Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells

RESEARCH ARTICLE Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells Daniela Pezzolla 1,3 , Javier López-Beas 1,3 , Christian C. Lachaud 1 , Alejandro DomínguezRodríguez 2 , Tarik Smani 2 , Abdelkrim Hmadcha 1,3 * ‡ , Bernat Soria 1,3‡ 1Department of Stem Cells, Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER)—Fundación Progreso y Salud (FPS), Sevilla, Spain, 2Cardiovascular Pathophysiology, Institute of Biomedicine of Seville (IBIS), Sevilla, Spain, 3Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Barcelona, Spain ‡These authors are joint senior authors on this work. *[email protected] Abstract Human embryonic stem cells (hESCs) retain the extraordinary capacity to differentiate into different cell types of an adult organism, including pancreatic β-cells. For this particular lineage, although a lot of effort has been made in the last ten years to achieve an efficient and reproducible differentiation protocol, it was not until recently that this aim was roughly accomplished. Besides, several studies evidenced the impact of resveratrol (RSV) on insulin secretion, even though the mechanism by which this polyphenol potentiates glucosestimulated insulin secretion (GSIS) is still not clear. The aim of this study was to optimize an efficient differentiation protocol that mimics in vivo pancreatic organogenesis and to investigate whether RSV may improve the final maturation step to obtain functional insulinsecreting cells. Our results indicate that treatment of hESCs (HS-181) with activin-A induced definitive endoderm differentiation as detected by the expression of SOX17 and FOXA2. Addition of retinoic acid (RA), Noggin and Cyclopamine promoted pancreatic differentiation as indicated by the expression of the early pancreatic progenitor markers ISL1, NGN3 and PDX1. Moreover, during maturation in suspension culture, differentiating cells assembled in islet-like clusters, which expressed specific endocrine markers such as PDX1,SST,GCG and INS. Similar results were confirmed with the human induced Pluripotent Stem Cell (hiPSC) line MSUH-001. Finally, differentiation protocols incorporating RSV treatment yielded numerous insulin-positive cells, induced significantly higher PDX1 expression and were able to transiently normalize glycaemia when transplanted in streptozotocin (STZ) induced diabetic mice thus promoting its survival. In conclusion, our strategy allows the efficient differentiation of hESCs into pancreatic endoderm capable of generating β-cell-like cells and demonstrates that RSV improves the maturation process. PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 1/21 OPEN ACCESS Citation: Pezzolla D, López-Beas J, Lachaud CC, Domínguez-Rodríguez A, Smani T, Hmadcha A, et al. (2015) Resveratrol Ameliorates the Maturation Process of β-Cell-Like Cells Obtained from an Optimized Differentiation Protocol of Human Embryonic Stem Cells. PLoS ONE 10(3): e0119904. doi:10.1371/journal.pone.0119904 Academic Editor: Angel Nadal, Universidad Miguel Hernández de Elche, SPAIN Received: December 5, 2014 Accepted: February 3, 2015 Published: March 16, 2015 Copyright: © 2015 Pezzolla et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data are publicly available within the paper as Supporting Information files: S1 Table. List of Antibodies used in this study; and S2 Table. Primer Sequence Sets. Funding: Authors are supported by the nonprofit Foundation (Fundación Progreso y Salud) Consejería de Salud, Junta de Andalucía (Grant PI-0022/2008); FEDER co-funded grants from Consejería de Innovación Ciencia y Empresa, Junta de Andalucía (Grant CTS-6505; INP-2011-1615-900000 and P10CVI-6095); FEDER co-funded grants from Instituto de Introduction Human embryonic stem cells (hESCs) display two important characteristics self-renewal and pluripotency [1]. Proof-of-concept experiments demonstrate that ESCs have the ability to differentiate into insulin-producing cells, but with a very low efficiency [2–4]. The use of gene selection procedure based on neomycin-resistance transgenes for the insulin and the Nkx6.1 genes allowed the achievement of a purified population that can mature and normalize glycaemia when transplanted in diabetic mice [2,5,6]. Improvement of the in vitro differentiation process has benefited from a deeper knowledge of islet development. Sequential expression of the transcription factors [7–9] and signaling pathways [10] involved in human β-cell genesis are instrumental to achieve in vitro differentiation processes. Hence, the common approach to differentiate hESCs is based on a multi-stages protocol attempting to reproduce in vivo pancreas development aiming to induce hESCs to follow a sequential transition through mesendoderm, definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor stages, finally obtaining functional insulin-expressing cells [11–13]. The major problems in directing hESCs differentiation to β-cell-like cells are the low reproducibility of the current differentiation protocols and the low amount of insulin-secreting cells obtained at the end of the differentiation processes. Protocols described so far generate PDX1 and/or insulin positive cells, which need further maturation when transplanted into immunocompromised mice [14–16]. Maturating endocrine precursors toward specialized and functional hormone-secreting cells, still the most problematic step for hESCs differentiation to insulin-producing cells [17,18]. Despite the great number of biologically active compounds that have been already tested for this purpose, none of them has successfully worked [19,20]. Cells obtained from in vitro differentiation strategies are not mature enough to be completely functional; although they express different markers of β-cells, such as insulin, GLUT2 or GK, they could show functional problems due to impairment of the glucose sensing pathway or the exocytotic machinery [21–24]. Hence, strategies to ameliorate the in vitro maturation process of endocrine precursors are needed and up quite recently were achieved [12,13]. On the other hand, several studies reported the beneficial impact of resveratrol (RSV) on insulin secretion and how this compound potentiates glucose-stimulated insulin secretion (GSIS), not only in rat insulinoma cell lines (INS-1E), but also in isolated human islets [25]. On this basis, we investigated whether RSV could improve the final maturation step of hESCs differentiation towards β-cells. RSV (3,5,40-trihydroxy-trans-stilbene) is a polyphenol that has been shown to activate SIRT1, a NAD + -dependent deacetylase [26,27]. We have recently shown that SIRT1 contributes to the establishment of specific developmental/ differentiation programs of hESCs [28]. Other studies demonstrated the effect of RSV on insulin secretion using INS-1E and human islet [25,29]. SIRT1 represses mitochondrial uncoupling protein 2 (Ucp2) transcription by binding directly to its promoter [30], resulting in increased ATP production and insulin secretion in INS-1E and in BESTO mice islets [31,32]. Additionally, RSV induced an up-regulation of key genes for β-cell function such as Pdx1,Glut2,Gk,Hnf1αand Tfam in both INS-1E cells and human islets [25], this upregulation has been described as a possible mechanism by which RSV potentiates metabolism-secretion coupling in β-cells and interestingly for the maintenance of the β-cell identity [33,34]. In the present study, we showed for the first time that RSV is a critical compound improving the maturation of hESCs-derived endocrine precursors towards insulinsecreting cells, thus proposing its use for a more efficient insulin-secreting cells differentiation strategy. Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 2/21 Salud Carlos III (Red TerCel-Grant RD06/0010/0025, RD12/0019/0028 and RD12/0042/0041; PI10/00964, PI14/01015 and PI10/00871) and the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Results Effects of resveratrol on insulin content and secretion in INS-1E cells INS-1E cells were treated with different concentrations of RSV (50–75 μM) or with sirtinol (SRT)-SIRT1 inhibitor50 μM during 48 hours, and their insulin content and secretion was then analyzed. Comparative immunofluorescence analysis indicated increased insulin content in INS-1E cells treated with 75 μM RSV compared to all other conditions (Fig. 1A). MetaMorph-based fluorescence signals quantification confirmed a 25% increase in insulin expression level in cells treated with 75 μM of RSV compared to control cells; however cells treated with 50 μM RSV or SRT showed no significant changes in insulin content (Fig. 1B). INS-1E cells pre-treated or not with RSV were challenged with 20 mM glucose and then insulin secretion was quantified using ELISA assay. INS-1E treated with 50 and 75 μM of RSV increased their insulin secretion by 3,2 and 15,6 fold respectively, compared to values found in glucose stimulated control cells (Fig. 1C). To test whether RSV treatment of the INS-1E cells could increase intracellular Ca 2+ concentration upon glucose stimulation, we monitored the dynamic changes in cytosolic free Ca 2+ using Fura-2AM loaded cells. Measurements of Ca 2+ influx indicated that intracellular Ca 2+ concentration was increased in RSV-treated cells in a dose-dependent manner (Figs. 1D and 1E). Quantification of the average maximal amplitude of Ca 2+ influx (peak Δratio) indicated how RSV pre-treated cells displayed significantly higher intracellular Ca 2+ mobilization than untreated cells. By contrast, and as expected, SRT pretreatment reduced Ca 2+ influx levels, which were much lower than those recorded in untreated cells (Fig. 1E). Taking into account these results, the rest of the study was carried out using the most effective RSV concentration of 75 μM. To shed light on a possible mechanism of action, we checked if the effect of RSV on insulin secretion was related to SIRT1 binding to Ucp2 promoter and inhibiting its expression, thus causing a better sensing of glucose-stimulated insulin secretion. ChIP experiments confirmed the binding of SIRT1 to the Ucp2 promoter in INS-1E cells and showed that this binding was significantly increased in cells pre-treated for 48 hours with RSV (Fig. 1F). Based on these results, we decided to investigate the effect of RSV on hESCs and hiPSCs differentiation towards insulin-producing cells. Recapitulation of pancreas organogenesis for efficient hESCs differentiation We sought to recapitulate all the factors and signaling pathways involved in β-cell formation during organogenesis (Fig. 2A) in order to develop a highly efficient step-wise protocol, as schematically described in Fig. 2B. To corroborate the efficiency of our differentiation protocol, we analyzed the temporal expression of some key transcription factors involved in pancreas organogenesis at different time-points during differentiation protocol. As shown in Fig. 3A, SOX17 and FOXA2 were detected after five days of culture, indicating the differentiation of hESCs to a definitive endoderm population. At the same time the expression of HNF1B and HNF4A, two factors respectively essential for NGN3 induction and hepatic formation, was increased. Their up-regulation decreased later in the differentiation protocol at day 14, indicating a pancreatic endoderm specification at the expense of other foregut endoderm lineages. The expression of PDX1 turned on showing a peak at day 11, then decreased during endocrine proliferation at day 14 and finally was re-expressed on mature β-cell-like cells. The step of endocrine induction occurred between days 11–14 as evidenced by the peak of expression of the endocrine progenitor marker NGN3 that started to decrease at day 14 in conjunction with the high expression of the β-cell precursor marker NKX2.2. Insulin, a late marker of pancreatic Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 3/21 Fig 1. Effects of resveratrol and sirtinol on insulin content and secretion in INS-1E cells. INS-1E cells were cultured under control conditions (CTR) or with the indicated concentrations of RSV and SRT in standard medium during 48 h. A: Immunofluorescence images showing representative insulin detection (green) after treatments with RSV and SRT. Nuclei are stained with Hoechst (blue). B: Quantification of insulin staining by MetaMorph analysis. Values are mean ±SE of 2 independent experiments. (**)p<0.01.C: Effects of RSV and SRT on insulin secretion. Insulin release was measured over a 30 min incubation period at a stimulatory glucose concentration of 20 mM. Values are mean ±SE of 4 independent experiments. (**)p<0.01,(***)p<0.001. D: Representative traces showing the changes in intracellular Ca 2+ concentration (presented as the ratio of fluorescence at 340 to 380 nm (F340/F380) in Fura-2-loaded cells. E: Average of amplitude of RSV and SRT induced Ca 2+ influx (ratio ±SE), (*)p<0.05,(***)p<0.001. Then number of single cells analyzed for each condition is shown below each bar (N). F: ChiP assay of SIRT1 binding to Ucp2 promoter. Chromatin immunoprecipitation was carried out Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 4/21 endocrine differentiation was first detected at day 14 and drastically increased at the end of the differentiation protocol (day 22). The graphical overview of the dynamic expression of the studied genes (Fig. 3B) allows a better appreciation of the different and sequential pancreatic developmental stages that could be successfully recapitulated by our multi-steps differentiation protocol. Characterization of β-cell-like cells derived from hESCs differentiation Given the effects of RSV observed in INS-1E cells we investigated if it could similarly improve the insulin secretion of hESCs-derived β-cell-like cells. hESCs were subjected to the using SIRT1 antibody and it was analyzed by q-PCR of a regulatory region (Intron2) in the Ucp2 promoter. Values are mean ±SE of 3 independent experiments. (**)p<0.01. doi:10.1371/journal.pone.0119904.g001 Fig 2. Recapitulation of pancreas organogenesis for efficient hESCs differentiation. A: Schematic representation of the steps involved in ESCs differentiation toward a β-cell fate and the factors and signaling pathways involved in this process. ESC, embryonic stem cells; ME, mesendoderm; DE, definitive endoderm; PG, primitive gut; PF, posterior foregut; PE, pancreatic endoderm; EP, endocrine precursors; BC, β-cells. B: Schematic representation summary of the step-wise differentiation protocol used to obtain hESC-derived insulin-producing cells. RA, retinoic acid; Fib, fibronectin; ITS, insulintransferrin-selenium. doi:10.1371/journal.pone.0119904.g002 Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 5/21 differentiation with or without RSV (75 μM) addition during the last two days of differentiation and they were finally characterized for the expression of β-cell markers. We examined a panel of islet cell specific marker genes, such as PDX1,GLUT2,ISL1,NGN3,NKX2.2,GK,PC1/ 3,GCG,SST and INS which were all clearly detected by RT-PCR in both conditions (-RSV/ +RSV) (Fig. 4A). The mature endocrine cells markers PDX1, GCG, INS and C-PEP were also analyzed by immunofluorescence (Fig. 4B). These markers could be detected in many of both RSV-treated and untreated cells, being insulin immunostaining levels higher in the RSVtreated condition. Similar results were obtained by applying this differentiation protocol to the hiPSC line MSUH-001. As shown in Fig. 4C, RT-PCR analysis of hiPSCs at the end of the differentiation protocol could confirm the expression of definitive endoderm markers (SOX17, FOXA2), endocrine precursor markers (PDX1,NKX2.2) and islet-specific markers (GLUT2, GK,PC1/3,PC2,INS and GCG). These results confirmed the efficacy and reproducibility of our optimized differentiation protocol for the achievement of β-cell-like cells both from hESCs and hiPSCs. Nevertheless, the efficiency of the differentiation protocol is not the same for hiPSCs, comparing by q-PCR analysis the expression levels of some β-markers we observed that insulin expression is extremely lower in hiPSCs-derived β-cell-like cells compared to hESCs-derived cells, as well as PDX1 and PAX4 expression (Fig. 4D). Effects of resveratrol on maturation of hESCs derived β-cell-like cells To better understand the effects of RSV on cell maturation we further characterized hESCsderived insulin-producing cells at the final stage of differentiation protocol (Fig. 5). We Fig 3. Temporal dynamics of gene expression during hESCs differentiation. A: HS181 cells were differentiated to β-cell-like cells as described above. Cell samples were collected at days 0, 5, 8, 11, 14 and 22 and were analyzed by q-PCR for SOX17,FOXA2,HNF1B,HNF4A,PDX1,NGN3,NKX2.2and INS gene expression. For each sample, relative expression was normalized to day 0. B: Overview of the results obtained from q-PCR analysis of the genes described above. The schematic representation shows the sequential expression and temporal variation of these genes during our differentiation protocol. doi:10.1371/journal.pone.0119904.g003 Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 6/21 Fig 4. Characterization of the differentiated islet-like clusters obtained at the end of the differentiation protocol. A: RT-PCR detection of definitive endoderm, endocrine precursors and islet cells specific markers. β-actin was used as the input control. SD: hESCs spontaneously differentiated; -RSV: hESCs subjected to our differentiation protocol without RSV addition; +RSV: hESCs subjected to our differentiation protocol with RSV addition. H Pancreas: human pancreas as positive control. B: Immunofluorescence analysis of PDX1, GCG, INS and C-PEP expression in spontaneous differentiated cells (SD) and in differentiated β-cell-like cells without (-RSV) or with RSV addition (+RSV). Nuclei are stained with Hoechst (blue). Scale bar 50 μm. C: RT-PCR Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 7/21 performed confocal double immunofluorescence analysis of spontaneously differentiated cells (SD) and hESCs derived β-cell-like cells without or with RSV addition (-RSV/+RSV) and we evidenced a perfect colocalization of insulin and C-peptide in cytoplasmic granules of RSVtreated cells (Fig. 5A). MetaMorph-based quantitative analysis of insulin immunofluorescence levels confirmed that RSV-treated cultures displayed a significantly higher number of insulin positive cells compared to RSV-untreated cultures (39,1% vs. 19,6% positive cells, respectively, p<0.05)(Fig. 5B). In addition, at the single cell level, RSV-treated cells displayed significantly higher insulin levels than RSV-untreated cells (p<0.05)(Fig. 5C). The results obtained with INS-1E cells indicate that the effect of RSV is principally associated with a more efficient glucose-stimulated insulin secretion. Hence, as expected, differentiated cells treated with RSV (+RSV) showed an increase of 1,6 and 4,2 fold in the secretion of insulin compared with nontreated cells (-RSV) and spontaneous differentiated cells (SD) respectively, as shown in Fig. 5D. To study in vivo functionality, differentiated cells treated with RSV were transplanted under the kidney capsule of STZ-induced diabetic NOD/SCID mice with damaged islets of Langerhans (Fig. 6A). Soon after transplantation, we observed a period of normoglycaemia within 8 days, which correlates with a subsequent period of maintenance of the body weight, in contrast with the constant hyperglycaemia and body weight loss observed in non-transplanted diabetic mice (Figs. 6B and 6C). The subsequent regression to a hyperglycaemic state could be ascribed to the inability of maintaining the transplanted cells in the site of engraftment, as shown by the immunohistological stain of the transplanted mice where, even if it is still possible to detect some insulin positive cells throughout the kidney section, they were in a very limited and scattered way (Fig. 6D). Mechanism of action of resveratrol PDX1 expression was modulated upon treatment with RSV, which may explain the increase of insulin-positive cells and theire functionality, we performed a q-PCR analysis and we found that it was increased by 3 fold in cells treated with RSV compared to RSV-untreated cells (Fig. 7A). PDX1 is indeed important to induce the expression of different β-cell genes such as GLUT2,GK and insulin that characterize a mature and functional cell. Hence, with the aim to understand the mechanism of action of RSV, we studied the phosphorylation status of several kinases involved in the signaling pathways that lead to PDX1 activation (Fig. 7B). We observed an increase of AMPK phosphorylation in cells treated with RSV compared with non-treated cells and, in the same way, the PI3K/AKT pathway was also found to be stimulated at higher extent in response to RSV treatment, as detected by western blot of the phosphorylated proteins (Figs. 7C and 7D). Thus, indicating that RSV induced activation of the two main signaling pathways involved in PDX1 transcription by increasing the phosphorylated status of its upstream kinases. As a consequence of this activation, we observed an increased expression of both PDX1 and its downstream target genes GLUT2,GK and INS (Fig. 7E). We postulated that these two kinase cascades are also responsible for the first PDX1 induction at day 11 of our differentiation protocol (see Fig. 3) and as expected, inhibition of AMPK or PI3K by specific inhibitors (Fig. 7F) caused a drastic decrease of PDX1 expression (Fig. 7G). detection of definitive endoderm, endocrine precursors and islet cells specific markers of hiPSCs-derived β-cell-like cells. β-actin was used as the input control. D: q-PCR comparison of the levels of expression of some β-markers in β-cell-like cells derived from hiPSCs (red bars) or hESCs (blue bars) without or with RSV treatment (-RSV, +RSV). doi:10.1371/journal.pone.0119904.g004 Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 8/21 Fig 5. Effects of resveratrol on maturation of hESCs-derived insulin-secreting cells. A: Comparative confocal immunofluorescence expression of insulin (red) and C-peptide (green) expression in hESCs spontaneously differentiated cells (SD) and in differentiated β-cell-like cells without (-RSV) or with (+RSV) RSV addition. Nuclei are stained with Hoechst (blue). Scale bar 25 μm. B: Quantification by MetaMorph analysis of insulin positive cells (%). C: Immunofluorescence analysis of insulin content. Nuclei are stained with Hoechst (blue). Scale bar 25 μm. Graphs show the quantification by MetaMorph analysis of Integrated Intensity of insulin-positive cells (upper graph) and Average Intensity of insulin-positive cells (lower graph). Values are mean ±SE of 3 Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 9/21 expression were quantified with Meta Imaging Software MetaMorph Offline version 7.5.1.0 (MDS Analytical technologies, Sunnyvale, CA, USA). ELISA assay For INS-1E assays, after two days of RSV or SRT treatments, medium was removed and cells were washed three times with warm Krebs buffer (NaCl 129 mM, NaHCO 3 5 mM, KCl 4.8 mM, KH 2 PO 4 1.2 mM, MgSO 4 1.2 mM, CaCl 2 1 mM, HEPES 10 mM, BSA 0.1% at pH 7.4). Cells were then incubated with Krebs solution during one hour at 37°C and, at the end of incubation, cells were stimulated with Krebs solution supplemented with 2,7 mM or 16,8 mM glucose for 30 min at 37°C. For hESCs assays, spontaneous differentiated clusters and islet-like clusters obtained at day 20 of the differentiation protocol were plated onto matrigel-coated dishes. Differentiated islet-like clusters were divided into two sub-conditions: one was treated with RSV and the other one not. After 48 hours, cells were treated according to the protocol described above, being the glucose stimulation period of 1 hour instead of 30 minutes. Supernatants were collected and insulin secretion was measured using a commercially available human insulin ELISA (Mercodia AB, Uppsala, Sweden), according to manufacturer’s specifications. Measurements of intracellular Calcium influx INS-1E cells were loaded with Fura-2AM 2 μM during 30 min at 37°C and following a 15 min wash quantitative changes in intracellular calcium (Ca 2+ ) (F340/F380 ratio) were monitored using dual-excitation fluorescence imaging system (InCyt Im2, Intracellular Imaging, Cincinnati, OH, USA) as previously described [67]. Experiments were performed in 0 mM Ca 2+ solution containing 120 mM NaCl, 4.7 mM KCl, 4 mM MgCl2, 0.2 mM EGTA, and 10 mM HEPES, and the Ca 2+ influx was determined from changes in Fura-2 fluorescence after the addition of Ca 2+ (2 mM). Changes in intracellular Ca 2+ were expressed as a change in ratio (ΔRatio), which was calculated as the difference between the peak F340/F380 ratio after extracellular Ca 2+ was added and its basal level immediately before Ca 2+ addition. Chromatin Immunoprecipitation (ChIP) assay ChIP assays were performed according to Upstate (Merck KGaA) protocol. ChIP data are the average of real-time PCR (q-PCR) quantifications from three independent experiments. Samples were immunoprecipitated overnight at 4°C with 2 μg of a polyclonal antibody specific for SIRT1 (Merck KGaA) and immunoprecipitated DNA was analyzed by q-PCR of a regulatory region in the Ucp2 promoter previously described in Bordone et al [30]. q-PCRs were performed in an Applied Biosystems 7500 Real-Time PCR System, using the PerfeCTa SYBR Green SuperMix Low ROX (Quanta Biosciences, Gaithersburg, MD, USA) and primers designed to span rat Ucp2 promotor (S2 Table). ChIP was quantified relative to inputs using the ΔΔCt method. RT-PCR and q-PCR analysis Total RNA was extracted with TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg total RNA by using MMLV reverse transcriptase (Promega, Madison, WI, USA). RT-PCR was performed using the BioTaq DNA Polymerase (Bioline, London, UK) following the manufacturer’s protocol. The cycle conditions were as follows: 94°C for 3 min followed by 35 cycles (94°C denaturation for 30 s, 55–68°C annealing for 1 min, 72°C for 1 min), with a final incubation at 72°C for 10 min. q-PCR analysis was performed on ABI Prism 7500 system (Applied Biosystems, Foster City, CA, USA) using the Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 16 / 21 SYBR Green PCR Master Mix (Quanta Biosciences). The expression level of each gene at every checkpoint was normalized to UBC as an endogenous control. All quantities were expressed as number of folds relative to the expression level at Day 0, using the ΔΔCt method. In the case of no expression at Day 0, the Ct was arbitrarily set as 40. Primers information is provided in S2 Table. Western blotting analysis Cells from the final step of differentiation were lysed in RIPA buffer (Sigma Aldrich) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) and phosphatase (Sigma Aldrich) inhibitors during 45 minutes, sonicated and then centrifuged at 14000 rpm for 20 min at 4°C. The supernatant was removed and the amount of protein was determined by Bradford method using the Bio-Rad protein dye reagent (Bio-Rad, Hercules, Ca, USA), protein lysates were subjected to electrophoresis separation (30–40 μg/lane) in 10% SDS-PAGE and transferred to a Hybond-P polyvinylidene difluoride (PVDF) membrane (Amersham, Buckinghamshire, UK). Membranes were blocked overnight at 4°C in Trisbuffered saline with 5% BSA (Sigma Aldrich) and 0.1% Tween 20 (Sigma Aldrich). Blots were incubated overnight with primary antibodies and immunoreactive bands were detected with horseradish peroxidase-conjugated secondary antibodies followed by ECL Prime detection system (Amersham) and exposed to photographic film (Amersham). Primary and secondary antibodies used are listed in S1 Table. For bands optical density determination, photographic films were scanned using an ImmageScanner and LabScan software version 5.0 (Amersham), and the resulting images were quantified using the ImmageQuant software version 5.2 (Molecular Dynamics, Sunnyvale, CA, USA). The data were expressed as relative densitometry units. STZ treatment for diabetes induction Male 8 weeks old NOD/SCID mice (NOD.CB17-Prkdc scid/J) were purchased from Charles River Laboratories (Charles River Laboratories International, Barcelona, Spain). Mice received a single intraperitoneal injection of STZ (Sigma Aldrich) 170 mg/Kg freshly dissolved in citrate buffer (pH 4.5). Animals were considered diabetic when they reached blood glucose levels >300 mg/dl for at least 3 consecutive days. Blood glucose was monitored daily from a tail blood sample and following 3 hours morning fast using ACCU-CHEK Compact Plus glucometer (Roche Diagnostics). In vivo cell Transplantation The day of transplantation, hESCs-derived islet-like clusters from day 22 of the differentiation protocol were recollected and injected under the kidney capsule of diabetic mice. Briefly, mice were anesthetized with Ketamine (100 mg/kg) and Xylazine (10 mg/kg), the kidney was exposed through a lumbar incision and slurry of 10–20 μl of aggregates (~2000 islet-like clusters) was delivered under the kidney capsule with a catheter connected to a micropipette. Body weight and blood glucose level were monitored every two days as indicated above. Grafts were removed 4–6 weeks after transplantation, fixed with 4% paraformaldehyde solution overnight at 4°C, washed with PBS and transferred to a 30% sucrose solution over night at 4°C. Finally grafts were mounted in frozen blocks with Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA) and were cut into 10 μm thick tissue sections using a cryostat. Frozen sections were subjected to immunohistochemistry or to haematoxylin/eosin staining. All mouse experiments complied with Institutional Animal Care and Use Committee guidelines of the Department of Agriculture and Fishery for the Regional Government of Andalucia (Directive 2010/ 63/EU). This study was carried out in strict accordance with the guidelines for animal research Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 17 / 21 protocols established and approved by Animal Experimentation and Ethics Committee of CABIMER (CEEA-CABIMER). This Committee CEEA-CABIMER specifically approved this study (Permit Number: CEEA no.1/2012). All efforts were made to minimize animals suffering. Statistical analysis Values are presented as mean ±SE. Statistical significance was calculated by using Student’sttest, p<0.05 was considered statistically significant. Supporting Information S1 Table. List of Antibodies used in this study. (PDF) S2 Table. Primer Sequence Sets. (PDF) Acknowledgments We thank Nuria Mellado and Yolanda Aguilera for the technical assistance. Author Contributions Conceived and designed the experiments: AH BS. Performed the experiments: DP JLB CCL ADR. Analyzed the data: TS AH BS. Contributed reagents/materials/analysis tools: TS AH BS. Wrote the paper: DP AH BS. References 1. Hmadcha A, Dominguez-Bendala J, Wakeman J, Arredouani M, Soria B. The immune boundaries for stem cell based therapies: problems and prospective solutions. J Cell Mol Med. 2009;13: 1464–1475. doi: 10.1111/j.1582-4934.2009.00837.x PMID: 19583810 2. Soria B, Roche E, Berna G, Leon-Quinto T, Reig JA, Martin F. Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes. 2000;49: 157–162. PMID: 10868930 3. Segev H, Fishman B, Ziskind A, Shulman M, Itskovitz-Eldor J. Differentiation of human embryonic stem cells into insulin-producing clusters. Stem Cells. 2004;22: 265–274. PMID: 15153604 4. Rajagopal J, Anderson WJ, Kume S, Martinez OI, Melton DA. Insulin staining of ES cell progeny from insulin uptake. Science. 2003;299: 363. PMID: 12532008 5. Leon-Quinto T, Jones J, Skoudy A, Burcin M, Soria B. In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells. Diabetologia. 2004;47: 1442–1451. PMID: 15309294 6. Vaca P, Martin F, Vegara-Meseguer JM, Rovira JM, Berna G, Soria B. Induction of differentiation of embryonic stem cells into insulin-secreting cells by fetal soluble factors. Stem Cells. 2006;24: 258–265. PMID: 16109755 7. Soria B. In-vitro differentiation of pancreatic beta-cells. Differentiation. 2001;68: 205–219. PMID: 11776473 8. Bonal C, Herrera PL. Genes controlling pancreas ontogeny. Int J Dev Biol. 2008;52: 823–835. doi: 10. 1387/ijdb.072444cb PMID: 18956314 9. Cano DA, Soria B, Martin F, Rojas A. Transcriptional control of mammalian pancreas organogenesis. Cell Mol Life Sci. 2014;71: 2383–2402. doi: 10.1007/s00018-013-1510-2 PMID: 24221136 10. Champeris Tsaniras S, Jones PM. Generating pancreatic beta-cells from embryonic stem cells by manipulating signaling pathways. J Endocrinol. 2010;206: 13–26. doi: 10.1677/JOE-10-0073 PMID: 20385725 11. Soria B, Pezzolla D, López J, Rojas A, Hmadcha A. Generation of Pancreatic Islets from Stem Cells. In: Lanza R, Langer R, Vacanti JP, editors. Principles of Tissue Engineering Fourth Edition. Elsevier B.V.; 2014. p. 837–847. Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 18 / 21 12. Rezania A, Bruin JE, Arora P, Rubin A, Batushansky I, Asadi A et al. Reversal of diabetes with insulinproducing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol. 2014;32: 1121– 1133. doi: 10.1038/nbt.3033 PMID: 25211370 13. Pagliuca FW, Millman JR, Gurtler M, Segel M, Van Dervort A, Ryu JH, et al. Generation of Functional Human Pancreatic beta Cells In Vitro. Cell. 2014;159: 428–439. doi: 10.1016/j.cell.2014.09.040 PMID: 25303535 14. Kroon E, Martinson LA, Kadoya K, Bang AG, Kelly OG, Eliazer S, et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol. 2008;26: 443–452. doi: 10.1038/nbt1393 PMID: 18288110 15. Rezania A, Bruin JE, Riedel MJ, Mojibian M, Asadi A, Xu J, et al. Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice. Diabetes. 2012;61: 2016–2029. doi: 10.2337/db11-1711 PMID: 22740171 16. Rezania A, Bruin JE, Xu J, Narayan K, Fox JK, O’Neil JJ, et al. Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulinsecreting cells in vivo. Stem Cells. 2013;31: 2432–2442. doi: 10.1002/stem.1489 PMID: 23897760 17. Champeris Tsaniras S. Generating mature beta-cells from embryonic stem cells: strategies for latestage differentiation. Vitam Horm. 2011;87: 79–92. doi: 10.1016/B978-0-12-386015-6.00025-1 PMID: 22127238 18. Nostro MC, Keller G. Generation of beta cells from human pluripotent stem cells: Potential for regenerative medicine. Semin Cell Dev Biol. 2012;23: 701–710. doi: 10.1016/j.semcdb.2012.06.010 PMID: 22750147 19. Baetge EE. Production of beta-cells from human embryonic stem cells. Diabetes Obes Metab. 2008; (10Suppl 4):186–194. doi: 10.1111/j.1463-1326.2008.00956.x PMID: 18834446 20. Schiesser JV, Wells JM. Generation of beta cells from human pluripotent stem cells: are we there yet? Ann N Y Acad Sci. 2014;1311: 124–137. doi: 10.1111/nyas.12369 PMID: 24611778 21. Shiroi A, Yoshikawa M, Yokota H, Fukui H, Ishizaka S, Tatsumi K, et al. Identification of insulinproducing cells derived from embryonic stem cells by zinc-chelating dithizone. Stem Cells. 2002;20: 284–292. PMID: 12110697 22. Basford CL, Prentice KJ, Hardy AB, Sarangi F, Micallef SJ, Li X, et al. The functional and molecular characterisation of human embryonic stem cell-derived insulin-positive cells compared with adult pancreatic beta cells. Diabetologia. 2012;55: 358–371. doi: 10.1007/s00125-011-2335-x PMID: 22075915 23. Hrvatin S, O’Donnell CW, Deng F, Millman JR, Pagliuca FW, Dilorio P, et al. Differentiated human stem cells resemble fetal, not adult, beta cells. Proc Natl Acad Sci U S A. 2014;111: 3038–3043. doi: 10. 1073/pnas.1400709111 PMID: 24516164 24. Bruin JE, Erener S, Vela J, Hu X, Johnson JD, Kurata HT, et al. Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells. Stem Cell Res. 2014;12: 194– 208. doi: 10.1016/j.scr.2013.10.003 PMID: 24257076 25. Vetterli L, Brun T, Giovannoni L, Bosco D, Maechler P. Resveratrol potentiates glucose-stimulated insulin secretion in INS-1E beta-cells and human islets through a SIRT1-dependent mechanism. J Biol Chem. 2011;286: 6049–6060. doi: 10.1074/jbc.M110.176842 PMID: 21163946 26. Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003;425: 191–196. PMID: 12939617 27. Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature. 2006;444: 337–342. PMID: 17086191 28. Calvanese V, Lara E, Suarez-Alvarez B, Abu Dawud R, Vazquez-Chantada M, Martinez-Chantar ML, et al. Sirtuin 1 regulation of developmental genes during differentiation of stem cells. Proc Natl Acad Sci U S A. 2010;107: 13736–13741. doi: 10.1073/pnas.1001399107 PMID: 20631301 29. Zhang Y, Jayaprakasam B, Seeram NP, Olson LK, DeWitt D, Nair MG. Insulin secretion and cyclooxygenase enzyme inhibition by cabernet sauvignon grape skin compounds. J Agric Food Chem. 2004;52: 228–233. PMID: 14733500 30. Bordone L, Motta MC, Picard F, Robinson A, Jhala US, Apfeld J, et al. Sirt1 regulates insulin secretion by repressing UCP2 in pancreatic beta cells. PLoS Biol. 2006;4: e31. PMID: 16366736 31. Affourtit C, Brand MD. Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion. Biochem J. 2008;409: 199–204. PMID: 17824844 32. Moynihan KA, Grimm AA, Plueger MM, Bernal-Mizrachi E, Ford E, Cras-Meneur C, et al. Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-stimulated insulin secretion in mice. Cell Metab. 2005;2: 105–117. PMID: 16098828 Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 19 / 21 33. Fiori JL, Shin YK, Kim W, Krzysik-Walker SM, Gonzalez-Mariscal I, Carlson OD, et al. Resveratrol prevents beta-cell dedifferentiation in nonhuman primates given a high-fat/high-sugar diet. Diabetes. 2013;62: 3500–3513. doi: 10.2337/db13-0266 PMID: 23884882 34. Xie S, Sinha RA, Singh BK, Li GD, Han W, Yen PM. Resveratrol induces insulin gene expression in mouse pancreatic alpha-cells. Cell Biosci. 2013;3: 47. doi: 10.1186/2045-3701-3-47 PMID: 24330680 35. D’Amour KA, Bang AG, Eliazer S, Kelly OG, Agulnick AD, Smart NG, et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nat Biotechnol. 2006;24: 1392–1401. PMID: 17053790 36. Shim JH, Kim SE, Woo DH, Kim SK, Oh CH, McKay R, et al. Directed differentiation of human embryonic stem cells towards a pancreatic cell fate. Diabetologia. 2007;50: 1228–1238. PMID: 17457565 37. Jiang W, Shi Y, Zhao D, Chen S, Yong J, Zhang J, et al. In vitro derivation of functional insulinproducing cells from human embryonic stem cells. Cell Res. 2007;17: 333–344. PMID: 17426693 38. Jiang J, Au M, Lu K, Eshpeter A, Korbutt G, Fisk G, et al. Generation of insulin-producing islet-like clusters from human embryonic stem cells. Stem Cells. 2007;25: 1940–1953. PMID: 17510217 39. Zhang D, Jiang W, Liu M, Sui X, Yin X, Chen S, et al. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res. 2009;19: 429–438. doi: 10.1038/ cr.2009.28 PMID: 19255591 40. Borriello A, Cucciolla V, Della Ragione F, Galletti P. Dietary polyphenols: focus on resveratrol, a promising agent in the prevention of cardiovascular diseases and control of glucose homeostasis. Nutr Metab Cardiovasc Dis. 2010;20: 618–625. doi: 10.1016/j.numecd.2010.07.004 PMID: 20850033 41. Szkudelski T, Szkudelska K. Anti-diabetic effects of resveratrol. Ann N Y Acad Sci. 2011;1215: 34–39. doi: 10.1111/j.1749-6632.2010.05844.x PMID: 21261639 42. Chen WP, Chi TC, Chuang LM, Su MJ. Resveratrol enhances insulin secretion by blocking K(ATP) and K(V) channels of beta cells. Eur J Pharmacol. 2007;568: 269–277. PMID: 17573071 43. Affourtit C, Brand MD. On the role of uncoupling protein-2 in pancreatic beta cells. Biochim Biophys Acta. 2008;1777: 973–979. doi: 10.1016/j.bbabio.2008.03.022 PMID: 18433713 44. Azzu V, Affourtit C, Breen EP, Parker N, Brand MD. Dynamic regulation of uncoupling protein 2 content in INS-1E insulinoma cells. Biochim Biophys Acta. 2008;1777: 1378–1383. doi: 10.1016/j.bbabio.2008. 07.001 PMID: 18692019 45. Brand MD, Parker N, Affourtit C, Mookerjee SA, Azzu V. Mitochondrial uncoupling protein 2 in pancreatic beta-cells. Diabetes Obes Metab. 2010;(12 Suppl 2):134–140. doi: 10.1111/j.1463-1326.2010. 01264.x PMID: 21029310 46. Hay DC, Fletcher J, Payne C, Terrace JD, Gallagher RC, Snoeys J, et al. Highly efficient differentiation of hESCs to functional hepatic endoderm requires ActivinA and Wnt3a signaling. Proc Natl Acad Sci U S A. 2008;105: 12301–12306. doi: 10.1073/pnas.0806522105 PMID: 18719101 47. Sulzbacher S, Schroeder IS, Truong TT, Wobus AM. Activin A-induced differentiation of embryonic stem cells into endoderm and pancreatic progenitors-the influence of differentiation factors and culture conditions. Stem Cell Rev. 2009;5: 159–173. doi: 10.1007/s12015-009-9061-5 PMID: 19263252 48. Loh KM, Ang LT, Zhang J, Kumar V, Ang J, Auyeong JQ, et al. Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations. Cell Stem Cell. 2014;14: 237–252. doi: 10.1016/j.stem.2013.12.007 PMID: 24412311 49. Guo S, Mao X, He F, Liu H, Ming L. Activin A supplement in the hESCs culture enhances the endoderm differentiation efficiency. Cell Biol Int. 2014;38: 849–856. doi: 10.1002/cbin.10274 PMID: 24604611 50. D’Amour KA, Agulnick AD, Eliazer S, Kelly OG, Kroon E, Baetge EE. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol. 2005;23: 1534–1541. PMID: 16258519 51. Johannesson M, Stahlberg A, Ameri J, Sand FW, Norrman K, Semb H. FGF4 and retinoic acid direct differentiation of hESCs into PDX1-expressing foregut endoderm in a timeand concentrationdependent manner. PLoS One. 2009;4: e4794. doi: 10.1371/journal.pone.0004794 PMID: 19277121 52. Ameri J, Stahlberg A, Pedersen J, Johansson JK, Johannesson MM, Artner I, et al. FGF2 specifies hESC-derived definitive endoderm into foregut/midgut cell lineages in a concentration-dependent manner. Stem Cells. 2010;28: 45–56. doi: 10.1002/stem.249 PMID: 19890880 53. Kim SK, Melton DA. Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor. Proc Natl Acad Sci U S A. 1998;95: 13036–13041. PMID: 9789036 54. Schulz TC, Young HY, Agulnick AD, Babin MJ, Baetge EE, Bang AG, et al. A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. PLoS One. 2012;7: e37004. doi: 10.1371/journal.pone.0037004 PMID: 22623968 Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 20 / 21 55. Hou X, Xu S, Maitland-Toolan KA, Sato K, Jiang B, Ido Y, et al. SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase. J Biol Chem. 2008;283: 20015–20026. doi: 10. 1074/jbc.M802187200 PMID: 18482975 56. Dasgupta B, Milbrandt J. Resveratrol stimulates AMP kinase activity in neurons. Proc Natl Acad Sci U S A. 2007;104: 7217–7222. PMID: 17438283 57. Crandall JP, Barzilai N. Exploring the promise of resveratrol: where do we go from here? Diabetes. 2013;62: 1022–1023. doi: 10.2337/db12-1788 PMID: 23520278 58. Price NL, Gomes AP, Ling AJ, Duarte FV, Martin-Montalvo A, North BJ, et al. SIRT1 is required for AMPK activation and the beneficial effects of resveratrol on mitochondrial function. Cell Metab. 2012;15: 675–690. doi: 10.1016/j.cmet.2012.04.003 PMID: 22560220 59. Elrick LJ, Docherty K. Phosphorylation-dependent nucleocytoplasmic shuttling of pancreatic duodenal homeobox-1. Diabetes. 2001;50: 2244–2252. PMID: 11574405 60. Watanabe H, Saito H, Nishimura H, Ueda J, Evers BM. Activation of phosphatidylinositol-3 kinase regulates pancreatic duodenal homeobox-1 in duct cells during pancreatic regeneration. Pancreas. 2008;36: 153–159. doi: 10.1097/MPA.0b013e318157753e PMID: 18376306 61. Patel KR, Scott E, Brown VA, Gescher AJ, Steward WP, Brown K. Clinical trials of resveratrol. Ann N Y Acad Sci. 2011;1215: 161–169. doi: 10.1111/j.1749-6632.2010.05853.x PMID: 21261655 62. Brasnyo P, Molnar GA, Mohas M, Marko L, Laczy B, Cseh J, et al. Resveratrol improves insulin sensitivity, reduces oxidative stress and activates the Akt pathway in type 2 diabetic patients. Br J Nutr. 2011;106: 383–389. doi: 10.1017/S0007114511000316 PMID: 21385509 63. Crandall JP, Oram V, Trandafirescu G, Reid M, Kishore P, Hawkins M, et al. Pilot study of resveratrol in older adults with impaired glucose tolerance. J Gerontol A Biol Sci Med Sci. 2012;67: 1307–1312. doi: 10.1093/gerona/glr235 PMID: 22219517 64. Bashmakov YK, Assaad-Khalil SH, Abou Seif M, Udumyan R, Megallaa M, Rohoma KH, et al. Resveratrol promotes foot ulcer size reduction in type 2 diabetes patients. ISRN Endocrinol. 2014;2014: 816307. doi: 10.1155/2014/816307 PMID: 24701359 65. Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod. 2003;18: 1404–1409. PMID: 12832363 66. Merglen A, Theander S, Rubi B, Chaffard G, Wollheim CB, Maechler P. Glucose sensitivity and metabolism-secretion coupling studied during two-year continuous culture in INS-1E insulinoma cells. Endocrinology. 2004;145: 667–678. PMID: 14592952 67. Lachaud CC, Pezzolla D, Dominguez-Rodriguez A, Smani T, Soria B, Hmadcha A. Functional vascular smooth muscle-like cells derived from adult mouse uterine mesothelial cells. PLoS One. 2013;8: e55181. doi: 10.1371/journal.pone.0055181 PMID: 23405120 Role of Resveratrol in hESC Differentiation PLOS ONE | DOI:10.1371/journal.pone.0119904 March 16, 2015 21 / 21