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

Abstract

Summary Human 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.

Nanopore Sequencing Indicates That Tandem Amplification of Chromosome 20q11.21 in Human Pluripotent Stem Cells Is Driven by Break-Induced Replication

“…Collectively, these findings suggest that this chromosomal region is predisposed to tandem amplification, which is driven by microhomology-mediated break-induced replication [ 58 ]. This mechanism is also consistent with the constitutive replication stress to which human PSCs are particularly susceptible during in vitro culture [ 54 ]. Associated replication fork stalling and collapse could be exacerbated by Alu elements, which might then initiate such mutations at Alu-rich regions of the genome.…”

The Impact of Acquired Genetic Abnormalities on the Clinical Translation of Human Pluripotent Stem Cells

“…As our understanding of genomic stability improves, methods could be found to decrease its frequency as well. For example, the addition of nucleosides to a culture medium has been demonstrated to partially alleviate replication stress [33], and targeted small-molecule-induced death of chromosomally abnormal cells has also previously been demonstrated [23,121]. If built upon, these findings could be used to improve culture methods by routinely purifying cell cultures and reducing the instance of de novo CNV formation.…”

“…The mechanisms driving the high instance of chromosomal abnormalities in hPSC are still poorly understood, but are generally thought to be the result of suboptimal culture techniques and high levels of DNA damage compared to somatic cells [31,32]. This increase in DNA damage is in part due to high replication stress in the cells [33,34]. Replication stress is known to lead to replication fork stalling, which can result in DNA doublestrand breaks [35] and subsequent chromosomal rearrangements as a result of errors in repair [36].…”

“…This may help explain why cell lines with multiple chromosomal aberrations are so frequently identified. Medium acidification and replication fork stalling can be relieved by more frequent media changes and the addition of nucleosides to the culture media, respectively [33][34][35][36][37][38][39] indicating that improvements to culture methods may help alleviate the issue.…”

Fitness selection in human pluripotent stem cells and interspecies chimeras: Implications for human development and regenerative medicine

“…A pertinent question regarding the appearance of culture-acquired genetic changes relates to the frequency of their appearance. Time-lapse tracking of mitoses revealed a relatively high frequency of mitotic errors occurring in PSCs ( Halliwell et al., 2020b ; Lamm et al., 2016 ; Zhang et al., 2019 ). This observation parallels findings of high rates of mosaic aneuploidy in early human embryos ( van Echten-Arends et al., 2011 ; Vanneste et al., 2009 ; Starostik et al., 2020 ), suggesting that susceptibility to mitotic errors may not be simply explained as an artefact of an in vitro environment, but also reflects an intrinsic property of early embryonic cells.…”