DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells
et al. (2012) DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-
Pluripotent Human Cells. PLoS ONE 7(3): e30541. doi:10.1371/journal.pone.0030541
DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells
Li Z. Luo 0
Sailesh Gopalakrishna-Pillai 0
Stephanie L. Nay 0
Sang-Won Park 0
Steven E. Bates 0
Xianmin Zeng 0
Linda E. Iverson 0
Timothy R. O'Connor 0
Costanza Emanueli, University of Bristol, United Kingdom
0 1 Department of Cancer Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, United States of America, 2 Department of Stem Cell Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, California, United States of America, 3 North Bay CIRM Shared Research Laboratory for Stem Cells and Aging, Buck Institute for Age Research , Novato, California , United States of America
The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use.
Funding: This work was supported by funding from the California Institute of Regenerative Medicine (RS1-00428-1 to TRO, http://www.cirm.ca.gov/content/
sources-genetic-instability-human-embryonic-stem-cells), the National Institutes of Health (SPORE P50 CA107399, S. Forman, http://www.nih.gov/index.html), the
Irell and Manella School of Biological Sciences of City of Hope, the City of Hope Comprehensive Cancer Center Grant (NCI 5P30CA033572-27, M. Friedman, http://
www.cancer.gov/), and the Beckman Research Institute of City of Hope. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
The self-renewal and differentiation properties of human
pluripotent stem cells (pluripotent cells), including both human
embryonic stem cells (hESCs) and induced pluripotent stem cells
(iPSCs), make them promising resources for regenerative medicine.
Nevertheless, before these cells can be used therapeutically, it is
critical to understand the potential risks linked to cellular
maintenance and transmission of genetic information. DNA repair
mechanisms are responsible for preserving genomic integrity in all
cell types. However, reduced repair capacities can lead to genomic
instability, which has been reported in some hESC lines [1,2] and
iPSC lines [3,4]. Therefore, determining the DNA repair capacities
for DNA repair pathways in pluripotent cells is a critical issue for
pre-clinical information, as well as for understanding how
pluripotent cells protect their genomes from damage.
Standard DNA repair pathways in mammalian cells include base
excision repair [5,6], nucleotide excision repair [7,8], homologous
repair, single-strand annealing, non-homologous end-joining repair,
mismatch repair , and direct DNA repair . Base excision
repair corrects small DNA alterations, such as oxidized bases, uracil
or alkylating agent damage. Nucleotide excision repair, on the other
hand, removes mainly bulky lesions (e.g., cyclobutane pyrimidine
dimers) by excision of 2729-mer oligodeoxyribonucleotides.
Nucleotide excision repair is further subdivided into global
genome-nucleotide excision repair and transcription
couplednucleotide excision repair. Homologous repair, non-homologous
end-joining, and single-strand annealing are three different
pathways that repair DNA double-strand breaks (DSBs)
[11,12,13]. Error-free homologous repair requires a homologous
DNA template, while non-homologous end-joining does not
necessarily require homology, making it error-prone. Although
single-strand annealing requires a homologous template, it is
mutagenic because it anneals two extensive regions of homology
that flank either side of a DSB, resulting in a deletion. Mismatch
repair scans the genome for mismatched bases or single-strand loops
and direct DNA repair primarily removes methylation adducts.
Although some repair pathways are error-prone, for all of these
mechanisms, inefficient repair can result in mutation or
translocation, thus reducing the fidelity of genomic information transfer.
Despite substantial progress in the field of pluripotent stem cells,
little is known about the response of pluripotent cells to mutagens
or their DNA repair capacities as compared to differentiated cells.
Furthermore, much of the available information concerning
mutation and DNA repair has been obtained using mouse
embryonic stem cells (mESCs) and not hESCs. mESCs have
some prominent differences that distinguish them from their
differentiated counterparts. mESCs lack a G1 checkpoint [14,15]
and more readily undergo P53-independent apoptosis than do
differentiated cells . Therefore, mESCs are more susceptible to
apoptosis than differentiated mouse cells . However, mESCs
are more resistant to and more efficient at repairing oxidative
damage than differentiated mouse cells . With respect to
mutagenesis, spontaneous mESC mutant frequencies are 100-fold
lower than those of mouse embryonic fibroblasts  indicating
that mESCs have enhanced genomic stability compared to
differentiated counterparts. These data suggest that there may
be differences in genomic stability and DNA repair between
hESCs and differentiated human cells.
On the other hand, as compared to mESCs, hESCs have a
functional CDK2-dependent G1/S checkpoint [20,21]. However,
exposure of hESCs to high energy ionizing radiation at fluxes less
than 1 Gy induced apoptosis that was associated with damage
responses mediated by ATM, NBS1, CHEK2, and P53, in hESCs,
but not in fibroblasts [22,23]. Additional studies also suggest that
hESCs  and fibroblasts  exhibit different repair responses to
ionizing radiation  and that hESCs use homologous repair, not
non-homologous end joining, as the dominant DSB repair pathway
[23,26,27]. Single cell gel electrophoresis (comet assay) comparing
the number of DNA strand breaks following exposure to hydrogen
peroxide (H2O2), ultraviolet C (UVC) radiation (254 nm),
cradiation, or DNA cross-linking agents, showed that breaks were
generally repaired faster in two hESC lines examined than in
differentiated human cell lines . Taken together, these results
support the need to investigate further the response to DNA
damage, as well as the pathway-specific DNA repair capacities of
human embryonic and induced pluripotent stem cells. In particular,
data are lacking on the comparison of DNA repair over a range of
pathways in multiple human pluripotent stem cell lines. More
importantly, minimal data exist for reprogrammed iPSCs, which
could serve as better candidates for clinical use if they have similar
genomic stability as hESCs and maintain their pluripotency.
In this study, DNA repair was monitored in several pluripotent
and differentiated cell lines and DNA repair pathways to examine
sources of genomic instability in hESCs and iPSCs. These assays
encompassed nucleotide excision repair, base excision repair,
nonhomologous end joining, single-strand annealing, and
microsatellite instability. Repair capacities from hESCs and iPSCs were
compared to each other and to those of non-pluripotent cells.
Evaluation upon exposure to DNA damaging agents such as
UVC, dimethylsulfate (DMS) and c-radiation indicated that
pluripotent cells exhibited less damage than non-pluripotent cells,
but despite lower damage levels, pluripotent cells were more prone
to a type of apoptosis that could be linked to anoikis . This
investigation provides a basis for evaluating DNA repair capacities
in pluripotent cells and emphasizes the need to evaluate the DNA
repair capacity of each pluripotent cell line prior to laboratory and
Materials and Methods
hESC lines H9, BG01 and BG01V were obtained from WiCell,
Bresagen (NovoCell) and GlobalStem, respectively. Neural stem
cell line NSC09, derived from H9, was obtained from Millipore.
Induced pluripotent stem cell line iPSC1, derived from human
foreskin fibroblast line CRL-2097 using lentiviral vectors, was
obtained from Dr. James A. Thomson (University of
WisconsinMadison) . iPSC2, derived from a human lung fibroblast line
using the same retroviral introduced factors, was obtained from
Dr. Jiing-Kuan Yee (Beckman Research Institute).
Non-pluripotent IMR90 lung fibroblasts and CRL-2097 human foreskin
fibroblasts were purchased from ATCC, GM03348E human
foreskin diploid fibroblasts (HF02) were obtained from the Coriell
Cell Repository and HF55 (HF01) and HF51 human neonatal
foreskin fibroblasts were derived from discarded tissue provided by
Arcadia Methodist Hospital from an approved protocol (City of
Hope IRB# 92006). Pluripotent stem cell characterization is
presented in Figures S1 and S2.
All cell lines were cultured as recommended. Specifically,
hESCs (H9, BG01 and BG01V) and iPSCs (iPSC1 and iPSC2)
were cultured in mTeSR1 (StemCell Technology) on
hESCqualified Matrigel (BD Biosciences) or on irradiated mouse
embryonic fibroblasts or human fibroblasts (HFs) in conditioned
hESC medium (DMEM/F12) (Cellgro, 10-092-CM4),
supplemented with 20% knock-out serum replacement, 0.1 mM
nonessential amino acids, 2 mM L-glutamine, 20 ng/mL fibroblast
growth factor (bFGF) and 0.1 mM 2-mecaptoethanol. Medium
was changed daily, and cells were either mechanically harvested,
or passaged with Accutase. Rho-associated kinase (ROCK)
inhibitor Y-27632 was added transiently at 10 mM to the culture
medium to improve iPSC2 cell survival during passaging. Prior to
exposure to DNA damaging agents or transfection, pluripotent
cells were transferred to Matrigel, unless otherwise noted, to
remove differentiated fibroblast feeder cells. NSC09 cells were
cultured in Neurobasal Medium (Invitrogen, 21103-049)
conditioned with 2 mM L-glutamine, 0.1 mM non-essential amino
acids, 1X B27, Leukemia inhibitory factor (LIF, 1,000 U/mL) and
bFGF (20 ng/mL). Similar to above, medium was changed daily
and cells were passaged with Accutase. GM03348E cells (HF02)
were cultured in Minimal Essential Medium (MEM) a with
Glutamax-1 (Gibco, 32571-036) supplemented with 15% fetal
bovine serum (FBS) and 0.1 mM non-essential amino acids.
Medium was changed daily and cells were passaged with 0.05%
Trypsin-EDTA. IMR90 lung fibroblasts were cultured in MEM
containing Earles Salts and L-glutamine (Cellgro, 10-010-CV),
supplemented with 10% FBS, and passaged with 0.25%
TrypsinEDTA. HF55 (HF01) and HF51 were cultured in Dulbeccos
Modified Eagles Medium (DMEM) (Cellgro, 15-017-CV)
supplemented with 10% FBS and 2 mM L-glutamine and passaged with
Plasmids and antibodies
pCMS-end, pCMS-hom-stop, pEGFP, and pEYFP-tub were
gifts from Dr. R.H. Schiestl (UCLA) [30,31]. pRL-CMV was
purchased from Promega and pM1-Luc from Roche. Antibodies
were purchased from vendors as follows: Santa Cruz: rabbit
antiOCT4; Millipore: mouse anti-Oct4, goat anti-SOX2, rabbit and
mouse anti-cH2AX; Abcam: rabbit anti-NANOG, mouse
antiDnmt3b; Developmental Studies Hybridoma Bank: mouse
antiSSEA4; Cell Signaling Technology: rabbit anti-caspase 3;
Kamiya: mouse anti-CPD, mouse anti-6,4 photoproducts; Sigma
Aldrich: mouse anti-actin; Invitrogen: Alexa 488, 568 and 647
donkey anti-mouse, rabbit and goat IgG(H+L); and LiCor
Biosciences: IR Dye800 and 680 goat anti-mouse and rabbit
(secondary antibodies for dot blots and Western blots).
Single Cell Gel Electrophoresis (Comet Assay). The comet assay
was performed using alkaline conditions, following the
recommended protocol of the Trevigen Comet Assay Kit. Images were
collected on an Olympus IX81 automated inverted fluorescence
microscope and comets (sample size = 100) were quantified by
measuring the %DNA in each comet tail, using CometScore
software (TriTek Corp).
DNA dot blot assay
Cells were exposed to UVC radiation (10 or 20 J/m2) from a
germicidal lamp as previously described . After treatment, cells
were either harvested immediately to determine DNA damage or
allowed to repair for defined periods. Genomic DNA was extracted
using the DNeasy DNA extraction kit (Qiagen), following the
manufacturers instructions or by standard phenol/chloroform
extraction methods, as described . Concentrations of
cyclobutane pyrimidine dimers (CPD) and 6,4 pyrimidine-pyrimidone
adducts (6,4-PP) were determined using immunological detection
with DNA South-Western dot blots [34,35]. Residual RNA was
removed by DNase-free RNase A (1 mg/mL), followed by a final
extraction with phenol:chloroform:isoamyl alcohol 1:1 and
centrifugation (6666 g, 5 min, room temperature [RT]). DNA in the
aqueous phase was then precipitated by addition of 3 volumes of
icecold 100% ethanol, followed by a 70% ethanol wash. Genomic
DNA was air-dried and dissolved in 10 mM Tris-EDTA buffer
(pH 8.0) (several h, RT or overnight, 4u C). Concentrations were
determined using a NanoDrop spectrophotometer. For the DNA
dot blot assay, DNA samples were prepared at 1 ng/mL in DNA
denaturing solution (1.5 M NaCl, 0.5 M NaOH). A
positivelycharged, nylon membrane (Roche) was hydrated and fixed in a dot
blot apparatus (BioRad) with a Convertible Filtration Manifold
System (Life Technologies). DNA (100 ng in 100 mL) was added
into three replicate wells for each sample and an equal volume of
150 mM NaCl, 50 mM Tris-HCl pH 7.6 (16 TBS) was added to
all other wells not containing sample. After incubation (30 min), a
vacuum was used to draw out samples and the membrane was
washed (365 min) with 16TBS, using a vacuum to remove each of
the washes. The membrane was then air dried for 15 min, after
which it was incubated with 2% blocking solution (Roche), diluted
in 16 TBS (1 h, RT). The membrane was incubated with primary
antibody (mouse anti-CPD or 6,4 photoproduct; 1:2000 (Kamiya)
prepared in 1% blocking solution (1 h, RT or overnight, 4u C),
washed 3 times for 5 min each with 16TBS-T (Tween-20, 1:1000),
and incubated with secondary antibody (near-IR dye 800 CW goat
anti-mouse IgG; 1:20,000) in 1% blocking solution (1 h, RT). After
incubation with secondary IR-antibody, the membrane was washed
again in 16 TBS-T (3 times, each for 5 min), and subjected to
infrared detection by a Li-Cor Odyssey Infrared Imager. The
images were quantified by TotalLab Analysis software (TotalLab
Ltd.). For DNA repair assays using antibody detection, the initial
ESS/Mb at time = 0 h obtained were: H9, 4.660.5; BG01,
6.360.1; iPSC1, 6.260.2; iPSC2, 3.260.2; human skin fibroblasts
(CRL-2097), 25.561.1; human lung fibroblasts (IMR90), 14.560.3;
and human foreskin fibroblasts (HF51), 13.960.4.
Live Cell Imaging. UVC-irradiated (or unirradiated) H9 cells,
on Matrigel-coated chamber slides, were imaged in the live cell
chamber (37u C; 5% CO2) of a Zeiss Axio Observer Z1 inverted
microscope and live-cell imaging station. DIC images were taken
at 30 min intervals. Images and movies were compiled with Image
Annexin V apoptosis assay
Apoptosis was assessed using the Annexin V-FITC Apoptosis
Detection Kit I (BD Pharmingen). H9 cells, in cold 16 PBS, were
irradiated with UVC and refreshed in mTeSR1 immediately after
exposure. Floating and adherent cells were collected separately at
3 and 22 h by centrifugation (5006 g, 5 min) or by exposure to
Accutase followed by centrifugation (5006 g, 5 min), respectively.
Cell pellets were washed once with and resuspended in 0.5 mL 16
PBS prior to addition of 5 mL Annexin V-FITC and 5 mL
propidium iodide (PI). Control unstained, Annexin V only and PI
only cells were also prepared to establish gating parameters. FACS
analysis was performed on a MoFloTM MLS cell sorter and data
processed with Summit v4.3.
DNA fragmentation to detect apoptosis
H9 or iPSC2 cells (126106 cells) in 35-mm culture dishes were
irradiated with UVC (0 or 10 J/m2) in 16 PBS and incubated in
fresh medium (3, 5, and 24 h, 37u C). Samples were collected as
control (0 J/m2) or treated (10 J/m2) at each time point.
Additionally, cells were treated with staurosporine (STS) (1 mM,
3 h, 37u C) as a positive control for apoptosis. Medium containing
floating cells and attached cells was centrifuged (10006 g, 5 min)
and collected as the floating fraction (F) or attached fraction (A).
DNA was isolated using DNeasy Blood and Tissue kit (Qiagen),
heated (10 min, 65u C), and immediately loaded onto a 1%
agarose gel for electrophoresis (100 V, 2 h).
Western blot analysis
H9 or iPSC2 cells (126106 cells) in 35-mm culture dishes were
irradiated in 16 PBS with UVC (0 or 10 J/m2) and incubated in
fresh medium (3, 5 and 24 h, 37u C). Samples were collected as
control (0 J/m2) or treated (10 J/m2) at each time point.
Additionally, cells were treated with STS (1 mM, 3 h, 37u C) as
a positive control for apoptosis. The medium containing
nonadherent cells was centrifuged (5006 g, 10 min) to pellet floating
cells. To harvest protein, 100200 mL RIPA buffer (50 mM
TrisHCl [pH 7.4], 150 mM NaCl, 1% NP40, 0.25%
Na-deoxycholate, 1 mM PMSF, protease inhibitor cocktail and phosphatase
inhibitor cocktail) was added to floating cell pellets and to the
remaining adherent cells, samples were incubated on ice (10 min)
and centrifuged (14,0006 g, 10 min, 4Cu). Protein concentrations
were determined using a Coomassie Blue protein assay (BioRad)
. Sample (50 mg) was combined with 56 SDS-PAGE loading
buffer and dH2O, heated at 95uC for 5 min and loaded onto a 4
15% Mini Protean TGX SDS-PAGE gel (BioRad). Samples were
transferred to a 0.2 mm PVDF membrane at 25 V for 3 h, using a
wet electro-transfer method (0.2 M glycine, 25 mM Tris and 20%
methanol). The membrane was blocked in Li-Cor Odyssey
Infrared Imaging System Blocking Buffer (Li-Cor) (1 h, RT or
overnight, 4u C), followed by incubation with anti-actin (1:20,000)
and anti-caspase 3 (1:1000) primary antibodies (2 h, RT or
overnight, 4uC) in blocking solution (50% [v/v] Odyssey Blocking
Buffer/16 TBS). After primary antibody incubation, membranes
were washed (365 min) in 16 TBS-T (Tris-buffered saline
containing Tween-20 [1:1000]) prior to addition of near-infrared
secondary antibodies, diluted 1:10,000, in blocking solution, as
described for the primary antibody. Incubation in secondary
antibody was conducted for 1 h at room temperature followed by
16 TBS-T washes (365 min). Detection was carried out using an
Odyssey Imaging Station (Li-Cor) and band intensities were
quantified with TotalLab Analysis software (TotalLab Ltd.).
Optimal transfection conditions for H9, neural stem cells, and
other pluripotent cells were determined empirically by at least
three different programs using the Amaxa Nucleofector Kit II
(Lonza) for hESCs. Cells were harvested with Accutase,
centrifuged (1006 g, 10 min) and washed once with mTeSR1. Cell
number was determined and cells were resuspended in 100 mL
hESC Nucleofection Solution 2, mixed with 12 mg DNA/16106
cells and nucleofected with a set program (A-23 for H9 and iPSC1,
A-13 for BG01, and B-16 for iPSC2 and BG01V). Cells were
incubated in 500 mL pre-warmed RPMI 160 medium and
immediately transferred to Matrigel pre-coated multi-well plates
containing 1 mL mTeSR1 medium. Transfection of fibroblasts
was performed using Lipofectamine 2000 according to
recommendations from the manufacturer (Invitrogen).
Microsatellite instability assay
Template DNA was prepared as described for the DNA Dot
Blot Assay. The primers used in the assay are listed in Table S1
[37,38]. PCR conditions were: 5 U/mL Taq polymerase (BioRad),
0.25 mM dNTP mix, 1 mM primers, 40 ng DNA template in 16
reaction buffer, run in 10 mL reactions for 30 cycles (94u C, 50 sec;
56u C, 50 sec; 72u C, 1 min) after denaturing at 95u C for 5 min.
Product was analyzed on an ABI Prism 377 Sequencer and results
were scored with GeneMapper software.
Plasmid lesion assay
To determine the number of UVC radiation-generated lesions
in plasmids, pM1-Luc plasmid (10 mg per sample) was irradiated
with 200 J/m2 UVC and incubated (2 h, 37u C) with or without
1 mL T4 UV endonuclease (laboratory stock, 40 mg/mL). To
determine levels of damage induced by reactive oxygen species
photosensitization, pM1-Luc plasmid (10 mg per sample) in
10 mM sodium phosphate buffer (pH 7.4), containing 10 mM
methylene blue was exposed to visible light (100 Watts, 10 cm
distance, 3 min). Following exposure, plasmid was ethanol
precipitated and incubated (2 h, 37u C) with 0.5 mL (2 U)
formamidopyrimidine-DNA glycosylase (Fpg) (Trevigen). After
incubation with T4 UV endonuclease or Fpg, samples were
analyzed on 1% agarose gel. Damage sites were quantified with
ImageJ software and the number of breaks (n) per molecule was
calculated by the formula n = 2ln e, e being the fraction of the
remaining supercoiled DNA molecules .
Host cell reactivation (HCR) assay for nucleotide excision
repair and base excision repair DNA repair capacities
The Dual Luciferase Assay (Promega, E1910) was used to monitor
DNA repair capacities for nucleotide or base excision repair. Cells
were transfected (fibroblasts) or nucleofected (pluripotent and NSCs)
with 2.4 mg pM1-Luc (damaged or undamaged with UVC or
reactive oxygen species photosensitization as described in the
previous section) and 0.24 mg pRL-CMV (internal control) per
16106 cells, and harvested after 24 h to quantify Firefly and Renilla
luciferase activities. Briefly, transfected cells were washed with 16
PBS and lysed in 16 PLB buffer (passive lysis buffer supplied by
Promega) (250 mL/well in 12-well plates for pluripotent cells and
6well plates for non-pluripotent cells) on a shaking platform (20 min,
RT). Triplicate samples from each lysate (20 mL per well) were
transferred to individual wells of a 96-well plate, sequentially mixed
with 100 mL Luciferase Assay Reagent II (LAR II) and 100 mL Stop
and Glo in 96-well plates. Samples were analyzed with a Fluoroskan
Ascent FL (Thermo Electron Corporation). Each assay was
performed independently three times and the data combined
according to the manufacturers instructions (Promega).
HCR assay for double-strand DNA repair capacity
Prior to the HCR assay, pCMS-end (non-homologous
endjoining) and pCMS-hom-stop (single-strand annealing) plasmids
were cleaved with Xho I and Apa I, or with Xho I and Sac II
[30,31], respectively. The double restriction-digested, linearized
plasmids were confirmed as linear by verifying that the Escherichia
coli transformation efficiency was less than 0.1% as compared to
uncleaved plasmids. Cells were transfected (fibroblasts) or
nucleofected (pluripotent cells and NSCs) with pEGFP, pEYFP,
pCMS-end, pCMS-hom-stop, and double-digested pCMS-end or
pCMS-hom-stop and harvested by trypsin or Accutase 24 h later.
Upon harvesting, cells were stained with SYTOX red, to assess
cell viability, resuspended in 0.5 mL cold 16PBS and subjected to
FACS analysis using a MoFloTM MLS cell sorter. For each assay
performed, an untransfected control and simultaneous transfection
controls (pEGFP plasmid only and pEYFP-tub plasmid only) were
analyzed to establish the correct gating and compensation settings.
The laser settings used for GFP/YFP/Sytox Red were as follows:
GFP: laser excitation wavelength 488 nm (500 mW) with an
HQ500/10 emission filter, YFP: laser excitation wavelength
530 nm (50 mW) with an HQ600/30 emission filter, Sytox Red:
laser excitation: 647 nm (60 mW) with an HQ680/30 emission
filter. Data were analyzed using Summit v4.3 software (Dako
Other techniques used are described in Materials and
DNA damage from UVC is less in pluripotent cells than in
As a prelude to determining the DNA repair capacity for
nucleotide excision repair in pluripotent cells, we examined DNA
damage induced by UVC radiation (short wavelength, 100
280 nm). The levels of cyclobutane pyrimidine dimer (CPD) DNA
adducts induced by UVC radiation were quantitatively measured
using antibodies. H9 and BG01 ES, iPSC1 and iPSC2
inducedpluripotent, and IMR90 and CRL-2097 fibroblast cells were
irradiated with UVC (10 or 20 J/m2), genomic DNA was isolated
immediately after UVC exposure, and CPD adduct densities
established (Figures 1A and S4A). CPD enzyme sensitive sites
per megabase (ESS/Mb), an indication of adduct levels, were
determined via alkaline gel analysis of UVC-irradiated l DNA
 to standardize DNA samples (Figures S3A and S3B). The
numbers of CPD-ESS/Mb induced in pluripotent cells were 40
50% less at 10 J/m2 and 5070% less at 20 J/m2 than those in
both fibroblast lines evaluated (Figures 1A and S4A). Therefore,
pluripotent cells manifest lower CPD levels than fibroblasts
exposed to equal UVC fluxes.
Reactive oxygen species-induced DNA damage is less in
hESCs than in iPSCs and fibroblasts
Since UVC damage induced in pluripotent cells was less than
that induced in fibroblasts, we examined the effect of treatment
with other DNA damaging agents that require different pathways
for repair, including hydrogen peroxide (H2O2), which causes
damage that is repaired by base excision repair. Initially, hESCs,
iPSCs and fibroblasts were treated using an H2O2 concentration
(100 mM) that is sub-lethal to fibroblasts. Immediately after
treatment, cells were harvested, lysed and analyzed by the alkaline
comet assay. The relative levels of single-strand DNA breaks
(SSBs), indicative of initial DNA repair were quantified as the
percentage of DNA in the comet tail (%DNA Tail). Fibroblasts
and iPSCs showed substantial increases in the number of SSBs
(8to 20-fold increase) after treatment as compared to untreated
controls, whereas H9 cells showed only a 3-fold increase
(Figures 1B and S4B). Similar to results for UVC radiation,
H9 ESCs exposed to H2O2 incurred less damage than fibroblasts,
but, in contrast, iPSCs had damage levels similar to those for
In addition to generation of adducts repaired by the base
excision repair pathway, treatment with H2O2 can lead to DSBs.
Phosphorylation of Ser139 on histone H2AX is an early indicator
of DSB repair that is formed at nuclear foci . Therefore, to
assess DSB formation as a result of H2O2 treatment in hESCs,
iPSCs and fibroblasts, immunohistochemistry was used to visualize
cH2AX foci formation. The number of cH2AX foci that were
observed in fibroblasts was greater than in hESCs and iPSCs,
indicating that fibroblasts had more strand breaks when exposed
to the same amount of H2O2 damage (Figure S5). In contrast to
results obtained with the comet assay, iPSCs showed 3- to 4-fold
fewer cH2AX foci than did fibroblasts. However, iPSCs exhibited
an ,5-fold increase in cH2AX foci, compared to untreated cells
and ,2-fold more cH2AX foci than hESCs. These data are
consistent with greater protection against reactive oxygen
speciesinduced damage in pluripotent cells than in fibroblasts, with the
highest protection observed in hESCs. The fold differences in
cH2AX foci observed between iPSCs and fibroblasts are greater
than those observed in the comet assay. This difference may be
because the cH2AX foci assay generally scores DNA DSBs,
whereas the alkaline comet assay monitors SSBs. These results
indicate that for the cell types examined, the number of DNA
strand breaks (either SSBs or DSBs) associated with base excision
repair caused by H2O2 exposure was less in hESCs than in iPSCs
or fibroblasts, and iPSCs had fewer or similar numbers of breaks as
fibroblasts, depending on the type of break.
Dimethyl sulfate (DMS)-induced DNA damage is variable
and dependent on the pluripotent cell line
In addition to repair of reactive oxygen species-induced damage
that occurs via base excision repair, we also evaluated damage
generated by DMS. DMS generates principally 7-methylguanine
and 3-methyladenine  DNA damage and these adducts also
generate single-strand DNA breaks as intermediates during base
excision repair. Therefore, hESCs, iPSCs and fibroblasts were
incubated with DMS (0100 mM) for 30 min and harvested
immediately for alkaline comet assay analysis. When treated with
10 mM DMS, pluripotent and differentiated cells exhibited similar
damage levels, quantified as %DNA Tail (Figures 1C and S4C).
However, at 50 mM DMS, the %DNA Tail differed between the
two iPSC lines, with iPSC1 producing larger comets than all other
cell lines evaluated, including the parental line CRL-2097,
whereas iPSC2 exhibited the lowest %DNA Tail. The %DNA
Tail of H9 cells was lower than those of iPSC1 and IMR90, but
comparable to that of CRL-2097 fibroblasts. At 100 mM DMS,
the differences observed in the %DNA Tail for all the cell lines
were less pronounced, but maintained a pattern similar to that at
50 mM DMS. Therefore, there were no clear differences in the
damage produced by DMS in pluripotent and non-pluripotent
cells. In contrast to H2O2-induced single-strand breaks, after DMS
treatment, the differences in single-strand breaks observed
depended on the cell line and not on whether the cells were
pluripotent or differentiated.
Global genome-nucleotide excision repair of
UVCinduced CPDs is faster in pluripotent cells than in
Most CPD damage (,70%) in humans is repaired by global
genome-nucleotide excision repair . To monitor global
genome-nucleotide excision repair, we exposed pluripotent cells
(H9, BG01, iPSC1 and iPSC2) and fibroblasts (IMR90,
CRL2097 and HF51) to 10 J/m2 UVC radiation, collected adherent
cells at 0, 6, 12, and 24 h post-treatment, and isolated genomic
DNA for immunoblot analysis (Figures 2 and S6). We observed
that over 90% of adherent cells maintained intact cell membranes,
as determined by Trypan blue exclusion (data not shown). Despite
the presence of fewer CPD-ESS/Mb in pluripotent cells than in
fibroblasts immediately after irradiation, the DNA repair rate in
pluripotent cells was greater. Specifically, H9 and BG01 hESCs
were almost two times faster at repair (Figure 2A), and iPSC1 and
2 three times faster, than were fibroblasts (Figure 2B).
Interestingly, for hESCs, less than 10% of CPD repair had occurred
within 6 h after irradiation, with most repair occurring between 6
and 12 h. This contrasts with the rate of repair in iPSCs, which
had repaired 20% of CPDs by 6 h, but had a linear type response
over the 24 h period examined. This difference in the CPD repair
kinetics could indicate differences in the mechanism of global
genome-nucleotide excision repair between hESCs and iPSCs. We
also monitored repair of 6,4 pyrimidine-pyrimidone
photoproducts (6,4 PP), another UVC-induced DNA adduct. Repair of 6,4
PP was rapid for fibroblasts and pluripotent cells, with all of the
adducts removed in under 2 h (data not shown). Therefore, global
genome-nucleotide excision repair of CPDs induced by UVC
damage was significantly greater in pluripotent cells than in
fibroblasts, whereas no difference among the cell lines was
observed for 6,4 PP repair rates.
Transcription coupled-nucleotide excision repair of
UVCinduced damage is faster in pluripotent cells than in
Since pluripotent cells exhibit low DNA damage in response to
direct UVC treatment, we used host cell reactivation assays to
evaluate transcription coupled-nucleotide excision DNA repair
capacity in H9, BG01, BG01V, iPSC1, iPSC2, CRL-2097,
IMR90 and HF02 cells. Firefly luciferase plasmid (pM1-Luc)
was damaged with UVC radiation and levels of CPD damage
were determined by cleavage of supercoiled DNA with T4 UV
endonuclease (Figure 3A). An undamaged Renilla
luciferaseexpressing plasmid (pRL-CMV) was used as a control to normalize
for transfection efficiency. The damaged firefly luciferase plasmid
and undamaged control Renilla luciferase-expressing plasmid
were co-transfected into the above-mentioned cells. At 24 h
posttransfection, cells were harvested, lysates prepared, and firefly and
Renilla luciferase activities determined using the cell extracts. The
relative luciferase activities were compared to those obtained using
undamaged pRL-CMV. The ratio of firefly and Renilla luciferase
activities generated in cells co-transfected with the damaged
pM1Luc and control pRL-CMV was compared to the luciferase
activities generated in cells co-transfected with the undamaged
plasmids to reflect the ratio of repaired plasmid to intact plasmid
(Figure 3B), which is related to the cellular DNA repair capacity.
After transfection, the CRL-2097, IMR90 and HF02 fibroblast
cell lines had relative luciferase activities just under 80, 60, and
70%, respectively, similar to that of BG01V (70%), while H9 and
BG01 hESCs had relative luciferase activities between 80100%.
In contrast, iPSC1 and iPSC2 induced pluripotent cells exhibited
significantly different relative luciferase activities, ,25% and 80%,
respectively. Therefore, a generalization on the
UVC-transcription coupled nucleotide DNA repair capacity with respect to
pluripotency is not possible. These results indicate that recovery of
the firefly luciferase activity is dependent on the cell line, with
BG01 and BG01V recovery slower than that for H9. Surprisingly,
although both iPSC lines were derived from fibroblasts and with
the same reprogramming factors, their DNA repair capacities
were notably different.
DNA repair capacity in base excision repair is cell line
Similar to UVC, little damage was observed following H2O2
exposure of hESCs. Therefore, to monitor transcription
coupledbase excision repair, we used a host cell reactivation assay
analogous to that used for transcription coupled-nucleotide
excision repair, described above, but using methylene blue and
visible light to generate principally 8-oxoguanine in vitro . Total
8-oxoguanine in the pM1-Luc plasmid used for transfection was
estimated based on the DNA strand break frequencies induced
using Fpg (Figure 3C). Twenty-four hours after DNA damage
induction, H9, BG01V and iPSC2 exhibited superior base
excision repair, with over 50% of relative firefly luciferase activity
recovered, compared to CRL-2097, IMR90 and HF02 fibroblasts,
which recovered between 15 and 40% of relative firefly luciferase
activity (Figure 3D). Similar to transcription coupled-nucleotide
excision repair activity, the iPSC1 cell line displayed the lowest
repair efficiency. Surprisingly, the base excision repair capacity of
hESC line BG01 was more similar to that of IMR90 and iPSC1
than that of the H9 hESC line. Therefore, simple classification of
base excision repair solely on pluripotency is not possible.
Non-homologous end joining DSB DNA repair capacities
in pluripotent cells and fibroblasts are comparable
Non-homologous end joining is an error prone pathway for
repair of DSBs. Non-homologous end joining was monitored using
a transient transfection assay that did not require integration and
selection (Figure 4A) . In this analysis the GFP+YFP
quadrant indicates cells that have undergone repair and produce
not only the control GFP, but also the protein from the repaired
YFP coding sequence. As a control, the FACS analysis of the
uncleaved pCMS-end plasmid transfected into BG01 cells showed
a strong GFP+YFP quadrant (Figure 4B, top panel). After
cleavage with Apa I and Xho I, the reporter plasmid was
transfected into BG01 cells, which showed significant YFP+GFP
signal recovery after repair (Figure 4B, bottom panel). The DNA
repair capacities associated with non-homologous end joining for
the different cell lines showed that aside from iPSC1, the percent
of non-homologous end joining repair in the cell lines investigated
was less than 60% (Figure 4C). In contrast, iPSC1
nonhomologous end joining repair was nearly 90%, a significant
difference when compared to the other pluripotent and fibroblasts
cells evaluated. The greater non-homologous end joining DNA
repair capacity of iPSC1 also differed from the lower DNA repair
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 mg/0.24 mg in 16106 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 mean6standard 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
posttransfection and the firefly and Renilla luciferase activities determined. Values are mean6SD (n = 3). TC-NER, transcription-coupled nucleotide excision
repair; BER, base excision repair.
capacities observed for this cell line in the nucleotide and base
excision repair host cell reactivation assays. Those lower DNA
repair capacities suggest that iPSC1 would manifest greater
genomic instability than the other pluripotent cell lines analyzed.
Taken together, the non-homologous end joining DNA repair
capacities indicate that non-homologous end joining is similar
among hESC and fibroblast cell lines.
Single-strand annealing DSB repair DNA repair capacities
are lower in pluripotent cells than in fibroblasts
Single-strand annealing is a form of homologous recombination
that involves annealing of extensive regions of homology that flank
a DSB , which causes a deletion between the homologous
segments, and hence is inherently mutagenic . Using an assay
similar to that described for non-homologous end joining,
singlestrand annealing was measured using a transfection-based assay in
which the YFP coding sequence was restored by homologous
regions spanning ,300 bp on either side of the YFP-coding
sequence (Figure 5A). Results from this assay using BG01 hESCs
showed that transfection with uncleaved control plasmids
generated almost no cells that co-expressed YFP and the control
GFP proteins (Figure 5B, top panel). But a significant number of
BG01 cells transfected with cleaved pCMS-hom-stop expressed
YFP and GFP, representative of their single-strand annealing
DNA repair capacity (Figure 5B, bottom panel). Comparison of
the FACS analyses yielded a measurement of the single-strand
annealing repair percentage (Figure 5C). The single-strand
annealing DNA repair capacities of all pluripotent cells were
consistently lower than those of fibroblasts. That DNA repair
capacity was significantly lower (,2-fold) in BG01 and both iPSC
lines, suggests that single-strand annealing was not a preferred
repair pathway for pluripotent cells. Therefore, the lower
singlestrand annealing repair capacities observed for pluripotent cells
suggest that pluripotent cells develop fewer mutations due to that
pathway as compared to differentiated cells.
One iPSC line manifests microsatellite instability
Microsatellite instability (MSI) is often associated with defects in
mismatch repair or DNA polymerase errors, which have been
closely linked to genetic diseases that predispose individuals to
cancer. Generally, identification of MSI requires comparison to
reference cells that serve as an indicator of change from a starting
point. Therefore, we surveyed five autosomal markers of MSI in
eight cell lines, as four groups based on the relation among the cell
lines (pairs consisted of CRL-2097/iPSC1, IMR90/iPSC2, H9/
NSC9, and BG01/BG01V) (Figure 6). To evaluate differences or
defects in microsatellites, the selected primer sets (Table S1)
spanning regions near either mismatch repair genes (MSH2 [MutS
Homologue 2], MLH1 [MutL Homologue 1]) or tumor
suppressor genes (NF1 [neurofibromin 1], APC [adenomatous
polyposis coli]) were used [37,38]. The iPSC1 line, at passage 24,
exhibited two loci with MSI, APC and hMLH1 (marked by black
arrows). Additionally, BG01V, an aneuploid hESC line, had a shift
in a microsatellite for the APC gene as compared to BG01 early
passage cells. Overall, these MSI data show that even in iPSC1,
which is karyotypically and spectral karyotypically normal (Figure
S2), MSI is observed. Therefore, using these MSI loci is a
potentially valuable tool for evaluating pluripotent cell genomic
stability, as both lines that had MSI also had associated differences
in either chromosomal segregation (BG01V) or in DNA repair
UVC-induced apoptosis in pluripotent cells
During pilot experiments for UVC radiation exposure, we
noted that pluripotent cells (hESCs and iPSCs) were more sensitive
than fibroblasts, and that by 24 h post-treatment many pluripotent
cell colonies had disappeared from the culture, whereas the
fibroblasts underwent arrested replication . In contrast to
pluripotent cells, the arrest of fibroblasts was not accompanied by
changes in cell death or morphology. Interestingly, the apparent
pluripotent cell death following UVC exposure was characterized
by detachment of cells from Matrigel, suggesting that UVC
irradiation disrupted cell-cell or cell- extra cellular matrix
interactions and that UVC fluxes of 10 J/m2 were lethal for
pluripotent cells, whereas fibroblasts could recover following arrest
after the same dose of UVC . To further examine this
phenomenon, we observed 10 J/m2 UVC-treated and untreated
colonies over 18 h using time-lapse microscopy. H9 cells that were
not irradiated with UVC proliferated and expanded, whereas
UVC-irradiated colonies showed increasing numbers of detached,
non-viable cells starting 3.5 h post-treatment (Figure 7A). Time
lapse movies of this process revealed floating cells after UVC
treatment (H9 hESCs) that were not observed in the controls
(Movies S1 and S2).
To study this observation in more depth and determine cell
fate after release of the H9 cells (80% confluent) from the
colonies, cells were exposed to 10 J/m2 UVC radiation and
harvested at 3 and 22 h post-treatment. Having reasoned that cell
death was possibly associated with cell surface death receptors
, we used FACS analysis to examine detached and adherent
cells stained with Annexin V-FITC and propidium iodide (PI).
Annexin V-FITC detects cell surface phosphatidylserines and
indicates early apoptosis, whereas PI detects DNA (Figure 7B).
The untreated H9 cells had minimal dead cells and therefore
were omitted from the analysis (,2%). At 3 h post-treatment,
17% of non-adherent cells were still viable and had intact
membranes, whereas 29.2% cells were entering early apoptosis.
However, from 322 h post-treatment, the percentage of
Annexin V-FITC-stained H9 cells increased in both floating and
adherent cells, indicative of apoptosis. We also investigated the
effect of ROCK inhibitor (10 mM) on the apoptotic response.
ROCK inhibitor enhances the survival of hPSCs by improving
cell-cell and cell-extra cellular matrix interactions [49,50]. At
22 h post-treatment with the ROCK inhibitor, treated and
untreated H9 cells were compared, and the adherent cells did not
show significant differences in apoptosis between the two groups
(Figure 7B). Similarly, there was no difference in the number of
floating cells, regardless of whether cells were treated with the
To confirm that pluripotent cells were undergoing apoptosis,
H9 and iPSC2 cells were exposed to 10 J/m2 UVC and genomic
DNA was isolated separately from floating (F) and adherent (A)
cells at 6, 12 and 24 h post-treatment. Genomic DNA was
examined by agarose gel electrophoresis, and compared to an
untreated negative control and a staurosporine-treated (1 mM, 3 h)
positive control (Figures 7C and S8A). At all time points,
floating cells, and adherent cells to a lesser extent, exhibited DNA
ladders, indicative of apoptosis caused by endonuclease cleavage of
The activated caspase 3 form is created by cleavage of
procaspase 3 into 12 kDa and 17 kDa forms. The production of
the cleaved procaspase 3 forms has been associated with anoikis.
Anoikis is a form of apoptosis that anchorage dependent cells
undergo when those detach from the extracellular matrix  and
has been observed in hESCs . Therefore, to examine further
the apoptotic pathway involved in this process, we investigated
procaspase 3 cleavage using Western blot analysis of protein
extracts derived from either adherent or floating cells at 3, 5, and
24 post-UVC treatment (Figures 7D and S8B). At all three time
points, the procaspase 3 in floating cells was completely cleaved
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.
into the active 12 kDa and 17 kDa forms. For the adherent cells,
background cleavage of procaspase was noted at 3 and 5 h
postUVC treatment. However, in contrast to the floating cells, even at
24 h post-UVC treatment, procaspase 3 still formed a significant
percentage of the total procaspase+caspase 3 in the adherent cells
(Figure 7D), indicating that the remaining adherent cells were
We also examined procaspase 3 cleavage in iPSC2 cells. As
anticipated, the non-adherent cells showed a high percentage of
caspase 3 cleavage (Figure S8B). However, surprisingly, a high
percentage of iPSC2 cells manifested caspase 3 cleavage products
at all time points in adherent cells, suggesting that the apoptotic
response in iPSC2 cells was more sensitive to UVC-induced
apoptosis than that of hESCs.
In addition to evaluating apoptotic response after UVC
damage, we also noted that cells exposed to 50 and 100 mM
DMS also detached underwent cell death associated with
apoptosis (data not shown). Furthermore, such changes observed
in hESCs and iPSCs exposed to c-radiation as low as 1 Gy were
recently reported . In contrast to the apoptotic response
caused by UVC, DMS, and c-radiation, there was no indication of
apoptosis upon H2O2 exposure in either pluripotent cells or
fibroblasts. Thus, the low tolerance for DNA damage that triggers
apoptosis is not observed for all damaging agents.
We have shown that the DNA repair capacities of hESCs and
iPSCs are greater for nucleotide excision repair and base excision
repair, than are those of non-pluripotent cells. However, when
evaluating DSB repair, the DNA repair capacities of
nonhomologous end-joining in pluripotent cells were statistically
indistinguishable from those for non-pluripotent cells, except for
one iPSC line. In contrast, the DNA repair capacity for
singlestrand annealing, which is inherently mutagenic, was lower for all
pluripotent cell lines and highest in the fibroblast lines. Moreover,
induction of DNA-damage in pluripotent cells by UVC and H2O2
was lower than in fibroblasts. However, in pluripotent cells, despite
the reduced level of DNA damage and the rapid repair kinetics in
the global genome-nucleotide excision repair pathway, exposure to
UVC and DMS initiated apoptotic cell death, resulting in cell
detachment at doses that are non-lethal to fibroblasts. The
summarized data, comparing only data for each cell line for each
assay (Figure 8), demonstrate the complexity of studying DNA
repair in different pluripotent cell lines, and the need for
characterization of these lines prior to experimental use.
DNA damage induced by UVC and H2O2 is lower in
pluripotent cells than in differentiated cells
Using either adduct detection by lesion specific antibodies or the
comet assay, we showed that UVC and H2O2 induce less DNA
damage in hESC lines as compared to differentiated fibroblasts
(Figure 8). iPSC lines, however, only had less damage after UVC
irradiation, and otherwise exhibited similar levels of damage to
those of differentiated fibroblasts. Despite the differences in DNA
damage caused by UVC and H2O2, little difference was noted for
DMS treatment. The reasons for the reduced damage levels in
hESCs are unclear at this time. One possible explanation is the
colony structure of pluripotent cells may shield some cells from
exposure to DNA damaging agents. The consequences of
oxidative and methylating agent exposure in pluripotent cells also
require further investigation, because, in addition to possible
exposure differences, apoptosis and repair could also depend on
colony architecture. Because repair metabolism causes DNA
damage due to the development of reactive oxygen species and
methylation [52,53,54,55], future characterization of the factors
that reduce DNA damage in pluripotent cells is an area that merits
Global genome-nucleotide excision repair capacity, but
not transcription-coupled nucleotide excision repair, is
enhanced in pluripotent cells as compared to fibroblasts
A previous study evaluating strand breaks, as assessed by comet
assay, showed that repair was faster in pluripotent than in
nonpluripotent cells , but that study did not quantify adduct levels
or viability after mutagen treatment. In this report, we have
quantified UVC adduct formation and repair, and showed that the
difference in the global genome-nucleotide excision repair rate is
linked mainly to faster CPD adduct repair in pluripotent cells,
because 6,4 PP was rapidly repaired in both pluripotent cells and
the control lines. In pluripotent cells, global genome-nucleotide
excision CPD repair rates were 2- to 4-fold higher than those in
fibroblasts (Figure 8). In fibroblasts, transcription-coupled
nucleotide excision repair rates are faster than global
genomenucleotide excision repair rates [56,57,58]. However, the
transcription coupled-nucleotide excision DNA repair capacity in
pluripotent cells did not exceed a 2-fold difference. Consequently,
because the increased global genome-nucleotide excision DNA
repair capacity is greater in pluripotent cells, the mutation
frequency in pluripotent cells should be lower compared to that
observed in fibroblasts. Because global genome-nucleotide excision
repair rates are increased relative to transcription-coupled
nucleotide excision repair rates in pluripotent cells, we anticipate
that factors specific for global genome-nucleotide excision repair,
possibly XPC-HR23 recognition, are responsible for faster repair
Base excision repair is faster in pluripotent cells than in
The base excision repair rates of the pluripotent cell lines
investigated demonstrated greater heterogeneity than did their
transcription-coupled nucleotide excision repair rates (Figure 8
and Table S2). Specifically, the lowest DNA repair rate was
13fold less than the highest. Whereas H9, BG01V, and iPSC2 had
base excision DNA repair capacities that were greater than those
of the differentiated cells, BG01 and iPSC1 had lower DNA repair
capacities than the differentiated cells. Moreover, the aneuploid
line BG01V manifested a base excision repair DNA repair
capacity that was 3-fold higher than that of the parental BG01
line. For both transcription-coupled nucleotide excision repair and
base excision repair, the DNA repair capacity values were lowest
in the iPSC1 line derived from CRL-2097, which indicates this
line is more subject to genomic instability. The variability in the
base excision repair capacities of the different lines was not
separable into predictable categories of pluripotent and
differentiated cells. Thus, that underscores the necessity to evaluate DNA
repair capacity for each cell line prior to use in research or clinical
DSB repair is a source of differences between hESCs and
Some studies have used stably-transfected DSB reporter assays
in pluripotent cells to assess DSB repair [26,27]. This type of
system has the advantage of monitoring chromosomal events, but
requires the generation of stable transfectants that host the
reporter assay. At this time, introduction and selection of the
stably-transfected reporter assay systems is still non-trivial in
pluripotent cells and has been used for only the aneuploid BG01V
line . The advantage of the host cell reactivation systems is that
less time is required for the assay, permitting a rapid comparison of
a larger number of cell lines with respect to non-homologous
endjoining and single-strand annealing. Currently, it is unclear if the
source tissue of the fibroblasts (foreskin and lung) or the vectors
used in re-programming account for the substantially different
DNA repair capacities observed in the two iPSC lines. This
highlights the importance of standardizing reprogramming
protocols and minimizing additional variables that could
contribute to differences exhibited during characterization.
In contrast to the results for transcription-coupled nucleotide
excision repair and base excision repair, all cell lines examined,
except for iPSC1, showed similar DNA repair capacities for
nonhomologous end-joining. Interestingly, despite having the lowest
DNA repair capacity for transcription coupled-nucleotide and
base excision repair, iPSC1 displayed the highest non-homologous
end-joining DNA repair capacity. More surprisingly, although
both iPSC1 and iPSC2 were obtained from human fibroblasts
(foreskin and lung, respectively) using the same transcription
factors to induce pluripotency, the non-homologous end-joining
DNA repair capacity of iPSC2 is ,3-fold less than that of iPSC1.
Therefore, although the same transcription factors were used to
induce both iPSC lines, each cell line had significantly different
non-homologous end-joining repair characteristics. The lower
values for single-strand annealing in all pluripotent cells could be
associated with those cells having reduced the mutagenic
consequences from repair using that pathway.
Two pluripotent cell lines with microsatellite instability
also manifest DNA repair capacity differences from other
MSI is generally considered a marker for mismatch repair
defects and/or DNA synthesis, which can lead to DNA mutations
that are linked to human disease [59,60,61,62,63]. Since the
various pluripotent lines exhibited such drastic repair capacity
differences, MSI was evaluated as a candidate for these
inconsistencies. Among the hESC lines, BG01V manifested MSI
at a single locus. The major differences observed in BG01V are at
the chromosomal level , but we also identified one locus
adjacent to APC, a tumor suppressor gene, that differed between
BG01 and BG01V. However, screening for short tandem repeat
(STR) sequences did not reveal differences between these lines
, which suggests that the MSI screening loci used in this report
are more sensitive than STR analysis for genomic stability
assessment. Although spectral karyotyping showed that the iPSC
lines used in these experiments are normal, additional analysis of
induced pluripotent lines showed that the iPSC1 line manifested
greater MSI at two loci as compared to the parental line.
Therefore, the differences in iPSC1 DNA repair capacity,
compared to the other cell lines investigated, could be due in
part to factors linked to MSI, emphasizing the need to examine
pluripotent cells through other methods.
The therapeutic merits of hESCs and iPSCs are currently under
evaluation and some differences among hESCs and iPSCs have
been identified. Reports have indicated that iPSCs have less
efficient growth and differentiation capacities than hESCs ,
distinct methylation and de-methylation patterns in non-coding
RNAs , and variations in X-chromosome reactivation ,
suggesting that hESCs and iPSCs also have epigenetic differences.
Another study showed that hESCs and iPSCs are heterogeneous,
depending on their derivation source , and it is possible that
DNA repair pathway reprogramming is dependent on the cells of
origin. Our data indicate that hESCs and iPSCs have differences
in DNA repair that can be monitored by a series of assays that
encompass a range of DNA repair pathways. Based on our results,
further work that determines the genomic stability of iPSCs is
required that evaluates methods for induction of iPSCs as well as
the cells of origin. The decreased repair capacities observed in the
pathways studied, along with the increased MSI, suggests that
iPSC1 is more prone to errors from nucleotide and base excision
repair than are the other lines investigated, including iPSC2. The
comparisons of MSI in the karyotypically abnormal line (BG01V)
and the karyotypically normal iPSC1, indicate that screening for
MSIs could provide rapid evaluation of genomic stability. The
reduced DNA repair capacities manifested mainly by iPSC1, track
with its MSI, suggesting that determination of MSI at these loci
could also help to illuminate defects in other repair pathways.
Pluripotent cells are subject to increased apoptosis after
exposure to DNA damaging agents
Apoptosis occurs naturally in pluripotent cells grown in
mTeSR1, and is enhanced by depletion of basic fibroblast growth
factor . Our study indicates that pluripotent cells also undergo
apoptosis after low levels of exposure (e.g., 5 J/m2 UVC) or
concentrations (e.g., 50 mM DMS) of some DNA-damaging
agents. However, for H2O2, no apoptotic response was observed,
indicating that not all damaging agents elicit the same
programmed cell death. In addition, we showed that for up to
24 h after UVC radiation the majority of the adherent hESCs
have intact membranes that lack evidence of apoptosis, but that
iPSCs exhibit increased apoptotic sensitivity within 3 h
postirradiation, resulting in cells being released from colonies. To the
best of our knowledge, although apoptosis has been reported in
human pluripotent cells in response to c-radiation [22,23,70] no
previous data have addressed low-level UVC induced apoptosis in
human pluripotent cells. Due to the lower energy of UVC
radiation compared to c-radiation, the observation of apoptosis
was unexpected. Moreover, although ROCK inhibitor can
enhance pluripotent stem cell attachment , its failure to rescue
UVC-induced apoptosis in H9 cells suggests that the
hypersensitivity to UVC-induced cell death does not involve the
Rhomyosin-actin-caspase pathway, but some other apoptotic trigger,
possibly CHK1 and/or CHK2 . Further research is warranted
to identify the mechanism in the pathways involved.
The apoptotic response of pluripotent cells to low level UVC
may protect the genomic stability of the entire population by
sacrificing damaged cells in a timely fashion and raises the
question of evolutionary preservation in this instance, because
damage tolerance in hESCs could lead to mutations if proliferation
continues. Therefore, there is a paradox involved in genomic
stability of pluripotent cells: DNA repair is often rapid in
pluripotent cells as compared to differentiated cells, but exposure
to relatively low levels of DNA-damaging agents results in cell
death. In fact, the low level of UVC-induced damage tolerated by
pluripotent cells shows that these cells are almost as sensitive to
UVC as some human fibroblasts from individuals with defective
nucleotide excision repair genes . Thus, our data suggest that
pluripotent cells efficiently repair damage, but will undergo cell
death in the short damage response interval identified in this study
rather than risk the possibility of transmitting a mutation or
genetic rearrangement if the damage is not repaired. That the
higher energy c-radiation also results in apoptosis at low radiation
fluxes (less than 2 Gy [22,23], and our unpublished results]) argue
against peripheral damage of colonies as a provocation of
apoptosis. In the future, identification of the agents that induce
apoptosis and apoptotic signaling will yield important insight
concerning mechanisms of cell death in pluripotent cells.
We have demonstrated that, in general, hESCs excel in global
genome-nucleotide excision repair as compared to non-pluripotent
cells. Using the assays described, we have shown that pluripotent
cells and differentiated cells have similar repair capacities in
nonhomologous end joining, whereas pluripotent cells have attenuated
DNA repair capacities in the single-strand annealing as compared
to differentiated cells. Despite these generalities, DNA repair
capacities for individual pluripotent cell lines show complexity that
requires inspection of these and any other lines considered for
clinical use. Furthermore, our investigation has shown that
pluripotent cells are more prone to apoptosis than their
differentiated progenitors, despite enhanced repair rates when
exposed to UVC. This is consistent with pluripotent cells limiting
mutations in their progeny. In the future, the identification of
factors that enhance pluripotent cell genomic stability while
limiting apoptosis will enable wider use of these cells. Most
importantly, our work highlights that even though iPSCs may
display a normal karyotype, microsatellite instability, which
indicates general genomic instability, may predict alterations in
DNA repair responses of pluripotent cells to various DNA
damaging agents as compared to karyotypically normal
counterparts that lack MSI. Taken together, these results identify a critical
area that must be studied before the use of induced pluripotent
cells can be explored further for regenerative medicine. Based on
our results, requirements for pre-clinical screening for genomic
instability in hESCs, and especially iPSCs, would benefit from the
inclusion of assays to monitor transcription-coupled nucleotide
excision repair, non-homologous end-joining, and single-strand
annealing, as well as microsatellite instability.
Figure S1 Characterization of hESCs and iPSCs. (A)
Immunohistochemical staining of pluripotent cell markers in
hESCs and iPSCs. The indicated cell colonies were
immunostained for SSEA4 (green), NANOG (red), SOX2 (purple), and
DAPI (blue). Bars are 50 mm. (B) Immunohistochemical staining
of bona fide pluripotent cell markers in iPSCs. iPSC colonies were
immunostained for DNMT3B (green), SOX2 (red), and DAPI
(blue). Bars are 30 mm. H9, iPSC1 (shown in 1A), as well as BG01
and iPSC2 (not shown) were all positively stained for the
pluripotency markers (ES cell-specific transcription factors) Nanog,
SOX2, and SSEA4. Both iPSC1 and iPSC2 also stained positive
for DNMT3B (1B), confirming that they are bona fide iPSCs .
Figure S2 Karyotypes of investigated pluripotent cell
lines. (A) H9 passage p 110, (B) BG01 p 54. BG01V (not shown)
is a karyotypically abnormal (49, +12, +17 and XXY) long term
cell culture variant originally isolated and characterized from
BG01 cultures , and (C) iPSC2 p 11, as assessed by G-banding
and (D) iPSC1 p 24, as assessed by spectral karyotyping (SKY)
analysis. Both iPSC1 and iPSC2 were derived from human skin
fibroblasts (CRL-2097)  or human lung fibroblasts (IMR90),
respectively. The karyotypes examined for all these cells
manifested 46 chromosomes in greater than 90% of the metaphase
cells analyzed until at least p 110 for H9, p 54 for BG01, p 24 for
iPSC1 and passage 11 for iPSC2.
Figure S3 Analysis of CPD incidence in UVC-irradiated
l DNA. UVC-irradiated Bacteriophage l DNA was subjected to
alkaline gel analysis (A) and quantification (B) of UVC-induced
enzyme sensitive sites per mega base (ESS/Mb) was conducted.
Hind III-digested lamda DNA are used as DNA markers.
Figure S4 UVC, H2O2 or DMS-induced damage in
hESC, iPSC and fibroblast cells. (A) Dot blot of
UVCinduced (10 or 20 J/m2) CPD adducts in pluripotent cells and
fibroblasts, quantified in TotalLab. (B) Comet assays of hESCs
(H9), iPSCs (iPSC1), or human skin fibroblasts (CRL-2097) treated
with H2O2 (100 mM). Untreated cells were used as controls. (C)
Comet assays of hESCs (H9), iPSCs (iPSC2), or human skin
fibroblasts (IMR90) treated with DMS (50 mM).
Figure S5 Evaluation of cH2AX foci formation in
response to treatment with H2O2. (A) Fluorescence images
of hESCs (H9), iPSCs (iPSC1) and fibroblasts (CRL-2097) stained
for cH2AX foci after treatment with 100 mM H2O2 (4uC for
30 min). The expanded cell shows the foci as examined in the
individual cells. Controls are untreated samples. Bars are 20 mm.
(B) Quantification of percent of cells with greater than 4 cH2AX
Figure S6 Dot blot assay data for global
genomenucleotide excision repair of UVC-induced cyclobutane
pyrimidine dimers. Dot blot images of CPD repair time course
in (A) hESC (H9 and BG01), (B) iPSC (iPSC1 and iPSC2) and (C)
fibroblast (CRL-2097 and IMR90) cells following 10 J/m2 UVC
treatment. Only adherent cells were used in the assay.
Quantification of enzyme sensitive sites per mega base was determined
using standards loaded on each individual blot.
Figure S7 FACS analysis of the H9 cell states post UVC
irradiation (10 J/m2). At the time points indicated, floating (F)
and adherent (A) H9 cells were collected by centrifugation or
accutase treatment followed by centrifugation and incubated with
Annexin V-FITC and/or PI. Cells are divided by quadrants into
live (FITC2, PI2), early apoptotic (FITC+, PI2), late apoptotic
(FITC+, PI+) or necrotic (FITC2, PI+) sections. The
quantification is shown in Figure 7B.
UVC-induced apoptosis in induced
pluripocells. (A) DNA fragmentation analysis of
UVCirradiated iPSC2 cells. STS, staurosporine; S, supernatant; F,
floating cells; A, adherent cells (B) Caspase 3 cleavage in adherent
and floating cells. Upper panel: Western blot of caspase 3 cleavage
in iPSC2 cells, treated with 10 J/m2 UVC (6, 12 and 24 h) or
staurasporine (3 h), using near-infrared detection. Uncleaved
(Uncl.); Cleaved (Cl.); Floating cells (F); Adherent cells (A). Note
that there are no floating cells prior to treatment. Lower panel:
analysis of Western blots comparing uncleaved (Uncl.) and cleaved
(Cl.) bands for caspase 3.
Microsatellite markers for MSI analysis.
Table S2 Summary of DNA repair rates/capacities of hPSCs
and HFs in multiple DNA repair pathways investigated. The
rates/capacities for all the lines are relative to the rates/capacities
in IMR-90 fibroblasts (1.0). Values are mean 6 Standard
Deviation. Note that the repair rates are directly comparable
down a column and not across rows.
The authors thank Dr. J. Thomsen (University of Wisconsin-Madison) for
the iPSC1 line and Dr. J.-K. Yee (Beckman Research Institute) for the
IMR90 derived iPSC2 line; Drs. R. Schiestl and Z. Scuric for supplying the
non-homologous end joining and single-strand annealing assay plasmids
and helping to establish the assays; Dr. J. Stark for reading the manuscript;
Dr. K. Walker for comments on the manuscript; and Dr. R. Jove (Director,
Beckman Research Institute) for assistance in organization of the project.
Conceived and designed the experiments: LZL SP XZ TRO. Performed
the experiments: LZL SLN SGP SEB. Analyzed the data: LZL SLN SP
TRO. Contributed reagents/materials/analysis tools: SGP SP SEB XZ
LEI TRO. Wrote the paper: LZL SLN SGP SEB XZ LEI TRO.
Intellectual Input: SGP.
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