Nucleosides rescue replication-mediated genome instability of human pluripotent stem cells

Abstract

<jats:p>Human pluripotent stem cells (PSC) often acquire genetic changes on prolonged culture, which pose concerns for their use in research and regenerative medicine (Amps et al., 2011, Seth et al., 2011). The acquisition of these changes during culture necessarily first requires mutation and then selection of those mutations that provide a growth advantage. Whilst selection accounts for the recurrent nature of the variants commonly reported (Draper et al., 2004, Olariu et al., 2010), the mechanisms of mutation in PSC remain largely elusive. Here we show that, in contrast to somatic cells, human PSC have an increased susceptibility to DNA damage and mitotic errors, both of which are caused by heightened replication stress in PSC and this can be alleviated by culture with exogenous nucleosides. These results reflect the requirement for rapid replication of human PSC enabled by a truncated G1 (Becker et al., 2006, Becker et al., 2010) that impairs the preparation of these cells for the ensuing DNA replication. A similar relationship has been shown in relation to chromosomal instability in cancer cells (Burrell et al., 2013, Wilhelm et al., 2019) but PSC differ by replication stress triggering apoptosis (Desmarais et al., 2012, Desmarais et al., 2016). Nevertheless, evasion of this response still leads to the appearance of genetic variants that are of concern for regenerative medicine. The inclusion of nucleosides into culture media greatly improves the efficiency of human PSC culture and minimises the acquisition of genomic damage.</jats:p>

Acquisition of chromosome 1q duplication in parental and genome‐edited human‐induced pluripotent stem cell‐derived neural stem cells results in their higher proliferation rate in vitro and in vivo

“…Scale bars: 100 µm prevent their occurrence as it was demonstrated in several previous studies. 43,[61][62][63][64][65] Nevertheless, the genomic integrity of all cell products used for regenerative approaches should be carefully assessed and monitored to ensure that they are safe and therapeutically effective.…”

DNA Fiber Assay for the Analysis of DNA Replication Progression in Human Pluripotent Stem Cells

“…DNA damage and genome stress, including replication stress, can lead to genetic instability like that often observed in cancer (Burrell et al., 2013). Previous studies have highlighted that human PSC are prone to replication stress and DNA damage (Halliwell et al., 2020, Simara et al., 2017, Vallabhaneni et al., 2018), which may also drive the high frequency of mitotic errors that has been reported elsewhere (Lamm et al., 2016, Zhang et al., 2019). Yet, despite this, the underlying mutation rate in these cells is low (Thompson et al., 2020).…”

“…The self‐renewal of human PSC is characterized by an abbreviated G1 phase that bypasses the Rb/E2F checkpoint and is driven by high expression of cyclin D2 and constitutive expression of cyclin E (Becker et al., 2006, Filipczyk, Laslett, Mummery, & Pera, 2007). Maintaining this rapid proliferation over extensive culture periods may expose these cells to replication stress, characteristics of which, such as reduced replication rates, have been defined in human PSC using the DNA fiber assay (Halliwell et al., 2020). Furthermore, we have recently identified regions of microhomology at the breakpoint of chromosome 20 tandem amplification, which implicates that template‐switching mechanisms at stalled or collapsed forks are responsible for these mutations (J.A.…”

“…The procedure described in this article has been optimized for use with human PSC, and has been successfully applied to several human iPSC and human ESC cell lines (Halliwell et al., 2020). We have utilized this assay to improve culture conditions: supplementing cultures with nucleosides alleviates replication stress and decreases the frequency of mitotic errors, highlighting that these events are linked in human PSC (Halliwell et al., 2020). The DNA fiber assay has revealed approaches that can be used to reduce the appearance of genetic instability in human PSC, which is necessary for the safe application of human PSC in regenerative medicine.…”

“…The protocols in this article have been successfully used to monitor replication dynamics in several human iPSC and ES cell lines cultured in feeder‐free conditions using mTeSR, Nutristem, or E8 on a matrix of recombinant vitronectin−coated plates (Halliwell et al., 2020).…”