutations in cultured hPSCs is not known. Here, we sequenced the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hESC) lines, including 26 lines prepared for potential clinical use4. We then applied computational strategies for identifying mutations present in a subset of cells5. Though such mosaic mutations were generally rare, we identified five unrelated hESC lines that carried six mutations in the TP53 gene that encodes the tumor suppressor P53. Notably, the TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We used droplet digital PCR to demonstrate that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that P53 mutation confers selective advantage. When we then mined published RNA sequencing data from 117 hPSC lines, we observed another nine TP53 mutations, all resulting in coding changes in the DNA binding domain of P53. Strikingly, in three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from loss of heterozygosity at the TP53 locus. As the acquisition and favored expansion of cancer-associated mutations in hPSCs may go unnoticed during most applications, we suggest that careful genetic characterization of hPSCs and their differentiated derivatives should be carried out prior to clinical use.

Putting p53 in Context

“…The most proliferative individuals outcompete populations of single cell organisms, while multicellular organisms require cellular cooperation, at the expense of competition, to maintain coordinated, specialized functions. The need for cooperation starts in embryonic development, where p53 restricts expansion of individual “cheater” cells, observed in chimeric blastocysts upon p53 knockdown (Dejosez et al, 2013) and following positive selection of spontaneous TP53 mutations detected in commonly used human ES lines (Merkle et al, 2017). Tight regulation of DNA methylation by DNMT and TET family enzymes requires p53 and it appears that epigenetic disorder contributes to this clonal heterogeneity in p53-deficient ESC colonies (Tovy et al, 2017).…”

Insights into the Mutational Burden of Human Induced Pluripotent Stem Cells from an Integrative Multi-Omics Approach

“…A study utilizing this approach showed that in normal skin cells, which are often used to derive iPSCs, many cancer genes harbor somatic mutations that were likely caused by ultraviolet (UV) light exposure (Martincorena et al, 2015). Additionally, a study on human embryonic stem cells (Merkle et al, 2017) found that ~20% of analyzed cell lines had deleterious subclonal mutations, including some known cancer drivers in TP53 . We hypothesize that due to their different origins, clonal and subclonal mutations have been under different selective pressures.…”

Lung cancer stem cells—origin, characteristics and therapy

“…Mutations are well known for causing cancer through inheritance, environmental carcinogens or DNA replication errors (23 (24).…”