Nitric Oxide And Hypoxia Response In Pluripotent Stem Cells

The expansion of pluripotent cells (ESCs and iPSCs) under conditions that maintain their pluripotency is necessary to implement a cell therapy program. Previously, we have described that low nitric oxide (NO) donor diethylenetriamine/nitric oxide adduct (DETA-NO) added to the culture medium, promote the expansion of these cell types. The molecular mechanisms are not yet known. We present evidences that ESC and iPSCs in normoxia in presence of low NO triggers a similar response to hypoxia, thus maintaining the pluripotency. We have studied the stability of HIF-1α (Hypoxia Inducible Factor) in presence of low NO. Because of the close relationship between hypoxia, metabolism, mitochondrial function and pluripotency we have analyzed by q RT-PCR the expression of genes involved in the glucose metabolism such as: HK2, LDHA and PDK1; besides other HIF-1α target gene. We further analyzed the expression of genes involved in mitochondrial biogenesis such as PGC1α, TFAM and NRF1 and we have observed that low NO maintains the same pattern of expression that in hypoxia. The study of the mitochondrial membrane potential using Mito-Tracker dye showed that NO decrease the mitochondrial function. We will analyze other metabolic parameters, to determinate if low NO regulates mitochondrial function and mimics Hypoxia Response. The knowledge of the role of NO in the Hypoxia Response and the mechanism that helps to maintain self-renewal in pluripotent cells in normoxia, can help to the design of culture media where NO could be optimal for stem cell expansion in the performance of future cell therapies

Nitric oxide prevents mouse embryonic stem cell differentiation through regulation of gene expression, cell signaling, and control of cell proliferation

Nitric oxide (NO) delays mouse embryonic stem cell (mESC) differentiation by regulating genes linked to pluripotency and differentiation. Nevertheless, no profound study has been conducted on cell differentiation regulation by this molecule through signaling on essential biological functions. We sought to demonstrate that NO positively regulates the pluripotency transcriptional core, enforcing changes in the chromatin structure, in addition to regulating cell proliferation, and signaling pathways with key roles in stemness. Culturing mESCs with 2 μM of the NO donor diethylenetriamine/NO (DETA/NO) in the absence of leukemia inhibitory factor (LIF) induced significant changes in the expression of 16 genes of the pluripotency transcriptional core. Furthermore, treatment with DETA/NO resulted in a high occupancy of activating H3K4me3 at the Oct4 and Nanog promoters and repressive H3K9me3 and H3k27me3 at the Brachyury promoter. Additionally, the activation of signaling pathways involved in pluripotency, such as Gsk3-β/β-catenin, was observed, in addition to activation of PI3 K/Akt, which is consistent with the protection of mESCs from cell death. Finally, a decrease in cell proliferation coincides with cell cycle arrest in G2/M. Our results provide novel insights into NO-mediated gene regulation and cell proliferation and suggest that NO is necessary but not sufficient for the maintenance of pluripotency and the prevention of cell differentiation.Ministerio de Economía y Competitividad, Secretaria de Estado de Investigacion Desarrollo e Innovacion co-funded by Fondos FEDER; Grant number: SAF2005-08014, SAF2006-06673, SAF2007/60105, CYT-836, IPT-2011-1615-900000; Grant sponsor: Ministerio de Economía y Competitividad, Instituto de Salud Carlos III co-funded by Fondos FEDER; Grant number: RED-TERCEL RD06/0010/0025, FIS-052106 and CIBERDEM initiative; Grant sponsor: Junta de Andalucía, Consejería de Economía, Innovacion, Ciencia y Empleo-CEICE; Grant number: PAI/CTS576, PI-0022/2008, Proyecto Motriz/CTS-7127/2011; Grant sponsor: Junta de Andalucía, Consejería de Salud; Grant number: PI-0105/2010, PI-0095/2007; Grant sponsor: Junta de Andalucía, Consejería de Salud, Servicio Andaluz de Salud; Grant number: SAS 11245.Peer Reviewe

Mesenchymal Stromal Cell-Based Therapies as Promising Treatments for Muscle Regeneration After Snakebite Envenoming

Snakebite envenoming is a global neglected disease with an incidence of up to 2.7 million new cases every year. Although antivenoms are so-far the most effective treatment to reverse the acute systemic effects induced by snakebite envenoming, they have a limited therapeutic potential, being unable to completely neutralize the local venom effects. Local damage, such as dermonecrosis and myonecrosis, can lead to permanent sequelae with physical, social, and psychological implications. The strong inflammatory process induced by snake venoms is associated with poor tissue regeneration, in particular the lack of or reduced skeletal muscle regeneration. Mesenchymal stromal cells (MSCs)-based therapies have shown both anti-inflammatory and pro-regenerative properties. We postulate that using allogeneic MSCs or their cell-free products can induce skeletal muscle regeneration in snakebite victims, improving all the three steps of the skeletal muscle regeneration process, mainly by anti-inflammatory activity, paracrine effects, neovascularization induction, and inhibition of tissue damage, instrumental for microenvironment remodeling and regeneration. Since snakebite envenoming occurs mainly in areas with poor healthcare, we enlist the principles and potential of MSCs-based therapies and discuss regulatory issues, good manufacturing practices, transportation, storage, and related-procedures that could allow the administration of these therapies, looking forward to a safe and cost-effective treatment for a so far unsolved and neglected health problem.The authors are supported by the University Pablo de Olavide (Sevilla), the University Miguel Hernández (Elche, Alicante), National University Toribio Rodriguez de Mendoza (Chachapoyas, Peru) Grants: Contrato N° 09-2019-FONDECYT-BM-INC.INV to JRT, JDRF 2-SRA-2019-837-S-B and AVI-GVA COVID-19-68 to BS, Fundación Andaluza de I+D and Al-Andalus Biopharma Project (FAID-2018-1). The authors CC-O, CG-D, and TCSA were supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq) (Process: 406163/2018-9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil - CAPES (Program COFECUB Process: 88881.191812/2018-01) and by Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil (FAPEMIG)

Pdx1 is transcriptionally regulated by EGR-1 during nitric oxide-induced endoderm differentiation of mouse embryonic stem cells

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages.C.S.-A. was a doctoral PFIS fellow of ISCIII (FI11/00301). This study was supported by grants from Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI105/2010, TCMR06/009, and PI-0022) to F.J.B. and F.M.; from Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (CTS-7127/2011) to F.J.B.; from ISCIII cofunded by Fondos FEDER (RED-TERCEL RD 16-011-0034, along with PICI PICI21/00016 and GVA-COVID19/2021/047 to B.S.); from Servicio Andaluz de Salud (SAS 11245), and from Ministerio de Economía y Competitividad- Secretaría de Estado de Investigación Desarrollo e Innovación (IPT-2011-1615-900000). Along with this, there was support to PAIDI group CTS576, and by the European Regional Development Fund (FEDER) and the Consejería de Economía, Conocimiento, Empresas y Universidades de la Junta de Andalucía, within the framework of the operational program FEDER Andalucía 2014–2020. Specific Objective 1.2.3 “Promotion and generation of frontier knowledge and knowledge oriented to the challenges of society, development of emerging technologies” led the reference research project (UPO-1381598) of J.R.T.Peer reviewe

Role of nitric oxide in the maintenance of pluripotency and regulation of the hypoxia response in stem cells

Stem cell pluripotency and differentiation are global processes regulated by several pathways that have been studied intensively over recent years. Nitric oxide (NO) is an important molecule that affects gene expression at the level of transcription and translation and regulates cell survival and proliferation in diverse cell types. In embryonic stem cells NO has a dual role, controlling differentiation and survival, but the molecular mechanisms by which it modulates these functions are not completely defined. NO is a physiological regulator of cell respiration through the inhibition of cytochrome c oxidase. Many researchers have been examining the role that NO plays in other aspects of metabolism such as the cellular bioenergetics state, the hypoxia response and the relationship of these areas to stem cell stemnessSupported by Grants from Consejería de Igualdad, Salud y Politicas Sociales, Junta de Andalucía, No. PI105/2010; and Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía, No. CTS-7127/2011 (to Bedoya FJ); Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía, ISCIII co-funded by Fondos FEDER (RED TERCEL), No. RD06/0010/0025, RD12/0019/0028 and PI10/00964; Consejería de Economía, Innovación, Ciencia y Empleo, No. P10.CTS.6505; and the Ministry of Health and Consumer Affairs (Advanced Therapies Program Grant TRA-120) (to Soria B); Consejería de Igualdad, Salud y Políticas Sociales, No. PI0022/2008; and Consejería de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (PAI, BIO311) (to Martín F); Servicio Andaluz de Salud (SAS 11245) and Ministerio de Economía y Competitividad-Secretaría de Estado de Investigación Desarrollo e Innovación, No. IPT-2011-1615-900000 (to Tejedo JR).Peer Reviewe

The Role of Nitric Oxide in Stem Cell Biology

Nitric oxide (NO) is a gaseous biomolecule endogenously synthesized with an essential role in embryonic development and several physiological functions, such as regulating mitochondrial respiration and modulation of the immune response. The dual role of NO in embryonic stem cells (ESCs) has been previously reported, preserving pluripotency and cell survival or inducing differentiation with a dose-dependent pattern. In this line, high doses of NO have been used in vitro cultures to induce focused differentiation toward different cell lineages being a key molecule in the regenerative medicine field. Moreover, optimal conditions to promote pluripotency in vitro are essential for their use in advanced therapies. In this sense, the molecular mechanisms underlying stemness regulation by NO have been studied intensively over the current years. Recently, we have reported the role of low NO as a hypoxia-like inducer in pluripotent stem cells (PSCs), which supports using this molecule to maintain pluripotency under normoxic conditions. In this review, we stress the role of NO levels on stem cells (SCs) fate as a new approach for potential cell therapy strategies. Furthermore, we highlight the recent uses of NO in regenerative medicine due to their properties regulating SCs biology.This study has been financed by support to PAIDI group CTS576, and European Regional Development Fund (FEDER) and by the Consejería de Economía, Conocimiento, Empresas y Universidades de la Junta de Andalucía, within the framework of the operational program FEDER Andalucía 2014–2020. Specific Objetive 1.2.3 “Promotion and generation of frontier knowledge and knowledge oriented to the challenges of society, development of emerging technologies” in the framework of the reference research project (iUPO-1381598 to J. R. Tejedo)

Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation

The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transitionThis work was supported by Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI0022/2008, PI105/ 2010, and PI10/00964), Consejer´ıa de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (CTS-7127/2011, P10.CTS.6505, and PAI BIO-311), Instituto de Salud Carlos III/FEDER (Red TerCel, RD06/0010/0025, RD12/0019/0028), Ministerio de Economía y Competitividad (IPT-2011-1615- 900000), Ministerio de Salud y Consumo (TRA-120), and Servicio Andaluz de Salud (SAS 11245)Peer Reviewe

Transient Downregulation of Nanog and Oct4 Induced by DETA/NO Exposure in Mouse Embryonic Stem Cells Leads to Mesodermal/Endodermal Lineage Differentiation

The function of pluripotency genes in differentiation is a matter of investigation. We report here that Nanog and Oct4 are reexpressed in two mouse embryonic stem cell (mESC) lines following exposure to the differentiating agent DETA/NO. Both cell lines express a battery of both endoderm and mesoderm markers following induction of differentiation with DETA/NO-based protocols. Confocal analysis of cells undergoing directed differentiation shows that the majority of cells expressing Nanog express also endoderm genes such as Gata4 and FoxA2 (75.4% and 96.2%, resp.). Simultaneously, mRNA of mesodermal markers Flk1 and Mef2c are also regulated by the treatment. Acetylated histone H3 occupancy at the promoter of Nanog is involved in the process of reexpression. Furthermore, Nanog binding to the promoter of Brachyury leads to repression of this gene, thus disrupting mesendoderm transitionThis work was supported by Consejería de Igualdad, Salud y Políticas Sociales, Junta de Andalucía (PI0022/2008, PI105/ 2010, and PI10/00964), Consejer´ıa de Economía, Innovación, Ciencia y Empleo, Junta de Andalucía (CTS-7127/2011, P10.CTS.6505, and PAI BIO-311), Instituto de Salud Carlos III/FEDER (Red TerCel, RD06/0010/0025, RD12/0019/0028), Ministerio de Economía y Competitividad (IPT-2011-1615- 900000), Ministerio de Salud y Consumo (TRA-120), and Servicio Andaluz de Salud (SAS 11245)Peer Reviewe

Pdx1 Is Transcriptionally Regulated by EGR-1 during Nitric Oxide-Induced Endoderm Differentiation of Mouse Embryonic Stem Cells

The transcription factor, early growth response-1 (EGR-1), is involved in the regulation of cell differentiation, proliferation, and apoptosis in response to different stimuli. EGR-1 is described to be involved in pancreatic endoderm differentiation, but the regulatory mechanisms controlling its action are not fully elucidated. Our previous investigation reported that exposure of mouse embryonic stem cells (mESCs) to the chemical nitric oxide (NO) donor diethylenetriamine nitric oxide adduct (DETA-NO) induces the expression of early differentiation genes such as pancreatic and duodenal homeobox 1 (Pdx1). We have also evidenced that Pdx1 expression is associated with the release of polycomb repressive complex 2 (PRC2) and P300 from the Pdx1 promoter; these events were accompanied by epigenetic changes to histones and site-specific changes in the DNA methylation. Here, we investigate the role of EGR-1 on Pdx1 regulation in mESCs. This study reveals that EGR-1 plays a negative role in Pdx1 expression and shows that the binding capacity of EGR-1 to the Pdx1 promoter depends on the methylation level of its DNA binding site and its acetylation state. These results suggest that targeting EGR-1 at early differentiation stages might be relevant for directing pluripotent cells into Pdx1-dependent cell lineages

Nitric oxide repression of Nanog promotes mouse embryonic stem cell differentiation

Exposure of mouse embryonic stem (mES) cells to high concentrations of chemical nitric oxide (NO) donors promotes differentiation, but the mechanisms involved in this process at the gene expression level are poorly defined. In this study we report that culture of mES cells in the presence of 0.25-1.0 mM diethylenetriamine nitric oxide adduct (DETA-NO) leads to downregulation of Nanog and Oct4, the two master genes involved in the control of the pluripotent state. This action of NO was also apparent in the human ES cell line, HS 181. The suppressive action of NO on Nanog gene depends on the activation of p53 repressor protein by covalent modifications, such as pSer15, pSer315, pSer392 and acetyl Lys 379. NO-induced repression of Nanog is also associated with binding of trimethylated histone H3 and pSer315 p53 to its promoter region. In addition, exposure to 0.5 mM DETA-NO induces early differentiation events of cells with acquisition of epithelial morphology and expression of markers of definitive endoderm, such as FoxA2, Gata4, Hfn1-Β and Sox 17. This phenotype was increased when cells were treated with valproic acid (VPA) for 10 days. © 2010 Macmillan Publishers Limited All rights reserved.his study was supported by grants from Dirección General de Investigación Científica y Técnica (SAF2007/60105), Instituto de Salud Carlos III (TERCEL RD06/0010/0025) and Junta de Andalucía (CTS576) to FJ Bedoya; from Fondo de Investigaciones Sanitarias (FIS-052106) and Dirección General de Investigación Científica y Técnica (CYT-836, SAF2005-08014) to B Soria; from Dirección General de Investigación Científica y Técnica (SAF2003-03307; SAF2006-06673), Junta de Andalucía (exp. 0009/06) and Instituto de Salud Carlos III (RCMN C03/08, RETIC RD06/0015/0013 and CIBERDEM) to F Martín; and from Consejería de Salud-Junta de Andalucía (PI-0095/2007) and Instituto de Salud Carlos III (CIBERDEM) to JR Tejedo.Peer Reviewe