Nucleosides Rescue Replication-Mediated Genome Instability of Human Pluripotent Stem Cells.

Oliver J. Bower,Ivana Barbaric,Paul J. Gokhale,Sherif F. El-Khamisy,Peter W. Andrews,Owen Laing,Thomas J.R. Frith,Dylan Stavish,Zoe Hewitt,Christopher J. Price,Jason A. Halliwell

doi:10.1016/j.stemcr.2020.04.004

Abstract

SummaryHuman pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have now found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. However, we have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth.

Highlights

Human pluripotent stem cells (PSCs) often acquire genetic changes on prolonged culture, which pose concerns for their use in research and regenerative medicine (Amps et al, 2011; Taapken et al, 2011)
Replication stress due to an abbreviated G1 is implicated in cancer development, wherein high levels of cyclin E drive the premature entry of cells into the S phase in the absence of sufficient nucleotide pools needed for normal progression of the replication fork (Bester et al, 2011)
Two other PSC lines that we examined, TC113 (Baghbaderani et al, 2015) and MShef11 (Thompson et al, 2020), showed a increased level of DNA damage compared with their differentiated derivatives (Figures 1A and S1)

Summary

SUMMARY

Human pluripotent stem cells (PSCs) are subject to the appearance of recurrent genetic variants on prolonged culture. We have found that, compared with isogenic differentiated cells, PSCs exhibit evidence of considerably more DNA damage during the S phase of the cell cycle, apparently as a consequence of DNA replication stress marked by slower progression of DNA replication, activation of latent origins of replication, and collapse of replication forks. As in many cancers, which, like PSCs, exhibit a shortened G1 phase and DNA replication stress, the resulting DNA damage may underlie the higher incidence of abnormal and abortive mitoses in PSCs, resulting in chromosomal non-dysjunction or cell death. We have found that the extent of DNA replication stress, DNA damage, and consequent aberrant mitoses can be substantially reduced by culturing PSCs in the presence of exogenous nucleosides, resulting in improved survival, clonogenicity, and population growth

INTRODUCTION

RESULTS AND DISCUSSION

EXPERIMENTAL PROCEDURES