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

Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabili-ties 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 over-come 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 iber 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 indings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA iber 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 iber 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 iber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC.

Human pluripotent stem cells (PSC) acquire recurrent chromosomal instabilities during prolonged in vitro culture that threaten to preclude their use in cellbased 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 iber 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 indings have expanded on previous work demonstrating that extensive DNA damage in human PSC is replication associated. In this capacity, the DNA iber 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 iber assay allows for the study of replication fork dynamics at singlemolecule 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 iber assay intended for use with human PSC, and describe the methods employed to analyze replication fork parameters. © 2020 Wiley Periodicals LLC.

INTRODUCTION
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. To capitalize on this potential, researchers may need to expand PSC for long periods of time in a genetically stable and undifferentiated state. However, recurrent genetic changes have been reported to arise during prolonged in vitro culture (Draper et al., 2004, Olariu et al., 2010. These changes occur through mutation and subsequent selection of variant cells in which the genetic change provide a growth advantage (Avery et al., 2013, Blum, Bar-Nur, Golan-Lev, CldU (20 minutes) IdU ( & Benvenisty, 2009). Although extensive resources have been applied to understanding the mechanism of selection, comparatively little is known about the mechanism of mutation in these 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). This apparent contradiction can be reconciled by observations that cell cycle checkpoints such as CHK1, responsible for relay signaling in response to replication stress, are relaxed in human PSC, causing these cells to die in response to genomic damage , Desmarais, Unger, Damjanov, Meuth, & Andrews, 2016. These characteristics may relect the requirements of early embryogenesis, where a relentless need to proliferate while maintaining genetic stability is critical to ensure successful development. Nevertheless, this system is not perfect and mutations still arise both in vivo and in vitro.
To better understand the origins of genome instability in human PSC, the appropriate assays must be optimized for use in these systems. The DNA iber assay has become the gold standard in directly monitoring DNA replication fork dynamics, and is thus an important tool for the understanding of DNA replication stress. However, its application has been primarily focused on understanding replication in cancer cell lines (Nieminuszczy, Schwab, & Niedzwiedz, 2016). This article describes the basic protocols required to perform the DNA iber assay in human PSC: the sequential pulse labeling of actively replicating DNA (Basic Protocol 1), spreading of labeled DNA ibers onto glass slides (Basic Protocol 2), immunolabeling of nascent DNA ibers (Basic Protocol 3) for visualization by confocal microscopy (Support Protocol 1), and data analysis and replication event characterization (Support Protocol 3) will be described ( Fig. 1). Using our protocol, DNA iber length, replication fork speed, inter-replication origin distance, and fork symmetry can be measured, and replication events including the detection of stalled/terminated forks, new origins, bidirectional forks, and fork terminations can be quantiied.
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).

DNA FIBER LABELING
This protocol describes the pulse labeling of DNA and harvesting of the labeled human PSC intended for use in the DNA iber assay. Human PSC should be seeded at least 48 hr before starting the experiment. Pulse labeling is performed sequentially with two thymidine analogs-chlorodeoxyuridine (CldU) and iododeoxyuridine (IdU)-for 20 min each (Fig. 1A). DNA labeling exploits the ability of in vitro−cultured cells to incorporate nucleotide analogs into newly replicating DNA strands. To ensure consistent and robust labeling, the cells should be at ∼50% conluency at the start of the experiment: this will ensure the cells are still in the growth phase and replication is not suppressed. Further, the labeling time should be strictly monitored. A deviation in timing will introduce inaccuracy and confound experimental results. Once labeled, the cells should be dissociated into single cells using TrypLE, to minimize stress and allow for accurate cell counting. Dissociated cells should be kept on ice to inhibit further enzymatic activity before spreading (Basic Protocol 2).

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Current Protocols in Stem Cell Biology This protocol should produce a homogenous single-cell suspension of pulse-labeled cells in ice-cold PBS for DNA spreading (Basic Protocol 2).
2. Aspirate the cell culture medium from the T-12.5 lask of human PSC.
3. Add 1 ml TrypLE to the T-12.5 lask and incubate for 3 min at 37°C in a 5% CO 2 incubator.
4. Add 4 ml of human PSC culture medium to the T-12.5 lask to detach the cells. Transfer the cell suspension into a 15-ml tube.
5. Count the total cell number and centrifuge the cells 3 min at 300 × g, room temperature, then resuspend the pellet in 1 ml human PSC culture medium.
6. Seed 80,000 cells per well of the 6-well plate prepared in step 1. Culture for 72 hr (minimum of 48 hr, although cell numbers will need to be adjusted accordingly) with daily batch feeding, removing the Y-27632 after 24 hr.
11. Add IdU stock solution to the seeded cells for a inal concentration of 250 µM (Fig. 1A).
The concentration of IdU is 10-fold higher than the concentration of CldU. This is to ensure that IdU displaces CldU during the second pulse-labeling step. The addition of the second analog must be performed promptly following the 20-min incubation with the irst analog.

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Current Protocols in Stem Cell Biology 13. Aspirate the medium.
14. Wash twice, each time with 2 ml ice-cold PBS, aspirating medium after each wash.
Steps 13 to 14 should be performed rapidly to avoid over-labeling of nascent DNA.
15. Add 1 ml pre-warmed TrypLE solution to each well. Coat the wells thoroughly and aspirate off the excess solution.
17. Aspirate off the excess TrypLE and add 0.5 ml ice-cold PBS to release the cells from the 6-well plate. Transfer the cell suspension to a 15-ml conical tube.
18. Count the total cell number and dilute with ice-cold PBS to 400,000 cells per ml.
19. Keep on ice and spread (Basic Protocol 2) within 30 min.

DNA SPREADING
This protocol aims to describe the technique of DNA spreading (Parra & Windle, 1993). Following DNA labeling (Basic Protocol 1), a small droplet of cell suspension is lysed on a glass slide ( Fig. 1B) and then tilted to allow spreading. The droplet of cell lysate solution generates cohesive tension with the slide as it runs its length, stretching the DNA into ibers that can be later stained and visualized by immunoluorescence (Basic Protocol 3 and Support Protocol 1).
The major factor requiring optimization in this protocol is the type of glass slide used. Before initiating this protocol, different brands and batches of slides should be tested to ensure the droplet runs slowly down the length of the glass slide when tilted at 25°-40°. If this protocol is conducted properly, a droplet of cell lysate solution should run the length of a glass slide in 3 to 5 min. Once dried, the path of the droplet should leave behind a cloudy precipitate. Depending on the temperature and humidity, it may be necessary to alter the volume of the spreading buffer or the angle at which the slide is tilted to ensure that the droplet runs at a slow, constant rate.

Super premium microscope slides (BDH Laboratory Supplies)
Other slides are also suitable, although each batch should be tested to optimize spreading.
The lid of a multi-well plate (or something similar) to hold the tilted slides in place Cell lysis 1. Working with one sample at a time, pipette 2 µl of ice-cold cell suspension at the top of each of 3-5 microscope slides, depending on the number of replicates to be performed (Fig. 1B).
Be sure that the droplet does not touch the frosted end of the slide, as this will inhibit its ability to spread. It is a good idea to mark with pencil the position of the cell suspension, to aid in inding the ibers on the microscope.
This step will require optimization. Look for the edges of the droplet beginning to dry and for the droplet to become tacky. The time that this takes may vary depending on room temperature and humidity. However, do not allow the droplet to completely dry.

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Current Protocols in Stem Cell Biology 3. Add ∼7 µl of spreading buffer and stir with a pipette tip (Fig. 1B).
To ensure that the droplet spreads down the slide slowly (step 5 to 6), the volume of spreading buffer may need to be adjusted.
IMPORTANT NOTE: Stir with the pipette tip; do NOT pipette up and down.

Incubate for 2 min.
Spreading 5. Tilt each slide at an angle of 25°to 40°and hold in place on the lid of a multi-well plate.
6. Let the droplet run down the slide slowly and at a constant speed. The droplet should reach the bottom edge after 3 to 5 min (Fig. 1C).
It is important to ensure that the droplet spreads slowly and at a constant speed, so that the ibers do not clump and become uncountable during the analysis steps.
7. Lay the slides lat and allow to air dry completely (approximately 15 min).
8. Go back to step 1 and prepare the next sample.
Fixation 9. Using a 1-ml pipette, gently add 1 ml methanol/acetic acid ixative to the bottom right-hand corner of the slide and allow it to spread over the entirety of the slide (Fig. 1D).
11. Tilt the slide to allow the excess ixative to run off, and allow to air dry completely.
At this point, the slides can be stored at 4°C overnight.

IMMUNOSTAINING
This protocol describes the immunolabeling of DNA ibers spread onto glass slides. The protocol begins with an acid treatment to denature the DNA ibers before blocking for unspeciic binding and labeling the CldU and IdU labeled ibers with rat and mouse anti-BrdU primary antibodies, respectively. Detection of the labeled DNA ibers is possible by immunostaining with luorescently labeled secondary antibodies (Fig. 1E).
Following this immunostaining protocol, it will be possible to detect the labeled DNA ibers by confocal microscopy. The labeled DNA should be detectable as bi-labeled ibers in the 555 and 488 Alexa Fluor visible spectrum. Further details and expected results from this protocol can be found in the "Understanding Results" section. Glass staining troughs with slide racks (e.g., Sigma, BR472200) Coplin staining jar (e.g., Sigma, S5766) Coverslips, 60 × 22 mm (ThermoFisher Scientiic, BB02200600A113MNT0)

Wash, denature, and block
1. Wash slides twice with double-distilled H 2 O in a staining trough.
All wash steps should be performed by placing slides in a slide rack, submerging in the wash solution within a staining trough, and agitating back and forth twice.
2. Rinse the slides once in 2.5 M hydrochloric acid in a staining trough.
All rinse steps should be performed by dipping the slides, held in a slide rack, in and out of the rinse solution within a staining trough.
3. Denature in 2.5 M hydrochloric acid in a staining trough for 1 hr.

Wash the slides twice with blocking solution in a Coplin jar.
To wash, place slide inside Coplin jar then add blocking solution; after irst wash, pour out the buffer and replace with fresh buffer.
6. Incubate the slides in fresh blocking solution in the Coplin jar for 1 hr.
Primary antibody immunolabeling 7. Make a fresh solution of primary antibodies by mixing rat monoclonal anti-BrdU antibody (1:500) and mouse monoclonal anti-BrdU antibody (1:500) in blocking solution.
Antibodies should be titered, as the optimal concentration will depend on the source and nature of the antibody.
8. Using a 1-ml pipette, gently add 750 µl of the antibody solution to the bottom righthand corner of the slide and allow it to spread over the entirety of the slide.
To stop the solution from evaporating, this step should be performed on a damp paper towel covered with a plastic lid. Alternatively, a slide staining tray can be used (e.g., Sigma, Z670146).
10. Rinse three times with 1× PBS in a staining trough. Remove the slides and stand them upright on a paper towel to remove any excess PBS.
11. Using a 1-ml pipette, gently add 1 ml of 4% PFA to the bottom right-hand corner of the slide and allow it to spread over the entirety of the slide.
13. Rinse three times with 1× PBS in a staining trough.
14. Wash three times with blocking solution in a Coplin jar.

Secondary antibody immunolabeling
All steps should be performed in the dark to reduce photobleaching of the luorophore labels. Use a dark staining trough and Coplin jar or wrap in aluminum foil to block the light.
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Current Protocols in Stem Cell Biology Antibodies should be titered, as the optimal concentration will depend on the source and nature of the antibody.
16. 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).

MICROSCOPY/DATA ACQUSITION
In support of the basic protocols above, we provide microscopy parameters and dataacquisition instructions to facilitate accurate data analysis (Support Protocol 2). It is important to take pictures of ibers from across the entire slide where ibers do not overlap, in order to ensure that robust measurements will be obtained. In total, 150-200 ibers should be acquired per experimental condition to enable reliable estimation of replication parameters. Three or more independent repeats should be performed.

Microscopy parameters
The parameters in Table 1 have provided robust measurements when using an Olympus FV1000 confocal microscope.

DATA ANALYSIS
This protocol will describe the data analysis and replication-event characterization that can be achieved from DNA iber assays as described above. In particular, we will describe how to measure DNA iber length, replication fork speed, fork symmetry, and interreplication origin distance, and how to detect and quantify different replication events.

Figure 2
Representative image showing a single field of DNA fibers. The image was taken using the described microscopy parameters (see Support Protocol 1 and Table 1). The fibers within the field show minimal overlap and can be easily measured. Analysis of fields with a high density of DNA fibers can confound measurements, as they are likely to overlap, hindering their measurement from end-to-end. The DNA fibers pictured were labeled sequentially with CldU and IdU for 20 min each. Scale bar = 50 µm.
In each case, a total of 150-200 ibers should be analyzed per condition for a reliable estimation of iber length, and to account for the range of iber lengths that can be attributed to the varied dynamics of replication forks. Only clear ibers that do not overlap should be included (Fig. 2). It is also important to image ibers from across the entire slide to ensure robust measurement of replication fork dynamics. Data should be plotted as scatter dot plots, or in box-and-whiskers plots. Statistical signiicance between two groups is normally assessed using the Mann-Whitney U test (unpaired and nonparametric).

Determining DNA iber length (µm)
The following steps are for determining total length covered by the replication fork within the 40-min pulse labelings.
1. Open each TIFF image from Support Protocol 1 in ImageJ.
2. In the Analyze menu, select measure.

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Current Protocols in Stem Cell Biology 3. For each iber, measure the length of the CldU (red) followed by the IdU (green).
4. Find the sum of the red and green lengths (Fig. 3A).

Determining replication fork speed of a progressing fork
The following steps are for determining the average speed of the replication fork over the two sequential 20-min pulse labelings.
6. Find the average length of each of CldU and IdU.
8. To convert from µm/min to kb/min, a commonly used conversion factor of 2.59 kb per µm (Jackson & Pombo, 1998; Fig. 3A) can be applied.

Fork symmetry
The following steps are for analysis of the synchronized progression of sister forks emanating from a single origin. Fork asymmetry suggests replication-fork stalling events. This analysis is performed on ibers that have contiguous IdU-CldU-IdU (green-redgreen) signals.
10. Measure the length of the two IdU (green) tracts emanating from a single CldU (red) origin (Fig. 3B).
11. Calculate the ratio between the two green tracts.

Inter-origin distance
The following steps are for determining distance between origins in two consecutive DNA tracts. Care should be taken when measuring the inter-origin distance using the DNA spreading technique, selecting only consecutive DNA tracts that are certain to be on the same DNA iber. Ideally, DNA should be counterstained with a luorescent dye, such as YOYO-1.

Replication events
The frequency of replication events, detailed in Figure 3, can be quantiied and normalized to the total number of ibers present. These measurements can provide insight into replication response to loss of protein or replication stress.

Blocking solution
Thoroughly dissolve 5 g BSA and 0.5 ml of Tween 20 in 500 ml of 1× Dulbecco's PBS (see recipe). Make fresh and chill to 4°C prior to use.  Figure 3 Replication dynamics analysis and replication event characterization. Representative images of DNA replication events with the directionality of the CldU and IdU labeling presented. A description of the replication event is shown, and where relevant the formula required in the calculation of the specified replication dynamic is shown. (A) Ongoing replication fork. Analysis of these forks can be used to determine the fiber length and replication fork speed using the formulae shown. (B) Example of a bi-directional replication fork. Analysis of these fibers allows for the quantification of fork symmetry; loss of symmetry indicates that replication fork stalling has occurred. (C) Double replication origins allow for the distance between replication initiation sites to be measured. Inter-origin distance is a measure of the density of origin firing, which is often a symptom of replication stress in response to oncogene activation. (D) A representative image of replication fork termination. Two forks travelling in opposite directions, which meet and terminate fork progression. (E) The image shows the firing of a replication fork from a new origin of replication, which has occurred during the second pulse labeling with IdU. The presence of IdU-only (See next page for legend) Halliwell et al.

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Current Protocols in Stem Cell Biology tracts is a sign of origin firing, as the fork was not progressing during the period of the first labeling.
(F) CldU-only labeled fibers indicate the stalling or collapse of replication forks prior to the addition of the second, IdU label. Quantification of CldU-only fibers, relative to the total number of fibers, can be used to indicate replication stress, and may be a prerequisite to genome instability. (G) Hydroxyurea arrests DNA replication in human PSC. The image shows the severely slowed or stalled progression of replication forks of hydroxyurea-treated human PSC. Scale bars = 10 µm.
Hydrochloric acid, 2.5 M Slowly add 83.2 ml hydrochloric acid (Sigma, 320331) to 316.8 ml double-distilled H 2 O. Prepare fresh for each use.

Methanol/acetic acid ixative
Mix methanol (Sigma, 179337) and acetic acid (Sigma, 695092) at a 3:1 ratio. For example, to make 50 ml of ixative, mix 37.5 ml of methanol with 12.5 ml acetic acid. Store at room temperature in an air-tight container for up to 1 year.

Paraformaldehyde solution, 8%
Add 40 g of paraformaldehyde (Sigma, P6148) to 400 ml double-distilled H 2 O. Heat the solution to 60°C and stir at a medium speed under a fume hood. Add ∼10 drops of 1 N NaOH to dissolve the PFA granules. Continue to mix for 2 hr. Allow the solution to cool, and add double-distilled H 2 O for a total volume of 500 ml. Aliquot and store at −20°C for up to 1 year.

Rho-associated, coiled-coil containing protein kinase 1 inhibitor (Y-27632)
Dissolve Y-27632 dihydrochloride (TOCRIS, 1254) in DMSO (Sigma, 472301) for a 10 mM stock solution. Store for up to 1 year at −20°C. Combine solution 1 and 2 and add 0.5 g SDS. Make the solution up to 100 ml with double-distilled H 2 O and vortex to dissolve. Store at room temperature for up to 1 year.

Vitronectin-coated 6-well plate
Thaw 120 µl vitronectin recombinant protein (ThermoFisher Scientiic, A14700) at room temperature and combine with 11.88 ml of 1× Dulbecco's PBS (see recipe). Mix well and add 2 ml to each well of a 6-well cell culture plate. Incubate at room temperature for 1 hr. For longer-term storage, keep at 4°C and use within 2 weeks.
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COMMENTARY Background Information
Human PSC hold great promise in the ield of regenerative medicine. Yet, in order to reach the full potential of these cells, we must irst capitalize on their ability to rapidly and endlessly renew to generate large numbers of genetically stable and undifferentiated cells. However, 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. The recurrent nature of certain karyotypic changes, such as ampliications to chromosomes 1q, 12p, 17q, and 20q, have highlighted that certain mutations provide a growth advantage to the variant cell, which becomes selected for in a culture over time (Amps et al., 2011, Baker et al., 2016, Olariu et al., 2010. Despite the mechanism of selection now being well deined, relatively little is known about the underlying mutational mechanisms, although 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). 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 deined in human PSC using the DNA iber assay (Halliwell et al., 2020). Furthermore, we have recently identiied regions of microhomology at the breakpoint of chromosome 20 tandem ampliication, which implicates that template-switching mechanisms at stalled or collapsed forks are responsible for these mutations (J.A. Halliwell, D. Baker, P.W. Andrews, I. Barbaric, unpub. observ.). Collectively, these studies have determined that genetic stability in human PSC is overtly linked to DNA replication. This article provides experimental details and protocols required to perform the DNA iber assay, which have been optimized for studying replication stress in human PSC.
The DNA iber assay has become the goldstandard assay in direct monitoring of DNA replication-fork dynamics. The protocols described here are based on the previously described DNA iber assay (Merrick, Jackson, & Difley, 2004), which itself was modiied from the original DNA iber labeling (DIRVISH) technique (Jackson & Pombo, 1998). Two distinguishable modiied nucleotides, CldU and IdU, are added sequentially to the cell culture medium to pulse label nascent DNA strands. The labeled nascent DNA is then removed from the cells by lysis and stretched onto glass slides. By tilting the glass slide, the droplet of DNA solution runs down its length under the force of gravity, stretching the ibers as it runs (Parra & Windle, 1993). Stretching the DNA by spreading is fast, and requires little material or preparation. It is therefore affordable and accessible to most research laboratories. However, the DNA iber assay described here has relatively low resolution and does not detect ssDNA discontinuities, which means that analysis of the results must be done carefully to ensure that broken ibers and crossed ibers are not included. The addition of replication-stalling and DNA-damaging agents prior to and during the DNA-labeling procedure can facilitate the study of fork progression following stalling, and when encountering DNA lesions. Human PSC are particularly sensitive to genotoxic agents, radiation, or agents used to induce replication block, even at doses that have little effect on cancer or somatic cells Desmarais et al., 2016;Hyka-Nouspikel et al., 2012;Luo et al., 2012;Simara et al., 2017). However, these experiments have been reviewed extensively elsewhere (Quinet, Carvajal-Maldonado, Lemacon, & Vindigni, 2017).
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 iber 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.
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Critical Parameters
We have observed little difference in iber assay results whether human PSC are cultured in mTeSR, E8, or Nutristem cell culture medium. Currently, feeder layer−dependent cell culture practices have not been tested. The whole assay can be done in a single day, although we have included a stop point following the ixation step in Basic Protocol 2, which allows the protocol to be run over a period of 2 days.
We advise allowing the human PSC to recover for at least 48 hr following plating. This will permit 24 hr in culture without Rho-associated, coiled-coil containing protein kinase 1 inhibitor (Y-27632). It will also allow recovery from stressful re-plating, and for the cells to re-enter logarithmic growth phase. It is critical, where the results of experiments are to be compared, that the density of cells initially seeded and the conluency at the point of starting the experiment be consistent. Higher conluency may cause cell proliferation and DNA replication to slow.
With regard to labeling of DNA, it is possible to increase or decrease the pulse labeling times, but this will result in longer and shorter DNA ibers, respectively. Again, the labeling time must be constant across comparable experiments. It is also important that the labeling time be accurately measured. Deviations one of 1 min will increase labeling by 5%, and will confound the results of the experiment. To ensure timely labeling, it is advised that the reagents be prepared well ahead of starting the experiment. Also, when adding the second nucleotide analog (IdU), the plate should be removed from the incubator 1 min before the end of the irst incubation period, to give a time buffer when preparing the second label.
Once harvested, the cells should be suspended in ice-cold PBS and kept on ice. DNA spreading should then be performed within 30 min, to minimize cell death.
When spreading DNA ibers, the user should select slides where the droplet spreads down the length of the slide in 3 to 5 min at a constant rate. In our experience, batches of slides can differ from one another, and each batch should be optimized.

Troubleshooting
Table 2 describes problems that can arise with various steps in the assay, along with their possible causes and Solutions.

Statistical Analysis
It is important to take images of ields with minimum iber cross overs across the whole slide to ensure a robust measurement of the progressing replication forks. A minimum of 150-200 individual ibers must be measured to account for the heterogeneity between progressing forks. The conversion factor for stretched DNA ibers is 2.59 kb/µm (Jackson & Pombo, 1998).
Consideration should be given to the presentation of data. Histograms and scatter plots are appropriate for capturing normal differences in replication fork progression. Statistical signiicance can be calculated using an unpaired Mann-Whitney U-test.

Understanding Results
The DNA ibers produced can be variable but, in our experience, under normal growth conditions, they should contain equal lengths of CldU and IdU staining. When measured, we ind the replication fork speed to vary between 0.5 and 0.75 kb/min. A reduced iber length or fork speed can indicate replication stress.
Monitoring the frequency of replication events can provide valuable information regarding the replication processes going on during the culture of human PSC (Fig. 3). A strict control of replication origin density is required to maintain chromosomal stability (Prioleau & MacAlpine, 2016). Excessive replication-origin iring can deplete necessary protein and metabolites required for eficient DNA replication (Sørensen & Syljuåsen, 2012). A decrease in inter-origin distance or a higher density of IdU-only labeled ibers is a measure of increased origin density, suggesting greater numbers of simultaneously iring origins of replication. Fork stalling is a prerequisite for DNA breakage, which can become a substrate for genetic instability (Toledo, Neelsen, & Lukas, 2017). Measuring the frequency of CldU-only ibers or the ratio of the IdU labels on a bi-directional fork can be used to measure fork stalling. A shorter IdU iber on one side of a bi-directional fork implies a fork-stalling event, and will result in a greater ratio between the IdU tracts.

Basic Protocol 1
Cells should be grown for a minimum of 48 hr following seeding; we have found 72 hr to be optimal. Steps 1 to 6: ∼40 min will be needed for cell seeding. ∼20 min per day will be needed to refresh the medium.
Steps 7 to 18: ∼1 hr will be needed to pulse label and harvest the cells.

Basic Protocol 2
Step 1 to 4: preparation of cell lysate for DNA spreading will require ∼15 min.
Step 5 to 7: DNA spreading will require ∼20 min for spreading one set of three slides simultaneously.

Support Protocol 1 and 2
Several hours will be required for image acquisition and data analysis, but this will be variable depending on the quality of the DNA ibers and the biological question being asked.