Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress

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Research Article

Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress Yingying Zhai a,b,1, Xi Chen a,b,1, Dehai Yu a,b, Tao Li b, Jiuwei Cui a, Guanjun Wang a, Ji-Fan Hu a,b,n, Wei Li a,nn a b

Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061, PR China Stanford University Medical School, Palo Alto Veterans Institute for Research, Palo Alto, CA 94304, USA

art ic l e i nf o

a b s t r a c t

Article history: Received 10 May 2015 Received in revised form 3 June 2015 Accepted 6 June 2015

Histone deacetylase inhibitor valproic acid (VPA) has been used to increase the reprogramming efficiency of induced pluripotent stem cell (iPSC) from somatic cells, yet the specific molecular mechanisms underlying this effect is unknown. Here, we demonstrate that reprogramming with lentiviruses carrying the iPSC-inducing factors (Oct4-Sox2-Klf4-cMyc, OSKM) caused senescence in mouse fibroblasts, establishing a stress barrier for cell reprogramming. Administration of VPA protected cells from reprogramming-induced senescent stress. Using an in vitro pre-mature senescence model, we found that VPA treatment increased cell proliferation and inhibited apoptosis through the suppression of the p16/p21 pathway. In addition, VPA also inhibited the G2/M phase blockage derived from the senescence stress. These findings highlight the role of VPA in breaking the cell senescence barrier required for the induction of pluripotency. & 2015 Elsevier Inc. All rights reserved.

Keywords: Valproic acid Stem cell Senescence iPSC Pluripotency p16 p21 Cell cycle

1. Introduction Terminally-differentiated somatic cells can be reprogrammed into pluripotent stem cells (iPSC) by inducing the expression of a combination of factors associated with pluripotency (Oct4, Sox2, Klf4, and c-Myc) [1]. The iPSCs provide a versatile and ethical alternative to create patient-specific stem cells for regenerative medicine and human disease research with a wide range of biotechnological and therapeutic applications [2]. However, generation of iPSCs from human somatic cells using defined factors is an extremely inefficient process [3,4]. Methods to improve reprogramming efficiency and to ensure genomic integrity and safety of iPSCs must be warranted before this technology can be translated into clinical application [5,6]. Numerous attempts have been made to improve the efficiency of iPSC induction, including the use of DNA methylation inhibitors [7], histone modification inhibitors [8], inhibition of tumor n Corresponding author at: Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061, PR China. nn Corresponding author. E-mail addresses: [email protected] (J.-F. Hu), [email protected] (W. Li). 1 Equal contribution to the work.

suppresser genes [9–11], antioxidants, vitamin C [12], and signaling pathway inhibitors [8,13–15]. Valproic acid (VPA), a well-established histone deacetylase inhibitor [16–18], has been demonstrated to have the ability of pluripotency-promoting activity [8]. In the presence of cytokine cocktails, VPA enhances long-term engraftment of hematopoietic stem cells [19] and stimulates selfrenewal [20,21]. In the clinic, VPA has been used for the treatment of epilepsy, bipolar mania and migraine prophylaxis [22,23]. However, little is known about the molecular mechanisms by which VPA mediates the promotion of pluripotency. Senescence and cellular reprogramming are deeply intertwined processes [5]. Fibroblasts cultured from old mice, which have high levels of the Ink4b/Arf/Ink4a, are less efficiently reprogrammed than are cells from young mice [11]. Lentiviral delivery of iPSCinducing factors (OSKM) often creates cell stress, cell cycle pause, and apoptosis. Suppression of cell cycle regulator genes, like p16, p21, and p53 [9,24,25], promotes cell reprogramming. In the process of cell reprogramming [26], we noticed that supplementation with VPA significantly increased cell proliferation in OSKM lentiviruses-transfected cells, suggesting a possible anti-senescence role of VPA in cellular reprogramming. In this study, we examined whether the VPA treatment improved iPSC induction through a

http://dx.doi.org/10.1016/j.yexcr.2015.06.003 0014-4827/& 2015 Elsevier Inc. All rights reserved.

Please cite this article as: Y. Zhai, et al., Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.003i

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2

2. Materials and methods

BSA, and incubated with primary antibodies for pluripotent markers (SSEA4, Nanog, Sox2). After washing with PBS, cells were incubated with Cy3 or Alexa Fluor 488 labeled secondary antibodies. Fluorescence images were acquired with a Zeiss AxioCam Camera.

2.1. Cell lines and cell culture

2.5. Reprogramming-induced cell senescence

MBW2 cells were fibroblast-like cells derived from culturing of M. spretus-Balb/c F1 mouse bone marrow mesenchymal stem cells [27,28]. Cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1% non-essential amino acid (NEAA) and 1% antibiotics (penicillin-streptomycin) at 37 °C in an atmosphere containing 5% CO2. Mouse muscle-derived fibroblasts (MDFs) and brain-derived fibroblasts (BDFs) were cultured from a CF-1 mouse fetus as previously described [27,28]. Briefly, fresh fetal tissues were minced with a razor and cultured in 6-well plates with minimum DMEM medium that just covered the tissue. Three days after culturing, fibroblast-like cells expanding around the tissue were digested with trypsin and passaged for reprogramming and senescence assays.

Reprogramming with OSKM cocktail lentiviruses induced cell senescence, particularly in primary fibroblasts when cultured in KSR-containing mES medium. To observe cell senescence, fibroblast-like cells were transfected with OSKM lentiviruses and cell reprogramming was induced in the presence of VPA. Seven days after OSKM lentiviral infection in the presence of VPA, cells were collected and seeded into new 6-well plates to get rid of feeder cells. The lentivirus-induced senescence was analyzed by the staining of senescence-associated β-galactosidase (SA β-gal).

mechanism involved in the suppression of reprogramming-induced senescence stress.

2.2. Promotion of mouse cell reprogramming by VPA Mouse cell reprogramming by iPSC-inducing factors was performed as previously described [26,29]. Briefly, the Oct4-Sox2Klf4-c-Myc (OSKM) lentiviruses were packaged and produced in 293T cells by co-transfecting the lenti-OSKM with viral packaging vectors using lipofectamine 2000 (Invitrogen, CA). The virus-containing supernatants were collected at 48 h and 72 h and concentrated with Amicon Ultra-15 Centrifugal Filter Units (Millipore, MA). For reprogramming, fibroblast-like cells were seeded into 12well plates at 3
104 cells per well in DMEM/F12 (Invitrogen, CA) supplemented with 20% knockout serum replacement (KSR), 0.1 mM beta-mercaptoethanol, L-Glutamine, and 1
10–4 M nonessential amino acids (Invitrogen, CA), and were infected with concentrated lentiviruses in the presence of polybrene (8 mg/ml). Three days after infection, the cells were digested and 3–4
104 cells were transferred to 100 mm dishes on mitomycin C-inactivated MEF feeder cells. The media were replaced with ES medium (DMEM/F12 supplemented with 20% KSR), 10 ng/ml Leukemia inhibitory factor (LIF, Sigma), 10 ng/ml β-FGF (PeproTech), 0.1 mM β-mercaptoethanol, L-Glutamine, and 1
10  4 M non-essential amino acids [30]. As previously described [8], 0.5–1.0 mM VPA was added to the medium to promote reprogramming. 2.3. Alkaline phosphatase staining Reprogramming efficiency was measured by staining the iPSC colonies using an alkaline phosphatase (AP) kit (Millipore, MA) following the manufacturer's instruction. The cells were fixed in 4% paraformaldehyde/PBS for 1–2 min, rinsed with PBS and then incubated with staining solution in the dark at room temperature. After 15 min, colonies of cells expressing AP (red colonies) were recorded using a microscope-mounted camera [26,29]. 2.4. Pluripotent marker staining After expansion, the isolated iPSC colonies were characterized by staining pluripotent markers using the method as previously described [18,20]. Briefly, cells were fixed with 4% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS containing 3%

2.6. Induction of premature senescence Cell reprogramming using OSKM lentiviruses is a time-consuming process and requires the support of mitomycin C-inactivated MEF feeder cells. To better study the role of VPA in cell senescence, we adopted a simple in vitro premature senescence cell model in the following study. In this model, premature senescence was induced in MBW2 cells and primary culture cells using a copper method as previously reported [31–34]. Copper, an essential micronutrient, plays a catalytic role in the activity of several enzymes through changes of its oxidation state (e.g. cytochrome c oxidase) and the generation of ROS [35]. Copper is implicated in the aging process and in age-associated disorders such as Alzheimer disease [36]. Briefly, fibroblast-like cells at 60% confluence were exposed to low doses of copper (400–600 mM CuSO4). After exposure, VPA (1 mM) was added to the medium and the cells were allowed to grow for 48 h. Cell senescence was determined by SA β-gal staining. 2.7. Staining of senescence-associated

β-galactosidase

Cell senescence was analyzed by staining of senescence-associated β-galactosidase (SA β-gal). The activity of SA β-gal was determined using the method as described by Beane et al. [37]. Briefly, cells were washed with PBS and fixed by 3% formaldehyde for 3–5 min at room temperature. After washing with PBS, cells were incubated at 37 °C with freshly prepared senescence-associated SA β-gal staining solution: 1 mg/ml 5-bromo-4-chloro-3indolyl-beta-D-galactopyranoside (X-Gal), 40 mM citric acid/sodium phosphate, pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, 2 mM MgCl2. After overnight incubation, cells demonstrating SA β-gal staining were counted using a microscope-mounted camera. 2.8. Cell Viability Cell survival was measured using the MTT (3-(4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide) assay [38,39]. Cells (1
104/well) were plated onto 96-well plates. After VPA treatment, cells were incubated with 20 ml 5 mg/ml MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide](Sigma, MO) per well at 37 °C for 4 h. After removal of the media, 150 ml DMSO was added, and the cells were shaken using an orbital shaker for 10 min. The cell absorbance was measured at 490 nm using a microplate reader (Bio-TEK Instruments, USA). Cells treated with equal volume of PBS were set the control. Cell viability (%) was calculated based on the following equation:

Please cite this article as: Y. Zhai, et al., Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.003i

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Cell viability (%)¼(Asample/Acontrol)
100%, where Asample and Acontrol represent the absorbance of the sample and control wells, respectively. 2.9. Cell proliferation assay The cell proliferation rate was measured using a sulforhodamine B (SRB) colorimetric assay. MBW2 cells and primary muscle cells (1x104/well) in 96-well plates were fixed with trichloroacetic acid for 1 h at 4 °C and were stained by 0.4% (w/v) SRB dissolved in 1% acetic acid for 30 min. After washing, the protein-bound dye was dissolved with 10 mM Tris base. The absorbance was read at a wavelength of 564 nm and recorded using a microplate reader (Molecular Devices, CA). 2.10. Cell cycle analysis Cell cycle was evaluated using Fluorescence-Activated Cell Sorter (FACS) analysis. Cells were seeded in 6-well plates at 3
105 cells per well. The cells were harvested by trypsin digestion, washed with cold PBS (4 °C), centrifuged and fixed in 70% ethanol at 4 °C for 24 h. The fixed cells were collected after brief centrifugation and suspended in 500 ml PBS containing 0.1% triton X-100, 200 mg/L RNaseA (Sigma) and 15 mg/L propidium iodide (Sigma, MO). After incubation for 30 min at 4 °C, samples were subjected to FACS analysis [42,43]. 2.11. RT-PCR Total RNA was extracted by TRIzol reagent (Sigma, MO) from cells and stored at  80 °C. The following primers were used for PCR: (1). β-Actin forward, 5′-CAG GTC ATC ACC ATT GGC AAT GAG C-3′ and reverse, 5′-CGG ATG TCC ACG TCA CAC TTC ATG A-3′; (2). Caveolin 1 (Cav-1) forward, 5′-TCC CAT CCG GGA ACA GGG CAA CAT-3′ and reverse, 5′-GTC CCT TCT GGT TCT GCA ATC-3′; (3). p16 forward, 5′-GTG TGC ATG ACG TGC GGG-3′ and reverse, 5′-GCA GTT CGA ATC TGC ACC GTA G-3′; (4). p21 forward, 5′-GTG GGC CCG GAA CAT CTC AGG-3′, and reverse, 5′-ATG GGG AAG AGG CCT CCT GA-3′; (5). Apolipoprotein J (Apo-J) forward, 5′-GGT CTC wGA CAA TGA GCT CCA-3′ and reverse, 5′-TCC CAG AGG GCC ATC ATG GTC-3′; and (6). Heme oxygenase 1 (OX-1) forward, 5′-CAG CAT GCC CCA GGA TTT GTC-3′ and reverse, 5′-CAk GTC CTG CTC CAG GGC AGC-3′. RT-PCR reaction was performed with a Bio-Rad Thermol Cycler. The amplification was composed of 1 cycle at 95 °C for 5 min, 33 cycles at 95 °C for 20s, 62 °C for 15 s and 72 °C for 15 s, and 1 cycle at 72 °C for 10 min. Results were normalized to the internal control β-Actin [40,41]. Quantitative real-time RT-PCR amplification was performed using SYBR GREEN PCR Master (Applied Biosystems, USA) as previously described [42,43]. The threshold cycle (Ct) values of target genes were assessed by quantitative PCR in triplicate using a sequence detector (ABI Prism 7900HT; Applied Biosystems) and were normalized over the Ct of the β-actin control. 2.12. Statistical analysis All experiments were performed in triplicate, and the data were expressed as mean 7SD. Data were analyzed using SPSS software (version 16.0; SPSS, Inc., IL). Student's t-test or one-way ANOVA (Bonferroni test) was used to compare statistical differences for variables among treatment groups. Results were considered statistically significant at p r 0.05.

3

3. Results 3.1. VPA enhances cell reprogramming The MBW2 cells were derived from continuous culturing of bone marrow mesenchymal stem cell (MSC) of the M. spretusBalb/c F1 mouse. To demonstrate if VPA is able to promote cell reprogramming in MBW2 cells, we constructed a polycistronic viral vector, containing the mouse Oct4-Sox2-Klf4-c-Myc (OSKM) [6,26]. Each of the polycistronic factors was separated by a linker sequence encoding a translation skipping peptide T2A, which allows translation to skip and produces two proteins from a single RNA transcript [44]. Three days after viral infection, MBW2 cells were collected and transferred onto mitomycin C-inactivated MEF feeder cells. The media were replaced with ES medium containing 1 mM VPA as previously reported [8], and iPSC-like colonies were identified by AP staining (Fig. 1A) [26,29]. We found that in MBW2 cells, VPA significantly increased the number of AP-positive iPSC colonies (Fig.1B and C) as has been previously reported [8]. The isolated iPSC cells were expanded on MEF feeders and were characterized by immunohistochemical staining of pluripotent markers. As seen in Fig. 1D, the isolated iPSC colonies were positive for pluripotency markers, including SSEA4, Nanog and Sox2. 3.2. VPA inhibits reprogramming-induced cell senescence Cell aging is a critical barrier to cell reprogramming [24]. In the process of iPSC induction, we noticed significant premature senescence after transduction of the OSKM viruses, particularly when the cells that had experienced more passages were used in the study. Therefore, we examined if VPA was able to inhibit reprogramming-induced senescence (Fig. 2A). After lentiviral infection, cells were treated with 1 mM VPA to enhance reprogramming as previously described [8]. Cell senescence was examined using the senescence-associated beta-galactosidase (SA β-gal) assay. The OSKM-transduced MBW2 cells grew poorly (Fig. 2B, top panel 1) and exhibited a significantly increased senescence (Fig. 2B, bottom panel 1) as defined by betagalactosidase (SA β-gal) staining. However, we noticed that treatment with VPA dramatically reduced cell senescence in virally-infected MBW2 cells and enhanced cell proliferation (Fig. 2B, panel 2). 3.3. Reduction of chemically-induced premature senescence by VPA treatment Cell reprogramming by OSKM cocktail factors is a time-consuming process and needs the niche support of the mitomycin C-inactivated MEF feeder cells. Thus, we adopted a simple in vitro premature senescence cell model, in which cells were treated with a low dose of copper. After copper treatment, cells underwent typical premature senescence in MBW2 cells, with altered morphology from small spindle to enlarged spread shape. The senescent cells were stained positively for SA β-gal (Fig. 3A, panel 1). After treatment with VPA, cells were collected and stained for the presence of SA β-gal. We found that VPA protected copper-treated cells from premature senescence and exhibited a better cell proliferation than that in copper-treated cells (Fig. 3A, panel 2). No significant senescence was noticed in the VPA and PBS control cells (Fig. 3A, panels 3–4). We also tested the protective role of VPA in muscle-derived fibroblasts and brain-derived fibroblasts. Treatment with copper significantly induced premature senescence in these two cells, and the degree of senescence was inhibited by the VPA treatment (Fig. 3B, C, panels 2 vs 1).

Please cite this article as: Y. Zhai, et al., Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.003i

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Fig. 1. VPA promotes the induction of iPSC in MBW2 cells. A. Schematic diagram of VPA to enhance iPSC induction. OSKM: Oct4-Sox2-Klf4-c-Myc. AP: alkaline phosphatase staining. B. Enhanced generation of AP-positive colonies by VPA. C. Quantitation of AP-positive colonies. The rate of iPSC induction was given as the average number of alkaline phosphatase (AP)-positive colonies per 10,000 transduced cells from three independent experiments. **p o 0.01 as compared with the OSKM group. D. Expanded iPSC colony. The isolated iPSC colonies were immunohistochemically stained for pluripotency markers SSEA-4, Nanog and Sox2.

Fibroblasts

Morphology OSKM retroviral infection VPA

β-gal staining

SA β-gal staining

OSKM

OSKM+VPA

copGFP

Fig. 2. VPA reduces senescence in retroviral OSKM-transfected cells. A: Schematic diagram of VPA treatment. B. SA β-galactosidase staining. Top panel: morphology of lentivirus-infected cells. Bottom panel: SA β-galactosidase staining. OSKM þ VPA: the OSKM-transfected cells were treated with 1 mM VPA for seven days; copGFP: the control cells that were transfected with the lentivirus carrying the copGFP reporter gene. Note the reduced cell proliferation and the increased β-galactosidase activity (arrowed) in lentivirus-transfected cells compared with that in the VPA control group.

3.4. Enhanced cell proliferation by VPA treatment Using the sulforhodamine B (SRB) assay, we found that exposure of MBW2 cells to copper significantly reduced cell proliferation. However, VPA protected cells from induced senescence, leading to increased cell growth (Fig. 4). This is particularly true for the muscle-derived fibroblasts, which were more sensitive to the chemical exposure. 3.5. VPA protects the senescent cells by inhibiting p16/p21 pathway genes We then examined the molecular mechanisms related to the protection by VPA in our senescence model. We collected cells from different treatment groups and used RT-PCR to examine the expression of genes that have been reported to associate with cell senescence. As shown in Fig. 5A, copper exposure upregulated the

expression of several senescence pathway genes, including p16, p21, Cav-1, Apo-J, and OX-1 (lanes 3–4). However, treatment with VPA attenuated the activation of the senescence pathway (lanes 5–6). The activation of the senescence pathway genes was also confirmed using the quantitative qPCR (Fig. 5B, p o0.01). 3.6. VPA releases the G2/M blockage As p16 and p21 are involved in the regulation of cell cycle, we examined if the VPA treatment would affect the cell cycle in the prematurely senescent cells. As compared with VPA and PBS controls (Fig. 6A–B), exposure to copper significantly inhibited cell division by blocking cells at the G2/M phases (Fig. 6C, 68.3%). However, treatment of cells with VPA diminished the G2/M blockage (Fig. 6D, 45.3%).

4. Discussion Valproic acid (VPA) has been used successfully to promote cell reprogramming of neonatal foreskin fibroblasts into iPSCs [8]. VPA is a potent histone deacetylase (HDAC) inhibitor that is widely used as an antiepileptic, anti-mania, and anti-migraine drug. VPA is also currently being evaluated for cancer treatment. In general, histone acetylation is associated with an active gene promoter. It is presumed that its role as an HDAC inhibitor would lead to increased histone acetylation in the promoter of genes that are involved in pluripotency. Surprisingly, VPA treatment alone failed to activate the promoters of several pluripotency-related genes, like Oct4, Sox2, and Nanog. When VPA was used in conjunction with the OSKM factors, it was unable to immediately activate these endogenous genes until iPSC colonies were formed. Clearly, activation of these pluripotency genes by the increased histone acetylation is not the immediate pathway, thus suggesting alternative mechanisms. Cell senescence induced by OSKM viral infection is a welldocumented barrier to iPSC reprogramming. In this report, we

Please cite this article as: Y. Zhai, et al., Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.003i

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5

Aging

Aging+VPA

Control

VPA

Aging

Aging+VPA

Control

VPA

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Fig. 3. VPA inhibited in vitro pre-mature senescence. Premature senescence was induced by CuSO4 in MBW2 (A), muscle-derived fibroblasts (B), and brain-derived fibroblasts (C) (the Aging group). After senescence induction, cells were treated with 1 mM VPA (the Agingþ VPA group). In the control group, cells were treated with 1 mM VPA only (the VPA group). Cell senescence was measured by SA β-galactosidase staining (red arrow). The cells in the Aging group exhibited cell enlargement and the increased βgalactosidase activity compared with cells in the Aging þVPA group and the control groups.

PBS Aging Aging+VPA VPA

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Fig. 4. Cell proliferation in VPA-treated cells. MBW2 and muscle-derived fibroblasts were induced into premature senescence. Cells were collected at different time points (24 h, 48 h, 72 h, and 96 h) and cell proliferation was assessed by the SRB assay. All data shown are mean 7 SEM from three independent. **p o 0.01 as compared with the PBS control group.

demonstrate that VPA treatment significantly attenuates cell senescence in the virally-reprogrammed fibroblasts as shown by the improvement of cell growth, the reduction of senescence-associated β-gal activity, and the increased generation of iPSC colonies. Similarly, in a chemically-induced senescence model, the administration of VPA also significantly protects against cell death and increases cell growth. Senescence leads to activation of cycle checkpoint genes, such as p16 and p21, as well as senescence genes, Apo-J and OX-1. In a parallelly-conducted study, we have also found that OSKM-reprogramming causes senescence in two human bone marrow MSC-derived cells (HSC-J2 and HSC-L1). Treatment with VPA attenuates this reprogramming-induced senescence stress (Chen et al. unpublished data). Taken together, these data suggest that VPA treatment promotes cell reprogramming in part by inhibiting the senescence barrier imposed by reprogramming-induced stress. The critical role of senescence during cell reprogramming has been well documented in other models. Banito et al. [24] noticed that expression of the reprogramming factors impaired cell proliferation and increased the percentage of cells arrested in G1, without inducing apoptosis. Knock-down of p16Ink4a/p19Arf, p21Cip1, or p53 by shRNAs increased reprogramming efficiency upon depletion of senescence effectors. Yi et al. [45] observed that decreased p53 activity favored the entire process of somatic cell reprogramming. Reactivation of p53 at any time point during the reprogramming process not only interrupted the formation of iPSCs, but also induced newly formed stem cells to differentiate. Senescence is associated with the irreversible cell cycle arrest

Please cite this article as: Y. Zhai, et al., Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.003i

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C

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ol A

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Fig. 5. VPA inhibits senescence pathway genes. (A) RT-PCR analyzes of senescence pathway genes. (B) Quantitation of senescence genes by qPCR. All data shown are mean 7SEM from three independent. *p o0.01 as compared with the control and the VPA groups; #po 0.01 as compared with the aging group.

G1

60.8%

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G1

22.9%

G2/M 68.3%

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66.2%

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Fig. 6. Attenuation of G2/M phase blockage by VPA. As compared to the control (A), MBW2 cells treated with copper (C) were arrested in G2/M phase, with the ratio increasing from 31.9% to 68.3%. The arrest was extenuated by the treatment of VPA (D). VPA itself (B) alone did not affect cell cycle obviously.

samples, VPA maintained a significantly higher proportion of CD34 þLPC and colony forming units compared to control cultures [49]. Quite recently, Jung et al. also demonstrated that VPA protected human bone marrow-mesenchymal stromal stem cells against oxidative stress by upregulating KRIT1 and its target proteins (SOD2, FoxO1 and cyclin D1) [50]. In this study, we found that VPA treatment increased cell proliferation and inhibited senescence through the suppression of the p16/p21 pathway and the G2/M phase blockage. Thus, it seems that the effect of VPA on cell proliferation may depend on cell types, VPA doses, and experimental models. In summary, ectopic expression of four defined cocktail factors (OSKM) in mouse fibroblast-like cells triggers significant premature senescence, particularly in those cells derived from adult skin or cells that have been passed in cell culture for many passages. This reprogramming-induced senescence impairs the induction of iPSCs. The data in this study suggest that VPA improves cell reprogramming at least partly through the suppression of the p16/p21 pathway and the inhibition of G2/M phase blockage.

Conflict of interest No conflicts of interest are declared by the authors.

during the G1 transition, which can be elicited by replicative exhaustion or in response to stresses, primarily through activation of p53 and the up-regulation of the cycle dependent kinase (CDK) inhibitors p16INK4a and p21CIP1. Among the p53 target genes, p21, a cyclin-dependent kinase inhibitor may also function in initiating senescence. In this study, we also observed that lentiviral infection of fibroblasts triggered the activation of p16 and p21. We also observed that senescent cells exhibited significant blockage of cell division at the G2/M phases. VPA inhibited the expression of these senescent genes and released the G2/M blockage, leading to the acceleration of cell reprogramming. It should be noted that the published data regarding the role of VPA on cell proliferation are quite controversial. For example, Fortson et al reported that VPA induced senescence and apoptosis in ERG-positive prostate cancer cells by upregulating the p21/ Waf1/CIP1 pathway [46]. Witt et al showed that VPA inhibited the proliferation and migration of primary murine prostate cancer cells by upregulating cyclin D2, but had no effect in fibroblasts, which typically have high basal levels of cyclin D2 [47]. In human mesenchymal stem cells, Lee et al reported that VPA flattened the morphology of MSCs and inhibited their growth by activating the p21(CIP1/WAF1)/p16(INK4A) pathway [48]. In contrast, two groups independently reported that VPA enhances proliferation and self-renewal in normal hematopoietic stem cells [20,21]. Similarly, Bug et al. reported that in acute myeloid leukemia (AML)

Acknowledgment This work was supported by California Institute of Regenerative Medicine (CIRM) Grant (RT2-01942), Jilin International Collaboration Grant (#20120720), the National Natural Science Foundation of China Grant (#81272294, #31430021) to J.F.H.; National Natural Science Foundation of China (#81372835) and Jilin Great Science Grant (#11ZDGG003) to W.L.; the National Natural Science Foundation of China grant (#81302380) and Development Foundation for Youths of Jilin Provincial Science & Technology Department grant (#20140520017JH) to D.Y.

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Please cite this article as: Y. Zhai, et al., Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.003i

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Please cite this article as: Y. Zhai, et al., Histone deacetylase inhibitor valproic acid promotes the induction of pluripotency in mouse fibroblasts by suppressing reprogramming-induced senescence stress, Exp Cell Res (2015), http://dx.doi.org/10.1016/j.yexcr.2015.06.003i