Graphical Abstract

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Highlights

     * •
       SIRT1 downregulation induces cell death in hESCs but not in
       differentiated cells
     * •
       SIRT1 inhibition increases DNA damage by reducing the level of DNA
       repair enzymes
     * •
       SIRT1 inhibition activates p53 and induces two pro-apoptotic genes,
       PUMA and BAX
     * •
       SIRT1 acts as a guardian of hESCs via genome stability
     __________________________________________________________________

   Jang and colleagues show that SIRT1 downregulation in hESCs decreased
   the levels of DNA repair enzymes such as MSH2, MSH6, and APEX1 and
   increased DNA damage (in both SSBs and DSBs). The authors also
   demonstrate that SIRT1 inhibition induced massive cell death, caused at
   least partially by DNA damage-mediated p53 activation. Thus, SIRT1
   contributes to genome stability and survival of hESCs.

Introduction

   Human pluripotent stem cells (hPSCs), such as human embryonic stem
   cells (hESCs) and induced pluripotent stem cells (iPSCs), are
   characterized by their capacity to proliferate indefinitely and to
   differentiate into most cell types in the human body. Because of these
   unique properties, hPSCs hold great promise for the treatment of many
   incurable diseases through cell replacement therapy ([36]Hwang et al.,
   2010, [37]Yoo et al., 2013).

   Despite more than a decade of intensive research, understanding of the
   physiology and functional regulation of hPSCs, especially hESCs,
   remains far from complete. The rapid proliferation of hESCs, which is
   caused primarily by a shortened G[1] phase and a rapid G[1]/S
   transition, increases the risk of replication errors and enhances the
   levels of reactive oxygen species within these cells ([38]Becker
   et al., 2006). A variety of internal factors and external environmental
   stimuli, such as genotoxic chemicals and physical stimuli (e.g.,
   ionizing radiation, UV light), are potential causes of different forms
   of DNA damage, such as single-strand breaks, abasic sites, DNA adducts,
   and double-strand breaks (DSBs) ([39]Houtgraaf et al., 2006). Such DNA
   damage in turn induces various intracellular responses, such as cell
   cycle changes, the activation of DNA repair machineries, and apoptosis.

   Sirtuin1 (SIRT1) is a nicotinamide adenine dinucleotide
   (NAD^+)-dependent class III histone deacetylase that is known to
   regulate stress responses, genomic stability, and cell survival
   ([40]Chung et al., 2006, [41]Terada et al., 2002, [42]Ying et al.,
   2002). Previous work has shown that the DNA damage repair system does
   not function properly in SIRT1 mutant mice ([43]Wang et al., 2008). In
   addition, several studies have implicated SIRT1 in DNA repair and cell
   survival in cancer cells ([44]Kalle et al., 2010, [45]Kojima et al.,
   2008, [46]Lin and Fang, 2013), which is consistent with the abnormal
   SIRT1 protein levels in many cancer cells ([47]Elangovan et al., 2011,
   [48]Huffman et al., 2007, [49]Kuzmichev et al., 2005). To date, the
   role of SIRT1 in hESCs has remained poorly understood. Recent studies
   have suggested a link between Sirt1 and the differentiation of mouse
   ESCs (mESCs), hematopoietic stem cells, and mesenchymal stem cells
   ([50]Calvanese et al., 2010, [51]Lee et al., 2012, [52]Ou et al., 2011,
   [53]Simic et al., 2013, [54]Tang et al., 2014). In particular, [55]Ou
   et al. (2011) have reported that Sirt1 knockout mice display defects in
   hematopoietic differentiation and the formation of a primitive
   vasculature. Sirt1 has also been shown to enhance the reprogramming of
   mouse fibroblasts ([56]Lee et al., 2012), facilitate telomere
   elongation in mouse iPSCs ([57]De Bonis et al., 2014), and maintain the
   pluripotency of hESCs ([58]De Bonis et al., 2014, [59]Zhang et al.,
   2014). In addition, SIRT1 has been shown to induce autophagy in
   response to oxidative stress in both mESCs and hESCs ([60]Ou et al.,
   2014). To date, most studies regarding the functions of Sirt1 in
   pluripotent stem cells have been performed using mESCs, and
   understanding of SIRT1 functions in hESCs remains far from complete. In
   particular, the roles of SIRT1 in hESC genome stability and survival
   have yet to be investigated in detail.

   In this study, we sought to examine the role of SIRT1 in hESC survival.
   Using proteomic analysis, we showed that SIRT1 in hESCs critically
   modulates the levels of two DNA mismatch repair enzymes, MSH2 and MSH3,
   and a DNA base excision repair enzyme, APEX1, and provided evidence
   that SIRT1 is critically implicated in DNA repair processes that in
   turn affect the genome stability and survival of hESCs.

Results

SIRT1 Is Abundantly Expressed in hESCs

   Previous reports have shown that Sirt1 protein regulates the
   pluripotency and differentiation of mESCs ([61]Ou et al., 2011,
   [62]Zhang et al., 2014). We first compared the level of SIRT1 among
   hESCs and their differentiated derivatives, such as embryoid bodies
   (EBs) and neural precursors (NPs). qRT-PCR showed that SIRT1 mRNA
   levels were at least 4-fold higher in hESCs than in EBs and NPs
   ([63]Figure 1A). Western blotting also showed that SIRT1 protein levels
   were significantly higher in hESCs than in EBs and NPs ([64]Figure 1B).
   Furthermore, immunocytochemistry clearly showed that SIRT1 was strongly
   expressed in undifferentiated (SSEA4^+) hESCs ([65]Figure 1C, top two
   panels), whereas its expression was drastically decreased in a large
   population of differentiated (SSEA4^−) cells ([66]Figure 1C, top
   panels, the areas surrounded by white dotted lines). A merged image
   clearly showed that most sites of SIRT1 and SSEA4 immunoreactivity were
   co-localized ([67]Figure 1C, bottom right panel, inside the white
   dotted circle).

Figure 1.

   [68]Figure 1
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   SIRT1 Levels Are High in hESCs and Are Greatly Decreased in
   Differentiated Cells

   (A) SIRT1 expression levels in hESCs and differentiated cells (EBs and
   NPs) were measured by qRT-PCR. Decreased SIRT1 expression was observed
   in EBs and NPs compared with hESCs. Data are shown as means ± SEM (n =
   6 independent experiments; ^∗p < 0.05).

   (B) SIRT1 expression levels in hESCs, EBs, and NPs were compared by
   western blotting.

   (C) Immunocytochemistry shows the co-localization of SSEA4 (green) and
   SIRT1 (red) immunoreactivity sites. The white dashed lines demarcate
   differentiated cells.

   (D) Flow cytometric analysis for SSEA-4 (phycoerythrin, PE) and SIRT1
   (fluorescein isothiocyanate, FITC) surface antigen expression was
   performed on hESCs (left panel) or NPs (right panel). Numbers shown in
   the individual quadrants indicate the percentage of cells of each type.

   Flow-cytometric analysis showed that most hESCs (∼91.5%) displayed
   immunoreactivity toward both SSEA4 and SIRT1, thus demonstrating
   co-expression of SSEA4 and SIRT1in hESCs at a cellular level
   ([70]Figure 1D, left panel). However, the expression of both markers
   was simultaneously downregulated (i.e., SSEA4^+ SIRT1^+ = ∼3.91%,
   SSEA4^− SIRT1^− = ∼75.4%) in NPs that were differentiated from hESCs
   ([71]Figure 1D, right panel).

   Together, our results demonstrated a high SIRT1 level in hESCs but not
   in differentiated cells (i.e., EB and NPs). This dramatic change in
   SIRT1 level suggested that Sirt1 is critically involved in the
   physiology of hESCs.

SIRT1 Inhibition Induced Apoptosis in hESCs

   SIRT1 has been reported to decrease apoptotic cell death in cancer
   cells ([72]Kalle et al., 2010, [73]Kojima et al., 2008). In this study,
   we hypothesized that one of the functions of SIRT1 in hESCs might be to
   protect the cells from apoptotic cell death. To elucidate the potential
   involvement of SIRT1 in hESC survival, we examined how knocking down
   the level or inhibiting the function of SIRT1 affected hESC
   survival/death. First, western blotting confirmed that hDFs, which were
   used as a negative control (i.e., differentiated cells), did not
   express SIRT1, whereas hESCs displayed significant SIRT1 expression
   ([74]Figure 2A).

Figure 2.

   [75]Figure 2
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   Tenovin-6 Treatment Leads to Cell Death in hESCs but Not in
   Differentiated Cells

   (A) A representative immunoblot showing the difference in SIRT1
   expression level between undifferentiated cells (hESCs) and fully
   differentiated cells (hDFs).

   (B) Typical phase-contrast images of hESCs and hDFs were taken after
   Tenovin-6 (5 μM) treatment for 12 hr.

   (C) Immunocytochemistry showed that cleaved caspase-3 (C-CAS 3)
   immunoreactivity dramatically increased after Tenovin-6 treatment
   (2 μM, 12 hr). The areas surrounded by white dashed lines demarcate the
   areas composed of undifferentiated hESCs.

   (D) Flow cytometric analysis of hESCs was performed after Tenovin-6
   treatment (5 μM, 12 hr). y Axis, propidium iodide (PI); x axis,
   annexin V (FITC-labeled).

   (E) Percentages of apoptotic cells obtained from flow-cytometric
   analysis of hESCs treated with Tenovin-6 (5 μM, 12 hr). Data are shown
   as means ± SEM (n = 6 independent experiments; ^∗p < 0.05).

   (F) Flow-cytometric analysis of hDFs was performed after Tenovin-6
   treatment (5 μM, 12 hr). y Axis, PI; x axis, annexin V (FITC-labeled).

   (G) Flow-cytometric analysis of apoptotic cells using PI and an annexin
   V antibody was performed at 48 hr after transfection with siRNA-αSIRT1.

   (H) The three histograms derived from each independent flow cytometric
   analysis are shown. In the experiments, the number of apoptotic cells
   was counted at 48 hr after transfection of hESCs with either
   siRNA-αSIRT1 or siRNA-αSIRT2. An FITC-conjugated annexin V antibody was
   used for the analysis (n = 3 independent experiments).

   When hESCs were treated for 12 hr with Tenovin-6, a SIRT1/2 inhibitor,
   hESCs in compact colonies were severely damaged ([77]Figure 2B, top
   right panel), whereas hDFs remained intact under the same conditions
   ([78]Figure 2B, bottom right panel). Next, we observed that a 12-hr
   treatment with Tenovin-6 induced a significant increase in cleaved
   caspase-3 (C-CAS3), an apoptotic marker, in hESCs, but not in hDFs
   ([79]Figure 2C). Flow-cytometric analysis using annexin V and propidium
   iodide (PI) to evaluate apoptosis showed a significant increase in
   apoptotic cells after Tenovin-6 (5 μM, 12 hr) treatment in hESCs (i.e.,
   from ∼4.86% to ∼22.54%) ([80]Figures 2D and 2E), but not in hDFs
   ([81]Figure 2F). Repeated experiments consistently showed that the
   percentage of apoptotic cells was significantly increased after
   Tenovin-6 treatment in a time-dependent manner, reaching nearly 40% at
   16 hr post treatment ([82]Figures S1A and S1B). Moreover, treatment of
   hESCs and hDFs with other SIRT1 inhibitors, such as Sirtinol (50 μM),
   Salermide (50 μM), and EX527 (5 μM), also induced cell death of only
   hESCs without affecting hDFs ([83]Figure S1C). Because EX527 is a
   selective SIRT1 inhibitor that does not block other SIRT family
   proteins, it is evident that SIRT1 inhibition induces hESC death
   ([84]Figure S1C, top rightmost panel).

   To further confirm the involvement of SIRT1 in the regulation of hESC
   death, we specifically knocked down SIRT1 using an SIRT1-specific small
   interfering RNA (siRNA-αSIRT1) and examined the degree of cell death by
   flow-cytometric analysis using an annexin V antibody and PI
   ([85]Figure 2G). The knockdown specificity of siRNA-αSIRT1 used in this
   experiment was confirmed because it decreased only the SIRT1 mRNA level
   ([86]Figure S2A). After transfection of hESCs with siRNA-αSIRT1, the
   SIRT1 mRNA level was decreased in a reverse time-dependent manner,
   probably because of the degradation of transfected siRNA-αSirt1 over
   time ([87]Figure S2B). At 48 hr after siRNA-αSIRT1 transfection, we
   still observed a significant number of apoptotic cells (2.7%,
   siRNA-Scrambled versus 28.9%, siRNA-αSIRT1) ([88]Figure 2G).

   When we examined the contribution of SIRT2 to hESC survival/death by
   using an siRNA specific for SIRT2 ([89]Figure S2C), no significant
   induction of cell death was detected ([90]Figure 2H), thus suggesting
   that SIRT2 plays only a marginal role in the regulation of hESC
   survival. This result also indicated that a large portion of cell death
   induced by Tenovin-6 treatment (i.e., inhibition of SIRT1 and 2) is
   caused by blocking SIRT1 ([91]Figure 2H).

   Together, our results demonstrated that either blocking the function or
   decreasing the level of SIRT1 robustly enhanced hESC death, thus
   indicating that SIRT1 is critical for the survival of hESCs.

Proteomic Analysis Showed that SIRT1 Inhibition Significantly Decreased DNA
Repair Proteins in hESCs

   To gain mechanistic insight into the early events occurring after SIRT1
   inhibition, we treated hESCs with Tenovin-6 for 2 hr and performed
   proteomic analysis. A total of 1,401 different proteins were identified
   using isobaric tags for relative and absolute quantification
   (iTRAQ)-based proteomic analysis. Of these, 140 and 79 proteins were
   downregulated and upregulated, respectively, in hESCs treated with
   Tenovin-6 compared with cells treated with DMSO (control)
   ([92]Figure 3A and [93]Tables S1–S4). The 140 downregulated proteins
   were divided into 46 stringently regulated proteins (red-brown squares)
   and 94 moderately regulated proteins (pear-green triangles)
   ([94]Figure 3A, left half; [95]Tables S1 and [96]S2). Likewise, the 79
   upregulated proteins were divided into 23 stringently regulated
   proteins (red-brown squares) and 56 moderately regulated proteins
   (pear-green triangles) ([97]Figure 3A, right half; [98]Tables S3 and
   [99]S4).

Figure 3.

   [100]Figure 3
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   Proteomic Analysis Showed that SIRT1 Inhibition Significantly Decreased
   DNA Repair Proteins in hESCs

   (A) Volcano plot of differentially expressed proteins (DEPs) between
   control and SIRT1-inhibited groups. Fold change (in log[2]) is plotted
   against the Edge R-generated p value (log base 2). Differential
   expression analysis identified 219 DEPs between the control and
   SIRT1-inhibited groups: a total of 140 and 79 proteins were
   downregulated and upregulated by SIRT1 inhibition, respectively. The
   140 downregulated proteins were divided into 46 stringently regulated
   proteins (red-brown squares, log[2] < −1, p < 1.0 × 10^−0.2) and 94
   moderately regulated proteins (pear-green triangles, −1 < log[2] < 0,
   p < 1.0 × 10^−0.2). Likewise, the 79 upregulated proteins were divided
   into 23 stringently regulated proteins (red-brown squares, log[2] > 1,
   p < 1.0 × 10^−0.2) and 56 moderately regulated proteins (pear-green
   triangles, 0 < log[2] < 1, p < 1.0 × 10^−0.2).

   (B) The protein-protein interaction biochemical network was generated
   using the STRING database (Search Tool for the Retrieval of Interacting
   proteins) ([102]http://string-db.org/) and provided information on
   proteins that either directly or indirectly interact with SIRT1.

   (C) Western blotting of SIRT1, MSH2, MSH6, and APEX1 was performed at
   12 hr post transfection.

   (D) Representative western blotting of hESCs treated with resveratrol
   (10 μM) for 24 hr was performed to examine the levels of SIRT1 and DNA
   repair proteins (MSH2 and MSH6).

   (E) Western blotting of MSH2, MSH6, and APEX1 was performed after hESCs
   were treated with a combination of a SIRT1 inhibitor and/or a
   proteasome inhibitor (DMSO only, Tenovin-6 only, Tenovin-6 + MG132, and
   MG132 only).

   Next, we performed KEGG pathway enrichment analysis and found that the
   enriched categories included pathways involved in DNA repair. Notably,
   MSH2 and MSH6, which are involved in repairing DNA base mismatches, and
   APEX1, which has been implicated in DNA excision repair, were found in
   the stringently downregulated group ([103]Figure 3A). The
   protein-protein interaction biochemical network generated using the
   STRING database (Search Tool for the Retrieval of Interacting proteins
   database) ([104]http://string-db.org/) provided information on proteins
   that either directly or indirectly interact with SIRT1. Analysis of
   this protein network showed that SIRT1 was closely linked to several
   important DNA repair proteins, including MSH2, MSH6, and APEX1
   ([105]Figure 3B).

   Together, these results suggest the intriguing possibility that SIRT1
   interacts with several DNA repair proteins and that these interactions
   may be responsible for SIRT1-mediated hESC survival.

Proteomic Analysis Data Were Validated by Western Blotting

   We first examined whether our proteomic data were reliable. To this
   end, we chose several up- and downregulated proteins and validated the
   expression of these proteins by using western blotting. All four
   upregulated proteins examined, PDI, T-PLASTIN, VDAC1, and VIMENTIN,
   were detected at a higher level in Tenovin-6-treated hESCs than in
   control (DMSO-treated) hESCs ([106]Figure S3A). In addition, the levels
   of four downregulated proteins, MTHF1, MYOSIN, FILAMIN1, and EZRIN,
   were lower in Tenovin-6-treated hESCs than in control hESCs
   ([107]Figure S3B). These western blot results confirmed the validity of
   our proteomic data.

   Next, we examined the levels of the three DNA repair proteins, MSH2,
   MSH6, and APEX1, which were shown to be downregulated by Tenovin-6
   treatment in our proteomic analysis. For western blotting, we
   transfected hESCs with siRNA-Scrambled and siRNA-αSIRT1 and collected
   samples at 12 hr post transfection. As expected, the protein levels of
   SIRT1, MSH2, MSH6, and APEX1 were decreased in cells transfected with
   siRNA-αSIRT1 ([108]Figure 3C).

   To further confirm the role of SIRT1 in regulating the protein
   expression of MSH2, MSH6, and APEX1, we elevated the SIRT1 protein
   level by treating hESCs with resveratrol, a SIRT1 activator. Our
   western blotting results showed that increasing SIRT1 by resveratrol
   treatment upregulated DNA repair proteins such as MSH2 and MSH6
   ([109]Figure 3D).

   Intriguingly, the MSH2, MSH6, and APEX1 mRNA levels did not decrease
   after Sirt1 knockdown (data not shown). This observation contrasted
   with the decreased protein levels of these DNA repair genes after SIRT1
   inhibition, thus suggesting the involvement of post-transcriptional
   regulation.

   To understand the mechanisms underlying the decrease in DNA repair
   proteins without the decrease in their respective mRNAs after SIRT1
   inhibition, we tested whether SIRT1 inhibition induced the proteasomal
   degradation of these DNA repair enzymes. To this end, we treated cells
   with Tenovin-6 along with a proteasome inhibitor, MG132. The
   significant decrease in DNA repair proteins after Tenovin-6 treatment
   did not occur when MG132 was also added, thus indicating that
   proteasomal degradation is involved in the SIRT1-mediated regulation of
   DNA repair enzyme levels ([110]Figure 3E). This result suggested that
   the decrease in the three DNA repair proteins, MSH2, MSH6, and APEX1,
   after SIRT1 inhibition was at least partly due to enhanced proteasomal
   degradation.

   Together, our data demonstrated that a high level of SIRT1 protein in
   hESCs increases several DNA repair proteins and the proteasomal
   degradation pathway is at least partly involved in the SIRT1-mediated
   regulation of the DNA repair proteins.

SIRT1 Inhibition Increased DNA Damage in hESCs

   Because we found a link between the levels of SIRT1 and DNA repair
   proteins, we examined whether blocking SIRT1 activity eventually leads
   to increased DNA damage. First, we performed apurinic/apyrimidinic
   (AP)-site staining of hESCs after Tenovin-6 treatment (5 μM, 12 hr): AP
   sites are the most common form of single-strand DNA damage ([111]Demple
   and Harrison, 1994). Tenovin-6-treated hESCs displayed a large number
   of AP-site-positive cells, whereas control (DMSO-treated) cells showed
   no prominent AP sites ([112]Figure 4A, upper two panels and graph). As
   a positive control, hESCs were treated with epigallocatechin gallate
   (EGCG), which reacts with the culture medium and generates hydrogen
   peroxide (H[2]O[2]) and further causes DNA damage ([113]Figure 4A,
   lower two panels). These results suggested that SIRT1 inhibition causes
   a concomitant increase in single-strand DNA breaks in hESCs.

Figure 4.

   [114]Figure 4
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   Tenovin-6 Treatment Induces DNA Damage in hESCs

   (A) Immunofluorescence analysis of AP (apurinic/apyrimidinic) sites in
   hESCs treated with Tenovin-6 (5 μM, 12 hr) and EGCG (5 μM, 12 hr) as a
   positive control. The data were quantified and are presented in a graph
   (lower right panel). Data are shown as means ± SEM (n = 3 independent
   experiments; ^∗∗p < 0.01).

   (B) Immunofluorescence analysis showed that the number of hESCs
   containing DSBs (γ-H2AX^+) also increased in a dose-dependent manner
   after Tenovin-6 treatment.

   (C) The number of γ-H2AX^+ cells per 70 μm^2 DAPI^+ area is presented
   as a bar graph. Data represent means ± SEM (n = 3 independent
   experiments; ^∗∗p < 0.01).

   (D) The numbers of Dead Green^+ and γ-H2AX^+ cells were counted at 2 hr
   and 6 hr after Tenovin-6 (5 μM) treatment and plotted in the graph.
   Data represent means ± SEM (n = 6 independent experiments; ^∗p < 0.05,
   ^∗∗p < 0.005).

   (E) The percentages of the total γ-H2AX^+ cells that were Dead Green^+
   were measured and presented as a bar graph. Data are shown as means ±
   SEM (n = 6 independent experiments; ^∗p < 0.01, ^∗∗p < 0.001).

   Next, we sought to examine whether SIRT1 inhibition in hESCs might
   promote more severe DNA damage such as DNA DSBs, which are known to
   cause apoptotic cell death ([116]Roos and Kaina, 2006). Phosphorylated
   H2AX (γ-H2AX) is a good indicator of DSBs, because DSBs are typically
   followed by histone H2AX phosphorylation ([117]Kinner et al., 2008).
   Treatment of hESCs with Tenovin-6 for 12 hr increased the number of
   γ-H2AX^+ cells in a dose-dependent manner ([118]Figures 4B and 4C).
   Nuclear staining with DAPI showed that the compact colony morphology
   was lost at high concentrations of Tenovin-6 (i.e., 5 μM and 10 μM)
   ([119]Figure 4B, lower panels). By contrast, hDFs did not display any
   morphological changes at the high concentrations of Tenovin-6 used in
   this study (i.e., 5 μM) ([120]Figure S4A), in agreement with the
   results in [121]Figures 2B and 2F. In addition, few γ-H2AX^+ cells were
   detected in hDFs after Tenovin-6 (5 μM) treatment ([122]Figure S4A).

   To examine the correlation between DNA damage and cell death, we
   examined the number of γ-H2AX^+ and Dead Green^+ (a marker for cell
   death) cells at both 2 hr and 6 hr. At 2 hr post treatment, some
   γ-H2AX^+ cells were observed, whereas few Dead Green^+ cells appeared
   ([123]Figures S4B [upper panels] and [124]4D). A significant number of
   Dead Green^+ cells and γ-H2AX^+ cells were detected at 6 hr, and
   γ-H2AX^+ cells outnumbered Dead Green^+ cells ([125]Figures S4B [lower
   panels] and [126]4D). Intriguingly, a large portion of γ-H2AX^+ cells
   were positive for Dead Green at 6 hr (>60%) ([127]Figure 4E), and no
   Dead Green^+ γ-H2AX^− cells were detected ([128]Figure S4B), thus
   indicating the close relationship between DSB DNA damage and cell death
   after Tenovin-6 treatment. In addition, H2AX phosphorylation preceded
   cell death in hESCs ([129]Figures 4D and [130]S4B), thus further
   suggesting that DNA DSBs were at least partly responsible for cell
   death after SIRT1 inhibition. These results confirmed that Tenovin-6
   treatment resulted in increased DNA damage (i.e., DSBs) and subsequent
   apoptotic cell death.

   Because Tenovin-6 inhibits the functions of both SIRT1 and SIRT2
   ([131]Lain et al., 2008), we examined the effect of only SIRT1 by
   specifically knocking down SIRT1 mRNA by using siRNA-αSIRT1. As
   expected, DNA DSBs were drastically increased at 24 and 48 hr after
   siRNA-αSIRT1 transfection in hESCs, but not in hDFs ([132]Figures 5A
   and 5B).

Figure 5.

   [133]Figure 5
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   SIRT1 Plays a Major Role in Genome Stability and Is Marginally Involved
   in hESC Differentiation

   (A) Immunofluorescence images showing the numbers of hESCs and hDFs
   with DSB DNA damage (γ-H2AX^+) at 24 hr and 48 hr after transfection
   with siRNAs (siRNA-Scrambled and siRNA-αSIRT1).

   (B) Percentages of total cells (DAPI^+ cells) that were γ-H2AX^+. Data
   are shown as means ± SEM (n = 6 independent experiments; ^∗p < 0.01,
   ^∗∗p < 0.001).

   (C) Histogram showing intracellular γ-H2AX^+ levels of hESCs treated
   with 2.5 mM H[2]O[2] in the absence or presence of resveratrol
   pretreatment (10 μM, 24 hr).

   (D) The level of SSEA-4 (phycoerythrin, PE), an undifferentiated cell
   marker, was examined using flow-cytometric analysis at 72 hr after
   transfection with siRNAs (siRNA-Scrambled and siRNA-αSIRT1).

   (E) Real-time qPCR analysis of the mRNA levels of SIRT1, NESTIN, SOX17,
   and BRACHYURY genes at 72 hr after transfection with siRNAs
   (siRNA-Scrambled [siCon] and siRNA-αSIRT1). Data are shown as means ±
   SEM (n = 3 independent experiments; ^∗∗p < 0.005).

   In addition, enhancement of SIRT1 expression by resveratrol treatment
   decreased the oxidative stress (H[2]O[2])-induced increase in DNA DSBs,
   thus indicating a clear role of SIRT1 in the decrease in DNA damage
   ([135]Figure 5C).

   Together, our results demonstrated that SIRT1 plays a critical role in
   preventing DNA damage in hESCs, which in turn protects hESCs from
   death.

SIRT1 Knockdown Marginally Induced hESC Differentiation

   To determine whether SIRT1 is involved in hESC differentiation, as
   previously shown ([136]De Bonis et al., 2014, [137]Zhang et al., 2014),
   we analyzed hESCs at 72 hr after transfection with siRNA-αSIRT1. Our
   flow cytometric analysis displayed a slight decrease in the SSEA4^+
   (undifferentiated) cell population after SIRT1 knockdown
   ([138]Figure 5D), thus indicating a slight induction of differentiation
   by SIRT1 knockdown. Furthermore, we detected an increase in NESTIN, a
   neuroectoderm marker, whereas mesoderm and endoderm lineage markers
   were not changed ([139]Figure 5E).

   Our results together showed some evidence of hESC neural
   differentiation, although the most dramatic effects detected after
   SIRT1 inhibition or knockdown were increased DNA damage and subsequent
   cell death.

Tenovin-6-Mediated p53 Acetylation Was Not Required for the Early Decrease in
DNA Repair Proteins

   We already showed that SIRT1 inhibition induced apoptotic cell death of
   hESCs ([140]Figure 2). Because DNA damage activates p53 through
   acetylation ([141]Sakaguchi et al., 1998), which in turn results in
   apoptosis ([142]Sykes et al., 2006, [143]Tang et al., 2006), we
   examined whether Tenovin-6 treatment might promote p53 acetylation with
   subsequent induction of apoptosis in hESCs. Our western blotting
   analysis showed that the levels of acetylated (activated) p53 and
   cleaved caspase-3 were significantly increased after Tenovin-6
   treatment (5 μM, 12 hr) in hESCs, but not in hDFs ([144]Figure 6A).
   This result was consistent with our previous immunostaining and flow
   cytometry data showing a significant increase in hESC death at 12 hr
   after Tenovin-6 treatment ([145]Figure 2). Our qRT-PCR results showed
   that SIRT1 inhibition increased the expression of PUMA and BAX,
   pro-apoptotic p53 target genes, in hESCs ([146]Figure 6B), thus
   indicating a connection between SIRT1 inhibition and p53-mediated
   apoptosis of hESCs.

Figure 6.

   [147]Figure 6
   [148]Open in a new tab

   Tenovin-6-Mediated p53 Acetylation Was Not Required for the Early
   Decrease in DNA Repair Proteins

   (A) Western blotting for p53 (a known target of SIRT1), acetylated-p53
   (an active form of p53), and cleaved caspase-3 (an apoptotic marker)
   was performed in cells with or without Tenovin-6 treatment (5 μM,
   12 hr) (left panel). The morphologies of the cells used for western
   blotting are shown on the right.

   (B) Expression levels of PUMA and BAX, two pro-apoptotic genes, were
   examined by real-time qPCR in cells with or without Tenovin-6 treatment
   (5 μM, 12 hr). Data represent means ± SEM (n = 3 independent
   experiments; ^∗p < 0.05, ^∗∗p < 0.005).

   (C) Western blotting for SIRT1, acetylated p53 (acetyl-p53), MSH2,
   MSH6, APEX1, and actin (a loading control) was performed at 2 hr after
   treatment with Tenovin-6 (5 μM).

   We next examined whether the decrease in the DNA repair proteins after
   Tenovin-6 treatment occurred through p53 acetylation. To detect which
   event occurs first, we performed western blotting at an early time
   point after Tenovin-6 treatment. In this experiment we examined only an
   acetylated form of p53, because [149]Lain et al. (2008) have shown that
   Tenovin-6 does not change the level of total p53 (as seen also in our
   data, [150]Figure 6A) and does not activate p53 by phosphorylation.
   Strikingly, treatment of hESCs with Tenovin-6 for 2 hr resulted in a
   decrease in MSH2, MSH6, and APEX1 proteins, whereas no acetylated form
   of p53 was apparent at this time point ([151]Figure 6C). This result
   suggested that the decrease in the DNA repair proteins does not require
   p53 activation, at least during the early stage.

   Together, our results indicated no evidence of a functional connection
   between acetylated p53 and the decrease in DNA repair proteins. It is
   plausible that the DNA damage-induced apoptosis after SIRT1 inhibition
   in hESCs occurs through a p53-independent mechanism, at least during
   early events (i.e., decrease in DNA repair proteins). This study
   provides valuable insights into the mechanism underlying the
   SIRT1-mediated survival of hESCs.

Discussion

   hESCs have unlimited proliferation potential and typically have a
   shorter cell cycle (approximately 15.8 hr) than most somatic cells
   (24–32 hr), owing to a shortened G[1] phase (approximately 2.5–3 hr)
   and a faster G[1]/S transition ([152]Becker et al., 2006). hESCs retain
   pluripotency because they are derived from the inner cell mass of
   blastocysts, which can generate any type of cell in the human body
   after full development.

   It may be reasonable to speculate that cells from early developmental
   stages (i.e., cells that constitute the inner cell mass and hESCs)
   retain efficient DNA repair systems, because the introduction of DNA
   defects during early developmental stages would be disastrous and lead
   to inherited mutations in differentiated cells. In support of this
   idea, mESCs have been reported to show more efficient DNA repair
   activity than fibroblasts after oxidative stress- and ionizing
   radiation-induced DNA damage ([153]Saretzki et al., 2004). Furthermore,
   hESCs are prone to undergo apoptosis at lower doses of IR exposure than
   somatic cells, thus decreasing the chances of propagating abnormal
   cells with DNA defects. These observations suggest that hESCs have a
   fail-safe strategy to decrease cells with defective genetic information
   by boosting DNA repair systems and/or inducing apoptosis of defective
   cells ([154]Filion et al., 2009, [155]Sokolov et al., 2012, [156]Wilson
   et al., 2010). These fail-safe strategies are of great importance in
   allowing hESCs to avoid any disastrous outcomes derived from the
   propagation of cells into different cell types with DNA abnormalities.
   Cells constantly undergo DNA damage in the form of nucleotide changes,
   abasic sites, and single-strand breaks. If these defects are not
   efficiently corrected, they eventually form lethal DSBs during DNA
   replication, when replicative DNA polymerases encounter single-strand
   breaks. DSBs are extremely detrimental to cells and often cause
   chromosomal translocations, which are associated with tumor formation
   and even cell death ([157]Kaina, 2003, [158]Khanna and Jackson, 2001,
   [159]Rich et al., 2000, [160]Richardson and Jasin, 2000). In response
   to DNA damage, hESCs arrest at the G[2]/M checkpoint, activate DNA
   repair systems such as BER, MMR, and DSB repair, and finally induce
   apoptosis, depending on the severity of damage or the success of the
   DNA repair processes ([161]Liu et al., 2014, [162]Momcilovic et al.,
   2010).

   In our study we found that SIRT1 was required for hESC survival,
   because SIRT1 inhibition robustly induced apoptotic cell death.
   Strikingly, we found that inhibiting SIRT1 activity dramatically
   decreased the level of three DNA repair proteins, MSH2, MSH6, and
   APEX1. Both MSH2 and MSH6 are principal components of MutS (e.g., the
   mismatch repair complex), and MutS dysfunction leads to single-strand
   DNA damage ([163]Westmoreland and Resnick, 2013). APEX1 is a major
   component of BER, and APEX1 malfunction also leads to single-strand DNA
   damage ([164]Hable et al., 2012). The single-strand DNA damage
   generated by SIRT1 inhibition became DSBs and eventually caused
   apoptosis. In accordance with this finding, resveratrol treatment
   increased the expression of SIRT1 and, hence, DNA repair proteins
   ([165]Figure 3D), thereby decreasing DSBs in response to genotoxic
   stress ([166]Figure 5C). Evidently, this observation was in agreement
   with findings from a previous report indicating that SIRT1
   overexpression leads to increased survival after oxidative stress
   ([167]Oberdoerffer et al., 2008). Consequently, our findings indicated
   that SIRT1 is a critical regulator in the orchestration of multiple DNA
   repair systems and is important for maintaining genomic fidelity and
   integrity in hESCs.

   One important issue to consider is whether p53 plays a critical role in
   the associations among SIRT1 inhibition, DNA damage induction, and DNA
   damage-mediated apoptosis. On the basis of previous reports showing
   that SIRT1 inhibition activates p53 through acetylation, it is
   conceivable that Tenovin-6 treatment enhances p53 acetylation in hESCs,
   which in turn leads to (1) a rapid decrease in DNA damage repair
   proteins, (2) a subsequent increase in DNA damage, and (3) DNA
   DSB-mediated apoptosis. To resolve this issue, we examined the levels
   of acetylated p53 and the three DNA repair proteins in hESCs at an
   early time point (i.e., 2 hr) after Tenovin-6 treatment. Strikingly,
   the levels of all three DNA repair proteins analyzed, MSH2, MSH6, and
   APEX1, were rapidly decreased in the absence of p53 acetylation, thus
   suggesting that acetylated p53 may not be responsible for the rapid
   decrease in the DNA repair proteins ([168]Figure 6C). Intriguingly, we
   observed DSBs as early as 2 hr after Tenovin-6 treatment
   ([169]Figure S4B), thus also indicating that DNA DSBs begin forming
   rapidly in the absence of acetylated p53. Therefore, it is plausible
   that activation of p53 by SIRT1 inhibition may not be involved in early
   events (i.e., decrease in DNA repair proteins and induction of DNA
   damage) but may be involved in the late stage (i.e., DSB-mediated
   apoptosis) of SIRT1/DNA damage-mediated apoptosis ([170]Figure 7).

Figure 7.

   [171]Figure 7
   [172]Open in a new tab

   The Mechanism Underlying the Role of SIRT1 in hESC Survival

   A high activity/level of SIRT1 in hESCs maintains sufficient levels of
   certain DNA repair enzymes. For example, we detected a significant
   decrease in MSH2 and MSH6 as well as APEX1 after inhibition or
   knockdown of SIRT1. The efficient repair of genetic aberrations by
   these DNA repair proteins prevents small mutations from progressing
   into hazardous DSBs, which eventually lead to cellular apoptosis. In
   the absence of SIRT1 activity in hESCs, the level of several DNA repair
   enzymes is rapidly decreased by the proteasome and/or another unknown
   pathway. The early rapid decrease in the DNA repair proteins does not
   appear to be mediated by acetylated p53. The lack of sufficient DNA
   repair allows DSB DNA damage to dramatically increase, and this is
   followed by increased cell death. Acetylation of p53 and concomitant
   BAX and PUMA overexpression have been implicated in the DNA
   damage-mediated apoptosis.

   At this stage, we do not fully understand how SIRT1 modulates the level
   of DNA repair proteins. However, our results demonstrated a connection
   between SIRT1 and the proteasome pathway. Although beyond the scope of
   this study, it would be interesting to unveil the detailed mechanism
   explaining the link between SIRT1 and the proteasome-mediated decrease
   in DNA repair proteins.

   In summary, our study showed that SIRT1 is indispensable for hESC
   survival and that the modulation of the levels of multiple DNA repair
   proteins is at least partially responsible for the SIRT1-mediated
   prevention of cell death ([173]Figure 7). Given the ever-increasing
   importance of hESCs as a cell source for replacement therapy,
   this study offers valuable insight into the growth and maintenance of
   DNA damage-free hESCs for clinical applications.

Experimental Procedures

Culture of hESCs

   The human ESC line H9 (WiCell) was cultured in hESC medium containing
   DMEM-F12 supplemented with 20% Knockout-Serum Replacement medium
   (Invitrogen), 1× non-essential amino acids (Invitrogen), 0.1 mM
   β-mercaptoethanol (Sigma), and 4 ng/mL basic fibroblast growth factor
   (bFGF) (Invitrogen) on a layer of mitotically arrested STO cells
   (ATCC). hESC colonies were transferred onto fresh feeder cells every
   7 days by either a mechanical method or enzymatic passaging using
   collagenase type IV (Sigma). For feeder-free culture, hESCs were
   cultured on Matrigel-coated dishes in TeSR-E8 medium (STEMCELL
   Technologies). All the hESC experiments were approved by the
   Institutional Ethical Committee, Yonsei University College of Medicine
   (4-2015-1097).

Differentiation of hESCs In Vitro

   hESC clumps were transferred to uncoated 35-mm bacterial dishes (SPL
   Life Science) to be cultured as EBs in suspension culture, then
   cultured on Matrigel-coated dishes in differentiation medium. The
   differentiation medium contained the same components as the hESC
   medium, except that bFGF was omitted and 2% fetal bovine serum was
   added (Invitrogen).

Transfection of Small Interfering RNAs

   The siRNA sequences used in this study are as follows: siRNA-αSIRT1
   (forward: 5′-GCA CAG AUC CUC GAA CAA UUC UU-3′, reverse: 5′-GAA UUG UUC
   GAG GAU CUG UGC UU-3′); siRNA-αSIRT2 (forward: 5′-GAA GAC AUU GCU UAU
   UGG AUU-3′, reverse: 5′-UCC AAU AAG CAA UGU CUU CUU-3′); and
   siRNA-Scrambled (forward: 5′-CCU CGU GCC GUU CCA UCA GGU AGU U-3′,
   reverse: 5′-CUA CCU GAU GGA ACG GCA CGA GGT T-3′). Double-stranded
   siRNAs were synthesized by Genolution. hESCs were treated with 10 μM
   ROCK inhibitor Y27632 (EMD Millipore) for 1 hr before harvesting. The
   cells were dissociated for 4 min using TrypLE Select (Invitrogen) and
   neutralized by TeSR-E8 cell culture medium. The cells were centrifuged
   at 100 × g for 3 min, washed with 1× PBS, and resuspended in R buffer
   from the Neon Transfection system (Invitrogen). The reaction contained
   100 nM siRNA, and transfections were performed using the Neon
   Transfection system at 900 V, 20 ms, and 1 pulse. The cells were then
   immediately plated onto Matrigel-coated plates with Y27632, followed by
   a medium change the next day.

Apoptosis Analysis

   The apoptosis of hESCs was detected using a fluorescein isothiocyanate
   (FITC) Annexin V Apoptosis Detection Kit (BD Biosciences). Cells were
   harvested and resuspended in binding buffer at a concentration of 1 ×
   10^6 cells/mL. A total of 100 μL of the solution was transferred to a
   5-mL culture tube and treated with 5 μL each of Annexin V-FITC and PI.
   The cell suspension was incubated for 15 min at room temperature in the
   dark and then analyzed by flow cytometry using a FACS LSRII (BD
   Biosciences).

Western Blotting

   Cells treated with various concentrations of Tenovin-6 were lysed with
   RIPA buffer (Sigma) containing 1× protease inhibitor cocktail (Roche
   Applied Science). The extracts (20 μg of total protein) were separated
   on polyacrylamide-SDS gels and transferred to polyvinylidene fluoride
   membranes (Amersham). The membranes were blocked with a solution
   containing 2% BSA/1× PBS and incubated overnight with a primary
   antibody at 4°C. Subsequently, the membranes were incubated with a
   horseradish peroxidase-conjugated secondary antibody (1:100)
   (Invitrogen). The reactive proteins were visualized using an ECL
   substrate solution (Pierce) according to the manufacturer's
   instructions. Western blotting was performed using antibodies
   ([174]Table S6).

Immunocytochemistry

   Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1%
   Triton X-100. The samples then were incubated with blocking buffer (2%
   BSA in 1× PBS). The cells were incubated overnight with primary
   antibodies diluted in blocking buffer at 4°C. Primary antibodies
   ([175]Table S6) to the following proteins were used: SSEA4 (Santa Cruz
   Biotechnology), cleaved caspase-3 (Cell Signaling Technology), γH2AX
   (Invitrogen), and SIRT1 (Cell Signaling). The samples were washed with
   1× PBS and incubated with fluorescently labeled secondary antibodies
   for 30 min at room temperature. The secondary antibodies used were
   Alexa Fluor 488- or Alexa Fluor 594-labeled donkey immunoglobulin G
   (IgG)/goat IgG (1:1,000; Invitrogen). A cover glass was mounted onto
   the slides using Vectashield Hardset mounting medium (Vector
   Laboratories) containing DAPI (Invitrogen). Images were obtained on
   a DP71 fluorescence microscope (Olympus) and an LSM700 confocal
   microscope (Carl Zeiss). The Image-iT Dead Green viability stain
   (Invitrogen) was used to stain dead cells by incubation of the test
   cells with Dead Green (1:1,000) complete medium.

RNA Preparation and Real-Time qRT-PCR

   Total mRNA was extracted using an Easy-Spin Total RNA Purification Kit
   (iNtRON Biotechnology) and was used for first-strand cDNA synthesis
   using a PrimeScript RT Reagent Kit (Takara Bio) according to the
   manufacturer's instructions. Real-time qRT-PCR was performed using SYBR
   PremixExTaq (Takara Bio). Reactions were performed on a CFX96 Real-Time
   System (Bio-Rad), and the results were evaluated with CFX real-time
   detection system software. Quantification was performed by determining
   the threshold cycle value (C[T]), and target genes were normalized to
   GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Relative target gene
   expression was quantified using the comparative C[T] method. All PCRs
   were performed in triplicate. Primer sequences are shown in [176]Table
   S5.

Proteomic Analysis and the Protein-Protein Interaction Network

   A proteomic tandem mass spectrometry approach was applied to reveal how
   proteins were differentially regulated by SIRT1 inhibition in hESCs. To
   this end, hESCs were treated with Tenovin-6 for 2 hr and the
   differentially regulated proteins were analyzed with respect to control
   (DMSO-treated) hESCs. iTRAQ analysis identified a total of 219
   differentially regulated proteins (>4-fold), of which 46 (stringent)
   and 94 (moderate) proteins were downregulated and 23 (stringent) and 56
   (moderate) proteins were upregulated. To display the SIRT1-dependent
   protein regulation in hESCs, we used the STRING database
   ([177]http:/string-db.org) and KEGG pathway enrichment analyses.

Apurinic/Apyrimidinic Site Analysis

   A DNA damage-AP site-Assay kit (Cell-Based) (Abcam) was used for the
   determination of abasic sites in genomic DNA, according to the
   manufacturer's instructions. In brief, hESCs were treated with vehicle
   (control), 100 μM EGCG (positive control), or 5 μM Tenovin-6 for 12 hr.
   The cells were then stained for AP sites with a DNA damage-AP
   site-Assay kit. AP sites were labeled with avidin-FITC and directly
   counted under an Olympus IX 71 microscope.

Author Contributions

   D.-Y.H. and D.-W.K. designed the study; J.J., Y.J.H., H.-J.C., J.P.,
   and B.L. performed the experiments; and D.-Y.H., J.J., and D.-W.K.
   wrote the manuscript.

Acknowledgments