Low rates of mutation in clinical grade human pluripotent stem cells under different culture conditions

Sian Gregory,Felix Krueger,Oliver Thompson,John Alexander,Richard Weightman,Andrew Wood,Peter W Andrews,Kosuke Yusa,Marta Milo,Wolf Reik,Paul J Gokhale,Serena Nik-Zainal,Zoe Hewitt,Ferdinand Von Meyenn,Simon Andrews,Ivana Barbaric,Harry D Moore

doi:10.1038/s41467-020-15271-3

Abstract

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, commonly 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.

Highlights

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application
Developing culture conditions that minimise change is important for improving safety in clinical applications of human pluripotent stem cells (PSC)-derived therapeutics
It is encouraging that following culture over an extended period of time and multiple passages, both the human PSC in this study acquired a relatively low burden of single-nucleotide base substitutions, and none of the common genetic variation seen in human PSC

Summary

Introduction

The occurrence of repetitive genomic changes that provide a selective growth advantage in pluripotent stem cells is of concern for their clinical application. Acquired genetic variants of human ES cells were first noticed as non-random gains of particular chromosomes or fragments of chromosomes detected by G-banding karyotypes[6,7]. Over succeeding years these observations were repeated in many laboratories as it became clear that certain chromosomal regions of human PSC, whether ES or iPS cells, were subject to gains, notably chromosomes 1q, 12p, 17q and 20q, and the X chromosome, or losses, notably 10p, 18q and 22p2. Single base-pair mutations have been reported in the TP53 gene of several human ES cell lines[11,12]

Methods

Results

Conclusion