Abstract
Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cell-based regenerative medicine. The rapid proliferation of pluripotent cells leads to constitutive replication stress, hindering the progression of DNA replication forks and in some cases leading to replication-fork collapse. Failure to overcome replication stress can result in incomplete genome duplication, which, if left to persist into the subsequent mitosis, can result in structural and numerical chromosomal instability. We have recently applied the DNA fiber assay to the study of replication stress in human PSC and found that, in comparison to somatic cells states, these cells display features of DNA replication stress that include slower replication fork speeds, evidence of stalled forks, and replication initiation from dormant replication origins. These findings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA fiber assay has enabled the development of an advanced nucleoside-enriched culture medium that increases replication fork progression and decreases DNA damage and mitotic errors in human PSC cultures. The DNA fiber assay allows for the study of replication fork dynamics at single-molecule resolution. The assay relies on cells incorporating nucleotide analogs into nascent DNA during replication, which are then measured to monitor several replication parameters. Here we provide an optimized protocol for the fiber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC. Basic Protocol 1: DNA fiber labeling Basic Protocol 2: DNA fiber spreading Basic Protocol 3: Immunostaining Support Protocol 1: Microscopy/data acquisition Support Protocol 2: Data analysis.
Highlights
Human pluripotent stem cells (PSC) possess the ability to endlessly renew and differentiate into any cell type of the body, making them a promising resource for cell-based regenerative medicine (Takahashi et al, 2007, Thomson et al, 1998)
It has become apparent that human PSC acquire genetic changes during long-term culture, which raise concerns over the safety of stem cell−derived products that are destined for the clinic (Draper et al, 2004, Olariu et al, 2010)
Despite the mechanism of selection being well deined, relatively little is known about the underlying mutational mechanisms, the observations of replication stress and genomic damage in human PSC are similar to the oncogene-induced model of genetic instability in cancer development and progression (Halazonetis, Gorgoulis, & Bartek, 2008)
Summary
Human pluripotent stem cells (PSC) possess the ability to endlessly renew and differentiate into any cell type of the body, making them a promising resource for cell-based regenerative medicine (Takahashi et al, 2007, Thomson et al, 1998). Following DNA labeling (Basic Protocol 1), a small droplet of cell suspension is lysed on a glass slide (Fig. 1B) and tilted to allow spreading. Slides with spread DNA ibers (Basic Protocol 2) 2.5 M hydrochloric acid (see recipe) 1× PBS (see recipe) Blocking solution (see recipe), pre-chilled Rat monoclonal anti-BrdU antibody [BU1/75 (ICR1)] (Abcam, ab6326) Mouse monoclonal anti-BrdU antibody (clone B44) Using a 1-ml pipette, gently add 750 μl of the secondary antibody mix to the bottom right-hand corner of the slide, and allow it to spread over the entirety of the slide (Fig. 1E) As before, this step should be performed on a damp paper towel covered with a plastic lid. A slide staining tray can be used (e.g., Sigma, Z670146)
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