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Low Rates of Mutation in Clinical Grade Human Pluripotent Stem Cells Under Different Culture Conditions

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Low Rates of Mutation in Clinical Grade Human Pluripotent Stem Cells Under Different Culture Conditions This is a repository copy of Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/159362/ Version: Published Version Article: Thompson, O., von Meyenn, F., Hewitt, Z. orcid.org/0000-0001-7519-7029 et al. (14 more authors) (2020) Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions. Nature Communications, 11 (1). 1528. https://doi.org/10.1038/s41467-020-15271-3 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ ARTICLE https://doi.org/10.1038/s41467-020-15271-3 OPEN Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions Oliver Thompson1, Ferdinand von Meyenn 2,3,6, Zoe Hewitt 1, John Alexander 1,7, Andrew Wood 1, Richard Weightman 1, Sian Gregory 1, Felix Krueger 4, Simon Andrews4, Ivana Barbaric1, Paul J. Gokhale 1, Harry D. Moore1, Wolf Reik2,5, Marta Milo 1, Serena Nik-Zainal 5,8,9, ✉ ✉ Kosuke Yusa 5,10 & Peter W. Andrews 1 1234567890():,; The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. However, the effect of different culture conditions on the underlying mutation rate is unknown. Here we show that the mutation rate in two human embryonic stem cell lines derived and banked for clinical application is low and not substantially affected by culture with Rho Kinase inhibitor, com- monly used in their routine maintenance. However, the mutation rate is reduced by >50% in cells cultured under 5% oxygen, when we also found alterations in imprint methylation and reversible DNA hypomethylation. Mutations are evenly distributed across the chromosomes, except for a slight increase on the X-chromosome, and an elevation in intergenic regions suggesting that chromatin structure may affect mutation rate. Overall the results suggest that pluripotent stem cells are not subject to unusually high rates of genetic or epigenetic alterations. 1 The Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield S10 2TN, UK. 2 Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK. 3 Department of Medical & Molecular Genetics, King’s College London, London SE1 9RT, UK. 4 Bioinformatics Group, Babraham Institute, Cambridge CB22 3AT, UK. 5 Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SA, UK. 6Present address: Institute of Food, Nutrition and Health, ETH Zurich, 8603 Schwerzenbach, Switzerland. 7Present address: Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London, UK. 8Present address: Academic Laboratory of Medical Genetics, Cambridge University Hospitals NHS Foundation Trust, Box 238, Lv6 Addenbrooke’ Treatment Centre, Cambridge Biomedical Research Campus, Cambridge CB2 0QQ, UK. 9Present address: MRC Cancer Unit, University of Cambridge, Hutchinson/MRC Research Centre, Box 1297, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK. 10Present address: Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, ✉ Japan. email: [email protected]; p.w.andrews@sheffield.ac.uk NATURE COMMUNICATIONS | (2020) 11:1528 | https://doi.org/10.1038/s41467-020-15271-3 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-15271-3 he presence of mutations in human pluripotent stem cells rearrangements or other mutations cannot be excluded. PSC are (PSC), whether embryonic stem (ES) cells or induced one of the few ‘normal’ diploid cell types that do not undergo T fi pluripotent stem (iPS) cells, is a concern for their safe use senescence and can be maintained inde nitely in vitro. Other in therapeutic applications. Indeed, in one case, a potential trial of diploid somatic cells undergo senescence, whereas other easily retinal pigment cells from an autologous iPS cell line was aban- accessible cells that can be grown indefinitely are likely to be doned because the cells carried a mutation of unknown sig- transformed cancer cells. Further, cell cycle control in PSC nificance1. Certainly some such variants are likely to have been differs with respect to the lack of key checkpoints, notably the present in the embryos or somatic cells from which particular G1/S checkpoint19, or the CHK1 checkpoint in S-Phase DNA PSC were derived and can be classed as ‘variants of origin’2,3. replication resulting in apoptosis of PSC in response to DNA However, the propensity of PSC to acquire genetic variants on replication stress, in contrast to somatic cells20.Thismight prolonged passage poses additional concerns, not only because of reflect the relation of PSC to the rapidly dividing pluripotent the difficulty for their early detection4, but also their selective cells of the early embryo for which there may be a survival growth advantages might presage malignant potential5. advantage if cells suffering DNA damage undergo apoptosis Acquired genetic variants of human ES cells were first noticed rather than repair the damage. as non-random gains of particular chromosomes or fragments of Most studies in human PSC to date have been concerned with chromosomes detected by G-banding karyotypes6,7. Over suc- mutations providing a selective advantage, as these are the most ceeding years these observations were repeated in many labora- frequently and easily detected when screening cell lines. However, tories as it became clear that certain chromosomal regions of estimating the underlying mutation rate is more difficult and can human PSC, whether ES or iPS cells, were particularly subject to be confounded by the emergence of an advantageous or cell-lethal gains, notably chromosomes 1q, 12p, 17q and 20q, and the X mutation, both of which could bias the mutational load of the chromosome, or losses, notably 10p, 18q and 22p2. At the same population. To de-couple mutation from selection it is necessary time, other chromosomes, notably chromosome 4, appeared to distinguish acquired de novo mutations, occurring during impervious to any gains or losses. More refined analyses, for prolonged cell culture, from variants-of-origin mutations (i.e. example using SNV arrays2,8, also began to reveal small structural those already present in the parental cells) and to estimate the variants not readily detectable by G-banding cytogenetics. For frequency of their appearance before the selective overgrowth of example, a small variable length CNV was found in the proximal any advantageous mutations. region of chromosome 20q in 20 human ES cell lines that were In this study, to determine the rate and types of mutation that otherwise scored as normal, diploid cells by G-banding2,9,10. occur during culture of human PSC, we have used a cloning More recently, single base-pair mutations have been reported in strategy coupled with whole-genome-, whole-genome bisulfite- the TP53 gene of several human ES cell lines11,12. and RNA-sequencing to compare two well characterised, clinical It seems likely that the repetitive, non-random nature of many, grade human ES cell lines, as well as the effects of culture in the if not all, acquired mutations observed in human PSC results presence of a Rho-kinase inhibitor, now commonly used in from their conferring a selective growth advantage. Certainly, routine culture of human PSC, and of culture under low oxygen chromosomal variants when initially observed in a small pro- (5% O2) conditions. We have tested whether the mutations are portion of cells in a culture commonly come to predominate randomly distributed throughout the genome or clustered in within very few passages, while experiments in which small regions related to chromatin structure and gene expression. Our numbers of variant cells have been mixed with their normal results indicate that the mutation rate is low compared to esti- counterparts confirm the strong selective growth advantage of the mates in somatic or cancer cells and can be further reduced by variants13. Time lapse imaging of the growth patterns of variant culture under low oxygen conditions. Furthermore, the muta- and normal cells also indicates marked effects on the ability of the tional signature of human PSC is similar to that of other cultured cells to form viable long-term colonies after passaging by over- human cells, but low oxygen culture does alter the frequency of coming multiple bottlenecks that restrict the ability of normal some types of base-pair change associated with oxidative damage. cells to proliferate14. Further, in the minimal amplicon of the chromosome 20 CNV, it has been possible to identify the Results likely driver gene, BCL2L1, overexpression of which inhibits Experimental design. For these experiments, we chose two male apoptosis and increases survival of the variant cells15,16. Likewise, human ES cell lines, MShef4 and MShef11 that we had derived the mutations in TP53 are likely to provide a growth advantage by under GMP-like conditions suitable for their potential clinical use suppressing apoptosis11,12. in regenerative medicine. During their initial derivation, banking There have been many estimates of mutation rate in the and characterisation, no karyotypic variants of MShef4 had been germline and soma, although finding consensus in the reported observed, and it was designated, a priori, as a genetically more rates is confounded by the variety of experimental and analytical stable line.
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