DNA replication is reprogrammed after human SCNT

Error-free duplication of the human genome is crucial to prevent genomic instability and mutations, which could lead to cell transformation or cell death (Aguilera and Gómez-González, 2008; Bandura and Calvi, 2002; Blow and Gillespie, 2008; Branzei and Foiani, 2010). Because we detected an increase in genomic instability, particularly after the induction of replicative stress, we next analyzed whether defects in the DNA replication could be present in these cells. Since no major differences in the replication timing were found previously among human PSCs (Ryba et al., 2010), we decided to analyze the DNA replication using a high-resolution single-molecule technique called SMARD (Fig. 2 A; and Fig. S1, B and C). Using SMARD, alterations in the DNA replication program can be visualized, which could be missed using bulk DNA studies. The progression of DNA replication was determined at specific genomic loci, where distinctive DNA replication fork profiles and differences were detected during cell development (Fig. S1 B; Gerhardt et al., 2016; Schultz et al., 2010). These specific developmentally regulated genomic loci, such as the Frataxin (FXN) and Nanog gene loci, show a different replication profile in hESCs in contrast to differentiated cells (Gerhardt et al., 2016; Schultz et al., 2010).

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Figure 2.

DNA replication in NT-hESCs is very similar to that in hESCs. (A) Schematic of the steps of SMARD that enable the visualization of single replicating DNA molecules. First, cells are pulsed with IdU (red) and CldU (green). Cells are then embedded in agarose and lysed. After digestion with PmeI enzyme DNA molecules are separated by PFGE. The DNA segment containing the FXN locus is then identified, excised from the gel, and stretched on silanized glass slides. The labeled DNA molecules are detected by immunostaining (red and green) and two FISH probes surrounding the endogenous genomic locus. (B–E) Top: Map of the PmeI segment containing the FXN gene (black) analyzed with SMARD. The positions of the FISH probes are marked in blue. Middle: Photomicrographs of labeled DNA molecules from hESCs, neonatal fibroblasts, NT-hESCs derived from neonatal (neo NT-hESCs), and adult cells (NT-hESCs) are ordered according to replication fork (yellow arrows) progression in the 5′-3′ and 3′-5′ directions, replication initiation; and termination. Bottom: The percentage of molecules with IdU incorporation (first pulse) in PSCs is calculated from the DNA molecules shown above.

First, we determined the replication in PSCs generated by SCNT at these loci (Fig. 2). NT-hESCs were made by replacing of a human oocyte nucleus with a somatic cell nucleus, followed by parthenogenesis (Fig. 1 A). As previously shown, in contrast to differentiated cells, we found active replication initiation sites within the FXN locus in 22% of the DNA molecules in unaffected control hESCs (Fig. 2 B; and Fig. S2, A and B; Gerhardt et al., 2016). In addition, the replication forks progressed in 3′-5′ and 5′-3′ directions through the FXN locus at normal speeds without fork stalling (Fig. 5, D and E). As previously observed in differentiated cells, we found only very few replication origins (7% of total DNA molecules) in the neonatal fibroblast line from which the neonatal NT-hESCs were derived (Fig. 2 C; Gerhardt et al., 2016). However, we found that the isogenic neonatal NT-hESCs and a second NT-hESC line (derived from adult fibroblast line 1018) had a similar DNA replication pattern at the FXN locus as hESCs (~23% of the DNA molecules contained active replication origins; Fig. 2, D and E). These results show that DNA replication in PSCs reprogrammed by SCNT was comparable to the replication seen in hESCs.

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Figure S2.

DNA replication program is not altered in a second neonatal iPSCs at the FXN and at the TCERG1L gene locus. Related to Figs. 2, ​,3,3, and ​and4.4. (A and B) Examples of SMARD molecules from each cell line are shown with higher resolution and separated by fluorescence colors. (A) Shown are molecules with replication origins. (B) Shown are molecules with replication forks in the 3′ to 5′ direction. (C) Table summarizing the total count of analyzed SMARD molecules at the FXN locus from three hESCs (WCMC4, Fig. 2 B; H9 and H14, Gerhardt et al., 2016), three iPSCs (iPSCs B and E, Fig. 3, A and B; and iPSCs NP, Gerhardt et al., 2016), two NT-hESCs (NT-hESCs, Fig. 2 D, and neonatal NT-hESCs, Fig. 2 E), two neonatal (neo) iPSCs (Fig. 3, C and D), and two diff. cells (neonatal fibroblasts, Fig. 2 C; and diff. H9 cells, Gerhardt et al., 2016; standard deviation and P values are indicated in the diagram) and percentage of molecules containing a replication origin per each cell line. (D and E) Top: Map of the PmeI segment containing the FXN gene or TCERG1L gene. The positions of the FISH probes are marked in blue. Middle: Photomicrographs of labeled DNA molecules from neonatal (neo) iPSCs M (D) and neonatal iPSCs (E) are ordered according to replication fork (yellow arrows) progression in the 5′-3′ and 3′-5′ directions; replication initiation; and termination. Bottom: The percentage of molecules with IdU incorporation (first pulse) is calculated from the DNA molecules shown above.

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Figure 5.

IPSCs show a decrease in DNA replication initiation and altered response to aphidicolin compared with hESCs. (A) Left: Diagram of the percentage of DNA molecules with replication initiation sites in unaffected hESCs, iPSCs, NT-hESCs, neonatal iPSCs, and differentiated cells (isogenic fibroblasts line BJ and H9 rosettes), as well as aphidicolin-treated hESCs, iPSCs, and NT-hESCs. Standard deviation and P values are indicated in the diagram (n ≥ 100). Right: Examples of DNA fibers containing replication origins from which the percentage of molecules with origins is calculated (see left) are shown for each cell line: hESCs, iPSCs, NT-hESCs, and differentiated cells. (B) SMARD; Top: Map of the PmeI segment containing the FXN gene. Middle: Photomicrographs of labeled DNA molecules from hESCs, iPSCs, and NT-hESCs treated with 0.4 µM aphidicolin are ordered according to replication fork (yellow arrows) progression in the 5′-3′ and 3′-5′ directions, replication initiation, and termination sites. (C) Diagram of the percentage of DNA molecules with replication initiation sites at the FXN locus in hESCs, iPSCs, and NT-hESCs treated with aphidicolin (calculated from the replication pattern shown in B and without aphidicolin (from Fig. 2, B and E; and Fig. 3 A). (D) Replication fork speed with aphidicolin (A) was calculated from the replication pattern shown in B as well as without aphidicolin from the replication pattern shown in Fig. 2, B and E; and Fig. 3 A in hESCs, NT-hESCs, and iPSCs. Fork speed calculation is described in the Materials and methods section. STD, standard deviation. (E–G) The percentage of molecules with replication forks for each 5-kb DNA interval was calculated at the FXN locus in hESCs (green, E), iPSCs (red, F) and NT-hESCs (blue, G) treated with (calculated from the replication pattern shown in B) and without aphidicolin (from Fig. 2, B and E; and Fig. 3 A). Black arrows point at stalled replication forks.

DNA replication is incompletely reprogrammed in iPSCs derived from adult cells

Next, we analyzed the DNA replications in iPSCs, which were generated from fibroblasts by the nonviral mRNA reprogramming technology (adult iPSCs E) or by retroviral transduction of the transcription factors Oct3/4, Sox2, Klf-4, and c-Myc in the presence of sodium butyrate (Ku et al., 2010; adult iPSCs B). The adult iPSCs E showed the lowest differentiation potential (Fig. 1 B) and is a line isogenic to adult NT-hESCs, as shown in Fig. 2 E. We found that in contrast to PSCs generated by SCNT, DNA replication in iPSCs differed from hESCs at the FXN locus (Fig. 3, A and B). SMARD results showed that very few replication initiation sites were activated at the FXN locus in these two iPSCs (4.3% or 7.4% of total DNA molecules, respectively) similar to our previous results (Gerhardt et al., 2016). Replication with few replication initiation sites in these two iPSCs resembled the replication seen in differentiated cells (Fig. 2 C; Gerhardt et al., 2016). In contrast, we found more active replication origins at the FXN locus in iPSCs derived from neonatal fibroblasts (27% of DNA molecules; Fig. 3 C) than in adult iPSCs. This cell line is isogenic to the neonatal NT-hESCs (Fig. 2 D, generated from the same neonatal fibroblast line, Fig. 2 C). Neonatal iPSCs and NT-hESCs had a higher differentiation potential than that of adult iPSCs (Fig. 1 B). We confirmed these results in a second neonatal iPSC line (21% of DNA molecules contained origins, Fig. S2 D). Thus, our results show that the reprogramming of DNA replication at the FXN locus in iPSCs, generated from adult fibroblasts using integrating and nonintegrating methods, was incomplete (Fig. 3 D and Fig. S2 C). Our results also show that the reprogramming of DNA replication is more efficient in iPSCs generated from neonatal cells.

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Figure 3.

The DNA replication is altered at the developmentally regulated FXN gene locus in iPSCs. (A–C) Top: Map of the PmeI segment containing the FXN gene (marked in black). The positions of the FISH probes are marked in blue, and the DNA segment (146 kb) was analyzed using SMARD. Middle: Photomicrographs of labeled DNA molecules from hESCs, iPSCs, and NT-hESCs are ordered according to replication fork (yellow arrows) progression in the 5′-3′ and 3′-5′ directions, replication initiation; and termination. Bottom: The percentage of molecules with IdU incorporation (first pulse) in hESCs, iPSCs, and NT-hESCs is calculated from the DNA molecules shown above. (D) Diagram of the percentage of DNA molecules with replication initiation sites from three hESCs (Fig. 2 B, H9 and H14; Gerhardt et al., 2016), three iPSCs (iPSCs B and E, Fig. 3, A and B; and iPSCs NP, Gerhardt et al., 2016), two NT-hESCs (NT-hESCs, Fig. 2 D; and neonatal NT-hESCs, Fig. 2 E), two neonatal iPSCs (see Fig. 3 C and Fig. S2 D), and two diff. cells (neonatal fibroblasts, Fig. 2 C; and diff. H9 cells, Gerhardt et al., 2016). Standard deviation and P values are indicated in the diagram. (E) CpG methylation analysis of the FXN gene locus using bisulfite sequencing.

We next analyzed DNA replication in hESCs and iPSCs at a second developmentally regulated gene locus containing the Nanog gene (Fig. 3, A–C). Differences in replication fork direction between hESCs and differentiated cells were observed at this locus (Schultz et al., 2010). Similar to the FXN gene locus, we found that the replication at the Nanog locus is altered in adult iPSCs compared with hESCs. More replication forks progressed in the 3′-5′ direction through the Nanog gene locus in iPSCs, similar to the replication program seen in differentiated cells (Fig. S3, B and C; Schultz et al., 2010). However, in isogenic NT-hESCs, the replication fork progressed equally in both directions through the Nanog locus, as in hESCs (Fig. S3, B and C).

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Figure S3.

DNA replication is altered at the developmentally regulated Nanog gene locus in iPSCs compared with hESCs. Related to Fig. 2. (A) Map of the Nanog segment with adjacent genes and DNA segment analyzed by SMARD (orange). (B) Top: Map of the Nanog segment with FISH probes. Middle: Photomicrographs of labeled DNA molecules from hESCs, iPSCs, and NT-hESCs are ordered according to replication fork (yellow arrows) progression in the 5′-3′ and 3′-5′ directions; replication initiation; and termination. Bottom: The percentage of molecules with IdU incorporation (first pulse) in hESCs, iPSCs, and NT-hESCs is calculated from the DNA molecules shown above. (C) Diagram of the percentage of DNA molecules with replication fork in 3′-5′ and 5′-3′ direction in hESCs, iPSCs, and NT-hESCs. Standard deviation and P values are indicated in the diagram (P values hESCs = 0.2 and NT-hESCs 0.48). (D) CpG methylation analysis of the Nanog gene locus using bisulfite sequencing (methylation in H9 cells was not analyzed at this locus).

In summary, our results show that the reprogramming of DNA replication was incomplete in human iPSCs in contrast to PSCs generated by SCNT (NT-hESCs). However, iPSCs reprogrammed from neonatal cells have a similar DNA replication program as hESCs, in contrast to the iPSCs reprogrammed from adult cells. These observations suggest that neonatal cells are more efficiently reprogrammed than adult cells. Moreover, incompletely reprogrammed DNA replication would explain the variability in the differentiation potential among these PSCs.

The reprogramming of the DNA replication at developmentally regulated and at abnormally methylated loci is not primarily determined by DNA methylation

To identify the cause of incompletely reprogrammed replication in iPSCs, we examined whether aberrant DNA methylation influences the DNA replication in iPSCs. Refined gene expression profiles have revealed small sets of differentially expressed genes and differentially methylated regions (DMRs) in genetic matching sets of hESCs and iPSCs (Liang and Zhang, 2013). To test whether the reprogramming of the DNA replication is influenced by the abnormal CpG methylation in reprogrammed iPSCs, we examined replication at the aberrant methylated TCERG1L gene locus (Fig. 4 A). The TCERG1L gene locus has been shown to have differential gene expression and differential DNA methylation (Choi et al., 2015). The TCERG1L promoter is methylated in the iPSCs but not in hESCs and NT-hESCs (Fig. 4 B). Using SMARD, we found that the replication fork direction, initiation, and termination at the TCERG1L gene locus in iPSCs (Fig. 4, D and F) did not differ significantly from the replication observed at the TCERG1L gene locus in hESCs and NT-hESCs (Fig. 4, C, E, and F). We detected a similar replication pattern at the TCERG1L locus in iPSCs and NT-hESCs despite differential methylation at this locus. The replication forks progressed from both directions through the locus, and only a few replication initiation and termination sites were detected in all cell lines. To summarize, no major alterations were detected in the DNA replication at the abnormally methylated TCERG1L gene locus in PSCs, suggesting that aberrant DNA methylation does not alter the replication fork progression and replication initiation at this genomic site.

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Figure 4.

DNA replication is similar at the differentially methylated TCERG1L regions in iPSCs and hESCs. (A) Map of the TCERG1L locus containing the positions of the FISH probes, which are marked in blue, and the DNA segment (118 kb) analyzed using SMARD (green). (B) CpG methylation analysis of the TCERG1L gene locus using bisulfite sequencing. (C–E) Top: Map of the TCERG1L segment with FISH probes. Middle: Photomicrographs of labeled DNA molecules from hESCs, iPSCs, and NT-hESCs are ordered according to replication fork (yellow arrows) progression in the 5′-3′ and 3′-5′ directions; replication initiation; and termination. Bottom: The percentage of molecules with IdU incorporation (first pulse) in hESCs, iPSCs, and NT-hESCs is calculated from the DNA molecules shown above. (F) Diagram of the percentage of DNA molecules with replication fork progression in the 3′-5′ and 5′-3′ directions in hESCs, iPSCs, and NT-hESCs. Standard deviation and P values are indicated in the diagram (P values: hESCs = 0.19, iPSCs = 0.068, NT-hESCs = 0.065).

Next, we determined whether differences in replication at the FXN and Nanog gene loci could be explained by differential DNA methylation. Thus, we examined DNA methylation at the FXN and Nanog loci as well. We did not find any differences in the DNA methylation pattern between PSCs at these genomic loci (Fig. 3 E and Fig. S3 D), indicating that DNA methylation is not the cause of incompletely reprogrammed DNA replication in adult iPSCs.

Cause of incomplete reprogrammed DNA replication

Studies by our laboratory and other groups have shown that the replication fork progression at certain specific genomic loci is cell type–specific and regulated during cell development such as at the FXN locus (Aladjem, 2007; Gerhardt et al., 2016; Ryba et al., 2011; Schultz et al., 2010). To better understand the reprogramming of DNA replication in iPSCs, we examined the FXN and Nanog loci, where the DNA replication is developmentally regulated in human cells (Gerhardt et al., 2016). Previously we found that aberrant replication and replication fork stalling at the FXN locus led to genomic instability in stem cells derived from Friedreich’s ataxia patients (Gerhardt et al., 2016). Our data showed incomplete reprogramming of the replication in these two genomic regions in reprogrammed iPSCs, in particular a decrease in origin activation. Although replication timing has been linked previously to DNA methylation, our results surprisingly show that alterations in the replication program seems not to be primarily determined by the DNA methylation at the developmental regulated loci that were examined.

In addition, it was previously reported that ESCs contain a higher density of origins than differentiated cells, which was suggested to be important for sufficient cell growth and genomic stability at this early developmental stage. Our data suggest that the low number of replication origins in iPSCs derived from adult cells is the result of an altered response to aphidicolin. This could impede the completion of DNA synthesis in these cells and cause genomic instability (Fig. S5). Indeed, we found a larger increase in micronuclei formation and DNA breaks in iPSCs after the induction of replicative stress.

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Figure S5.

Model for replication defects in PSCs that lead to genomic instability and altered cell differentiation potential. Related to Figs. 1, ​,2,2, ​,3,3, ​,4,4, and ​and5.5. ESCs have been shown to have a higher density of replication origins, which has been suggested to be an important factor for sufficient cell growth and genomic stability at early developmental stages. In addition, it has been shown that ESCs have more replication origins than adult cells because of a less rigid checkpoint response. Our data show fewer replication initiation sites in iPSCs, which seems to be the result of an altered response to aphidicolin. A decrease in replication initiation sites could impede the completion of DNA synthesis in these cells, causing genomic instability and lowering the differentiation potential of PSCs. However, reprogrammed NT-hESCs and neonatal iPSCs with a similar number of replication origins as hESCs are less prone to genomic instabilities.

Cell cycle analysis

To perform EdU (5-ethynyl-2'-deoxyuridine) staining, we used the Click-iT^TM EdU Alexa Fluor 488 Imaging Kit (C10337; Invitrogen) according to the manufacturer’s instructions. The cells were cultured for 1 h in EdU. Next for nuclear staining, the samples were washed with cold PBS and centrifuged (5 min, 1,000 rpm) at 4°C. RNase was added to a final concentration of 0.2 μg/μl, and incubated at room temperature (RT) for 30 min. Then, 1 ml of 2 μg/ml Hoechst 33342 was added to the samples, which were incubated at RT for 30 min. After washing, each sample was analyzed using the BioRad ZE5 at the Columbia University Stem Cell Initiative Flow Cytometry Core.

SMARD

The cells were grown at 37°C for 4 h in the presence of 25 μM 5-iodo-2′-deoxyuridine (Sigma-Aldrich). After washing cells with PBS, hESCs medium with 25 μM 5-chloro-2′-deoxyuridine (Sigma-Aldrich) was added to the cultures, and the cells were incubated for an additional 4 h. The cells were lifted with Accutase or Trypsin. Following centrifugation, the cells were resuspended at 3 × 10⁷ cells per ml in PBS. Melted 1% InCert agarose (Lonza Rockland, Inc.) in PBS was added to an equal volume of cells at 42°C. The cell suspension was pipetted into a chilled plastic mold with 0.5 by 0.2–cm wells with a depth of 0.9 cm for preparing DNA gel plugs. The gel plugs were allowed to solidify on ice for 30 min. Cells were lysed in buffer containing 1% n-lauroylsarcosine (Sigma-Aldrich), 0.5 M EDTA, and 20 mg/ml proteinase K. The gel plugs remained at 50°C for 64 h and were treated with 20 mg/ml proteinase K (Roche Diagnostics), every 24 h. Gel plugs were then rinsed several times with Tris-EDTA (TE) and once with phenylmethanesulfonyl fluoride (Sigma-Aldrich). The plugs were washed with 10 mM MgCl₂ and 10 mM Tris-HCl (pH 8.0).

The genomic DNA in the gel plugs was digested with 40 units of PmeI (New England BioLabs Inc.) at 37°C overnight. The digested gel plugs were rinsed with TE and cast into a 0.7% SeaPlaque GTG agarose gel (Lonza Rockland, Inc.). A gel lambda ladder PFG marker and yeast chromosome PFG marker (both from New England BioLabs, Inc.) were cast next to the gel plugs. Gel slices from the appropriate positions in the pulsed-field electrophoresis gel (PFGE) were cut and melted at 72°C for 20 min. GELase enzyme (Epicentre Biotechnologies 1 unit per 50 μl of agarose suspension) was carefully added to digest the agarose and incubated at 45°C for a minimum of 2 h. The resulting DNA solutions were stretched on 3-aminopropyltriethoxysilane (Sigma-Aldrich)–coated glass slides. The DNA was pipetted along one side of a coverslip that had been placed on top of a silane-treated glass slide and allowed to enter by capillary action. The DNA was denatured with sodium hydroxide in ethanol and then fixed with glutaraldehyde.

The slides were hybridized overnight with a biotinylated probe (the blue bars diagrammed on the maps indicate the positions of the probes used). The following day, the slides were rinsed in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) 1% SDS and washed in 40% formamide solution containing 2 × SSC at 45°C for 5 min and rinsed in 2 × SSC-0.1% IGEPAL CA-630. Following several detergent rinses (four times in 4× SSC-0.1% IGEPAL CA-630), the slides were blocked with 1% BSA for at least 20 min and treated with Avidin Alexa Fluor 350 (Invitrogen Molecular Probes) for 20 min. The slides were rinsed with PBS containing 0.03% IGEPAL CA-630, treated with biotinylated anti-avidin D (Vector Laboratories) for 20 min, and rinsed again. The slides were then treated with Avidin Alexa Fluor 350 for 20 min and rinsed again, as in the previous step. The slides were incubated with the IdU antibody, a mouse anti-bromodeoxyuridine (Becton Dickinson Immunocytometry Systems), the antibody specific for CldU, a monoclonal rat anti-bromodeoxyuridine (anti-BrdU; Accurate Chemical and Scientific Corporation) and biotinylated anti-avidin D for 1 h. This was followed by incubation with Avidin Alexa Fluor 350 and secondary antibodies, Alexa Fluor 568 goat anti-mouse IgG (H+L; Invitrogen Molecular Probes), and Alexa Fluor 488 goat anti-rat IgG (H+L;Invitrogen Molecular Probes) for 1 h. After a final PBS/CA-630 rinse, the coverslips were mounted with ProLong gold antifade reagent (Invitrogen). A fluorescent microscope (Axioscop 2 M2 with Plan Apochromat 63×/1.4 NA oil differential interference contrast objective; Carl Zeiss) with a camera (CoolSNAP HQ; Photometrics) and IPlab 4.0.8 software (BD) was used to detect the nucleoside incorporation into the DNA molecules. Images were processed with Photoshop CS5 software. Image processing involves alignment of the DNA molecules according to the FISH probes, adjusting of the contrast, and removing unspecific background signal. Total number of DNA molecules for each cell line were captured from three blinded experiments. The molecules presented are the complete dataset. We used Student’s t test with two-tailed distribution for P value calculation.

Replication fork speed at the FXN locus was calculated using the following equation: Average kb /min = [Length of segment (kb) / Td (min)] / Average number of replication forks for this segment (Norio and Schildkraut, 2001). Td; duplication time of the fragment; the time required for the segment to duplicate (min), which is calculated by the following equation: Td= Tp1 x NRG / (NR + NRG) or Td= Tp1 x NRG / (NG + NRG). Tp1 = Time for the first or second labeling (4 hours). NR or NG = the number of molecules fully stained in red or green. NRG = the number of molecules fully stained in both red and green in the segment.

DNA fiber analysis

To analyze origin density, cells were pulsed labeled with 25 μM 5-iodo-2′-deoxyuridine (Sigma-Aldrich) and with 25 μM 5-chloro-2′-deoxyuridine (Sigma-Aldrich). The DNA was stretched on glass slides and the incorporated nucleotides were detected by IdU antibody (mouse anti-bromodeoxyuridine, Becton Dickinson Immunocytometry Systems) and CldU antibody (monoclonal rat anti-bromodeoxyuridine [anti-BrdU], Accurate Chemical and Scientific Corporation) as described above. We used Student’s t test with two-tailed distribution for P value calculation.

DNA methylation analysis

DNA extraction was performed using the Qiagen DNeasy Blood and Tissue kit (69506). One microgram of genomic DNA was spiked with 5 ng/ul of unmethylated cl857 Sam7 Lambda DNA (D1501; Promega) to later compute non-conversion rate. The DNA was fragmented to 150–200 bp using a Covaris S2 and ligated with mC adapters provided by Illumina. The adaptor-ligated DNA underwent a sodium bisulphite conversion using the MethylCode kit from Life Technologies and then PCR. The samples were sequenced on Illumina HiSeq 2500. The methylation reads were mapped to hg19, and methylation sites called using methylpy (Schultz et al., 2015). The non-conversion rate was computed from each sample by analyzing the cytosine mischaracterization rate on the lambda DNA sequence. Experiments were performed blind.

Micronuclei assay

For the micronuclei assay, cells were dissociated into single cells using tryptase (Gibco, 10 min). The cells were harvested and resuspended in PBS-EDTA (1 mM EDTA). The cell count was adjusted to 1 million cells/ml, and cells were harvested (~50–100 µl/ slide) by centrifugation on positively charged slides (VWR) using CytoSpin 4 (Thermo Scientific). The centrifugation was done at 500 rpm for 5 min. The slides were air dried and mounted with Antifade Mountant with DAPI (Invitrogen). The slides were allowed to dry before scoring for micronuclei. The slides were read on Zeiss fluorescence microscopes at 100X magnification. The nuclei were assessed for the presence of micronuclei, and micronuclei bridges. All results were performed in triplicates, and ≥120 cells were scored for each experiment. We used Student’s t test with two-tailed distribution for P value calculation.

γH2AX detection

Cells were gently placed on polysine-coated slides (Shandon; catalog no. 6776216; lot no. 112216-9). The cells were allowed to settle for 20 min at RT. Liquide was removed by gently tilting the slide and the cells were fixed-permeabilized in PMTEF buffer for 20 min at RT (4% paraformaldehyde, 200 mM PIPES, pH 6.8, 200 mM MgCl2, 10 mM EGTA, 0.2% Triton X-100). After fixation-permeabilization, slides were washed three times with PBS. Immunostaining was performed using the primary rabbit monoclonal anti-γ-H2AX antibody (9718T; Cell Signaling Technology) and the secondary goat anti-rabbit Alexa-488-conjugated IgG (44125; Cell Signaling Technology). Nuclei were stained with DAPI. We performed three independent blinded experiments. Fluorescence microscopy was used to visually and record cells with positive foci (n ≥ 150). We used Student’s t test with two-tailed distribution for P value calculation.

We thank Alexandra MacWade for proofreading the manuscript.

This work was funded by the Perelman research recruitment gift (J. Gerhardt) and NYSTEM IDEA Award (C32564GG to D. Egli). The studies at the FXN locus in iPSCs were supported by National Institutes of Health grant R03 NS106216 (J. Gerhardt).

The authors declare no competing financial interests.

Author contributions: J. Gerhardt and D. Egli conceived and designed the study. M. Smith, D. James, and N. Wang grew PSCs. J. Gerhardt, T. Paniza, and M. Deshpande performed SMARD and γH2AX experiments and analyzed the results. N. Wang and M.V. Zuccaro performed cell cycle analysis. R. O’Neil and J. Ecker performed the DNA methylation analysis. J. Gerhardt wrote the manuscript. D. Egli, Z. Rosenwaks, and A. Madireddy edited and/or discussed the manuscript.