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The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs Mi-Ok Lee a, *, Yee Sook Cho b, c a
Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea c Department of Bioscience, KRIBB School, University of Science & Technology (UST), Daejeon, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2019 Accepted 27 September 2019 Available online xxx
Selenium (Se) plays a vital role in reactive oxygen species (ROS) homeostasis and redox regulation in intracellular signaling via selenocysteine (Sec), known as the 21st proteinogenic amino acid, but its specific biological functions in development and disease remain undiscovered. In this study, we explored the role of selenophosphate synthetase 1 (SEPHS1) in the pluripotency maintenance and reprogramming. We found that high level of SEPHS1 is retained in undifferentiated embryonic stem cells (ESCs), which is decreased during their differentiation. SEPHS1 knockdown significantly reduced reprogramming efficiency, proving that SEPHS1 is required for acquisition of pluripotency. However, SEPHS1 knockdown did not affect the expression of significant pluripotency genes, suggesting that SEPHS1 may be involved in the survival of pluripotent stem cells rather than in the regulation of pluripotency genes. Transcriptome analysis revealed altered expression of the gene set related to the ROS pathway and apoptosis in SEPHS1-knockdown cells. We also demonstrated the role of SEPHS1 in human ESC clonogenicity, and we found improved single-cell survival of hESCs by selenium treatment in a concentrationdependent manner. Our study implies that hSEPHS1 is a regulator of selenium-mediated redox-signaling in human pluripotent stem cells and plays a role in their survival. © 2019 Elsevier Inc. All rights reserved.
Keywords: ESCs hPSCs Pluripotency Selenium SEPHS1 ROS signaling
1. Introduction Reactive oxygen species (ROS), a chemically reactive species containing oxygen, plays a critical role in diverse cellular signaling pathways and in homeostasis. However, excessive ROS cause oxidative stress through 1) DNA or RNA damage, 2) lipid peroxidation, 3) oxidation of amino acids, and 4) oxidative deactivation of a specific enzyme. In particular, the redox state in human embryonic stem cells (hESCs) is known to play an essential role in stem cell maintenance and lineage differentiation [1]. An imbalance between ROS production and the ROS scavenging system results in apoptosis or loss of pluripotency in hESCs. Primarily, hESCs are prone to apoptosis through high priming mitochondria, which are involved in sensitization to DNA-damage-induced apoptosis [2]. Therefore, redox regulation is vital for maintaining healthy hESCs. Endogenous ROS are produced via single-electron reduction of oxygen during oxidative phosphorylation (OXPHOS) to produce
* Corresponding author. E-mail address: [email protected] (M.-O. Lee).
ATP in mitochondria. In pluripotent stem cells (PSCs), mitochondria have globular immature mitochondria, and glycolysis takes precedence over OXPHOS, resulting in low ROS production [3e5]. Changes in metabolism during the differentiation of pluripotent stem cells lead to increased ROS production [4,6,7]. On the contrary, high anti-oxidant activity results in low ROS levels in hESCs through some anti-oxidant proteins such as superoxide dismutase 2 (SOD2), glutathione peroxidase 2 (GPX2), and glutathione reductase (GSR), which are highly expressed in undifferentiated cells [6,7]. However, contrasting results on the expression of oxidative stress response genes have also been reported [8], leading to an incomplete understanding of the mechanism of ROS scavenging to maintain redox homeostasis in human PSCs (hPSCs). Selenium is an essential micronutrient for most organisms and influences human development and health through various cellular effects including anti-oxidant, anti-inflammatory effects, and production of thyroid hormone [9]. The primary role of Selenium is mediated by selenocysteine (Sec), which is called the 21st proteinogenic amino acid, and has biological activity through incorporation into polypeptides of selenoproteins, whose genes have a specific secondary structure termed as the SECIS element in their
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Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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30 -untranslated region (UTRs) [10]. SEPHS1 is an enzyme that uses ATP and selenide to synthesize selenophosphate, which acts as a selenium donor [11]. In Drosophila, SEPHS1 mRNA is highly expressed during embryogenesis, which is especially enriched in highly proliferating cells and plays an essential role in cell proliferation and survival during embryogenesis [11]. SEPHS1 knockout in Drosophila has been shown to cause embryonic lethality [11,12]. In humans, expression of SEPHS1 is specifically found in mature oocytes and embryonic stem cells [13], but its role is completely unknown. In this study, we examine the expression of SEPHS1 of undifferentiated ESCs and its role in the acquisition of pluripotency in the context of the ROS pathway and apoptosis. We also examine whether selenium affects the single-cell survival of hESCs.
Sigma-Aldrich) and then selected the transduced cells by treatment with 0.5 mg/ml puromycin. 2.4. RNA extraction and quantitative RT- PCR
2. Materials and methods
Total RNA was extracted using the easy-BLUE™ Total RNA Extraction Kit (Cat# 17061, iNtRON Biotechnology) according to the manufacturer’s protocol. The amount of total RNA was quantified using a spectrophotometer (NanoDrop 2000, Thermo Fisher scientific) and cDNA was synthesized for 2 mg of total RNA using SuperScript™ IV First-Strand Synthesis System (Cat# 8091050, Invitrogen™, Thermo Fisher Scientific). The expression of each gene was compared by real-time PCR using Fast SYBR™ Green Master Mix (Cat# 4385612, Applied Biosystems™, Thermo Fisher scientific). The gene-specific primer sequences used in the experiments are summarized in Supplemental Table 1.
2.1. hESC culture
2.5. Immunocytochemistry and immuno-peroxidase detection
This research using hESCs and hiPSCs was approved by the Public Institutional Bioethics Committee designated by the Ministry of Health and Welfare (MoHW) (Seoul, Republic of Korea; IRB no.P01-201409-ES-01 and P01-201609-31-002). hESCs (H9, HUES15, HUES16, WiCell Research Institute, USA) were cultured as described previously [14]. Briefly, we prepared feeder cells with irradiated CF1-MEFs at a density of 5 105 cells per 60 mm-dish and then transferred mechanically detached clumps of hESCs on to the feeder layers with daily replacement of hESC culture medium [DMEM/F12 media supplemented with 20% KnockOut™ Serum Replacement (CAT# 10828-028, Thermo Fisher/Gibco), 1% MEMNEAA (CAT# 11140-050, Thermo Fisher Scientific), 1% penicillinstreptomycin (15140-122, Thermo Fisher Scientific), 1% GlutaMAX™ (CAT# 35050-061, Thermo Fisher Scientific), 0.1% betamercaptoethanol (CAT# 21985-023, Thermo Fisher Scientific), and 10 ng/ml FGF-basic]. The hESCs were passaged every week.
Cells were washed with PBS and fixed with 4% PFA solution for 10 min. After washing twice with PBS, cells were permeabilized in 0.2% Triton x-100 solution for 15 min and then blocked with 3% BSA solution for 30 min at room temperature (RT). The primary antibody to OCT4 (Cat#SC-8629, Santacruz) and SEPHS1 (Cat#SC365945,Santacruz) was bound overnight at 4 C, and cells were then washed three times with TBST buffer. Cell were then stained with fluorescence-conjugated secondary antibody at RT for 1 h. After washing three times with TBST buffer, cells were mounted with fluorescence mounting medium (Cat#S3023, Dako). Immuno-peroxidase staining was performed to verify the number of TRA-1-60þ colonies. Like in ICC, cells were fixed, permeabilized, blocked, and bound with the primary antibody. After binding with the biotinylated secondary antibody, streptavidinconjugated peroxidase from the Vectastain Universal Elite ABC kit (Cat# PK-6200, Vector Laboratories) was added and the cells were then stained using DAB (3,30 -diaminobenzidine) HRP substrate solution (Cat# SK-4100, Vector Laboratories).
2.2. iPSC reprogramming and AP staining Reprogramming factors, OSKM (OCT4, SOX2, KLF4, C-Myc), were delivered to human fibroblasts (CRL2097, 1 105 cells/well) by infecting 5MOI of retrovirus or by electroporation of episomal vector DNA (Neon Transfection System, Thermo Fisher Scientific). After four days, cells were transferred to 6-well plates at a 1:6 ratio and the medium (mTESR™1, Cat. No. 85850, STEMCELL Technologies) was changed every other day. On day 21, cells were fixed and stained using the Leukocyte Alkaline Phosphatase Kit (Sigma Aldrich, 86R) according to the manufacturer’s protocol. 2.3. Knock-down of SEPHS1 in hESCs To transiently inhibit the expression of SEPHS1, SEPHS1-targeting siRNA (siSEPHS1, CAT# 22929-1, BIONEER) and non-target control siRNA (siNC, CAT# SN-1001-CFG, BIONEER) were used. hESCs were transferred to Matrigel-coated plates with mTESR™1 medium and grown for seven days. The hESCs were then dissociated into small clumps using ReLeSR™ (CAT# 05872, STEMCELL technologies) and transferred to a Matrigel-coated dish. The next day, siRNA mixed with Lipofectamine RNAiMAX siRNA transfection reagent (CAT# 13778, Invitrogen, Thermo Fisher Scientific) was added to the hESCs at a concentration of 20 nM. After 24 h, the medium was replaced with fresh medium, and the cells were harvested for RNA extraction at the indicated times. To knockdown SEPHS1 stably, we infected hESCs and human fibroblasts with the lentivirus for SEPHS1-targeting shRNA (shSEPHS1, Sigma-Aldrich) and non-target control shRNA (shNC,
2.6. Transcriptome analysis and informatics analysis To analyze the global transcriptome differences, a microarray was performed using the one-color (Cy3) Whole Human Genome Microarray 4 44K, according to the manufacturer’s protocol (Agilent Technologies, Inc., Santa Clara, CA, USA) in Ebiogen (http:// www.e-biogen.com/index.php) as described previously [15]. Global-scale normalization was performed using GeneSpring software version 11.0 (Agilent Technologies, Inc.). Geneset Enrichment analysis was performed using GSEA Desktop v3.0 (http://software. broadinstitute.org/gsea/index.jsp, Broad Institute, Inc) [16,17]. The RNA sequencing data for comparing the transcriptome of hPSCs and hFBs in Fig. 3c was obtained from a previous report [18]. 3. Results 3.1. High expression of SEPHS1 in human pluripotent stem cells To compare the expression pattern in various cell types, we examined the expression of SEPHS1 and pluripotent stem cellspecific markers on the Amazonia web site (http://amazonia. transcriptome.eu/), an online resource to visualize the publicly available human whole-genome expression data. As expected, the expression of typical pluripotency markers, POU5F1, NANOG, Lin28, and N-myc was highly selective to hESCs and hiPSCs. In case of SEPHS1, very high levels of transcript expression were found in hESC and hiPSC lines, with relatively high expression in oocytes or
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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Fig. 1. SEPHS1 retained a higher expression level in undifferentiated hPSCs than in differentiated cells. (a) Relative mRNA expression levels during spontaneous EB differentiation, hPSC-specific marker; POU5F1, differentiated-cell markers; NES, PAX6, AFP. Data are represented as mean values of replicates with ±SD, **p < 0.01 (b) Semi-quantitative RT-PCR for SEPHS1 transcription levels compared to POU5F1 and AFP during spontaneous EB differentiation (c) Immune-blot of hSEPHS1 and hOCT4 in hFBs (CRL2097,MRC5) and hPSC lines (H9, HUES15, HUES16). Alpha-tubulin levels were used as the internal control, (d) Co-immunostaining of hSEPHS1 and hOCT4 in partially-differentiated hESC lines. DAPI; nuclear staining in all cells. Scale bar ¼ 100 mm.
Fig. 2. Knockdown of SEPHS1 inhibits iPSC reprogramming. (a) Relative mRNA expression levels of SEPHS1 and POU5F1 in the original hFBs, reprogrammed iPS, and differentiated iPS-EB. (bec) AP staining and immunoperoxidase detection of TRA-1-60 positive colonies in reprogramming of SEPHS1 shRNA-treated cells (d) Reprogramming efficiency of SEPHS1 shRNA lentivirus-treated cells to iPS cells (e) Relative mRNA levels of NANOG expression during iPS reprogramming with or without SEPHS1 shRNA lentivirus transduction.
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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ovaries, but low levels in other human tissues or cells (supp Fig. 1), as described previously [13]. To determine whether SEPHS1 is specifically expressed in undifferentiated pluripotent stem cells, we spontaneously differentiated hESCs through embryonic body (EB) formation and compared the transcriptional changes by real-time PCR (Fig. 1a). As the differentiation progressed, POU5F1 expression was decreased, and the expression of differentiation marker genes (PAX6 and NES; ectoderm specific markers, AFP; endoderm specific marker) was increased (Fig. 1a). SEPHS1 expression was also dramatically reduced during differentiation similar to POU5F1, indicating undifferentiated-PSC specific expression of SEPHS1 (Fig. 1a and b). To compare the levels of SEPHS1 protein in the undifferentiated pluripotent stem cells and differentiated cells, the relative amount of SEPHS1 protein in human fibroblasts (hFBs) and hESC lines was verified by immunoblotting. The results showed that SEPHS1 was highly expressed in undifferentiated hESCs relative to hFBs (Fig. 1c). Furthermore, we observed that SEPHS1 was expressed only in OCT4-positive cells in the partially differentiated hESC colonies
(Fig. 1d). These results demonstrated that human SEPHS1 was selectively expressed in the undifferentiated-state of hESCs at both transcript and protein levels, implying its possible role in pluripotency. 3.2. Intrinsic SEPHS1 is required for iPSC-reprogramming To analyze the expression of SEPHS1 during iPSC reprogramming, human foreskin fibroblasts were reprogrammed to iPSCs through ectopic expression of the canonical Yamanaka reprogramming factors (OCT4, SOX2, KLF4, C-Myc). As shown in Fig. 2a, SEPHS1 expression was increased in hiPSCs, and was decreased by EB differentiation of hiPSCs, similar to POU5F1 expression, suggesting functional associations of SEPHS1 in reprogramming. To determine the role of SEPHS1 in iPSC-reprogramming, we reprogrammed hFBs to hiPSCs along with shNC or shSEPHS1. Reprogramming efficiency was then evaluated by alkaline phosphatase (AP) staining (Fig. 2b) and immunoperoxidase staining of TRA-160þ colonies (Fig. 2c). The results showed that SEPHS1-knockdown
Fig. 3. Transient knockdown of SEPHS1 in hESCs induces the molecular signature of the ROS pathway. (a) Relative mRNA levels of pluripotency genes; POU5F1, REX1, KLF4 in siSEPHS1-treated hESCs (b) Enrichment plot of ROS pathway in the transcriptome data of siSEPHS1-treated cells compared to siNC-treated cells (c) Enrichment plot of the ROS pathway of hFBs compared to hPSCs (d) List of Gene set enrichment pathway in the siSEPHS1-treated cells. NES; Normalized Enrichment score, FDR; False discovery rate (e) Enrichment plot of apoptosis in SEPHS1 siRNA treated cells.
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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reduced the reprogramming efficiency significantly (Fig. 2bed). Moreover, induction of NANOG, a master transcription regulators of pluripotency, was also dramatically diminished by shSEPH1 treatment on day 16 of the reprogramming process. Overall, these results demonstrate that SEPHS1 expression is required for hiPSC reprogramming.
particular, we found that expression of the ROS pathway genes in SEPHS1-knockdown cells showed a pattern similar to that of the ROS pathway genes in differentiated hFBs compared to that in undifferentiated hPSCs (Fig. 3b and c). This result implies that SEPHS1 could regulate the hPSC-specific molecular signature of the ROS pathway.
3.3. Transient knockdown of SEPHS1 in hESCs activates reactive oxygen species pathways
3.4. Selenium treatment increases single-cell survival of hESCs
To verify the role of SEPHS1 in the maintenance of hPSCs, SEPHS1 expression was transiently knocked down in hESCs by transfection with SEPHS1-targeting siRNA (siSEPHS1). SEPHS1 expression in hESCs treated with two independent siSEPHS1 was reduced to less than 20% of that in non-target siRNA (siNC)-treated cells on day 1 and day 3 after siRNA treatment (Fig. 3a). However, there was no significant difference in the expression of pluripotency markers (POU5F1, REX1, and KLF4) in the cells treated with siSEPHS1 #1 and siSEPHS1 #2, respectively, compared to the siNCtreated cells. These results suggest that SEPHS1 is not directly involved in pluripotency-regulatory networks. To define the mechanistic effect of SEPHS1 in hESCs, we performed total transcriptome profiling of SEPHS1 knock-down cells via microarray analysis. Gene set enrichment analysis (GSEA) showed an altered molecular signature of Myc targets, the ROS pathway, G2M checkpoints, apoptosis, and so on in SEPHS1-knockdown cells (Fig. 3d). In
Furthermore, transcriptome data showed a different apoptotic signature in SEPHS1 knock-down cells (Fig. 3e). When hESCs were transduced with lentiviruses encoding non-target shRNAs or SEPHS1-targeting shRNA, stably integrated colonies were significantly reduced in shSEPHS1-treated cells (Fig. 4aec), suggesting the role of SEPHS1 in the clonogenicity of hESCs. Given that singlecell survival is related to the clonogenicity of hESCs, we tested the effect of selenium on the single-cell survival of hESCs. Selenium treatment of single-cell dissociated hESCs increased single cell survival in a concentration-dependent manner. Because Rho-kinase inhibitor, Y27632, is well-known as a chemical that inhibits singlecell dissociation-induced apoptosis of hPSCs, we tested the synergistic effects of sodium selenite with Y27632. When Y27632 was treated only for the first 24 h with continuous treatment of sodium selenite, single-cell survival of hESCs was increased by the selenium treatment in a dose-dependent manner, demonstrating a synergistic effect of selenium with Y27632.
Fig. 4. Treatment with selenium increased the single cell survival of hPSCs. (aeb) AP staining and immune-peroxidase detection of TRA-1-60 positive colonies after infection with the lentivirus for shNC and shSEPHS1. (c) The number of hESC colonies generated from single cells after infection with the lentivirus for shNC and shSEPHS1 (d) Quantitative graph (left panel) and AP staining for single cell survival by sodium selenite treatment at the indicated concentrations.
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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4. Discussion
analysis, decision to publish, or preparation of the manuscript.
Pluripotent stem cells have tremendous potential in the development of regenerative therapeutics, drug screening platforms, and in vivo mimics of human tissue models for studying human development and disease. With the potential for infinite availability of human pluripotent stem cells in regenerative medicine, it is crucial to understand the mechanistic details involved in acquiring and maintaining pluripotency. In this study, we analyzed the expression of undifferentiated stem cell-specific expression of SEPHS1, which is an essential regulator of selenium metabolism, and suggested its novel role in reprogramming and pluripotent stem cell maintenance. Selenium is an essential micronutrient, but its role in pluripotent stem cells is entirely unknown. We verified the undifferentiated-state specific expression of SEPHS1 in hPSCs, suggesting that selenium-mediated biological processes might play an important role in these cells. Reduced SEPHS1 expression blocked the formation of iPSC colonies during reprogramming of human dermal fibroblasts by the ectopic expression of OSKM, demonstrating its fundamental role in reprogramming. However, siSEPHS1-treated hESCs were still pluripotent, implying that SEPHS1 does not regulate pluripotency-regulating networks directly. Therefore, the mechanism of reprogramming inhibition by SEPHS1 knock-down is yet unknown. Because transcriptome analysis suggests that SEPHS1 plays a role in ROS signaling and apoptosis, it can be considered that SEPHS1-KD cells do not overcome reprogramming barrier. In particular, our results revealed that SEPHS1 is involved in the clonogenicity of hESCs, which is probably related to its role in reprogramming. Human pluripotent stem cells show unique apoptotic programs [2,19]. They are prone to apoptosis in response to genotoxic stress or oxidative stress [19] and they die after single-cell dissociation [20]. We showed that selenium treatment slightly increased the hESC survival in single-cell dissociation, indicating the involvement of selenium metabolism in single cell survival. Although Rhokinase inhibitors can completely inhibit the apoptosis of hESCs that have been dissociated to single cells [20], it is reported that single-cell passaging with Y27632 can accumulate DNA damage and genomic integrity rapidly, even within five passages, leading to anomalies such as copy-number variations [21]. The cause of DNA damage and genomic abnormalities due to single-cell passaging is unknown. In our study, selenium increased single cell survivals of hPSCs, although had low efficiency. Based on these result, a relation between single-cell dissociation and ROS signaling can be inferred. The selenium is probably responsible for protecting cells from ROSrelated cell damage caused by single cell dissociation. Since largescale production of hPSCs is required for regenerative medicine [22], further research into the association between single-cell dissociation and ROS will facilitate the development of safe culture methods that maintain genomic integrity. In conclusion, we provide novel information on the undifferentiation-state-specific expression and role of SEPHS1 in hESCs and reprogramming. SEPHS1 might contribute to the distinct properties of hESCs in ROS signaling and apoptosis. However, detailed mechanism of SEPHS1 in pluripotency maintenance is unknown.
Declaration of Competing interests
Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.NRF-2018R1C1B6008256, NRF-2018M3A9H3023077, NRF2017R1A2B2012190), and the KRIBB Research Initiative Program. The funders had no role in the study design, data collection or
The authors declare no competing financial interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.123. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.123 References [1] A.R. Ji, S.Y. Ku, M.S. Cho, Y.Y. Kim, Y.J. Kim, S.K. Oh, S.H. Kim, S.Y. Moon, Y.M. Choi, Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage, Exp. Mol. Med. 42 (2010) 175e186. [2] J.C. Liu, X. Guan, J.A. Ryan, A.G. Rivera, C. Mock, V. Agrawal, A. Letai, P.H. Lerou, G. Lahav, High mitochondrial priming sensitizes hESCs to DNA-damageinduced apoptosis, Cell Stem Cell 13 (2013) 483e491. [3] C.D. Folmes, T.J. Nelson, A. Martinez-Fernandez, D.K. Arrell, J.Z. Lindor, P.P. Dzeja, Y. Ikeda, C. Perez-Terzic, A. Terzic, Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming, Cell Metabol. 14 (2011) 264e271. [4] J. Zhang, E. Nuebel, G.Q. Daley, C.M. Koehler, M.A. Teitell, Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal, Cell Stem Cell 11 (2012) 589e595. [5] T.S. Cliff, T. Wu, B.R. Boward, A. Yin, H. Yin, J.N. Glushka, J.H. Prestegaard, S. Dalton, MYC controls human pluripotent stem cell fate decisions through regulation of metabolic flux, Cell Stem Cell 21 (2017) 502e516 e509. [6] G. Saretzki, L. Armstrong, A. Leake, M. Lako, T. von Zglinicki, Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells, Stem Cells 22 (2004) 962e971. [7] G. Saretzki, T. Walter, S. Atkinson, J.F. Passos, B. Bareth, W.N. Keith, R. Stewart, S. Hoare, M. Stojkovic, L. Armstrong, T. von Zglinicki, M. Lako, Downregulation of multiple stress defense mechanisms during differentiation of human embryonic stem cells, Stem Cells 26 (2008) 455e464. [8] A. Prigione, B. Fauler, R. Lurz, H. Lehrach, J. Adjaye, The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells, Stem Cells 28 (2010) 721e733. [9] M.P. Rayman, Selenium and human health, Lancet 379 (2012) 1256e1268. [10] E. Zoidis, I. Seremelis, N. Kontopoulos, G.P. Danezis, Selenium-dependent antioxidant enzymes: actions and properties of selenoproteins, Antioxidants 7 (2018). [11] J. Na, J. Jung, J. Bang, Q. Lu, B.A. Carlson, X. Guo, V.N. Gladyshev, J. Kim, D.L. Hatfield, B.J. Lee, Selenophosphate synthetase 1 and its role in redox homeostasis, defense and proliferation, Free Radic. Biol. Med. 127 (2018) 190e197. [12] B. Alsina, F. Serras, J. Baguna, M. Corominas, patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis, Mol. Gen. Genet. 257 (1998) 113e123. [13] S. Assou, D. Cerecedo, S. Tondeur, V. Pantesco, O. Hovatta, B. Klein, S. Hamamah, J. De Vos, A gene expression signature shared by human mature oocytes and embryonic stem cells, BMC Genomics 10 (2009) 10. [14] K.B. Jung, O. Kwon, M.O. Lee, H. Lee, Y.S. Son, O. Habib, J.H. Oh, H.S. Cho, C.R. Jung, J. Kim, M.Y. Son, Blockade of STAT3 causes severe in vitro and in vivo maturation defects in intestinal organoids derived from human embryonic stem cells, J. Clin. Med. 8 (2019). [15] M.O. Lee, K.B. Jung, S.J. Jo, S.A. Hyun, K.S. Moon, J.W. Seo, S.H. Kim, M.Y. Son, Modelling cardiac fibrosis using three-dimensional cardiac microtissues derived from human embryonic stem cells, J. Biol. Eng. 13 (2019) 15. [16] V.K. Mootha, C.M. Lindgren, K.F. Eriksson, A. Subramanian, S. Sihag, J. Lehar, P. Puigserver, E. Carlsson, M. Ridderstrale, E. Laurila, N. Houstis, M.J. Daly, N. Patterson, J.P. Mesirov, T.R. Golub, P. Tamayo, B. Spiegelman, E.S. Lander, J.N. Hirschhorn, D. Altshuler, L.C. Groop, PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes, Nat. Genet. 34 (2003) 267e273. [17] A. Subramanian, P. Tamayo, V.K. Mootha, S. Mukherjee, B.L. Ebert, M.A. Gillette, A. Paulovich, S.L. Pomeroy, T.R. Golub, E.S. Lander, J.P. Mesirov, Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 15545e15550. [18] D.H. Phanstiel, J. Brumbaugh, C.D. Wenger, S. Tian, M.D. Probasco, D.J. Bailey,
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
M.-O. Lee, Y.S. Cho / Biochemical and Biophysical Research Communications xxx (xxxx) xxx D.L. Swaney, M.A. Tervo, J.M. Bolin, V. Ruotti, R. Stewart, J.A. Thomson, J.J. Coon, Proteomic and phosphoproteomic comparison of human ES and iPS cells, Nat. Methods 8 (2011) 821e827. [19] H.C. Jeong, S.J. Cho, M.O. Lee, H.J. Cha, Technical approaches to induce selective cell death of pluripotent stem cells, Cell. Mol. Life Sci. 74 (2017) 2601e2611. [20] K. Watanabe, M. Ueno, D. Kamiya, A. Nishiyama, M. Matsumura, T. Wataya, J.B. Takahashi, S. Nishikawa, S. Nishikawa, K. Muguruma, Y. Sasai, A ROCK inhibitor permits survival of dissociated human embryonic stem cells, Nat.
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Biotechnol. 25 (2007) 681e686. [21] Q. Bai, J.M. Ramirez, F. Becker, V. Pantesco, T. Lavabre-Bertrand, O. Hovatta, J.M. Lemaitre, F. Pellestor, J. De Vos, Temporal analysis of genome alterations induced by single-cell passaging in human embryonic stem cells, Stem Cells Dev. 24 (2015) 653e662. [22] M.O. Lee, H. Jeon, M.Y. Son, S.C. Lee, Y.S. Cho, Clump-passaging-based efficient 3D culture of human pluripotent stem cells under chemically defined conditions, Biochem. Biophys. Res. Commun. 493 (2017) 723e730.
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs Mi-Ok Lee a, *, Yee Sook Cho b, c a
Stem Cell Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Republic of Korea c Department of Bioscience, KRIBB School, University of Science & Technology (UST), Daejeon, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 August 2019 Accepted 27 September 2019 Available online xxx
Selenium (Se) plays a vital role in reactive oxygen species (ROS) homeostasis and redox regulation in intracellular signaling via selenocysteine (Sec), known as the 21st proteinogenic amino acid, but its specific biological functions in development and disease remain undiscovered. In this study, we explored the role of selenophosphate synthetase 1 (SEPHS1) in the pluripotency maintenance and reprogramming. We found that high level of SEPHS1 is retained in undifferentiated embryonic stem cells (ESCs), which is decreased during their differentiation. SEPHS1 knockdown significantly reduced reprogramming efficiency, proving that SEPHS1 is required for acquisition of pluripotency. However, SEPHS1 knockdown did not affect the expression of significant pluripotency genes, suggesting that SEPHS1 may be involved in the survival of pluripotent stem cells rather than in the regulation of pluripotency genes. Transcriptome analysis revealed altered expression of the gene set related to the ROS pathway and apoptosis in SEPHS1-knockdown cells. We also demonstrated the role of SEPHS1 in human ESC clonogenicity, and we found improved single-cell survival of hESCs by selenium treatment in a concentrationdependent manner. Our study implies that hSEPHS1 is a regulator of selenium-mediated redox-signaling in human pluripotent stem cells and plays a role in their survival. © 2019 Elsevier Inc. All rights reserved.
Keywords: ESCs hPSCs Pluripotency Selenium SEPHS1 ROS signaling
1. Introduction Reactive oxygen species (ROS), a chemically reactive species containing oxygen, plays a critical role in diverse cellular signaling pathways and in homeostasis. However, excessive ROS cause oxidative stress through 1) DNA or RNA damage, 2) lipid peroxidation, 3) oxidation of amino acids, and 4) oxidative deactivation of a specific enzyme. In particular, the redox state in human embryonic stem cells (hESCs) is known to play an essential role in stem cell maintenance and lineage differentiation [1]. An imbalance between ROS production and the ROS scavenging system results in apoptosis or loss of pluripotency in hESCs. Primarily, hESCs are prone to apoptosis through high priming mitochondria, which are involved in sensitization to DNA-damage-induced apoptosis [2]. Therefore, redox regulation is vital for maintaining healthy hESCs. Endogenous ROS are produced via single-electron reduction of oxygen during oxidative phosphorylation (OXPHOS) to produce
* Corresponding author. E-mail address: [email protected] (M.-O. Lee).
ATP in mitochondria. In pluripotent stem cells (PSCs), mitochondria have globular immature mitochondria, and glycolysis takes precedence over OXPHOS, resulting in low ROS production [3e5]. Changes in metabolism during the differentiation of pluripotent stem cells lead to increased ROS production [4,6,7]. On the contrary, high anti-oxidant activity results in low ROS levels in hESCs through some anti-oxidant proteins such as superoxide dismutase 2 (SOD2), glutathione peroxidase 2 (GPX2), and glutathione reductase (GSR), which are highly expressed in undifferentiated cells [6,7]. However, contrasting results on the expression of oxidative stress response genes have also been reported [8], leading to an incomplete understanding of the mechanism of ROS scavenging to maintain redox homeostasis in human PSCs (hPSCs). Selenium is an essential micronutrient for most organisms and influences human development and health through various cellular effects including anti-oxidant, anti-inflammatory effects, and production of thyroid hormone [9]. The primary role of Selenium is mediated by selenocysteine (Sec), which is called the 21st proteinogenic amino acid, and has biological activity through incorporation into polypeptides of selenoproteins, whose genes have a specific secondary structure termed as the SECIS element in their
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Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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30 -untranslated region (UTRs) [10]. SEPHS1 is an enzyme that uses ATP and selenide to synthesize selenophosphate, which acts as a selenium donor [11]. In Drosophila, SEPHS1 mRNA is highly expressed during embryogenesis, which is especially enriched in highly proliferating cells and plays an essential role in cell proliferation and survival during embryogenesis [11]. SEPHS1 knockout in Drosophila has been shown to cause embryonic lethality [11,12]. In humans, expression of SEPHS1 is specifically found in mature oocytes and embryonic stem cells [13], but its role is completely unknown. In this study, we examine the expression of SEPHS1 of undifferentiated ESCs and its role in the acquisition of pluripotency in the context of the ROS pathway and apoptosis. We also examine whether selenium affects the single-cell survival of hESCs.
Sigma-Aldrich) and then selected the transduced cells by treatment with 0.5 mg/ml puromycin. 2.4. RNA extraction and quantitative RT- PCR
2. Materials and methods
Total RNA was extracted using the easy-BLUE™ Total RNA Extraction Kit (Cat# 17061, iNtRON Biotechnology) according to the manufacturer’s protocol. The amount of total RNA was quantified using a spectrophotometer (NanoDrop 2000, Thermo Fisher scientific) and cDNA was synthesized for 2 mg of total RNA using SuperScript™ IV First-Strand Synthesis System (Cat# 8091050, Invitrogen™, Thermo Fisher Scientific). The expression of each gene was compared by real-time PCR using Fast SYBR™ Green Master Mix (Cat# 4385612, Applied Biosystems™, Thermo Fisher scientific). The gene-specific primer sequences used in the experiments are summarized in Supplemental Table 1.
2.1. hESC culture
2.5. Immunocytochemistry and immuno-peroxidase detection
This research using hESCs and hiPSCs was approved by the Public Institutional Bioethics Committee designated by the Ministry of Health and Welfare (MoHW) (Seoul, Republic of Korea; IRB no.P01-201409-ES-01 and P01-201609-31-002). hESCs (H9, HUES15, HUES16, WiCell Research Institute, USA) were cultured as described previously [14]. Briefly, we prepared feeder cells with irradiated CF1-MEFs at a density of 5 105 cells per 60 mm-dish and then transferred mechanically detached clumps of hESCs on to the feeder layers with daily replacement of hESC culture medium [DMEM/F12 media supplemented with 20% KnockOut™ Serum Replacement (CAT# 10828-028, Thermo Fisher/Gibco), 1% MEMNEAA (CAT# 11140-050, Thermo Fisher Scientific), 1% penicillinstreptomycin (15140-122, Thermo Fisher Scientific), 1% GlutaMAX™ (CAT# 35050-061, Thermo Fisher Scientific), 0.1% betamercaptoethanol (CAT# 21985-023, Thermo Fisher Scientific), and 10 ng/ml FGF-basic]. The hESCs were passaged every week.
Cells were washed with PBS and fixed with 4% PFA solution for 10 min. After washing twice with PBS, cells were permeabilized in 0.2% Triton x-100 solution for 15 min and then blocked with 3% BSA solution for 30 min at room temperature (RT). The primary antibody to OCT4 (Cat#SC-8629, Santacruz) and SEPHS1 (Cat#SC365945,Santacruz) was bound overnight at 4 C, and cells were then washed three times with TBST buffer. Cell were then stained with fluorescence-conjugated secondary antibody at RT for 1 h. After washing three times with TBST buffer, cells were mounted with fluorescence mounting medium (Cat#S3023, Dako). Immuno-peroxidase staining was performed to verify the number of TRA-1-60þ colonies. Like in ICC, cells were fixed, permeabilized, blocked, and bound with the primary antibody. After binding with the biotinylated secondary antibody, streptavidinconjugated peroxidase from the Vectastain Universal Elite ABC kit (Cat# PK-6200, Vector Laboratories) was added and the cells were then stained using DAB (3,30 -diaminobenzidine) HRP substrate solution (Cat# SK-4100, Vector Laboratories).
2.2. iPSC reprogramming and AP staining Reprogramming factors, OSKM (OCT4, SOX2, KLF4, C-Myc), were delivered to human fibroblasts (CRL2097, 1 105 cells/well) by infecting 5MOI of retrovirus or by electroporation of episomal vector DNA (Neon Transfection System, Thermo Fisher Scientific). After four days, cells were transferred to 6-well plates at a 1:6 ratio and the medium (mTESR™1, Cat. No. 85850, STEMCELL Technologies) was changed every other day. On day 21, cells were fixed and stained using the Leukocyte Alkaline Phosphatase Kit (Sigma Aldrich, 86R) according to the manufacturer’s protocol. 2.3. Knock-down of SEPHS1 in hESCs To transiently inhibit the expression of SEPHS1, SEPHS1-targeting siRNA (siSEPHS1, CAT# 22929-1, BIONEER) and non-target control siRNA (siNC, CAT# SN-1001-CFG, BIONEER) were used. hESCs were transferred to Matrigel-coated plates with mTESR™1 medium and grown for seven days. The hESCs were then dissociated into small clumps using ReLeSR™ (CAT# 05872, STEMCELL technologies) and transferred to a Matrigel-coated dish. The next day, siRNA mixed with Lipofectamine RNAiMAX siRNA transfection reagent (CAT# 13778, Invitrogen, Thermo Fisher Scientific) was added to the hESCs at a concentration of 20 nM. After 24 h, the medium was replaced with fresh medium, and the cells were harvested for RNA extraction at the indicated times. To knockdown SEPHS1 stably, we infected hESCs and human fibroblasts with the lentivirus for SEPHS1-targeting shRNA (shSEPHS1, Sigma-Aldrich) and non-target control shRNA (shNC,
2.6. Transcriptome analysis and informatics analysis To analyze the global transcriptome differences, a microarray was performed using the one-color (Cy3) Whole Human Genome Microarray 4 44K, according to the manufacturer’s protocol (Agilent Technologies, Inc., Santa Clara, CA, USA) in Ebiogen (http:// www.e-biogen.com/index.php) as described previously [15]. Global-scale normalization was performed using GeneSpring software version 11.0 (Agilent Technologies, Inc.). Geneset Enrichment analysis was performed using GSEA Desktop v3.0 (http://software. broadinstitute.org/gsea/index.jsp, Broad Institute, Inc) [16,17]. The RNA sequencing data for comparing the transcriptome of hPSCs and hFBs in Fig. 3c was obtained from a previous report [18]. 3. Results 3.1. High expression of SEPHS1 in human pluripotent stem cells To compare the expression pattern in various cell types, we examined the expression of SEPHS1 and pluripotent stem cellspecific markers on the Amazonia web site (http://amazonia. transcriptome.eu/), an online resource to visualize the publicly available human whole-genome expression data. As expected, the expression of typical pluripotency markers, POU5F1, NANOG, Lin28, and N-myc was highly selective to hESCs and hiPSCs. In case of SEPHS1, very high levels of transcript expression were found in hESC and hiPSC lines, with relatively high expression in oocytes or
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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Fig. 1. SEPHS1 retained a higher expression level in undifferentiated hPSCs than in differentiated cells. (a) Relative mRNA expression levels during spontaneous EB differentiation, hPSC-specific marker; POU5F1, differentiated-cell markers; NES, PAX6, AFP. Data are represented as mean values of replicates with ±SD, **p < 0.01 (b) Semi-quantitative RT-PCR for SEPHS1 transcription levels compared to POU5F1 and AFP during spontaneous EB differentiation (c) Immune-blot of hSEPHS1 and hOCT4 in hFBs (CRL2097,MRC5) and hPSC lines (H9, HUES15, HUES16). Alpha-tubulin levels were used as the internal control, (d) Co-immunostaining of hSEPHS1 and hOCT4 in partially-differentiated hESC lines. DAPI; nuclear staining in all cells. Scale bar ¼ 100 mm.
Fig. 2. Knockdown of SEPHS1 inhibits iPSC reprogramming. (a) Relative mRNA expression levels of SEPHS1 and POU5F1 in the original hFBs, reprogrammed iPS, and differentiated iPS-EB. (bec) AP staining and immunoperoxidase detection of TRA-1-60 positive colonies in reprogramming of SEPHS1 shRNA-treated cells (d) Reprogramming efficiency of SEPHS1 shRNA lentivirus-treated cells to iPS cells (e) Relative mRNA levels of NANOG expression during iPS reprogramming with or without SEPHS1 shRNA lentivirus transduction.
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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ovaries, but low levels in other human tissues or cells (supp Fig. 1), as described previously [13]. To determine whether SEPHS1 is specifically expressed in undifferentiated pluripotent stem cells, we spontaneously differentiated hESCs through embryonic body (EB) formation and compared the transcriptional changes by real-time PCR (Fig. 1a). As the differentiation progressed, POU5F1 expression was decreased, and the expression of differentiation marker genes (PAX6 and NES; ectoderm specific markers, AFP; endoderm specific marker) was increased (Fig. 1a). SEPHS1 expression was also dramatically reduced during differentiation similar to POU5F1, indicating undifferentiated-PSC specific expression of SEPHS1 (Fig. 1a and b). To compare the levels of SEPHS1 protein in the undifferentiated pluripotent stem cells and differentiated cells, the relative amount of SEPHS1 protein in human fibroblasts (hFBs) and hESC lines was verified by immunoblotting. The results showed that SEPHS1 was highly expressed in undifferentiated hESCs relative to hFBs (Fig. 1c). Furthermore, we observed that SEPHS1 was expressed only in OCT4-positive cells in the partially differentiated hESC colonies
(Fig. 1d). These results demonstrated that human SEPHS1 was selectively expressed in the undifferentiated-state of hESCs at both transcript and protein levels, implying its possible role in pluripotency. 3.2. Intrinsic SEPHS1 is required for iPSC-reprogramming To analyze the expression of SEPHS1 during iPSC reprogramming, human foreskin fibroblasts were reprogrammed to iPSCs through ectopic expression of the canonical Yamanaka reprogramming factors (OCT4, SOX2, KLF4, C-Myc). As shown in Fig. 2a, SEPHS1 expression was increased in hiPSCs, and was decreased by EB differentiation of hiPSCs, similar to POU5F1 expression, suggesting functional associations of SEPHS1 in reprogramming. To determine the role of SEPHS1 in iPSC-reprogramming, we reprogrammed hFBs to hiPSCs along with shNC or shSEPHS1. Reprogramming efficiency was then evaluated by alkaline phosphatase (AP) staining (Fig. 2b) and immunoperoxidase staining of TRA-160þ colonies (Fig. 2c). The results showed that SEPHS1-knockdown
Fig. 3. Transient knockdown of SEPHS1 in hESCs induces the molecular signature of the ROS pathway. (a) Relative mRNA levels of pluripotency genes; POU5F1, REX1, KLF4 in siSEPHS1-treated hESCs (b) Enrichment plot of ROS pathway in the transcriptome data of siSEPHS1-treated cells compared to siNC-treated cells (c) Enrichment plot of the ROS pathway of hFBs compared to hPSCs (d) List of Gene set enrichment pathway in the siSEPHS1-treated cells. NES; Normalized Enrichment score, FDR; False discovery rate (e) Enrichment plot of apoptosis in SEPHS1 siRNA treated cells.
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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reduced the reprogramming efficiency significantly (Fig. 2bed). Moreover, induction of NANOG, a master transcription regulators of pluripotency, was also dramatically diminished by shSEPH1 treatment on day 16 of the reprogramming process. Overall, these results demonstrate that SEPHS1 expression is required for hiPSC reprogramming.
particular, we found that expression of the ROS pathway genes in SEPHS1-knockdown cells showed a pattern similar to that of the ROS pathway genes in differentiated hFBs compared to that in undifferentiated hPSCs (Fig. 3b and c). This result implies that SEPHS1 could regulate the hPSC-specific molecular signature of the ROS pathway.
3.3. Transient knockdown of SEPHS1 in hESCs activates reactive oxygen species pathways
3.4. Selenium treatment increases single-cell survival of hESCs
To verify the role of SEPHS1 in the maintenance of hPSCs, SEPHS1 expression was transiently knocked down in hESCs by transfection with SEPHS1-targeting siRNA (siSEPHS1). SEPHS1 expression in hESCs treated with two independent siSEPHS1 was reduced to less than 20% of that in non-target siRNA (siNC)-treated cells on day 1 and day 3 after siRNA treatment (Fig. 3a). However, there was no significant difference in the expression of pluripotency markers (POU5F1, REX1, and KLF4) in the cells treated with siSEPHS1 #1 and siSEPHS1 #2, respectively, compared to the siNCtreated cells. These results suggest that SEPHS1 is not directly involved in pluripotency-regulatory networks. To define the mechanistic effect of SEPHS1 in hESCs, we performed total transcriptome profiling of SEPHS1 knock-down cells via microarray analysis. Gene set enrichment analysis (GSEA) showed an altered molecular signature of Myc targets, the ROS pathway, G2M checkpoints, apoptosis, and so on in SEPHS1-knockdown cells (Fig. 3d). In
Furthermore, transcriptome data showed a different apoptotic signature in SEPHS1 knock-down cells (Fig. 3e). When hESCs were transduced with lentiviruses encoding non-target shRNAs or SEPHS1-targeting shRNA, stably integrated colonies were significantly reduced in shSEPHS1-treated cells (Fig. 4aec), suggesting the role of SEPHS1 in the clonogenicity of hESCs. Given that singlecell survival is related to the clonogenicity of hESCs, we tested the effect of selenium on the single-cell survival of hESCs. Selenium treatment of single-cell dissociated hESCs increased single cell survival in a concentration-dependent manner. Because Rho-kinase inhibitor, Y27632, is well-known as a chemical that inhibits singlecell dissociation-induced apoptosis of hPSCs, we tested the synergistic effects of sodium selenite with Y27632. When Y27632 was treated only for the first 24 h with continuous treatment of sodium selenite, single-cell survival of hESCs was increased by the selenium treatment in a dose-dependent manner, demonstrating a synergistic effect of selenium with Y27632.
Fig. 4. Treatment with selenium increased the single cell survival of hPSCs. (aeb) AP staining and immune-peroxidase detection of TRA-1-60 positive colonies after infection with the lentivirus for shNC and shSEPHS1. (c) The number of hESC colonies generated from single cells after infection with the lentivirus for shNC and shSEPHS1 (d) Quantitative graph (left panel) and AP staining for single cell survival by sodium selenite treatment at the indicated concentrations.
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
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4. Discussion
analysis, decision to publish, or preparation of the manuscript.
Pluripotent stem cells have tremendous potential in the development of regenerative therapeutics, drug screening platforms, and in vivo mimics of human tissue models for studying human development and disease. With the potential for infinite availability of human pluripotent stem cells in regenerative medicine, it is crucial to understand the mechanistic details involved in acquiring and maintaining pluripotency. In this study, we analyzed the expression of undifferentiated stem cell-specific expression of SEPHS1, which is an essential regulator of selenium metabolism, and suggested its novel role in reprogramming and pluripotent stem cell maintenance. Selenium is an essential micronutrient, but its role in pluripotent stem cells is entirely unknown. We verified the undifferentiated-state specific expression of SEPHS1 in hPSCs, suggesting that selenium-mediated biological processes might play an important role in these cells. Reduced SEPHS1 expression blocked the formation of iPSC colonies during reprogramming of human dermal fibroblasts by the ectopic expression of OSKM, demonstrating its fundamental role in reprogramming. However, siSEPHS1-treated hESCs were still pluripotent, implying that SEPHS1 does not regulate pluripotency-regulating networks directly. Therefore, the mechanism of reprogramming inhibition by SEPHS1 knock-down is yet unknown. Because transcriptome analysis suggests that SEPHS1 plays a role in ROS signaling and apoptosis, it can be considered that SEPHS1-KD cells do not overcome reprogramming barrier. In particular, our results revealed that SEPHS1 is involved in the clonogenicity of hESCs, which is probably related to its role in reprogramming. Human pluripotent stem cells show unique apoptotic programs [2,19]. They are prone to apoptosis in response to genotoxic stress or oxidative stress [19] and they die after single-cell dissociation [20]. We showed that selenium treatment slightly increased the hESC survival in single-cell dissociation, indicating the involvement of selenium metabolism in single cell survival. Although Rhokinase inhibitors can completely inhibit the apoptosis of hESCs that have been dissociated to single cells [20], it is reported that single-cell passaging with Y27632 can accumulate DNA damage and genomic integrity rapidly, even within five passages, leading to anomalies such as copy-number variations [21]. The cause of DNA damage and genomic abnormalities due to single-cell passaging is unknown. In our study, selenium increased single cell survivals of hPSCs, although had low efficiency. Based on these result, a relation between single-cell dissociation and ROS signaling can be inferred. The selenium is probably responsible for protecting cells from ROSrelated cell damage caused by single cell dissociation. Since largescale production of hPSCs is required for regenerative medicine [22], further research into the association between single-cell dissociation and ROS will facilitate the development of safe culture methods that maintain genomic integrity. In conclusion, we provide novel information on the undifferentiation-state-specific expression and role of SEPHS1 in hESCs and reprogramming. SEPHS1 might contribute to the distinct properties of hESCs in ROS signaling and apoptosis. However, detailed mechanism of SEPHS1 in pluripotency maintenance is unknown.
Declaration of Competing interests
Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No.NRF-2018R1C1B6008256, NRF-2018M3A9H3023077, NRF2017R1A2B2012190), and the KRIBB Research Initiative Program. The funders had no role in the study design, data collection or
The authors declare no competing financial interests. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.123. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.123 References [1] A.R. Ji, S.Y. Ku, M.S. Cho, Y.Y. Kim, Y.J. Kim, S.K. Oh, S.H. Kim, S.Y. Moon, Y.M. Choi, Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage, Exp. Mol. Med. 42 (2010) 175e186. [2] J.C. Liu, X. Guan, J.A. Ryan, A.G. Rivera, C. Mock, V. Agrawal, A. Letai, P.H. Lerou, G. Lahav, High mitochondrial priming sensitizes hESCs to DNA-damageinduced apoptosis, Cell Stem Cell 13 (2013) 483e491. [3] C.D. Folmes, T.J. Nelson, A. Martinez-Fernandez, D.K. Arrell, J.Z. Lindor, P.P. Dzeja, Y. Ikeda, C. Perez-Terzic, A. Terzic, Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming, Cell Metabol. 14 (2011) 264e271. [4] J. Zhang, E. Nuebel, G.Q. Daley, C.M. Koehler, M.A. Teitell, Metabolic regulation in pluripotent stem cells during reprogramming and self-renewal, Cell Stem Cell 11 (2012) 589e595. [5] T.S. Cliff, T. Wu, B.R. Boward, A. Yin, H. Yin, J.N. Glushka, J.H. Prestegaard, S. Dalton, MYC controls human pluripotent stem cell fate decisions through regulation of metabolic flux, Cell Stem Cell 21 (2017) 502e516 e509. [6] G. Saretzki, L. Armstrong, A. Leake, M. Lako, T. von Zglinicki, Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells, Stem Cells 22 (2004) 962e971. [7] G. Saretzki, T. Walter, S. Atkinson, J.F. Passos, B. Bareth, W.N. Keith, R. Stewart, S. Hoare, M. Stojkovic, L. Armstrong, T. von Zglinicki, M. Lako, Downregulation of multiple stress defense mechanisms during differentiation of human embryonic stem cells, Stem Cells 26 (2008) 455e464. [8] A. Prigione, B. Fauler, R. Lurz, H. Lehrach, J. Adjaye, The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells, Stem Cells 28 (2010) 721e733. [9] M.P. Rayman, Selenium and human health, Lancet 379 (2012) 1256e1268. [10] E. Zoidis, I. Seremelis, N. Kontopoulos, G.P. Danezis, Selenium-dependent antioxidant enzymes: actions and properties of selenoproteins, Antioxidants 7 (2018). [11] J. Na, J. Jung, J. Bang, Q. Lu, B.A. Carlson, X. Guo, V.N. Gladyshev, J. Kim, D.L. Hatfield, B.J. Lee, Selenophosphate synthetase 1 and its role in redox homeostasis, defense and proliferation, Free Radic. Biol. Med. 127 (2018) 190e197. [12] B. Alsina, F. Serras, J. Baguna, M. Corominas, patufet, the gene encoding the Drosophila melanogaster homologue of selenophosphate synthetase, is involved in imaginal disc morphogenesis, Mol. Gen. Genet. 257 (1998) 113e123. [13] S. Assou, D. Cerecedo, S. Tondeur, V. Pantesco, O. Hovatta, B. Klein, S. Hamamah, J. De Vos, A gene expression signature shared by human mature oocytes and embryonic stem cells, BMC Genomics 10 (2009) 10. [14] K.B. Jung, O. Kwon, M.O. Lee, H. Lee, Y.S. Son, O. Habib, J.H. Oh, H.S. Cho, C.R. Jung, J. Kim, M.Y. Son, Blockade of STAT3 causes severe in vitro and in vivo maturation defects in intestinal organoids derived from human embryonic stem cells, J. Clin. Med. 8 (2019). [15] M.O. Lee, K.B. Jung, S.J. Jo, S.A. Hyun, K.S. Moon, J.W. Seo, S.H. Kim, M.Y. Son, Modelling cardiac fibrosis using three-dimensional cardiac microtissues derived from human embryonic stem cells, J. Biol. Eng. 13 (2019) 15. [16] V.K. Mootha, C.M. Lindgren, K.F. Eriksson, A. Subramanian, S. Sihag, J. Lehar, P. Puigserver, E. Carlsson, M. Ridderstrale, E. Laurila, N. Houstis, M.J. Daly, N. Patterson, J.P. Mesirov, T.R. Golub, P. Tamayo, B. Spiegelman, E.S. Lander, J.N. Hirschhorn, D. Altshuler, L.C. Groop, PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes, Nat. Genet. 34 (2003) 267e273. [17] A. Subramanian, P. Tamayo, V.K. Mootha, S. Mukherjee, B.L. Ebert, M.A. Gillette, A. Paulovich, S.L. Pomeroy, T.R. Golub, E.S. Lander, J.P. Mesirov, Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 15545e15550. [18] D.H. Phanstiel, J. Brumbaugh, C.D. Wenger, S. Tian, M.D. Probasco, D.J. Bailey,
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123
M.-O. Lee, Y.S. Cho / Biochemical and Biophysical Research Communications xxx (xxxx) xxx D.L. Swaney, M.A. Tervo, J.M. Bolin, V. Ruotti, R. Stewart, J.A. Thomson, J.J. Coon, Proteomic and phosphoproteomic comparison of human ES and iPS cells, Nat. Methods 8 (2011) 821e827. [19] H.C. Jeong, S.J. Cho, M.O. Lee, H.J. Cha, Technical approaches to induce selective cell death of pluripotent stem cells, Cell. Mol. Life Sci. 74 (2017) 2601e2611. [20] K. Watanabe, M. Ueno, D. Kamiya, A. Nishiyama, M. Matsumura, T. Wataya, J.B. Takahashi, S. Nishikawa, S. Nishikawa, K. Muguruma, Y. Sasai, A ROCK inhibitor permits survival of dissociated human embryonic stem cells, Nat.
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Biotechnol. 25 (2007) 681e686. [21] Q. Bai, J.M. Ramirez, F. Becker, V. Pantesco, T. Lavabre-Bertrand, O. Hovatta, J.M. Lemaitre, F. Pellestor, J. De Vos, Temporal analysis of genome alterations induced by single-cell passaging in human embryonic stem cells, Stem Cells Dev. 24 (2015) 653e662. [22] M.O. Lee, H. Jeon, M.Y. Son, S.C. Lee, Y.S. Cho, Clump-passaging-based efficient 3D culture of human pluripotent stem cells under chemically defined conditions, Biochem. Biophys. Res. Commun. 493 (2017) 723e730.
Please cite this article as: M.-O. Lee, Y.S. Cho, The role of selenium-mediated redox signaling by selenophosphate synthetase 1 (SEPHS1) in hESCs, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.09.123