Figure 1.
UVC-, hydrogen peroxide (H2O2)- and dimethylsulfate (DMS)-induced damage in pluripotent cells and fibroblasts.
(A) Quantification of enzyme sensitive sites per mega base (ESS/Mb) in dot blot analysis of UVC-induced (10 or 20 J/m2) CPD adducts in pluripotent cells and fibroblasts. Values are mean±standard error of the mean (SEM) (n = 3). (B) Quantification of the percent DNA in comet tails for hESCs, iPSCs and fibroblasts treated with 100 µM H2O2. The sample size is 100 cells for each cell type and treatment; values are mean±SEM (n = 3). (C) Quantification of the percent of DNA in comet tails for hESCs and human skin fibroblasts treated with the indicated concentrations of DMS. The sample size is 100 cells for each cell type and treatment; values are mean±SEM (n = 3).
Figure 2.
Global-genome nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers (CPDs) in pluripotent cells.
Quantification of global genome-nucleotide excision repair of UVC damage as percent of CPD repair in (A) ESCs and fibroblasts, and (B) iPSCs and their parental fibroblast lines. Values are mean±SEM (n = 3). The initial number of ESS/Mb following 10 J/m2 UVC treatment in each cell line were: H9, 4.6±0.5; BG01, 6.3±0.1; iPSC1, 6.2±0.2; iPSC2, 3.2±0.2; human skin fibroblasts (CRL-2097), 25.5±1.1; human lung fibroblasts (IMR90), 14.5±0.3; and human foreskin fibroblasts (HF51), 13.9±0.4.
Figure 3.
Transcription-coupled nucleotide and base excision repair in pluripotent cells determined using host cell reactivation.
(A) Determination of the number of ESS/pM1-Luc plasmid induced by 200 J/m2 UVC (see Materials and Methods for details). The weak band seen between supercoiled (SC) and nicked (N) DNA is the linear form. (B) Host cell reactivation assay for CPD repair. Unirradiated or UVC irradiated (200 J/m2) pM1-Luc plasmid was co-transfected with untreated pRL-CMV plasmid (ratio of pM1-Luc/pRL-CMV was 2.4 µg/0.24 µg in 1×106 cells). Dual firefly and Renilla luciferase activities were performed at 24 h post-transfection. The relative luciferase activities were compared to undamaged pRL-CMV activities. Values are mean±standard deviation (SD) (n = 3). (C) Determination of the number of ESS/pM1-Luc plasmid induced by methylene blue and visible light treatment (see Material and Methods for details). (D) Host cell reactivation assay for 8-oxo-G repair. pM1-Luc treated with methylene blue/visible light was co-transfected with undamaged pRL-CMV using the same conditions as described in (B). Cells were isolated 24 h post-transfection and the firefly and Renilla luciferase activities determined. Values are mean±SD (n = 3). TC-NER, transcription-coupled nucleotide excision repair; BER, base excision repair.
Figure 4.
Double-strand break repair assay for non-homologous end joining using HCR.
(A) Schematic drawing of host cell reactivation assay for non-homologous end joining repair. Non-homologous end joining is assessed by cleaving the pCMS-end plasmid (Xho I/Apa I) to generate a non-religatable DSB between the promoter and the YFP-coding region prior to transfection. Repair of the DSB by non-homologous end joining reconstitutes a link between the promoter and YFP coding region while GFP serves as an expression control. Uncleaved pCMS-end served as a positive control (100% recombination efficiency). The absolute recombination efficiency was calculated as the fraction of cells that recombined (YFP+ and GFP+YFP cells) over the total number of transfectants (YFP+,GFP+, plus GFP+YFP cells), then corrected for the “recombination efficiency.” The efficiency for non-homologous end joining was calculated as follows: ([GFP+]+[YFP+])/([GFP+]+[YFP+]+[GFP+YFP])end-cleaved divided by ([GFP+]+[YFP+])/([GFP+]+[YFP+]+[GFP+YFP])end. (Adapted from [31]) (B) Representative FACS data for determination of the non-homologous end-joining DNA repair capacity in BG01. Transfected cells were harvested and assayed 24 h post-transfection. The upper panel shows FACS data for the control plasmid transfection data compared to the dual cleaved (Xho I/Apa I) plasmid in the bottom panel. (C) Quantification of non-homologous DSB repair capacity using host cell reactivation assays in hESCs, hESC-derived NSCs, iPSCs and fibroblasts. Values are mean±SEM (n = 3). *, statistically significant as determined by unpaired Student's t-test between iPSC1 and IMR90/iPSC2 with 2-tailed P value = 0.02 and 0.03, individually.
Figure 5.
Double-strand break repair assay for single-strand annealing using host cell reactivation.
(A) Schematic drawing of host cell reactivation assay for single-strand annealing repair. Single-strand annealing is assessed by cleaving the pCMS-hom-stop plasmid (Xho I/Sac II) to generate a DSB with incompatible ends between the 5′ and 3′ YFP coding regions prior to transfection. YFP signal was observed only when the correct reading frame was restored by single-strand annealing (adapted from [31]) (B) Representative FACS data for determination of single-strand annealing DNA repair capacity in BG01. The single-strand annealing efficiency is calculated as follows: ([GFP+]+[YFP+])/([GFP+]+[YFP+]+[GFP+YFP])hom-stop subtracted from ([GFP+]+[YFP+])/([GFP+]+[YFP+]+[GFP+YFP])hom-stop-cleaved. Uncleaved pCMS-hom-stop served as zero percent recombination efficiency in the single-strand annealing assay. The absolute recombination efficiency was calculated as the fraction of cells that recombined (YFP+ and GFP+YFP+ cells) over the total number of transfectants (YFP+,GFP+, plus GFP+YFP+ cells), then corrected for the “recombination efficiency.” (C) Quantification of DSB repairs in hESCs, hESC-derived neural stem cells, iPSCs and fibroblasts through the single-strand annealing repair pathway by HCR assay. Values are mean±SEM (n = 3). *, statistically significant as determined by unpaired Student's t-test between IMR90 and iPSC1, iPSC2, BG01 with 2-tailed P values of 0.007, 0.006, and 0.004, respectively.
Figure 6.
Microsatellite instability assay in pluripotent cells and differentiated cells.
Comparable cell lines are grouped in black boxes, and shifts in the peaks corresponding to microsatellite instability (MSI) are marked by black arrows. The X axis shows the scan number and the Y axis shows intensity of 6-FAM. Red peaks are internal controls to indicate the locations of microsatellites. CRL-2097 are human skin fibroblasts used to generate iPSC1, IMR90 are human lung fibroblasts used to generate iPSC2, H9 are hESCs used to generate NSC9, and BG01V are an aneuploid variant hESC isolated from BG01.
Figure 7.
UVC-induced apoptosis in hESCs.
H9 cells were treated (or not) with UVC radiation (10 J/m2). (A) Time lapse photomicrographs (differential interference contrast) of live cell cultures at 30 min intervals. Bars are 50 µm. The 3 upper panels show a control colony that was not exposed to UVC radiation. The 3 lower panels show a colony exposed to UVC radiation. The dark field in the upper panels represents the adherent attached colonies. The lower panels show that, at 3.5 h, a large number of cells from the colony are no longer adherent but float in the media. (B) FACS quantification of viable, Annexin V-FITC stained (early and late apoptotic) cells, and necrotic cells from either floating or adherent cells at either 3 or 22 h post-irradiation. The effect of ROCK inhibitor (Ri; 10 µM) on apoptosis was evaluated at 22 h in both adherent and floating cells. There were few if any floating cells prior to UVC exposure. Both floating and adherent cells were collected at indicated time points, labeled with the apoptosis markers Annexin V-FITC and propidium iodide and subjected to FACS analysis. Representative FACS data is in Figure S7, along with gating to indicate the different quadrants as live, early apoptotic, late apoptotic, and necrotic. (C) DNA fragmentation analysis of UVC-irradiated H9 cells. M, DNA markers; STS, staurosporine; F, floating cells; A, adherent cells. (D) Caspase 3 cleavage in adherent and floating cells. Upper panel: Western blot of caspase 3 cleavage in H9 cells treated with 10 J/m2 UVC (3, 5, and 24 h) or staurosporine (3 h. uncleaved (Uncl.); cleaved (Cl.); floating cells (F); adherent cells (A). Note that there were no floating cells prior to treatment. Lower panel: analysis of Western blots comparing uncleaved procaspase 3 (Uncl.) and cleaved (Cl.) bands for caspase 3.
Figure 8.
Summary of DNA repair rates/capacity in multiple DNA repair pathways in all cell lines investigated.
Y axis shows the logarithmic phase of fold difference of pluripotent cells over IMR90 fibroblasts. Dotted lines are used to separate the repair pathways and direct comparisons should be limited to within the pathways. Values are mean±SD. DNA repair capacities were evaluated at 24 h after treatment or transfection.