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. 2017 May 11;545(7653):229-233.
doi: 10.1038/nature22312. Epub 2017 Apr 26.

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

Florian T Merkle #  1   2   3   4 , Sulagna Ghosh #  1   2   3   4 , Nolan Kamitaki  3   5   6 , Jana Mitchell  1   2   3   4 , Yishai Avior  7 , Curtis Mello  3   5   6 , Seva Kashin  3   5   6 , Shila Mekhoubad  1   2   4 , Dusko Ilic  8 , Maura Charlton  1   2   3   4 , Genevieve Saphier  1   3   4 , Robert E Handsaker  3   5   6 , Giulio Genovese  3   5   6 , Shiran Bar  7 , Nissim Benvenisty  7 , Steven A McCarroll  3   5   6 , Kevin Eggan  1   2   3   4
Affiliations

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

Florian T Merkle et al. Nature. .

Abstract

Human pluripotent stem cells (hPS cells) can self-renew indefinitely, making them an attractive source for regenerative therapies. This expansion potential has been linked with the acquisition of large copy number variants that provide mutated cells with a growth advantage in culture. The nature, extent and functional effects of other acquired genome sequence mutations in cultured hPS cells are not known. Here we sequence the protein-coding genes (exomes) of 140 independent human embryonic stem cell (hES cell) lines, including 26 lines prepared for potential clinical use. We then apply computational strategies for identifying mutations present in a subset of cells in each hES cell line. Although such mosaic mutations were generally rare, we identified five unrelated hES cell lines that carried six mutations in the TP53 gene that encodes the tumour suppressor P53. The TP53 mutations we observed are dominant negative and are the mutations most commonly seen in human cancers. We found that the TP53 mutant allelic fraction increased with passage number under standard culture conditions, suggesting that the P53 mutations confer selective advantage. We then mined published RNA sequencing data from 117 hPS cell lines, and observed another nine TP53 mutations, all resulting in coding changes in the DNA-binding domain of P53. In three lines, the allelic fraction exceeded 50%, suggesting additional selective advantage resulting from the loss of heterozygosity at the TP53 locus. As the acquisition and expansion of cancer-associated mutations in hPS cells may go unnoticed during most applications, we suggest that careful genetic characterization of hPS cells and their differentiated derivatives be carried out before clinical use.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1
Extended Data Figure 1. Replicates of cell competition assays carried out at earlier starting passages.
Note that while the mutant allelic fractions for lines CHB11 and WA26 approach fixation, that the fraction of mutant cells unexpectedly decreases for ESI035 over several passages, indicating a potential selective disadvantage that co-segregates with the TP53 mutation in this experiment. The number of replicate wells is indicated in each graph. Error bars depict SEM.
Extended Data Figure 2
Extended Data Figure 2. Summary of all observed P53 mutations.
a-b, Graphical representation of each of the 9 mutated bases in P53 observed across the 252 whole exome sequenced (WES) and RNA sequenced (RNAseq) hPSC lines depicting their allele frequency in ExAC (a) and the incidence with which the relevant codons are mutated in human cancer (b). c, The 15 instances of these mutations in 12 distinct cell lines is represented along with whether the mutation was seen by WES or RNAseq. Although the M237I event is seen in two distinct iPSC lines, it is conservatively counted here as a single event since the hiPSC clones may be related.
Extended Data Figure 3
Extended Data Figure 3. Analysis of loss of heterozygosity in RNA sequencing samples.
a, Polymorphic sites on chromosome 17 in different hPSCs with mutations in TP53. WIBR3 cells with H193R mutation and H9 cells with both P151S and R248Q mutations show less polymorphism in the distal part of chromosome 17p compared to the proximal part of 17p and 17q. *samples with less than 25 reads. b, Summation of the polymorphic sites in the distal part of chromosome 17p compared to the proximal part of 17p and 17q, divided by the overall frequency of polymorphic sites along chromosome 17. WIBR3 cells with H193R mutation and H9 cells with both P151S and R248Q mutations have a significantly different proportion between the two parts of the chromosome, implying loss of heterozygosity (LOH). Error bars depict SEM, ***p < 0.001. c, A schematic representation of possible allele states of TP53 in cultured hPSCs with all observed mutations depicted. Depending on the percentage of mutant reads in a culture, one can deduce if the culture is homogenous or mosaic for a mutation, and whether, in addition to a point mutation, LOH has occurred in the TP53 locus. MAF, minor allele frequency.
Extended Data Figure 4
Extended Data Figure 4. Culture and passaging method employed for samples bearing P53 mutations.
a, P53 mutations were observed in hPSCs grown in a broad array of culture media including home-made medium supplemented with knockout serum replacement (KOSR), and defined, commercial media such as E8. b, Similar numbers of P53 mutations were observed from cells grown with feeder cells or under feeder-free conditions. c, Since passaging hPSCs can introduce stresses or clonal bottlenecks, we examined whether P53 mutations were consistently seen when a particular passaging method was used, but we observed a wide variety of passaging methods associated with these mutations. Note that the interpretation of these data are complicated by the fact that the culture methods employed in the final published study may not reflect the previous culture history of that cell line, which may have previously passed through multiple laboratories, as well as by the lack of detail about culture methods present in some published studies. d, The addition of supplements such as rock inhibitor at passages does not appear to be sufficient to prevent P53 mutations in hPSCs.
Figure 1
Figure 1. Acquisition and WES of 140 hESC lines.
a, Schematic workflow for hESC line acquisition and sequencing. b,c, 114 hESC lines were obtained, banked (b), and analyzed by WES along with 26 GMP-prepared cell lines (c). d, 45 hESC lines were excluded due to use restrictions. e, 140 hESC lines were banked and/or sequenced (see also Supplementary Table 1 and Materials and Methods). f, HESCs were minimally cultured before banking and sequencing. g, Cumulative passage number of hESCs was moderate. h, WES coverage for sequenced hESC lines. IRB, institutional review board; MTA, material transfer agreement; PGD, pre-implantation genetic diagnosis.
Figure 2
Figure 2. Identification of recurrent, cancer-associated TP53 mutations in hESCs.
a, Some heterozygous variants are present at low allelic fractions (boxed left) in hESCs. b,c Likely mosaic variants (P<0.01, red shading), include six mutations in TP53 (b, Supplementary Table 3) that are rare in ExAC (<0.0001) (c). d, The four affected P53 residues are commonly mutated in human tumors. e, On a crystal structure of P53 bound to DNA, the affected residues map to the DNA binding domain, including to arginine residues that directly interact with DNA. f) The residues mutated in hESCs disrupt DNA binding by P53.
Figure 3
Figure 3. TP53 mutations in hESCs are mosaic and confer strong selective advantage.
a, Droplet digital PCR (ddPCR) assay schematic. b, Representative ddPCR data showing droplets containing the reference allele (gray), mutant allele (red), both alleles (pink), or neither allele (black). c, Estimated fraction of mutant cells (red) in affected hESC lines. d, Mutant allelic fraction rapidly increases during standard hESC culture. Error bars depict SEM and numbers indicate replicate wells. Note further allele-fraction expansion (after P17) for WA26, likely involving LOH. e, Model of P53’s role in both cancer and stem cell biology.
Figure 4
Figure 4. A substantial fraction of hPSCs in published studies harbor TP53 mutations.
a-e, Published RNAseq data show that 7/117 (6%) unique hPSC lines harbor P53 mutations. f, Combined DNAseq and RNAseq analysis reveals 12/252 (5%) distinct cell lines affected by 15 TP53 mutations (Supplementary Table 3). g,h,i, P53 mutant WA01 was seen in three (g), WA09 acquired four distinct TP53 mutations in three groups (h), and WIBR3 lost all normal copies of TP53 after gene editing (i). j,k, TP53 mutant cells could be differentiated (j,k), and expanded relative to WT cells (k). Error bars depict SEM. l,m, Model of TP53 mutation enrichment during hPSC culture (l) or during clonal bottlenecks (m).
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