TY - JOUR
AU - Lee, Dong-Seok
AB - Abstract Redox balance has been suggested as an important determinant of “stemness” in embryonic stem cells (ESCs). In this study, we demonstrate that peroxiredoxin (Prx) plays a pivotal role in maintenance of ESC stemness during neurogenesis through suppression of reactive oxygen species (ROS)-sensitive signaling. During neurogenesis, Prx I and Oct4 are expressed in a mutually dependent manner and their expression is abruptly downregulated by an excess of ROS. Thus, in Prx I−/− or Prx II−/− ESCs, rapid loss of stemness can occur due to spontaneous ROS overload, leading to their active commitment into neurons; however, stemness is restored by the addition of an antioxidant or an inhibitor of c-Jun N-terminal kinase (JNK). In addition, Prx I and Prx II appear to have a tight association with the mechanism underlying the protection of ESC stemness in developing teratomas. These results suggest that Prx functions as a protector of ESC stemness by opposing ROS/JNK cascades during neurogenesis. Therefore, our findings have important implications for understanding of maintenance of ESC stemness through involvement of antioxidant enzymes and may lead to development of an alternative stem cell-based therapeutic strategy for production of high-quality neurons in large quantity. Stem Cells 2014;32:998–1011 Reactive oxygen species, Antioxidants, Embryonic stem cells, Neurogenesis, Peroxiredoxin Introduction Embryonic stem cells (ESCs) possess the unique ability for self-renewal and differentiation into multiple cell lineages, giving them unique therapeutic potential. Investigations into the loss of stemness and the subsequent initiation of differentiation have been increasing; however, relatively little is known about the underlying mechanisms. One recently suggested mechanism invokes the redox state as a central modulator for stemness [1-4] and differentiation [5-9]. Indeed, essential roles for reactive oxygen species (ROS) in the loss of stemness have been identified by differentiation experiments involving a variety of stem cells. In stem or precursor cells, differentiation stimuli cause elevation of ROS levels, thus inducing differentiation of the cells into specific lineages [1, 6-8, 10-13]. These observations imply a tight association of the physiological mechanism regulating ESC differentiation with signaling pathways governed by the balance between ROS generation and neutralization. Although differentiated cells frequently exhibit increased levels of ROS [7, 14, 15], antioxidant enzymes do not always exhibit expression kinetics in good accordance with intracellular ROS levels. For example, transcript levels of superoxide dismutase-2 (SOD2), glutathione peroxidases (Gpxs), and catalase are lower in mouse embryoid bodies than in undifferentiated ESCs [14, 16], whereas expression of Gpx1 is upregulated in spontaneously differentiated human ESCs [17]. Similar to the above mentioned antioxidant enzymes, peroxiredoxins (Prxs), a newly discovered family of antioxidant enzymes responsible for the removal of hydrogen peroxides (H2O2) for cellular defense [18-20] have been found to differ in self-renewing stem cells versus differentiated cells [14, 17, 21], indicating possible involvement in ROS-mediated differentiation. Therefore, the role of specific Prx isotypes in these cell types merits detailed investigation. The signaling pathways leading to the activation of mitogen-activated protein kinases (MAPKs) known as c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 MAPK, and involvement of these MAPKs in the differentiation process represent a paradigm in cellular redox signaling. During neurogenesis, activation of the ERK1/2 and JNK pathways is required for loss of stemness and neurogenesis, respectively [22-24]. In contrast, a high level of neurogenesis is attained when p38 MAPK pathways are inhibited or deleted [25, 26]. However, there have been few, if any, proposals for the underlying mechanism governing MAPK activation during loss of stemness and neurogenesis. Therefore, the specific involvement of antioxidants in modulation of redox signaling during ESC-mediated neurogenesis needs clarification. In this study, we demonstrate that ROS levels are increased during neural differentiation of ESCs and that Prx I−/− or Prx II−/− cells fail to reduce ROS levels in both ESCs and differentiated cells. ROS escaping from Prx I- or Prx II-mediated antioxidative surveillance destroy ESC stemness, subsequently leading to accelerated neuronal commitment, which appears to depend primarily on activation of JNK signaling. Collectively, Prx I and Prx II attenuate the tempo of ESC-mediated neurogenesis, and they act by suppressing the loss of stemness mediated by ROS/JNK signaling. These findings may contribute to development of stem cell therapies for currently incurable human diseases, and they increase our understanding of oxidative stress-associated developmental events. Therefore, our findings will provide important implications for understanding of oxidative stress-associated developmental events including ESC stemness maintenance and may lead to development of an alternative stem cell-based therapeutic strategy for production of high-quality neurons in large quantity. Materials and Methods Cell Culture For maintenance of J1 ESCs and for generation of ESCs from blastocysts, cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Grand Island, NY) containing 10% ESC-qualified fetal bovine serum (FBS; Invitrogen, Grand Island, NY), 5% FBS (HyClone, Logan, UT), 1× nonessential amino acids (Invitrogen, Grand Island, NY), 500 U/ml leukemia inhibitory factor (Chemicon, Temecula, CA), and antibiotics (Invitrogen, Grand Island, NY) on mitotically inactivated mouse embryonic fibroblasts (MI-MEFs). The culture medium for P19 mouse embryonic carcinoma (EC) cells and CRL-2073 human ESCs (ATCC, Manassas, VA) was DMEM containing 10% FBS (HyClone, Logan, UT) and antibiotics. Derivation of Prx I−/− or Prx II−/− ESCs For establishment of ESC lines, blastocysts were harvested from wild-type, Prx I−/−, and Prx II−/− 129/SvJae mice and cultured for the formation of inner cell mass clumps. These clumps were passaged and cultured until the appearance of colonies showing typical ESC morphology, such as a large nucleus/cytoplasm ratio and a shiny colony boundary. The individual colonies were cloned, proliferated until passage 7 or 8, and cryopreserved at −196°C. Differentiation of ESCs and Chemical Treatment Exponentially growing mouse ESCs were treated with trypsin and separated from MI-MEFs by virtue of their preferential anchorage on the surface of the culture dish. Following centrifugation, the ESCs were resuspended with neurobasal medium (NBM; DMEM/F12 containing 20 µl/ml N2 and 10 µl/ml B27 supplements [all from Invitrogen, Grand Island, NY]) and seeded on 0.1% gelatin-coated culture dishes. In a few experiments, B27 without antioxidant components (B27-AO; Invitrogen, Grand Island, NY) was used to examine the effect of ROS on neural differentiation of ESCs. The cells were passaged using 0.25% trypsin/5.3 mM EDTA (Invitrogen, Grand Island, NY), transferred to fibronectin (5 µg/ml)-coated culture dishes (Invitrogen, Grand Island, NY) at 4 days of neural differentiation, and cultured in NBM at 37°C under a 5% CO2 atmosphere. For chemical treatment during neurogenesis from ESCs, N-acetyl-l-cysteine (2.5 mM NAC; Sigma-Aldrich, St. Louis, MO), H2O2 (0.05 and 1 mM; Sigma-Aldrich, St. Louis, MO), and MAPK inhibitors (all from Calbiochem, San Diego, CA) for p38 MAPK (5 µM of SB203580), ERK (10 µM of PD98059), and JNK (2.5 or 5 µM of SP600125) were used. ROS Measurement Cells were incubated with 5 µM CM-H2DCF-DA (Invitrogen, Grand Island, NY), a ROS indicator, at 37°C for 15 minutes and washed twice with phosphate-buffered saline (PBS) by centrifugation at 200g for 5 minutes. The cells were resuspended with PBS, and the analysis of ROS levels was performed using a FACSCalibur instrument (BD Biosciences, San Jose, CA). Alkaline Phosphatase Activity Assay All activity assay procedures were performed according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO, Leukocyte Alkaline Phosphatase Kit, Cat# 86R). Briefly, after the culture medium had been discarded, the cells were fixed with citrate–acetone–formaldehyde fixative for 30 seconds at room temperature, and rinsed with calcium and magnesium PBS (Invitrogen, Grand Island, NY). They were then incubated with 200 µg/ml Naphthol AS-MX phosphate (Sigma-Aldrich, St. Louis, MO) and 1 mg/ml Fast Red TR salt (Sigma-Aldrich, St. Louis, MO) in 100 mM Tris buffer at pH 8.2 for 30 minutes at room temperature. The staining reaction was terminated by rinsing with PBS. Semi-qPCR and Real-Time RT-qPCR Total RNA was isolated using Trizol reagent (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions and quantified by spectrophotometry. The first-strand cDNA was synthesized from 0.75 µg of DNase-treated total RNA using 0.5 µg random hexamers (Invitrogen, Grand Island, NY) and 7.5 U AMV reverse transcriptase (Promega, Madison, WI) in a volume of 20 µl at 37°C for 60 minutes. Polymerase chain reaction (PCR) was performed using 22–28 cycles of 94°C for 30 seconds, 55–60°C for 30 seconds, and 72°C for 30 seconds, and a final incubation for 10 minutes at 72°C. PCR primers for amplification of human and mouse cDNAs were designed in silico using Primer3 software http://frodo.wi.mit.edu/primer3/input.htm; Supporting Information Table S2). Double-Label Immunocytochemistry Cells were fixed in 2% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) solution for 30 minutes, washed with PBS containing 0.02% Tween-20 (PBST) for 30 minutes, and then washed with PBS containing 0.26% ammonium chloride (Sigma-Aldrich, St. Louis, MO) for 30 minutes. Nonspecific binding was blocked by incubation with 10% normal goat serum (Invitrogen, Grand Island, NY) for 1 hour at room temperature. The cells were then incubated with polyclonal rabbit antibodies against Prx I (Alexis, San Diego, CA) or Prx II (Proteintech Group, Chicago, IL) overnight at 4°C. After washing with PBST for 1 hour, the cells were incubated with Alexa488- or Alexa598-conjugated goat anti-rabbit antibody (Invitrogen, Grand Island, NY) at 37°C for 1.5 hours, and the excess antibodies were removed by washing with PBST for 1 hour. For double-labeling, cells were incubated with monoclonal antibodies against SSEA1, Nestin, Tuj1, MAP2, GFAP (all from Chemicon, Temecula, CA), and Oct4 (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C and then with Alexa 488- or Alexa 598-conjugated goat anti-mouse antibody for 1 hour at 37°C. Nuclei were stained with 4 µg/ml of 4′,6-diamino-2-phenylindole (Sigma-Aldrich, St. Louis, MO) for 30 minutes at room temperature. Western Blotting Analysis Cellular proteins (30 µg/lane) were separated on 12% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore, Bedford, MA). The membranes were blotted with polyclonal rabbit antibodies specific for Prx I, Prx II (both from Abfrontier, Seoul, Korea), Oct4 (Santa Cruz Biotechnology, Santa Cruz, CA), Nanog, MAP2 (both from Chemicon, Temecula, CA), pJNK, JNK (both from Cell Signaling Technology), α-tubulin (Sigma-Aldrich, St. Louis, MO), and GAPDH (Abfrontier, Seoul, Korea) at 4°C overnight. The membranes were washed five times with Tris-buffered saline (TBS; 10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.2% Tween-20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO) for 1 hour at RT. Following removal of excess antibodies by washing with TBS, specific binding was detected using a chemiluminescence detection system (Amersham, Berkshire, U.K.) according to the manufacturer’s instructions. siRNA-Mediated Knockdown of mRNA The following small interfering RNA (siRNA) target sequences were used against human Oct4, Prx I, and Prx II. Oct4, 5′-AGC AGC UUG GGC UCG AGA A-3′; Prx I, 5′-AAA CUC AAC UGC CAA GUG A-3′; Prx II, 5′-CGC UUG UCU GAG GAU UAC GUU-3′. The following siRNA target sequences were used against mouse Oct4, Prx I, and Prx II. Oct4, 5′-AGG UGU UCA GCC AGA CCA C-3′; Prx I, 5′-GCG CUU CUG UGG AUU CUC AUU-3′; Prx II, 5′-AAA UCA AGC UUU CGG ACU AUU-3′. A scrambled siRNA sequence, 5′-UUC UUC GAA CGU GUG UCA CGU UU-3′, was used as a negative control. All siRNA oligonucleotide duplexes were purchased from Dharmacon (Lafayette, CO). The siRNAs (5 nM) were transfected into P19 or CRL-2073 ESCs using a Lipofectamine RNAi Max Transfection Reagent kit (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions. Prx I, Prx II, and Oct4 mRNA and protein levels were evaluated by both quantitative PCR (qPCR; 48 hours posttransfection) and immunoblotting assays (72 hours posttransfection), correspondingly. Teratoma Formation Following isolation of exponentially growing ESCs from MI-MEFs, 0.5–1.0 × 107 cells were loaded into a 1-ml syringe fitted with a 26-gauge needle and injected into the epidermis of athymic nude mice. The resulting tumors were surgically isolated 3–5 weeks later and analyzed using semi-qPCR and immunohistochemical staining. Immunohistochemistry Teratomas were fixed overnight with 10% neutral buffered formalin, embedded in paraffin, and processed as 5-µm-thick sections. For antigen retrieval, deparaffinized sections were briefly heated for 4 minutes in a pressure cooker containing 10 mM citrate buffer (pH 6.0). Subsequent procedures were performed at room temperature. Sections were pretreated with 3% H2O2 in 0.1 M TBS for 30 minutes to quench endogenous peroxidases, treated with protein block solution (Dako, Carpinteria, CA) for 20 minutes, and incubated with antibodies against Prx I, Prx II, Oct4, or proliferating cell nuclear antigen (PCNA; Santa Cruz Biotechnology, Santa Cruz, CA) for 30 minutes in a humidified chamber. Sections were washed with TBS containing 0.01% Tween-20, followed by incubation with EnVision anti-rabbit (Dako, Carpinteria, CA) polymer for 30 minutes. Peroxidase bound to the antibody complex was visualized by treatment with 3,3′-diaminobenzidine chromogen substrate solution (Dako, Carpinteria, CA). The color reaction was monitored under a microscope to determine optimal incubation time and was stopped using several washes with TBS. Immunolabeled sections were dehydrated in a graded ethanol series, defatted in xylene, and mounted. Sections were examined under bright-field using an Olympus BX51 microscope (Olympus, Tokyo, Japan), and the images were acquired using an Olympus DP 70 camera (Olympus, Tokyo, Japan). Statistical Analysis The data represent the mean and SD from three independent experiments (n = 3). Experimental differences were tested for statistical significance using ANOVA. A p < .05 was considered statistically significant and is indicated on graphs by an asterisk. p < .01 and < .001 are indicated by two and three asterisks, respectively. Results ROS Negatively Regulate ESC Stemness During Neurogenesis To examine the involvement of ROS in neurogenesis, ESCs were differentiated by cultivation in differentiation medium supplemented with either B27 or B27-AO (NBM or NBM–AO, respectively), incubated with the ROS indicator CM-H2DCF-DA, and subjected to flow cytometry (Fig. 1A). ROS levels increased during neurogenesis in both NBM and NBM–AO; however, a slightly greater increase was observed in NBM–AO. Open in new tabDownload slide Reactive oxygen species (ROS) cause loss of stemness during embryonic stem cell (ESC)-mediated neurogenesis. (A): Effect of antioxidant depletion on ROS generation during neurogenesis. J1 ESCs were differentiated in neurobasal medium (NBM) or NBM-antioxidant (AO) for 5 days, and DCF fluorescence intensities were measured by flow cytometry. (B, C): Effect of antioxidant depletion on the expression of stemness-associated genes during neurogenesis. J1 ESCs cultured in NBM or NBM-AO for 5 days were subjected to quantitative polymerase chain reaction (qPCR) (B) and Western blotting (C) analyses of Oct4 and Nanog. Relative transcript levels were normalized to the expression of glyceraldehyde phosphate dehydrogenase (GAPDH). Error bars indicate mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001. (D): Effect of antioxidant depletion on changes in alkaline phosphatase (AP) activity during neurogenesis. J1 ESCs were differentiated in NBM or NBM-AO for 5 days and stained for the determination of AP activity. The black arrows, white arrows, and white arrowheads indicate colonies with strong, moderate, and weak activities, respectively. The results were quantified by scoring the staining intensities of at least 100 colonies from three independent experiments. Error bars indicate mean ± SD (n = 3). Scale bar = 100 µm. (E): Effect of the addition of oxidant on ESC stemness-associated gene expression during neurogenesis. J1 ESCs were differentiated in NBM in the presence or absence of hydrogen peroxide (0.05 and 1 mM) for 5 days and subjected to qPCR analysis of Oct4 and Nanog. Error bars indicate mean ± SD (n = 3). **, p < .01; ***, p < .001 compared with untreated cells of neural differentiation 0-day. Abbreviations: AO, antioxidant; DCF-DA, 2′, 7′-dichlorofluorescin diacetate; ESCs, embryonic stem cells; H2O2, hydrogen peroxide; NBM, neurobasal medium. Open in new tabDownload slide Reactive oxygen species (ROS) cause loss of stemness during embryonic stem cell (ESC)-mediated neurogenesis. (A): Effect of antioxidant depletion on ROS generation during neurogenesis. J1 ESCs were differentiated in neurobasal medium (NBM) or NBM-antioxidant (AO) for 5 days, and DCF fluorescence intensities were measured by flow cytometry. (B, C): Effect of antioxidant depletion on the expression of stemness-associated genes during neurogenesis. J1 ESCs cultured in NBM or NBM-AO for 5 days were subjected to quantitative polymerase chain reaction (qPCR) (B) and Western blotting (C) analyses of Oct4 and Nanog. Relative transcript levels were normalized to the expression of glyceraldehyde phosphate dehydrogenase (GAPDH). Error bars indicate mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001. (D): Effect of antioxidant depletion on changes in alkaline phosphatase (AP) activity during neurogenesis. J1 ESCs were differentiated in NBM or NBM-AO for 5 days and stained for the determination of AP activity. The black arrows, white arrows, and white arrowheads indicate colonies with strong, moderate, and weak activities, respectively. The results were quantified by scoring the staining intensities of at least 100 colonies from three independent experiments. Error bars indicate mean ± SD (n = 3). Scale bar = 100 µm. (E): Effect of the addition of oxidant on ESC stemness-associated gene expression during neurogenesis. J1 ESCs were differentiated in NBM in the presence or absence of hydrogen peroxide (0.05 and 1 mM) for 5 days and subjected to qPCR analysis of Oct4 and Nanog. Error bars indicate mean ± SD (n = 3). **, p < .01; ***, p < .001 compared with untreated cells of neural differentiation 0-day. Abbreviations: AO, antioxidant; DCF-DA, 2′, 7′-dichlorofluorescin diacetate; ESCs, embryonic stem cells; H2O2, hydrogen peroxide; NBM, neurobasal medium. Next, we performed alkaline phosphatase (AP) activity assays and Western blotting and qPCR analyses for investigation of the effect of the increase in ROS level on modulation of ESC stemness. Alkaline phosphatase activity and expression levels of Oct4 and Nanog showed a more rapid decline in cells cultured with NBM–AO than in those cultured with NBM, whereas MAP2 level was increased in the NBM–AO treatment group (Fig. 1B–1D). Consistent with this result, supplementation of H2O2 (0.05 and 0.1 mM) into NBM resulted in acceleration of the decrease of Oct4 and Nanog transcript levels in a dose-dependent manner during neural differentiation of ESCs (Fig. 1E). Intercellular Distribution of Prx I and Prx II in Mouse ESC-Derived Neural Cells To examine the possible involvement of Prxs in ESC-mediated neurogenesis, we determined the expression profiles and intercellular distributions of the representative Prx isotypes Prx I and Prx II. According to data from semi-qPCR (Fig. 2A) and qPCR (Fig. 2B) analyses, the Prx II transcript level showed a gradual increase during neurogenesis in a time-dependent manner, as did the neural markers Nestin, NeuroD1, and MAP2. In contrast, Prx I expression showed a continuous decrease during neurogenesis, as did the stemness markers Oct4 and Nanog (Fig. 2A, 2B). Consistent with these results, the differential expression kinetics of Prx I and Prx II were detected by Western blotting analysis (Fig. 2C). Open in new tabDownload slide Prx I and Prx II are expressed reciprocally during neural differentiation of embryonic stem cells (ESCs). (A–C): Expression kinetics of Prx I and Prx II during ESC-mediated neural differentiation (ND). J1 ESCs were cultured in neurobasal medium (NBM) for the indicated days and subjected to semi-quantitative polymerase chain reaction (qPCR) (A), qPCR (B), and Western blotting (C) analyses of Oct4, Nanog, Prx I, Prx II, Nestin, NeuroD1, or MAP2. Error bars indicate mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001 compared with cells of 0-day of ND. (D, E): Double-label immunocytochemical analysis of Prxs and stemness (SSEA1) or neural differentiation markers (Nestin, Tuj1, MAP2, and GFAP) using J1 ESCs differentiated in NBM for 7 or 14 days. Regions of colocalization as determined by image merging are shown in yellow. Images were counterstained with 4′,6-diamino-2-phenylindole. Scale bar = 25 µm. Abbreviation: ND, neural differentiation. Open in new tabDownload slide Prx I and Prx II are expressed reciprocally during neural differentiation of embryonic stem cells (ESCs). (A–C): Expression kinetics of Prx I and Prx II during ESC-mediated neural differentiation (ND). J1 ESCs were cultured in neurobasal medium (NBM) for the indicated days and subjected to semi-quantitative polymerase chain reaction (qPCR) (A), qPCR (B), and Western blotting (C) analyses of Oct4, Nanog, Prx I, Prx II, Nestin, NeuroD1, or MAP2. Error bars indicate mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001 compared with cells of 0-day of ND. (D, E): Double-label immunocytochemical analysis of Prxs and stemness (SSEA1) or neural differentiation markers (Nestin, Tuj1, MAP2, and GFAP) using J1 ESCs differentiated in NBM for 7 or 14 days. Regions of colocalization as determined by image merging are shown in yellow. Images were counterstained with 4′,6-diamino-2-phenylindole. Scale bar = 25 µm. Abbreviation: ND, neural differentiation. Results of double-label immunocytochemical experiments indicated that the opposite expression kinetics of Prx I and Prx II were derived from their specific distributions to undifferentiated ESCs and neuron-lineage cells, respectively. Strong Prx II immunoreactivity clearly colocalized with Nestin, Tuj1, and MAP2, whereas Prx I immunoreactivity was exclusively detected in SSEA1-positive cells (Fig. 2D, 2E; Supporting Information Fig. S1), indicating that Prx I and Prx II may be markers for stemness and neuronal differentiation, respectively. Prx II-Deficient ESCs Differentiate Rapidly into Neuronal Cells To investigate the role of Prxs in the differentiation process, ESC lines were established from wild-type and Prx II−/− 129/SvJae blastocysts at 3.5 days post-coitum (Supporting Information Figs. S2, S3) and allowed to differentiate into neuronal lineages. Of particular interest, Prx II−/− ESCs showed rapid differentiation into neuronal cells over 7 days of neural differentiation and ultimately generated a more enriched population of Nestin- and MAP2-positive cells than that generated by wild-type cells (Fig. 3A), indicating an active neurogenesis from Prx II−/− ESCs. In contrast, colonies positive for both Prx I and SSEA1 were frequently found in wild-type cells during neurogenesis, whereas no immunoreactivity for the two antibodies was detected in differentiating Prx II−/− cells (Fig. 3B), suggesting an abrupt loss of stemness during neural differentiation of Prx II−/− ESCs. Consistent with this result, higher expression level of the neural markers Nestin and MAP2 were observed in Prx II−/− cells than in wild-type cells after 7 days of neural differentiation, whereas the expression of the stemness-associated genes Oct4 and Nanog was much lower in Prx II−/− ESCs than in wild-type cells (Fig. 3C). Western blotting analysis also showed a rapid decrease in Prx I and Oct4 and elevation of MAP2 in Prx II−/− ESCs during neural differentiation compared with wild-type (Fig. 3D). Open in new tabDownload slide Prx II deficiency leads to downregulation of Prx I and Oct4 expression and causes loss of embryonic stem cell (ESC) stemness during neurogenesis. (A): Immunocytochemical analysis of differentiation markers at 7 days of neural differentiation from wild-type (+/+) and Prx II−/− ESCs. The immunostained and 4′,6-diamino-2-phenylindole stained images were merged. The white arrow indicates a typical undifferentiated colony photographed under bright and fluorescent field. Scale bar = 50 µm. (B): Double-label immunocytochemical analysis of Prx I and differentiation markers using wild-type and Prx II−/− ESCs differentiated in neurobasal medium (NBM) for 14 days. Regions of colocalization as determined by image merging are shown in yellow. Scale bar = 25 µm. (C, D): Quantitative polymerase chain reaction (qPCR) (C) and Western blotting (D) analyses of upregulated and downregulated genes (UPGs and DRGs, respectively) using wild-type (+/+) and Prx II−/− ESCs differentiated in NBM for 7 and 14 days. Error bars indicate mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001 compared with cells of 0-day of neural differentiation. (E, F): Effect of Oct4 knockdown on Prx transcript levels. P19 (E) and CRL-2073 (F) ESCs were transfected with siOct4, harvested at 48 hours posttransfection, and subjected to Western blotting (left panel) and qPCR (right panel) analyses of Prx I, Prx II and Oct4. Error bars indicate mean ± SD (n = 3). **, p < .01; ***, p < .001 compared with control siRNA (siCon). (G, H): Effect of Prx knockdown on stemness marker transcript level. P19 (G) and CRL-2073 (H) ESCs were transfected with either siPrx I or siPrx II, harvested at 48 hours posttransfection, and subjected to Western blotting (upper panel) and qPCR (lower panel) analyses of Prx I, Prx II, Oct4, and Nanog. Error bars indicate mean ± SD (n = 3); ***, p < .001 compared with siCon. (I): Effect of Prx I deficiency on stemness marker transcript level. Exponentially growing wild-type (+/+), Prx I−/−, and Prx II−/− ESCs were harvested and subjected to Western blotting (upper panel) and qPCR (lower panel) analyses of Prx I, Prx II, Oct4, and Nanog. Error bars indicate mean ± SD (n = 3); ***, p < .001 compared with wild-type (+/+). Abbreviations: DAPI, 4′,6-diamino-2-phenylindole; DRG, downregulation gene; ESC, embryonic stem cell; ND, neural differentiation; URG, upregulation gene. Open in new tabDownload slide Prx II deficiency leads to downregulation of Prx I and Oct4 expression and causes loss of embryonic stem cell (ESC) stemness during neurogenesis. (A): Immunocytochemical analysis of differentiation markers at 7 days of neural differentiation from wild-type (+/+) and Prx II−/− ESCs. The immunostained and 4′,6-diamino-2-phenylindole stained images were merged. The white arrow indicates a typical undifferentiated colony photographed under bright and fluorescent field. Scale bar = 50 µm. (B): Double-label immunocytochemical analysis of Prx I and differentiation markers using wild-type and Prx II−/− ESCs differentiated in neurobasal medium (NBM) for 14 days. Regions of colocalization as determined by image merging are shown in yellow. Scale bar = 25 µm. (C, D): Quantitative polymerase chain reaction (qPCR) (C) and Western blotting (D) analyses of upregulated and downregulated genes (UPGs and DRGs, respectively) using wild-type (+/+) and Prx II−/− ESCs differentiated in NBM for 7 and 14 days. Error bars indicate mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001 compared with cells of 0-day of neural differentiation. (E, F): Effect of Oct4 knockdown on Prx transcript levels. P19 (E) and CRL-2073 (F) ESCs were transfected with siOct4, harvested at 48 hours posttransfection, and subjected to Western blotting (left panel) and qPCR (right panel) analyses of Prx I, Prx II and Oct4. Error bars indicate mean ± SD (n = 3). **, p < .01; ***, p < .001 compared with control siRNA (siCon). (G, H): Effect of Prx knockdown on stemness marker transcript level. P19 (G) and CRL-2073 (H) ESCs were transfected with either siPrx I or siPrx II, harvested at 48 hours posttransfection, and subjected to Western blotting (upper panel) and qPCR (lower panel) analyses of Prx I, Prx II, Oct4, and Nanog. Error bars indicate mean ± SD (n = 3); ***, p < .001 compared with siCon. (I): Effect of Prx I deficiency on stemness marker transcript level. Exponentially growing wild-type (+/+), Prx I−/−, and Prx II−/− ESCs were harvested and subjected to Western blotting (upper panel) and qPCR (lower panel) analyses of Prx I, Prx II, Oct4, and Nanog. Error bars indicate mean ± SD (n = 3); ***, p < .001 compared with wild-type (+/+). Abbreviations: DAPI, 4′,6-diamino-2-phenylindole; DRG, downregulation gene; ESC, embryonic stem cell; ND, neural differentiation; URG, upregulation gene. Based on the similarity in expression patterns between Prx I and stemness-related transcription factors, particularly Oct4, we used small-interfering RNA (siRNA) to knock down the expression of Oct4 and Prx isotypes in P19 and CRL-2073 embryonic carcinoma (EC) cells. Using Western blotting and qPCR analyses, we found that knockdown of Oct4 resulted in reduced expression of Prx I, but not Prx II (Fig. 3E, 3F), indicating that Oct4 is an upstream regulator of Prx I transcription. In contrast, knockdown of Prx I and Prx II did not affect expression levels of Oct4 and Nanog (Fig. 3G, 3H). To more closely examine the effect of Prx isotypes on ESC stemness, we established Prx I−/− ESC lines (Supporting Information Figs. S2, S3) and compared them with wild-type and Prx II−/− ESCs. Consistent with the results from siRNA experiments, no changes in expression levels of SSEA1, Oct4, or Nanog and AP activity were observed among wild-type, Prx I−/−, or Prx II−/− ESCs (Fig. 2I; Supporting Information Fig. S2D, S2E). Prx I- and Prx II-Null ESCs Generate High ROS Levels and Abruptly Lose Their Stemness for Active Neurogenesis To determine the effect of Prx deficiency on ROS generation during neurogenesis, we compared the levels of intracellular ROS in wild-type, Prx I−/−, and Prx II−/− ESCs during neural differentiation using flow cytometry. Although neurogenesis led to an increase of ROS levels in cells of all three genotypes, ROS were continuously generated at a higher level in Prx I−/− or Prx II−/− cells than in wild-type cells (Fig. 4A). In addition, high levels of AP activity and stemness-associated transcription factor expression were maintained in differentiating wild-type cells, but showed a rapid decline in Prx I−/− or Prx II−/− cells or when antioxidants were depleted from the NBM (Fig. 4B, 4C). Open in new tabDownload slide Prx deficiency enhances reactive oxygen species (ROS) generation and accelerates loss of stemness during neurogenesis. (A): Effect of Prx deficiency on ROS generation during neurogenesis from embryonic stem cells (ESCs). Wild-type (+/+), Prx I−/−, and Prx II−/− ESCs were differentiated in neurobasal medium (NBM) for 5 days and subjected to flow cytometry for measurement of DCF fluorescence intensities. Dashed lines indicate the mean DCF intensities of wild-type undifferentiated (green) and differentiated (violet) cells. Error bars represent mean ± SD (n = 3). (B): Accelerated loss of alkaline phosphatase (AP) activity during neurogenesis from Prx I−/− and Prx II−/− ESCs. ESCs of the indicated genotype were differentiated in NBM or NBM-antioxidant (AO) for 5 days and analyzed for AP activity. Error bars represent mean ± SD (n = 3). Scale bar = 100 µm. (C): Semi-quantitative polymerase chain reaction analysis of stemness-associated or neuronal marker transcripts using wild-type, Prx I−/−, and Prx II−/− ESCs differentiated in NBM for 7 days. (D): Double-label immunocytochemical analysis of Prxs and differentiation markers (right panel) using wild-type (+/+), Prx I−/−, and Prx II−/− ESCs. ESCs of the indicated genotype were differentiated in NBM for 7 days, photographed under bright-field (left panel), and immunostained with antibodies against Prx I, Prx II, SSEA1, and MAP2 (right panel). Regions of colocalization as determined by image merging are shown in yellow. Scale bar = 100 µm. Abbreviations: AO, antioxidant; AP, alkaline phosphatase; DCF dichlorofluorescin; ESC, embryonic stem cell; ND, neural differentiation; ROS, reactive oxygen species. Open in new tabDownload slide Prx deficiency enhances reactive oxygen species (ROS) generation and accelerates loss of stemness during neurogenesis. (A): Effect of Prx deficiency on ROS generation during neurogenesis from embryonic stem cells (ESCs). Wild-type (+/+), Prx I−/−, and Prx II−/− ESCs were differentiated in neurobasal medium (NBM) for 5 days and subjected to flow cytometry for measurement of DCF fluorescence intensities. Dashed lines indicate the mean DCF intensities of wild-type undifferentiated (green) and differentiated (violet) cells. Error bars represent mean ± SD (n = 3). (B): Accelerated loss of alkaline phosphatase (AP) activity during neurogenesis from Prx I−/− and Prx II−/− ESCs. ESCs of the indicated genotype were differentiated in NBM or NBM-antioxidant (AO) for 5 days and analyzed for AP activity. Error bars represent mean ± SD (n = 3). Scale bar = 100 µm. (C): Semi-quantitative polymerase chain reaction analysis of stemness-associated or neuronal marker transcripts using wild-type, Prx I−/−, and Prx II−/− ESCs differentiated in NBM for 7 days. (D): Double-label immunocytochemical analysis of Prxs and differentiation markers (right panel) using wild-type (+/+), Prx I−/−, and Prx II−/− ESCs. ESCs of the indicated genotype were differentiated in NBM for 7 days, photographed under bright-field (left panel), and immunostained with antibodies against Prx I, Prx II, SSEA1, and MAP2 (right panel). Regions of colocalization as determined by image merging are shown in yellow. Scale bar = 100 µm. Abbreviations: AO, antioxidant; AP, alkaline phosphatase; DCF dichlorofluorescin; ESC, embryonic stem cell; ND, neural differentiation; ROS, reactive oxygen species. Wild-type ESCs frequently formed ESC-like colonies with high cell density during neurogenesis, whereas breakdown of the colony-like morphology and extensive neurite formation were notable in Prx I−/− or Prx II−/− cells (Fig. 4D, bright-field). Double-label immunocytochemistry showed that Prx I and SSEA1 were expressed at high levels throughout the wild-type ESC-derived colonies, and Prx II and MAP2 were expressed only at the colony peripheries (Fig. 4D, fluorescence field). In contrast, large populations of Prx-knockout cells were showed immunostaining by anti-MAP2 and/or anti-Prx II antibodies and showed a dispersed distribution rather than maintaining a colony state (Fig. 4D). ROS Are Involved in Accelerated Neural Differentiation of Prx-Deficient ESCs To determine whether ROS are directly involved in ESC-mediated neurogenesis, wild-type, Prx I−/−, and Prx II−/− ESCs were differentiated in NBM in the presence or absence of the antioxidant NAC, and their ROS levels and neuronal gene expression profiles were determined using flow cytometry and semi-qPCR, respectively. Treatment of differentiating Prx I−/− or Prx II−/− cells with NAC resulted in efficient alleviation of the increase in ROS levels (Fig. 5A), resulting in reduced generation of neuron-like cells (Fig. 5B). Consistent with this result, treatment with NAC also resulted in a significant decrease in the enhanced expression of the neuronal genes Nestin, MAP2, NeuroD1, and Ngn1, but restored the decreased levels of Oct4 and Nanog in differentiating Prx I−/− or Prx II−/− cells (Fig. 5C). Consistent with this result, Western blotting analysis showed that the abruptly decreased Oct4 level in differentiating Prx-deficient ESCs was restored to the level of wild-type cells by supplementation with NAC during neural differentiation (Fig. 5D). Open in new tabDownload slide Acceleration of the neural differentiation process in Prx I−/− and Prx II−/− embryonic stem cells (ESCs) results from modulation of reactive oxygen species (ROS)-dependent signal pathways. (A): Effect of NAC supplementation on ROS generation during neurogenesis from Prx I−/− and Prx II−/− ESCs. ESCs of the indicated genotypes were differentiated in the presence or absence of NAC for 3 and 5 days and subjected to flow cytometry for measurement of DCF fluorescence intensities. Dashed lines indicate the mean DCF intensities of wild-type undifferentiated (green) and differentiated (violet) cells. Error bars represent mean ± SD (n = 3); *, p < .01. (B): Bright-field images of wild-type (+/+), Prx I−/−, and Prx II−/− ESCs differentiated in the presence or absence of NAC for 7 days. Scale bar = 50 µm. (C): Effect of NAC supplementation on transcription of stemness-associated or neuronal marker genes during neurogenesis from Prx I−/− and Prx II−/− ESCs. Band intensities were normalized to those corresponding to GAPDH and the error bars represent mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001. (D): Restoration of Oct4 and Prx protein levels by supplementation with NAC in differentiating Prx I−/− and Prx II−/− ESCs. Western blotting analysis was performed using ESCs of the indicated genotype differentiated in the presence or absence of 2.5 mM NAC during the indicated times, and band intensities were normalized to those of α-tubulin. Abbreviations: DCF, dichlorofluorescin; ESC, embryonic stem cell; NAC, N-acetyl-l-cysteine; ND, neural differentiation; ROS, reactive oxygen species; WB, Western blotting. Open in new tabDownload slide Acceleration of the neural differentiation process in Prx I−/− and Prx II−/− embryonic stem cells (ESCs) results from modulation of reactive oxygen species (ROS)-dependent signal pathways. (A): Effect of NAC supplementation on ROS generation during neurogenesis from Prx I−/− and Prx II−/− ESCs. ESCs of the indicated genotypes were differentiated in the presence or absence of NAC for 3 and 5 days and subjected to flow cytometry for measurement of DCF fluorescence intensities. Dashed lines indicate the mean DCF intensities of wild-type undifferentiated (green) and differentiated (violet) cells. Error bars represent mean ± SD (n = 3); *, p < .01. (B): Bright-field images of wild-type (+/+), Prx I−/−, and Prx II−/− ESCs differentiated in the presence or absence of NAC for 7 days. Scale bar = 50 µm. (C): Effect of NAC supplementation on transcription of stemness-associated or neuronal marker genes during neurogenesis from Prx I−/− and Prx II−/− ESCs. Band intensities were normalized to those corresponding to GAPDH and the error bars represent mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001. (D): Restoration of Oct4 and Prx protein levels by supplementation with NAC in differentiating Prx I−/− and Prx II−/− ESCs. Western blotting analysis was performed using ESCs of the indicated genotype differentiated in the presence or absence of 2.5 mM NAC during the indicated times, and band intensities were normalized to those of α-tubulin. Abbreviations: DCF, dichlorofluorescin; ESC, embryonic stem cell; NAC, N-acetyl-l-cysteine; ND, neural differentiation; ROS, reactive oxygen species; WB, Western blotting. Loss of Stemness in Prx I−/− or Prx II−/− ESCs During Neurogenesis Requires ROS-Dependent Activation of JNK To determine the involvement of MAPK signaling pathways in ROS-mediated neurogenesis, wild-type, Prx I−/−, and Prx II−/− ESCs were differentiated in NBM in the presence or absence of NAC and subjected to Western blotting with antibodies against phosphorylated ERK1/2, p38 MAPK, and JNK. During neurogenesis, phosphorylation of these three MAPKs showed a greater increase (pERK1/2 and pJNK) or occurred earlier (pp38 MAPK) in Prx I−/− or Prx II−/− ESCs than in wild-type cells. In contrast, NAC inhibited or retarded the phosphorylation (Fig. 6A; Supporting Information Fig. S4). Open in new tabDownload slide Abrupt loss of embryonic stem cell (ESC) stemness requires JNK activation governed by the Prx/reactive oxygen species (ROS) balance. (A): Effect of NAC supplementation on activation of JNK during neurogenesis from wild-type, Prx I−/−, and Prx II−/− ESCs. The pJNK band intensities were normalized to the intensity of total JNK. (B): Wild-type, Prx I−/−, and Prx II−/− ESCs were differentiated in the presence or absence of the MAPK inhibitors SP600125 (for JNK), PD98059 (for ERK), and SB203580 (for p38 MAPK). Levels of Oct4 and Nanog transcripts were determined by Semi-quantitative polymerase chain reaction (qPCR; upper panel) and qPCR (lower panel) analyses. Error bars represent mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001 compared with untreated cells of neural differentiation 7-day. (C): ESCs of the indicated genotypes were differentiated in the presence or absence of SP600125 or NAC for 3 days, and Prx I and Oct4 expression was examined by double-label immunocytochemical analysis. Scale bar = 25 µm. Abbreviations: NAC, N-acetyl-l-cysteine; NBM, neurobasal medium; ND, neural differentiation; PD, PD98059; pJNK, phospho-JNK; SB, SB203580; SP, SP600125; WB, Western blotting. Open in new tabDownload slide Abrupt loss of embryonic stem cell (ESC) stemness requires JNK activation governed by the Prx/reactive oxygen species (ROS) balance. (A): Effect of NAC supplementation on activation of JNK during neurogenesis from wild-type, Prx I−/−, and Prx II−/− ESCs. The pJNK band intensities were normalized to the intensity of total JNK. (B): Wild-type, Prx I−/−, and Prx II−/− ESCs were differentiated in the presence or absence of the MAPK inhibitors SP600125 (for JNK), PD98059 (for ERK), and SB203580 (for p38 MAPK). Levels of Oct4 and Nanog transcripts were determined by Semi-quantitative polymerase chain reaction (qPCR; upper panel) and qPCR (lower panel) analyses. Error bars represent mean ± SD (n = 3). *, p < .05; **, p < .01; ***, p < .001 compared with untreated cells of neural differentiation 7-day. (C): ESCs of the indicated genotypes were differentiated in the presence or absence of SP600125 or NAC for 3 days, and Prx I and Oct4 expression was examined by double-label immunocytochemical analysis. Scale bar = 25 µm. Abbreviations: NAC, N-acetyl-l-cysteine; NBM, neurobasal medium; ND, neural differentiation; PD, PD98059; pJNK, phospho-JNK; SB, SB203580; SP, SP600125; WB, Western blotting. To confirm the involvement of ROS-sensitive MAPK pathways in the loss of ESC stemness during neurogenesis, Prx I−/− or Prx II−/− ESCs were differentiated with NBM in the presence or absence of signaling inhibitors for JNK (SP600125), ERK1/2 (PD98059), and p38 MAPK (SB203580) and subjected to semi-qPCR and qPCR analyses for Oct4 and Nanog expression. ERK and p38 MAPK inhibitors did not affect Oct4 or Nanog transcript levels in differentiating Prx I−/− or Prx II−/− cells, whereas treatment with the JNK inhibitor resulted in notable recovery (Fig. 6B). Double-label immunocytochemistry also showed that the decrease of Oct4- and Prx I-positive cells in differentiating Prx I−/− or Prx II−/− colonies was reversed by treatment with NAC and by JNK inhibition (Fig. 6C). Long-Term Survival of ESC Stemness in Developing Teratomas Requires Cytoplasmic Prxs To further examine the regulatory role of the ROS/Prx axis in ESC differentiation, we generated teratomas in athymic nude mice by inoculation of wild-type, Prx I−/−, or Prx II−/− ESCs into their epidermis. The teratomas were isolated surgically for semi-qPCR, qPCR, and histological analyses. Prx I, Oct4, and Nanog transcript levels were maintained in wild-type teratomas but not in Prx I−/− or Prx II−/− teratomas (Fig. 7A, 7B). Consistent with this result, according to immunohistochemial analysis, Oct-4 positive cells were only detected in wild-type teratoma (Fig. 7C). In addition, immunohistochemistry using serial sections of the wild-type teratoma indicated strong immunoreactivity for Prx I and Prx II in Oct4-positive cells and neural tissue, respectively, accounting for the entire spectrum of neurogenesis (Fig. 7C, 7D; Supporting Information Fig. S5A). In addition, most immunoreactivity for PCNA was detected in Oct4-positive cells of wild-type teratomas and neural rosettes of Prx I−/− teratomas, whereas little or no PCNA immunoreactivity was found in Prx II−/− teratomas, with the exception of a small population of respiratory epithelium cells (Fig. 7C; Supporting Information Fig. S5A). In particular, Prx I and Prx II appeared to be shuttled from the cytoplasm of Oct4-positive cells to the nuclei of the other types of cells (Fig. 7D), as shown in vivo and in vitro (Supporting Information Fig. S5B, S5C). Open in new tabDownload slide Prx is essential for preservation of embryonic stem cell (ESC) stemness in developing teratomas. (A, B): Teratomas of the indicated genotypes were analyzed for expression of ESC stemness-associated genes and Prx isotypes using semi-quantitative polymerase chain reaction (qPCR) (A) and qPCR (B). (C): Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), Oct4, Prx I, and Prx II expressions in serial sections of wild-type teratoma. The Prx immunoreactivity intensity gradient is shown beneath the images. Scale bar = 200 µm. (D): Immunohistochemical analysis of PCNA, Oct4, Prx I, and Prx II expressions in serial sections of wild-type, Prx I−/−, and Prx II−/− teratomas. Undifferentiated cells, neural rosettes, and respiratory epithelium are indicated by dotted white lines, black arrows, and white arrows, respectively. Scale bar = 100 µm. (E): Schematic illustration of a hypothetical model for ESC-mediated neurogenesis governed by the Prx/ROS/JNK axis. The ROS/JNK cascade is required for ESC stemness loss during neural differentiation, and cytoplasmic Prx contributes to suppression of ROS/JNK pathways. Abbreviations: ESC, embryonic stem cell; H&E, hematoxylin and eosin staining; JNK, c-Jun N-terminal kinase; ND, neural differentiation; NSC, neural stem cell; PCNA, proliferating cell nuclear antigen; ROS, reactive oxygen species; Ud-ESC, undifferentiated ESC. Open in new tabDownload slide Prx is essential for preservation of embryonic stem cell (ESC) stemness in developing teratomas. (A, B): Teratomas of the indicated genotypes were analyzed for expression of ESC stemness-associated genes and Prx isotypes using semi-quantitative polymerase chain reaction (qPCR) (A) and qPCR (B). (C): Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA), Oct4, Prx I, and Prx II expressions in serial sections of wild-type teratoma. The Prx immunoreactivity intensity gradient is shown beneath the images. Scale bar = 200 µm. (D): Immunohistochemical analysis of PCNA, Oct4, Prx I, and Prx II expressions in serial sections of wild-type, Prx I−/−, and Prx II−/− teratomas. Undifferentiated cells, neural rosettes, and respiratory epithelium are indicated by dotted white lines, black arrows, and white arrows, respectively. Scale bar = 100 µm. (E): Schematic illustration of a hypothetical model for ESC-mediated neurogenesis governed by the Prx/ROS/JNK axis. The ROS/JNK cascade is required for ESC stemness loss during neural differentiation, and cytoplasmic Prx contributes to suppression of ROS/JNK pathways. Abbreviations: ESC, embryonic stem cell; H&E, hematoxylin and eosin staining; JNK, c-Jun N-terminal kinase; ND, neural differentiation; NSC, neural stem cell; PCNA, proliferating cell nuclear antigen; ROS, reactive oxygen species; Ud-ESC, undifferentiated ESC. Discussion In this study, we demonstrated that high levels of Prx I and Prx II are expressed in undifferentiated ESCs and in differentiating neuronal cells, respectively. The high expression of Prx II in neurons is in good accordance with result of our previously reported study [27, 28], whereas the distribution of Prx II in neural stem cells and early neurons is a novel finding of this study and is indicative of a broad spectrum of Prx II function from early- to late-stage neurogenesis. On the other hand, Prx I is highly expressed in ESCs and undifferentiated cells, and its expression pattern is similar to those of stemness-supporting transcription factors, such as Oct4 and Nanog. The difference in the two Prx isotypes results in their reciprocal expression pattern during neural differentiation of ESCs. These findings may provide a fundamental basis for understanding the role of antioxidants in ESC differentiation. The antioxidant content of ESC-derived cells is higher than that of tissue-derived primary cells, reflecting the higher demand for ESC-derived cells for protection against oxidative damage [14, 29]. Numerous studies have demonstrated that overexpression of Prx II results in reduction of oxidative stress in rat cortical neurons [30] and that the reduced peroxidase activity causes dopamine neurons to be vulnerable to oxidative insult [31, 32]. Prx II−/−, but not Prx I−/−, mice also exhibit an age-related decline in synaptic plasticity and spatial learning ability as a result of cumulative oxidative stress in hippocampal neurons [27]. Therefore, upregulation of Prx II in neuron-lineage cells may contribute to the ability of ESC-derived neurons to resist oxidative overload during neurogenesis. Prx I is also known to play an anti-apoptotic role in neurons. Increased expression of Prx I protects neuronal cells against oxidative stress-mediated cell death [33-35]. However, the results of earlier studies conflict with the idea of a defensive role for Prx I in neurons; Overexpression of Prx I causes accelerated induction of primary neuronal cell death by nerve growth factor deprivation [36]. In addition, in the mammalian central nervous system, Prx I has been reported to be restricted to glial cells, including oligodendrocytes, astrocytes, and microglia [28, 37-40], a finding suggestive of an obligatory absence of Prx I from neuronal expression. These preceding observations described in the latter case lead us to propose a hypothesis for the role of Prx I in neuron development. A decreased Prx I level may be essential for the entry of neurons into a specific phase of the cell cycle and for maintenance of cell cycle arrest. This hypothesis is supported by cell cycle-related studies of Prx I in mammalian cells. Human Prx I was cloned primarily as a proliferation-associated gene [41], and it is frequently expressed at high levels in malignant cells requiring rapid division [42-45]. Unlike malignant cells, which have a shorter than normal G1 arrest phase, mature neurons begin to function after arresting at G1. We noted G0/G1 arrest predominantly in neurogenesis from Prx I−/− or Prx II−/− ESCs and not from wild-type cells (Sun Uk Kim, unpublished data). Therefore, the decrease in Prx I content in ESC-derived neurons may help to diminish unfavorable side effects in neuronal cell cycle regulation, and elucidation of the molecular mechanism for the effects of ectopic expression of Prx I on neuron development is important. Data of this study conclusively demonstrate that Prx I and Prx II modulate the tempo of neural differentiation by neutralizing ROS during neural differentiation. In addition to the requirement for antioxidative defense in differentiating or differentiated cells, an essential role for intracellular ROS in propelling ESCs into a specific lineage has recently been suggested [7, 8, 46]. At high levels, intracellular ROS participate actively in the loss of stemness by oxidative inactivation of the DNA-binding activity of Oct4 [2], and thioredoxin can restore the DNA-binding activity of Oct4 through physical interaction with its oxidized cysteine [2]. This finding is indicative of ROS involvement in the loss of pluripotency in self-renewing ESCs. In addition, ROS are utilized as a stimulant for cardiomyogenesis, cardiovascular differentiation, and glial precursor cell differentiation [1, 7, 46]. Therefore, ESC differentiation kinetics are dependent on the orchestration between ROS action and the corresponding antioxidant defense system. However, most studies using alterations of the ROS-producing system, such as overexpression or knockdown of NADPH oxidase, have involved treatment with exogenous chemical antioxidants and pro-oxidants, such as NAC and H2O2, respectively. Therefore, ESCs established from specific antioxidant-knockout blastocysts provide a model that may lead to achievement of a novel understanding of ROS-mediated differentiation processes. In this study, we found that intracellular MAPK signaling is a regulator of ESC stemness and neural differentiation. In accordance with the previously reported findings showing that inhibition of ERK pathways promotes self-renewal of mouse ESCs [47], and that ERK is required for retinoic acid-mediated neurogenesis [22, 48], we found that Prx I−/− or Prx II−/− ESCs with high levels of ERK phosphorylation during neurogenesis exhibited explosive differentiation into neurons, and that the NAC-induced delay of ERK activation of these cells appeared to show correlation with inhibition of stemness loss (Supporting Information Fig. S4). However, we did not find any detectable recovery of stemness upon treatment with an ERK inhibitor during neurogenesis from Prx I−/− or Prx II−/− ESCs, although the ERK inhibitor was effective against wild-type ESCs (unpublished data). Inhibition of JNK signaling, rather than ERK signaling, provided the most notable protection against ROS-mediated loss of stemness in differentiating Prx I−/− or Prx II−/− cells. Thus, we presume that hyper-activation of JNK may override the stemness-regulating action of ERK during neurogenesis from Prx-null ESCs. An intriguing aspect of our study is the finding that Prx I−/− and Prx II−/− ESCs appear to be phenotypically similar with regard to ROS generation, stemness regulation, and neural differentiation kinetics although Prx I and Prx II are preferentially expressed in dissimilar cell types. Our results showed similarly enhanced neurogenesis in both Prx−/− ESCs. However, results of further experiments showed that oligodendrogenesis was increased in differentiating Prx I−/− ESCs compared with wild-type, whereas it was greatly suppressed in Prx II−/− ESCs, although astrogliogenesis appeared to be suppressed in both mutant ESCs (Supporting Information Fig. S7). Other dissimilarities were also observed in teratoma experiments. Among the three genotypes, Prx I−/− teratomas included the highest proportion of neural tissues, whereas the other types of cells or tissues, such as muscles and ectodermal epithelial cells, were frequently detected in Prx II−/− teratomas (data not shown), suggesting the preferential role of Prx isotypes in the differentiation process. In addition, relatively mature cell types were dominantly detected in Prx II−/− teratomas, whereas Prx I−/− teratoma contained a large proportion of immature tissues including neuroepithelium. This finding is indicative of possible roles of Prx isotypes in the regulation of differentiation tempo. In addition, Prx I−/− ESCs generated malignant teratomas with a high proportion of PCNA-positive cells (data not shown), leading to the largest teratoma in volume among the three genotypes (Supporting Information Fig. S6); this result is in good accordance with studies concerning the function of Prx I as a tumor suppressor [49, 50]. In contrast, the smallest teratomas were generated by Prx II ESCs (Supporting Information Fig. S6). Collectively, these results suggest possible involvement of Prx I and Prx II in a variety of differentiation events, such as stemness maintenance, lineage specification, and differentiation tempo, and proliferation. Currently, we are attempting to further investigate the differential roles of Prx I and Prx II in detail. Although wild-type, Prx I−/−, or Prx II−/− teratomas in neural rosettes differed considerably in size and Prx I/PCNA immunoreactivity, we noted an important consensus in cytoplasm-nucleus shuttling during neurogenesis. Strong Prx I and moderate Prx II immunoreactivity was found preferentially in the cytoplasm of undifferentiated ESCs, however, the immunoreactivity either disappeared or was predominantly found in the nuclei of differentiating or differentiated cell types, especially neural tissue components (Fig. 7D; Supporting Information Fig. S5, Table S1). These results suggest that cytoplasmic Prx I and Prx II may actively participate in the mechanism that maintains ESC stemness through opposing ROS/JNK activation during neurogenesis. Our attempts to find direct evidence of a relationship between cytoplasmic Prx and stemness regulation have led us to propose a comprehensive model for the mechanism by which ESC stemness is regulated by redox signaling (Fig. 7E). In self-renewing ESCs, cytoplasmic Prx I and Prx II contribute to suppression of JNK activation by reducing the ROS level. Upon detection of a differentiation stimulus, ESCs ramp up production of ROS; however, cytoplasmic Prx promptly scavenges the surplus ROS, allowing the ESCs to maintain their undifferentiated state (Ud-ESCs). Accumulation of ROS occurs at an even higher level during neurogenesis; these ROS are protected from Prx-mediated antioxidative surveillance and thus activate the JNK cascade. Subsequently, activated JNK inhibits expression of Oct4 and Prx I and activates several neural genes, causing Ud-ESCs to commit to becoming neural stem cells (NSCs). NSCs have higher ROS levels due to their severely reduced Prx I levels. At this point, translocation of Prx I and Prx II into the NSC nucleus occurs by an as-yet-unknown mechanism; this translocation increases the vulnerability of NSCs to cytoplasmic activation of the ROS/JNK cascade. Ultimately, many more neural genes are turned on by activated JNK, and the NSCs differentiate into neurons. Therefore, the neural differentiation process is highly dependent on the finely tuned interaction between nuclear-cytoplasmic Prx shuttling and the resulting local bias of ROS content. We are currently working to elucidate the direct involvement of Prx surveillance of local ROS content in ROS/JNK-mediated regulation of stemness. In summary, Prx I and Prx II have a unique function in regulating ESC stemness during neurogenesis. In ESCs, null Prx mutations cause ROS levels to rise in both differentiating and nondifferentiating cells, resulting in rapid loss of stemness and accelerated neural differentiation. The JNK pathway appears to be a downstream regulator of ROS, which is regulated by Prx I and Prx II. Ultimately, the effect of Prx deficiency exceeded our expectations, and we were able to produce a much higher neuronal population from Prx I−/− or Prx II−/− ESCs than from wild-type ESCs. Our results demonstrate the precise function of Prx isotypes in the complex redox-signaling pathways involved in maintenance of ESC stemness during neurogenesis and may lead to development of an alternative stem cell-based therapeutic strategy for producing high-quality neurons in large quantity. Conclusion Levels of ROS and JNK activation during neurogenesis were much higher in Prx I−/− or Prx II−/− ESCs than in wild-type ESCs. Of particular interest, Prx I−/− and Prx II−/− ESCs rapidly lost their stemness through hyperactivation of the ROS/JNK cascade during neurogenesis and showed rapid differentiation into neurons, which was restored by inhibition of the JNK/ROS pathway. Collectively, we conclude that Prx I and Prx II act as protectors of ESC stemness against oxidative stress during neurogenesis. Acknowledgments This work was supported by Grant No. 2013003473 from the SRC program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST), by Grant No. 112020-03-2-SB020 from the ARPC program of the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries, by Grant No. 2012M3A9B6055362 from the Bio and Medical Technology Development Program through NRF funded by MEST, by Grant No. 2013025702 and 2007-0054932 from the NRF funded by MEST, by Grant No. 0720560 from the National R&D Program for Cancer Control Ministry of Health and Welfare, and by Grant No. OGM0021312 from the Mid-career Researcher Program through NRF funded by the MEST, Republic of Korea. Author Contributions S.-U.K., Y.-H.P., and J.-M.K.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript; J.-I.C., S.H., S.S.C., and S.-C.C., collection and/or assembly of data and data analysis and interpretation; H.-N.S., I.-S.S., S.M.H., J.-M.K., and S.-H.L.: collection and/or assembly of data; T.-H.L., S.W.K., S.G.R., and K.-T.C.: conception and design and financial support; S.H.L., D.-Y.Y., and D.-S.L.: conception and design, financial support, data analysis and interpretation, manuscript writing, and final approval of manuscript. S.-U.K., Y.-H.P., and J.-M.K. contributed equally to this work. Disclosure of Potential Conflicts of Interest The authors indicate no potential conflicts of interest. References 1 Smith J , Ladi E, Mayer-Proschel M et al. Redox state is a central modulator of the balance between self-renewal and differentiation in a dividing glial precursor cell . Proc Natl Acad Sci U S A 2000 ; 97 : 10032 – 10037 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Guo Y , Einhorn L, Kelley M et al. Redox regulation of the embryonic stem cell transcription factor oct-4 by thioredoxin . Stem Cells 2004 ; 22 : 259 – 264 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Le Belle JE , Orozco NM, Paucar AA et al. Proliferative neural stem cells have high endogenous ROS levels that regulate self-renewal and neurogenesis in a PI3K/Akt-dependant manner . Cell Stem cell 2011 ; 8 : 59 – 71 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Zhang J , Nuebel E, Daley GQ et al. Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal . Cell Stem cell 2012 ; 11 : 589 – 595 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Ogasawara MA , Zhang H. Redox regulation and its emerging roles in stem cells and stem-like cancer cells . Antioxidant Redox Signal 2009 ; 11 : 1107 – 1122 . Google Scholar Crossref Search ADS WorldCat 6 Lee NK , Choi YG, Baik JY et al. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation . Blood 2005 ; 106 : 852 – 859 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Schmelter M , Ateghang B, Helmig S et al. Embryonic stem cells utilize reactive oxygen species as transducers of mechanical strain-induced cardiovascular differentiation . FASEB J 2006 ; 20 : 1182 – 1184 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Li J , Stouffs M, Serrander L et al. The NADPH oxidase NOX4 drives cardiac differentiation: Role in regulating cardiac transcription factors and MAP kinase activation . Mol Biol Cell 2006 ; 17 : 3978 – 3988 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Wang K , Zhang T, Dong Q et al. Redox homeostasis: The linchpin in stem cell self-renewal and differentiation . Cell Death Disease 2013 ; 4 : e537 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Vieira HL , Alves PM, Vercelli A. Modulation of neuronal stem cell differentiation by hypoxia and reactive oxygen species . Prog Neurobiol 2011 ; 93 : 444 – 455 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Chaudhari P , Ye Z, Jang YY. Roles of reactive oxygen species in the fate of stem cells . Antioxid Redox Signal . 2012 Nov 19. [Epub ahead of print]. OpenURL Placeholder Text WorldCat 12 Pervaiz S , Taneja R, Ghaffari S. Oxidative stress regulation of stem and progenitor cells . Antioxidant Redox Signal 2009 ; 11 : 2777 – 2789 . Google Scholar Crossref Search ADS WorldCat 13 Owusu-Ansah E , Banerjee U. Reactive oxygen species prime Drosophila haematopoietic progenitors for differentiation . Nature 2009 ; 461 : 537 – 541 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Saretzki G , Armstrong L, Leake A et al. Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells . Stem Cells 2004 ; 22 : 962 – 971 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Won H , Lim S, Jang M et al. Peroxiredoxin-2 upregulated by NF-kappaB attenuates oxidative stress during the differentiation of muscle-derived C2C12 cells . Antioxidant Redox Signal 2012 ; 16 : 245 – 261 . Google Scholar Crossref Search ADS WorldCat 16 Valle-Prieto A , Conget PA. Human mesenchymal stem cells efficiently manage oxidative stress . Stem Cells Dev 2010 ; 19 : 1885 – 1893 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Cho YM , Kwon S, Pak YK et al. Dynamic changes in mitochondrial biogenesis and antioxidant enzymes during the spontaneous differentiation of human embryonic stem cells . Biochem Biophys Res Commun 2006 ; 348 : 1472 – 1478 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Ishii T , Yamada M, Sato H et al. Cloning and characterization of a 23-kDa stress-induced mouse peritoneal macrophage protein . J Biol Chem 1993 ; 268 : 18633 – 18636 . Google Scholar Crossref Search ADS PubMed WorldCat 19 Kang SW , Baines IC, Rhee SG. Characterization of a mammalian peroxiredoxin that contains one conserved cysteine . J Biol Chem 1998 ; 273 : 6303 – 6311 . Google Scholar Crossref Search ADS PubMed WorldCat 20 Peskin AV , Cox AG, Nagy P et al. Removal of amino acid, peptide and protein hydroperoxides by reaction with peroxiredoxins 2 and 3 . Biochem J 2010 ; 432 : 313 – 321 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Hoffrogge R , Mikkat S, Scharf C et al. 2-DE proteome analysis of a proliferating and differentiating human neuronal stem cell line (ReNcell VM) . Proteomics 2006 ; 6 : 1833 – 1847 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Li Z , Theus MH, Wei L. Role of ERK 1/2 signaling in neuronal differentiation of cultured embryonic stem cells . Dev Growth Differ 2006 ; 48 : 513 – 523 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Amura CR , Marek L, Winn RA et al. Inhibited neurogenesis in JNK1-deficient embryonic stem cells . Mol Cell Biol 2005 ; 25 : 10791 – 10802 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Zhao L , Jiao Q, Yang P et al. Metabotropic glutamate receptor 5 promotes proliferation of human neural stem/progenitor cells with activation of mitogen-activated protein kinases signaling pathway in vitro . Neuroscience 2011 ; 192 : 185 – 194 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Aouadi M , Bost F, Caron L et al. p38 mitogen-activated protein kinase activity commits embryonic stem cells to either neurogenesis or cardiomyogenesis . Stem Cells 2006 ; 24 : 1399 – 1406 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Bain J , McLauchlan H, Elliott M et al. The specificities of protein kinase inhibitors: An update . Biochem J 2003 ; 371 : 199 – 204 . Google Scholar Crossref Search ADS PubMed WorldCat 27 Kim SU , Jin MH, Kim YS et al. Peroxiredoxin II preserves cognitive function against age-linked hippocampal oxidative damage . Neurobiol Aging 2011 ; 32 : 1054 – 1068 . Google Scholar Crossref Search ADS PubMed WorldCat 28 Jin MH , Lee YH, Kim JM et al. Characterization of neural cell types expressing peroxiredoxins in mouse brain . Neurosci Lett 2005 ; 381 : 252 – 257 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Yin X , Mayr M, Xiao Q et al. Proteomic analysis reveals higher demand for antioxidant protection in embryonic stem cell-derived smooth muscle cells . Proteomics 2006 ; 6 : 6437 – 6446 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Boulos S , Meloni BP, Arthur PG et al. Peroxiredoxin 2 overexpression protects cortical neuronal cultures from ischemic and oxidative injury but not glutamate excitotoxicity, whereas Cu/Zn superoxide dismutase 1 overexpression protects only against oxidative injury . J Neurosci Res 2007 ; 85 : 3089 – 3097 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Fang J , Nakamura T, Cho DH et al. S-nitrosylation of peroxiredoxin 2 promotes oxidative stress-induced neuronal cell death in Parkinson’s disease . Proc Natl Acad Sci U S A 2007 ; 104 : 18742 – 18747 . Google Scholar Crossref Search ADS PubMed WorldCat 32 Qu D , Rashidian J, Mount MP et al. Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson’s disease . Neuron 2007 ; 55 : 37 – 52 . Google Scholar Crossref Search ADS PubMed WorldCat 33 Zhang L , Yu H, Sun Y et al. Protective effects of salidroside on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells . Eur J Pharmacol 2007 ; 564 : 18 – 25 . Google Scholar Crossref Search ADS PubMed WorldCat 34 Lee YM , Park SH, Shin DI et al. Oxidative modification of peroxiredoxin is associated with drug-induced apoptotic signaling in experimental models of Parkinson disease . The Journal of biological chemistry . 2008 ; 283 : 9986 – 9998 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Kim SU , Hwang CN, Sun HN et al. Peroxiredoxin I is an indicator of microglia activation and protects against hydrogen peroxide-mediated microglial death . Biological Pharmaceutical Bull 2008 ; 31 : 820 – 825 . Google Scholar Crossref Search ADS WorldCat 36 Zhou Y , Zhang W, Easton R et al. Presenilin-1 protects against neuronal apoptosis caused by its interacting protein PAG . Neurobiol Dis 2002 ; 9 : 126 – 138 . Google Scholar Crossref Search ADS PubMed WorldCat 37 Sarafian TA , Verity MA, Vinters HV et al. Differential expression of peroxiredoxin subtypes in human brain cell types . J Neurosci Res 1999 ; 56 : 206 – 212 . Google Scholar Crossref Search ADS PubMed WorldCat 38 Mizusawa H , Ishii T, Bannai S. Peroxiredoxin I (macrophage 23 kDa stress protein) is highly and widely expressed in the rat nervous system . Neurosci Lett 2000 ; 283 : 57 – 60 . Google Scholar Crossref Search ADS PubMed WorldCat 39 Nakaso K , Kitayama M, Mizuta E et al. Co-induction of heme oxygenase-1 and peroxiredoxin I in astrocytes and microglia around hemorrhagic region in the rat brain . Neurosci Lett 2000 ; 293 : 49 – 52 . Google Scholar Crossref Search ADS PubMed WorldCat 40 Jarvela S , Rantala I, Rodriguez A et al. Specific expression profile and prognostic significance of peroxiredoxins in grade II-IV astrocytic brain tumors . BMC Cancer 2010 ; 10 : 104 . Google Scholar Crossref Search ADS PubMed WorldCat 41 Prosperi MT , Ferbus D, Karczinski I et al. A human cDNA corresponding to a gene overexpressed during cell proliferation encodes a product sharing homology with amoebic and bacterial proteins . J Biological Chem 1993 ; 268 : 11050 – 11056 . Google Scholar Crossref Search ADS WorldCat 42 Park YH , Kim SU, Lee BK et al. Prx I suppresses K-ras-driven lung tumorigenesis by opposing redox-sensitive ERK/cyclin D1 pathway . Antioxidants & redox signaling . 2013 ; 19 : 482 – 496 . Google Scholar Crossref Search ADS WorldCat 43 Chang JW , Jeon HB, Lee JH et al. Augmented expression of peroxiredoxin I in lung cancer . Biochem Biophys Res Commun 2001 ; 289 : 507 – 512 . Google Scholar Crossref Search ADS PubMed WorldCat 44 Noh DY , Ahn SJ, Lee RA et al. Overexpression of peroxiredoxin in human breast cancer . Anticancer Res 2001 ; 21 : 2085 – 2090 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 45 Kinnula VL , Lehtonen S, Sormunen R et al. Overexpression of peroxiredoxins I, II, III, V, and VI in malignant mesothelioma . J Pathol 2002 ; 196 : 316 – 323 . Google Scholar Crossref Search ADS PubMed WorldCat 46 Buggisch M , Ateghang B, Ruhe C et al. Stimulation of ES-cell-derived cardiomyogenesis and neonatal cardiac cell proliferation by reactive oxygen species and NADPH oxidase . J Cell Sci 2007 ; 120 : 885 – 894 . Google Scholar Crossref Search ADS PubMed WorldCat 47 Kim MO , Kim SH, Cho YY et al. ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4 . Nature Structural & Molecular Biology 2012 ; 19 : 283 – 290 . Google Scholar Crossref Search ADS WorldCat 48 Sartore RC , Campos PB, Trujillo CA et al. Retinoic acid-treated pluripotent stem cells undergoing neurogenesis present increased aneuploidy and micronuclei formation . Plos One 2011 ; 6 : e20667 . Google Scholar Crossref Search ADS PubMed WorldCat 49 Park YH , Kim SU, Lee BK et al. Prx I suppresses K-ras-driven lung tumorigenesis by opposing redox-sensitive ERK/cyclin D1 pathway . Antioxidant Redox Signal 2013 ; 19 : 482 – 496 . Google Scholar Crossref Search ADS WorldCat 50 Cao J , Schulte J, Knight A et al. Prdx1 inhibits tumorigenesis via regulating PTEN/AKT activity . EMBO J 2009 ; 28 : 1505 – 1517 . Google Scholar Crossref Search ADS PubMed WorldCat © 2013 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Dominant Role of Peroxiredoxin/JNK Axis in Stemness Regulation During Neurogenesis from Embryonic Stem Cells
JF - Stem Cells
DO - 10.1002/stem.1593
DA - 2014-04-01
UR - https://www.deepdyve.com/lp/oxford-university-press/dominant-role-of-peroxiredoxin-jnk-axis-in-stemness-regulation-during-cwMH2kfAO7
SP - 998
EP - 1011
VL - 32
IS - 4
DP - DeepDyve
ER -