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STEM CELL EPIGENETICS, GENOMICS, AND PROTEOMICS

Human Embryonic Stem Cells Have Enhanced Repair of Multiple Forms of DNA Damage

^aLaboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, Maryland, USA;
^bBuck Institute for Age Research, Novato, California, USA;
^cCrystallography and Recombinant Enzyme Art Unit, United States Patent and Trademark Office, Alexandria, Virginia, USA;
^dInvitrogen Corporation, Stem Cell and Regenerative Medicine, Carlsbad, California, USA

Key Words. Human embryonic stem cells • DNA repair • Genomic maintenance • Comet assay • Microarray

Correspondence: Correspondence: Vilhelm A. Bohr, M.D., Ph.D., Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Box 1, 5600 Nathan Shock Drive, Baltimore, Maryland 21224-6825, USA. Telephone: 410-558-8162; Fax: 410-558-8157; e-mail: bohrv{at}grc.nia.nih.gov

Received on December 8, 2007; accepted for publication on June 8, 2008.

First published online in STEM CELLS EXPRESS  June 19, 2008.

	ABSTRACT

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Embryonic stem cells need to maintain genomic integrity so that they can retain the ability to differentiate into multiple cell types without propagating DNA errors. Previous studies have suggested that mechanisms of genome surveillance, including DNA repair, are superior in mouse embryonic stem cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H₂O₂, UV-C, ionizing radiation, or psoralen) than human primary fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic maintenance pathways are enhanced in human embryonic stem cells, relative to differentiated human cells.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
DNA is subject to damage from endogenous and exogenous sources on a continuous basis. Cells have evolved multiple mechanisms, including several DNA repair pathways, to remove DNA mismatches and lesions and prevent the deleterious consequences of DNA damage [1–4]. In the case of persistent unrepaired DNA damage, cells rely on complex signaling pathways to induce senescence or apoptosis; these pathways are induced when cell death is a more suitable outcome than proliferation of damaged cells. Defects in DNA repair, cellular senescence, and/or apoptosis have been implicated in cancer and aging [5–7].

There are several DNA repair pathways to correct DNA damage. Three of these DNA repair pathways, nucleotide excision repair (NER), base excision repair (BER), and mismatch repair (MMR), use DNA excision mechanisms to maintain genomic stability. NER is specific for bulky, helix-distorting DNA lesions [5, 8]; BER corrects small base modifications, such as oxidative and alkylation damage, and DNA single-strand breaks [7]; and MMR corrects base-base mismatches and insertion/deletion loops formed by misincorporation or strand slippage during DNA replication [9, 10]. In addition, double-strand break repair (DSBR) uses nonhomologous end joining or homologous recombination repair pathways to repair double-strand DNA breaks (DSBs) [11, 12]. Repair of interstrand cross-links (ICLs) is poorly understood in mammalian cells but is thought to involve a combination of NER, translesion DNA synthesis, and/or recombination [13–16]. ICLs are extremely toxic lesions that block DNA replication and transcription [15, 17] and are generated by a number of antitumor agents [18, 19].

Embryonic stem cells (ESCs) have the capacity to differentiate to all cell types in the mammalian embryo, including germ line cells. Therefore, the maintenance of genomic stability, by stress defense and DNA repair, in human embryonic stem cells (hESCs) should be particularly stringent, because any genetic alterations in ESCs can compromise the genomic stability and functionality of entire cell lineages. Recent studies show that mouse embryonic stem cells (mESCs) are more resistant to oxidative stress and ionizing radiation (IR)-induced DNA damage than differentiated mouse fibroblasts [20]. However, mESCs are hypersensitive to several DNA-damaging agents and readily undergo apoptosis [21–23]. Furthermore, the efficiency of repair of UV-induced DNA damage is decreased when mouse embryocarcinoma stem cells are induced to differentiate [24]. Lastly, the mutation rate and the frequency of mitotic recombination are also approximately 100-fold lower in mESCs than in adult somatic cells or isogenic mouse embryonic fibroblasts (MEFs) [25, 26]. These data are consistent with the hypothesis that mESCs have superior DNA maintenance response (DNA repair, apoptosis). Recent work [27] has now given evidence that differentiation of hESCs leads to downregulation of multiple stress defense mechanisms, such as telomerase activity and the expression of major antioxidant genes and some genes involved in DNA repair. However, no work has assessed the rate or efficiency of DNA repair in hESCs.

In the present study, we compared DNA repair activity in hESCs (BG01, I6) with that of the differentiated cell lines WI-38, hs27, and HeLa. WI-38 cells are primary human fetal lung fibroblasts, hs27 cells are human newborn foreskin fibroblasts, and HeLa cells are highly proliferative transformed human cervical carcinoma cells. The average doubling time of the hESCs used in this study (30–40 hours) is slightly longer than for WI-38, hs27 (both 24–30 hours) and HeLa (22–24 hours) cells. Thus, any enhanced repair seen in hESCs would not be a reflection of faster proliferation.

Single-cell gel electrophoresis (comet assay) is a rapid and very sensitive fluorescent microscopy method to examine DNA damage and repair at the individual cell level. In this study, DNA repair was measured using the comet assay on cells treated with the DNA-damaging agents H₂O₂, UV-C, IR, and psoralen. The expression profile of DNA repair genes was also assessed, using cDNA-based microarray. Preliminary studies on relative stress response in terms of DNA repair protein expression was also done, by Western blot analysis of cell extracts. The results show that hESCs repair several types of DNA damage more efficiently than WI-38, hs27, and HeLa cells and have higher expression of several DNA repair genes. These observations are consistent with the hypothesis that hESCs have enhanced surveillance pathways to maintain genomic stability for proper development of adult tissues and organs.

	MATERIALS AND METHODS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Cell Lines and Cell Culture
hESCs were cultured as previously described [28, 29]. Briefly, BG01 and I6 hESC lines were maintained on Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com)-coated dishes in medium conditioned with MEFs for 24 hours. Cells were passaged every 4–6 days with collagenase IV (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Under such culture conditions, no karyotypic abnormality was observed. The hESC lines used in this study were in passages 18–70 (BG01) and 40–80 (I6).

HeLa and hs27 (passage 18) cells were grown at 37°C and 5% CO₂ in 60-mm culture dishes in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) and 50 µg/ml penicillin and streptomycin and passaged every 4–6 days. The same conditions were used for WI-38 cells (passage 20), except that the medium was supplemented with 0.1 mM nonessential amino acids and 1% minimal essential medium vitamin solution (Gibco-BRL).

Immunofluorescence
hESCs were fixed with 4% paraformaldehyde for 10 minutes. Fixed cells were permeabilized with 0.05% saponin in phosphate-buffered saline (PBS) for 5 minutes and fixed in 100% acetone for 15 minutes. Primary antibody was applied for 1.5 hours, and appropriately coupled secondary antibodies (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) were used for single and double labeling for 1.5 hours. All secondary antibodies were tested for cross-reactivity and nonspecific immunoreactivity. The following antibodies were used: Oct4, Nanog, and Sox2 (Research Diagnostics, Concord, MA, http://www.researchd.com). Hoechst 33342 stain was used to identify the nuclei. Images were captured on an Olympus fluorescence microscope (Olympus, Tokyo, http://www.olympus-global.com).

Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA was extracted from undifferentiated hESCs using RNA STAT-60 (Tel-Test, Friendswood, TX, http://www.isotexdiagnostics.com). cDNA was synthesized by using a reverse transcription kit (RETROscript; Ambion, Austin, TX, http://www.ambion.com) according to the manufacturer's recommendations. Primers used were as follows: OCT3/OCT4 (171 base pairs [bp]): forward (Fwd), 5' ctt gct gca gaa gtg ggt gga gga a; reverse (Rev), 5' ctg cag tgt ggg ttt cgg gca; SOX2 (437 bp): Fwd, 5' atg cac cgc tac gac gtg a; Rev, 5' ctt ttg cac ccc tcc cat tt; and NANOG (158 bp): Fwd, 5' caa agg caa aca acc cac tt; Rev, 5' tct gct gga ggc tga ggt at.

Single-Cell Gel Electrophoresis (Comet) Assays
Comet assays [30, 31] were performed on hESCs (BG01, I6) and WI-38, hs27, and HeLa cells. Cells were grown in 60-mm dishes and treated as follows: either 100 µM H₂O₂ for 30 minutes at 37°C; 20 J/m² UV-C; 5 Gy IR (Gammacell 40, ¹³⁷Cs source; MDS Nordion, Ottawa, http://www.mds.nordion.com) or 0.2 µg/ml psoralen (30 minutes) followed by UV-A for 5 minutes (to activate the psoralen). After treatment, cells were either immediately harvested (0-hour time point) or incubated in fresh medium before being harvested at the following time points, in preparation for comet assays: H₂O₂, 6 and 24 hours; UV-C, 6 hours; IR, 1 and 3 hours; and psoralen, 4 hours. Control assays were performed using untreated cells.

Harvested cells were collected and suspended in 250 µl of PBS. Approximately 5 x 10³ cells in 6–10 µl were mixed with 80 µl of 0.5% low-melting-point agarose in PBS, spread on a microscope slide precoated with 1% agarose in PBS, and allowed to cool for 5 minutes. Finally, 100 µl of low-melting-point agarose was applied on top of the sample layer. Slides prepared from cells treated with H₂O₂, UV, or IR were placed in cold lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100), and slides for measuring ICL repair were placed in cold lysis buffer plus 10% dimethyl sulfoxide. Slides were incubated overnight at 4°C. Slides for measuring ICL were then incubated in lysis solution plus 1 mg/ml proteinase K for 2 hours at 37°C.

Cells were rinsed three times, 5 minutes each, in neutralization buffer (0.4 M Tris-HCl, pH 7.4). The H₂O₂-comet slides were then treated with 100 U of formamidopyrimine DNA glycosylase (Fpg) (100 U/µl; Trevigen, Gaithersburg, MD, http://www.trevigen.com) per slide (1 µl of Fpg in 99 µl of Fragment Length Analysis using Repair Enzyme (FLARE) buffer I [Trevigen] containing 0.1 mg/ml bovine serum albumin [BSA]) and incubated for 1 hour at 37°C. Control slides were treated with FLARE buffer plus BSA. The UV-C-comet slides were each treated with 20 U of T4 endonuclease V (1,000 U/µl; Trevigen) in T4 endonuclease dilution buffer (25 mM NaPO₄, 1 mM EDTA, 100 mM NaCl, 1 mM dithiothreitol [DTT], 0.1 mg/ml BSA) for 1 hour at 37°C. Control slides were treated with T4 endonuclease dilution buffer only. Slides were washed in PBS three times before incubation in cold unwinding solution (300 mM NaOH, 1 mM EDTA, pH 12.1, or pH 13 in the case of ICL comet assay) in the dark at 4°C for 45 minutes.

Electrophoresis was carried out in tris-borate EDTA buffer at 25 V for 30 minutes (or in unwinding solution, pH 13, for 45 minutes for ICL comet assay). Slides were fixed in 100% ethanol and stained with ethidium bromide. Assay results (50–100 cells per sample) were visualized on a Zeiss Axiovert 200 M fluorescenct microscope (Carl Zeiss, Thornwood, NY, http://www.zeiss.com). The analysis of the comet tail length was performed using Komet 5.5 software (Andor Technology, South Windsor, CT, http://www.andor.com). The mean of >50 olive tail moments (OTM) was calculated. For Fpg or T4 endonuclease V-treated slides, assays were corrected for background signal by subtracting OTM of buffer-treated cells from that of enzyme-treated cells.

Bead Array Gene Expression Analysis
Our repair gene expression data were derived from the microarray analysis reported previously [32]. This gene expression analysis was carried out on three hESC lines (BG01, BG02, BG03) plus a pooled sample of H1, H7, and H9 hESC lines; three human embryoid body (EB) samples (EBs differentiated from BG01, BG02, and BG03) plus a pooled EB sample derived and differentiated from H1, H7, and H9 hESC lines; one human fibroblast line (HS27-hfibroblasts); one human embryonic carcinoma (hEC) line (NTera2); and one human neural stem cell (NSC) line (NSC-fetal-1, derived from fetal tissues; as described by Shin et al. [33]).

Western Analysis
hESCs (BG01) and WI-38 cells were treated with 100 µM H₂O₂ for 30 minutes and allowed to repair for 1 and 3 hours. Untreated and treated cells were collected and lysed in buffer containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% Triton X-100. Proteins were separated by 12% SDS-polyacrylamide gel electrophoresis in Tris-glycine (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). Approximately 20 µg of whole cell extracts was loaded per lane. Transfer to polyvinylidene difluoride membranes (Invitrogen) was carried out by electroblotting in transfer buffer (12 mM Tris, pH 8.3, 96 mM glycine, 20% methanol) for 1 hour at 100 V. Membranes were preincubated for 1 hour at room temperature in 5% nonfat dry milk (Bio-Rad, Hercules, CA, http://www.bio-rad.com) in 20 mM Tris-HCl, pH 7.2, 137 mM NaCl, 0.1% Tween-20 (TBST). All antibodies were diluted in 5% fresh milk in TBST and incubated with the membrane. Antibodies used were as follows: anti-actin (Abcam, Cambridge, MA, http://www.abcam.com), anti--oxoguanine DNA glycosylase (anti-OGG1) (Assay Designs, Ann Arbor, MI, http://www.assaydesigns.com), and anti-apurinic/apyrimidinic endonuclease-1 (APE-1) (Trevigen). Immunoblot signals were visualized with horseradish peroxidase-coupled secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) and ECL Plus (GE Healthcare, Little Chalfont, U.K., http://www.gehealthcare.com).

Quantification of 8-Hydroxydeoxyguanosine
8-Hydroxydeoxyguanosine (8-oxoG) was quantified as previously described [34]: DNA was extracted using a salting out procedure [35, 36]. Specifically, cell pellets were resuspended in lysis buffer (0.5 M Tris-HCl, pH 8, 20 mM EDTA, 10 mM NaCl, 1% SDS, and 0.5 mg/ml proteinase K) and incubated overnight at 37°C. One-fourth volume of saturated NaCl was then added, and the samples were centrifuged at 500g for 45 minutes. The supernatant was transferred to a new tube, and the DNA was precipitated with ethanol. The DNA was resuspended in lysis buffer and incubated for 3 hours at 37°C with 100 µg/ml RNase. After reprecipitation with ethanol, the DNA was resuspended in water, and the concentration was determined. DNA was treated with nuclease P1 and calf alkaline phosphatase. The digested material was then filtered and analyzed by high-performance liquid chromatography (HPLC) with electrochemical detection using the ESA Biosciences Coularray system (ESA Biosciences Inc., Chelmsford, MA, http://www.esainc.com). The 8-oxo-G concentration was estimated as described previously [37], except desferoxamine was omitted from all procedures.

OGG1 Activity Assay
OGG1 activity was measured using a double stranded 30mer oligonucleotide with a single 8-oxodG (5'-ATATACCGCGOGCCGGCCGATCAAGCTTATT-3'), as described by Souza-Pinto et al. [38]. The lesion-carrying DNA strand was 5'-end-labeled using T4 polynucleotide kinase (Promega, Madison, WI, http://www.promega.com) and [-³²P]ATP (PerkinElmer New England Nuclear, Waltham, MA, http://www.las.perkinelmer.com) before hybridization to a complementary oligonucleotide. OGG1 activity was assayed in a reaction containing 20 mM Hepes-KOH, pH 7.6, 1 mM EDTA, 1 mM DTT, 50 mM KCl, 10% glycerol, 100 fmol of oligonucleotide, and 30 µg of protein extract. Reactions were incubated for 2 hours at 37°C, terminated with 1 µl of 5 mg/ml proteinase K and 1 µl of 10% SDS, incubated at 55°C for 30 minutes, and mixed with an equal volume of formamide loading dye (90% formamide, 1 mM EDTA, 0.1% bromphenol blue). Samples were analyzed by 20% polyacrylamide/7M urea gel electrophoresis. Reaction products were visualized using the Molecular Dynamics Storm imaging system (GE Healthcare) and quantified using ImageQuant 5.2 software (GE Healthcare). Percentage of incision was calculated by normalizing the amount of radioactivity in the reaction product to the total radioactivity per assay.

	RESULTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
Expression of Pluripotency Markers in Undifferentiated hESCs
Proper hESC culture for this study required that the hESCs were maintained in an undifferentiated state. Therefore we tested the BG01 hESCs line for pluripotency markers by immunofluorescence (Fig. 1). Oct3/4 and Sox2 were highly abundant, even on the colony periphery (Fig. 1A, 1B). Reverse transcription-polymerase chain reaction supported these observations, showing that Oct3/4 and Sox2, as well as another pluripotency marker, Nanog, were abundantly expressed in the BG01 cells (Fig. 1F). Moreover, from visual observation (Fig. 1E), it is apparent that no significant differentiation had occurred, even on the colony periphery.

View larger version (74K): [in this window] [in a new window]   Figure 1. Expression of pluripotency markers in undifferentiated human embryonic stem cells (hESCs). (A–D): Immunocytochemical analysis of expression of pluripotent markers in hESC line BG01, passage 38. Shown are Oct3/4 (A), Sox2 (B), nuclear Hoechst staining (C), and the merged picture (D). (E): Morphology of a typical undifferentiated BG01 hESC colony grown in mouse embryonic fibroblast-conditioned medium. (F): Reverse transcription-polymerase chain reaction on BG01 hESCs with pluripotency markers Oct3/4, Sox2, and Nanog. Abbreviation: bp, base pairs.

View larger version (29K): [in this window] [in a new window]   Figure 2. Illustration of comet assay design: repair of hydrogen peroxide-induced Fpg-sensitive sites. (A): Olive tail moment (OTM) value is the product of the amount of DNA in the tail and the mean distance of migration in the tail. The values for OTM also incorporate subtraction of local background. OTM values were measured as described in Materials and Methods. (B): Representative assays showing hESC (BG01) and WI-38 cells treated with 100 µM H2O2 and with or without 100 U of Fpg, as described in Materials and Methods. Abbreviations: Fpg, formamidopyrimine DNA glycosylase; hESC, human embryonic stem cell; hr, hours; UT, untreated.

View larger version (32K): [in this window] [in a new window]   Figure 3. Comet assays for repair of DNA damage. The indicated cell types were treated with 100 µM H2O2 (A), 20 J/m2 UV-C (B), 5 Gy of ionizing radiation (C), or 0.2 µg/ml psoralen (30 minutes)/UV-CA (5 minutes) (D). Comet assays were then performed as described in Materials and Methods. All 0-hour time points were normalized to OTM values of 100%. After the indicated amounts of time, H2O2- and UV-C-treated cells were treated with 100 U of formamidopyrimine DNA glycosylase (Fpg) and 20 U of T4 endonuclease V, respectively. Each data point is an average of 50–100 cells calculated as follows, where t represents a given time point after treatment, endo refers to T4 endonuclease, and UT refers to cells not treated with the stress agent indicated: H2O2, (OTM Fpgt – OTM no Fpgt) – (OTM FpgUT – OTM no FpgUT); UV-C, (OTM endot – OTM no endot) – (OTM endoUT – OTM no endoUT); IR and psoralen-UV-CA, OTMt – OTMUT. Data are represented as mean ± 2x SEM of 50–100 cells. Triple asterisks (***) indicate p < .01 in human embryonic stem cells (hESCs) relative to all three other cell lines. Double asterisks (**) indicate p < .01 in hESCs relative to only the two fibroblasts cell lines (WI-38 and hs27). Abbreviation: OTM, olive tail moments.

In the case of IR treatment, cells were exposed to 5 Gy of IR and allowed to recover for 1 or 3 hours, and IR-induced DNA strand breaks were measured directly by comet assay (Fig. 3C). Under the comet assay conditions used (alkali unwinding buffer, pH 12.1), both single- and double-strand breaks were detected. When cells were allowed 1 hour to recover from IR treatment, the two hESC lines had significantly (p < .01) lower OTM values than the other three cell lines. At the 3-hour time point, all cell lines except WI-38 had a reduction in OTM value of more than 75% (Fig. 3C). These data suggest that hESCs have faster repair of IR-induced DNA strand breaks.

Repair of psoralen-induced ICLs is initiated by the enzymatic unhooking of the DNA ICLs [14, 41]. Therefore, in this comet assay, higher ICL unhooking (first step in repair of ICLs) correlates with a longer comet tail. When cells were allowed 4 hours to recover after psoralen treatment, ICL unhooking was significantly (p < .01) more progressed in hESCs than in the other three cell lines (Fig. 3D). These data suggest that hESCs have faster DNA ICL unhooking, suggesting more efficient repair of ICLs. Together, these data suggest that hESCs have a higher capacity for repair of all four types of DNA lesions compared with WI-38 and hs27 cells and a slightly higher capacity for repair of three of the lesion types (the exception being UV-C-induced lesions) compared with HeLa cells.

8-OxoG and OGG1 Activity in hESCs
8-OxoG is a common "highly mutagenic" oxidative lesion in DNA and is repaired by BER [42, 43]. 8-OxoG accumulates in DNA with aging, and it can mispair with adenine, leading to G:C to T:A transversion mutations [44]. The level of 8-oxoG in untreated cells, as measured on isolated chromosomal DNA by HPLC-electrochemical detection, was significantly lower in hESCs than in WI-38 cells (p = .02; Fig. 4A), suggesting either faster removal or slower accumulation of 8-oxoG in hESCs. Because OGG1 is the primary enzyme responsible for repair of 8-oxoG in mammalian cells, we hypothesized that the lower levels of 8-oxoG might be due to higher OGG1 activity in hESCs. However, a direct assay for OGG1 activity, on an 8-oxoG-containing DNA substrate, showed a similar level of activity in extracts from hESCs, WI-38 cells, and HeLa cells (Fig. 4B). We had previously compared the OGG1 activity in hESCs with that in the human embryonic kidney cell line HEK293 and found that although there was a trend for higher OGG1 activity in hESCs, the difference was not quite significant (p = .13; Fig. 4C). These results suggest either that another component or modifier of BER capacity may be altered in hESC (resulting in faster repair) or that the hESCs have higher antioxidant activity (resulting in slower accumulation of 8-oxoG).

View larger version (29K): [in this window] [in a new window]   Figure 4. Quantification of 8-oxoG and oxoguanine DNA glycosylase (OGG1) incision activity in hESCs. (A): DNA was isolated from hESCs and WI-38, digested with nuclease P1 and alkaline phosphatase, and analyzed by high-performance liquid chromatography as described in Materials and Methods. The value for 8-oxoG per E6 bases is shown. The asterisk (*) indicates significant difference (p = .02) from the hESC level. (B): OGG1 incision assay was performed in duplicate using the indicated cells. Fpg was the positive control. Percentage incision was calculated as the amount of radioactivity in the product relative to total radioactivity per assay. Background correction was performed using a no-enzyme control. (C): Incision assay was performed in triplicate using extracts from hESCs and HEK293. The average incision value ± SEM was calculated. Abbreviations: C, negative control (no enzyme); E6, 106; Fpg, formamidopyrimine DNA glycosylase; h, hours; hESC, human embryonic stem cell; 8-oxoG, 8-hydroxydeoxyguanosine.

View larger version (31K): [in this window] [in a new window]   Figure 5. Graph of BER gene expression in hESCs relative to embryoid bodies and fibroblasts. Expression levels of repair genes important in BER (A), NER (B), double-strand break repair (C) and ICL repair (D) are shown. hESC = average expression level of all hESC lines: three hESC lines (BG01, BG02, BG03) and a pool of three other hESC lines (H1, H7, and H9). hEB indicates the average expression level of all hEB cell lines: three hEB lines (differentiated from BG01, BG02, and BG03) and a pool of hEB lines differentiated from hESCs H1, H7, and H9. Data are represented as mean ± SEM. Abbreviations: BER, base excision repair; DSBR, double-strand break repair; hEB, human embryoid body; hESC, human embryonic stem cell; ICL, interstrand cross-link; NER, nucleotide excision repair.

To gain some insight into the effects of stress on expression of DNA repair proteins in hESCs relative to differentiated cells, we treated hESCs (BG01) and WI-38 fibroblasts with H₂O₂ and examined protein levels of two important BER proteins, OGG1 and APE-1 (Fig. 6). We did not use this blot to compare protein levels between BG01 and WI-38 since the loading controls were either higher (actin, lamin B) or lower (tubulin) in the BG01 cells even though Bradford assay and Ponceau S staining (data not shown, except for actin in Fig. 6) of the same Western blot indicated even loading (Fig. 6 actin bands; Discussion). Therefore, we compared only the change in band intensities after oxidative stress (Fig. 6, bar graph [adjusted for actin loading]). Quantitation of the band intensities, relative to actin loading control, showed that the OGG1 and APE-1 protein levels increased 1.5- and 1.7-fold, respectively, in hESCs relative to WI-38 cells, 3 hours after treatment with H₂O₂. This indicates that hESCs may upregulate DNA repair pathways more strongly than differentiated cells after stress

View larger version (46K): [in this window] [in a new window]   Figure 6. Western blot analysis of extracts from hESC, WI-38, and HeLa cells. Shown is a Western blot of hESCs and WI-38 cells treated with 100 µM H2O2 for 30 minutes. Cells were allowed to recover for the indicated amount of time before preparation of cell extract. The blots were probed with antibodies to -OGG1, APE-1, and actin (loading control) (A), and band intensities were quantitated relative to actin (B). Since actin intensity was higher in the hESC line, we compared only the change in band intensities. Abbreviations: -OGG1, -oxoguanine DNA glycosylase; APE-1, apurinic/apyrimidinic endonuclease-1; h, hours; hESC, human embryonic stem cell; UT, untreated.

	DISCUSSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

Specific strategies were implemented in the comet assays to give insight into the types of damage being repaired. In the case of H₂O₂ and UV-C treatments, we introduced the extra step of digesting the nucleoids with an enzyme that recognizes a particular kind of damage and creates a break [45]: Fpg and T4 endonuclease V treatments enabled the comet assay to detect the major purine oxidation product 8-oxoG (as well as other altered purines) and UV-C-induced cyclobutane dimers, respectively. For the psoralen treatment, the specificity is for psoralen cross-links and so the comet assay measured the ability to unhook the cross-links from the DNA, an important early step in ICL repair [15]. In the case of IR damage, the pH of the unwinding buffer was set to 12.1 to promote specific detection of strand breaks; this does not distinguish between single-strand breaks and DSBs [46]. However, it is thought that DSBs are the major lesion induced by IR [47]. DSBR is know to be rapid, with most DSBs rejoined within 2 hours [48]. The rapid repair kinetics observed in our IR-comet assay suggests that the assay is measuring DSBR.

It is important to note that the rate of DNA damage-induced apoptosis may also influence the results of these studies. In this study the only treatment condition that produced observable apoptosis was UV-C: detached cells became apparent first in hESCs, at 12–18 hours after UV-C treatment (well after the 6-hour repair period studied). To minimize any potential effects of cell death or slowed growth, the doses of all stress agents used were kept to the minimum levels necessary to obtain the robust comet tails necessary for accurate determinations of differences or changes in mean OTM values.

Additional markers of repair were compared among hESCs and differentiated cells, including 8-oxoG levels, OGG1 incision activity, and expression of several DNA repair genes. The observation that the level of 8-oxoG was lower in hESCs than in WI-38 cells suggests a higher degree of 8-oxoG lesion repair. However, another explanation could be that there is less oxidative damage occurring in hESCs, possibly due to higher levels of antioxidants. In fact, recent work with two hESC lines (H1, hES-NCL1) showed that major antioxidant genes were downregulated during differentiation, accompanied by an increase in reactive oxygen species levels [27]. Earlier work with mESCs had demonstrated superior antioxidant capacity in mESC compared with various differentiated murine cells [20]. Since we have now shown that hESCs have enhanced DNA repair of oxidative damage relative to various differentiated human cells, it is likely that the lower 8-oxoG levels observed in the hESCs are at least partially due to superior DNA repair capacity. In addition, we found that the OGG1 incision activity was not significantly higher in hESC line BG01 than fibroblasts (WI-38) or transformed cells (HeLa, HEK293) (Fig. 4). There was a trend for higher OGG1 mRNA levels in hESCs (1.5-fold) relative to their differentiated counterparts (embryoid bodies); however, this difference was not significant (Fig. 5). Interestingly, OGG1 and APE-1 proteins were induced by exposure to H₂O₂ more strongly in hESCs (BG01) than WI-38 cells (Fig. 6). It is possible that in untreated cells higher expression levels of multiple BER genes, as seen in Figure 5A, combine to give enhanced repair of inherent 8-oxoG in hESCs. After exposure to oxidative agents, a more intense induction of OGG1 and other repair enzymes may mediate a even stronger BER response in hESCs. Further work in this regard should entail measurements of markers for damage and stress defense in hESCs and various differentiated cells at different stages of the repair processes. Interestingly, we have consistently found in hESCs (BG01) altered expression levels of various nuclear matrix proteins commonly used as loading controls (data not shown, except for actin in Fig. 6): in three different Western blotting experiments, the levels of lamin B2 and actin were always higher, whereas the level of -tubulin was always lower. Therefore, in the experiment shown in Figure 6, we made use of actin only as a loading control within each cell type. We are in the process of investigating nuclear matrix protein expression further in other hESC cell lines relative to differentiated cells.

The mRNA expression data presented here suggest that several DNA repair genes, including BER, NER, DSBR, and ICL repair genes, are expressed at higher levels in hESCs relative to differentiated cells. Although these genes are not strikingly overexpressed, the cumulative effect of higher expression of several DNA repair genes (and their encoded proteins) could account for the higher rates of repair seen in the comet cell assays reported here. Recently it was demonstrated that several stress defense mechanisms are downregulated during differentiation of hESCs [27]. Although the DNA repair rate was not measured, the report showed that H2AX foci frequency increased and expression of several DNA repair genes decreased during differentiation. The identity of one of these DNA repair genes overlapped with the genes we analyzed, namely BRAC1. In our study, BRAC1 showed a significantly higher expression level (1.7-fold) than differentiated counterpart embryoid bodies.

It is currently thought that adult tissues are maintained by a small number of slowly proliferating tissue-specific stem cells, which provide a source of daughter cells to replace damaged or senescent terminally differentiated cells. It is also thought that adult stem cells are themselves subject to aging and time-dependent loss of function, which may be associated with DNA damage [49, 50]. Moreover, recent reports indicate that defects in DNA repair can have deleterious effects on the function of adult stem cells [51, 52]. In future studies, it may be of interest to compare DNA repair capacity in embryonic, young, and old stem cells or in aged stem cells and age-matched differentiated cells.

	CONCLUSION

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
ESCs have the capacity to differentiate into any cell type in the adult organism. Therefore it is predicted that they would have superior genomic maintenance systems, including efficient DNA repair. Here we showed that hESCs have more efficient DNA repair than several differentiated cells in response to various DNA-damaging agents (H₂O₂, UV-C, IR, psoralen). Microarray analysis indicated that hESCs have higher expression of several DNA repair genes spanning a range of DNA repair pathways. In addition, expression levels of two BER repair proteins (OGG1 and APE-1) were upregulated by oxidative stress in hESCs more than in differentiated fibroblasts. The level of 8-oxoG oxidative lesions was lower in hESCs (relative to fibroblasts), but OGG1 incision activity was not significantly higher. On the basis of our data, we propose that a factor contributing to the superior DNA repair in hESCs is a combined effect of several DNA repair proteins being more highly expressed in hESCs before and after acute damage.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 
We are indebted to Daniel R. McNeill for HPLC analysis of 8-oxoG levels, Patrice Cook for technical assistance on the OGG1 activity assays, and Al May for helpful instruction on many aspects of the comet assays. We also thank Dr. Jason Aulds and Dr. Tomasz Kulikowicz for the critical reading of the manuscript and Dr. Christopher Morrell for assistance with the statistical analysis. This work was supported by the Intramural Research Program of the National Institute on Aging, NIH.

	FOOTNOTES

 
Author contributions: S.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; A.M.S., J.W.L., Y.L., S.-T.L., A.D.C., and M.R.: collection and/or assembly of data, data analysis and interpretation; N.D.S.-P. and X.Z.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; V.A.B.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript.

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Top Footnotes Abstract Introduction Materials and Methods Results Discussion Conclusion Disclosure of Potential... Acknowledgments References

 

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