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Yehya A, Azar J, Al-Fares M, Boeuf H, Abou-Kheir W, Zeineddine D, Hadadeh O. Cardiac differentiation is modulated by anti-apoptotic signals in murine embryonic stem cells. World J Stem Cells 2024; 16:551-559. [PMID: 38817332 PMCID: PMC11135258 DOI: 10.4252/wjsc.v16.i5.551] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/30/2023] [Revised: 01/29/2024] [Accepted: 04/01/2024] [Indexed: 05/24/2024] Open
Abstract
BACKGROUND Embryonic stem cells (ESCs) serve as a crucial ex vivo model, representing epiblast cells derived from the inner cell mass of blastocyst-stage embryos. ESCs exhibit a unique combination of self-renewal potency, unlimited proliferation, and pluripotency. The latter is evident by the ability of the isolated cells to differentiate spontaneously into multiple cell lineages, representing the three primary embryonic germ layers. Multiple regulatory networks guide ESCs, directing their self-renewal and lineage-specific differentiation. Apoptosis, or programmed cell death, emerges as a key event involved in sculpting and forming various organs and structures ensuring proper embryonic development. However, the molecular mechanisms underlying the dynamic interplay between differentiation and apoptosis remain poorly understood. AIM To investigate the regulatory impact of apoptosis on the early differentiation of ESCs into cardiac cells, using mouse ESC (mESC) models - mESC-B-cell lymphoma 2 (BCL-2), mESC-PIM-2, and mESC-metallothionein-1 (MET-1) - which overexpress the anti-apoptotic genes Bcl-2, Pim-2, and Met-1, respectively. METHODS mESC-T2 (wild-type), mESC-BCL-2, mESC-PIM-2, and mESC-MET-1 have been used to assess the effect of potentiated apoptotic signals on cardiac differentiation. The hanging drop method was adopted to generate embryoid bodies (EBs) and induce terminal differentiation of mESCs. The size of the generated EBs was measured in each condition compared to the wild type. At the functional level, the percentage of cardiac differentiation was measured by calculating the number of beating cardiomyocytes in the manipulated mESCs compared to the control. At the molecular level, quantitative reverse transcription-polymerase chain reaction was used to assess the mRNA expression of three cardiac markers: Troponin T, GATA4, and NKX2.5. Additionally, troponin T protein expression was evaluated through immunofluorescence and western blot assays. RESULTS Our findings showed that the upregulation of Bcl-2, Pim-2, and Met-1 genes led to a reduction in the size of the EBs derived from the manipulated mESCs, in comparison with their wild-type counterpart. Additionally, a decrease in the count of beating cardiomyocytes among differentiated cells was observed. Furthermore, the mRNA expression of three cardiac markers - troponin T, GATA4, and NKX2.5 - was diminished in mESCs overexpressing the three anti-apoptotic genes compared to the control cell line. Moreover, the overexpression of the anti-apoptotic genes resulted in a reduction in troponin T protein expression. CONCLUSION Our findings revealed that the upregulation of Bcl-2, Pim-2, and Met-1 genes altered cardiac differentiation, providing insight into the intricate interplay between apoptosis and ESC fate determination.
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Affiliation(s)
- Amani Yehya
- Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Beirut 1107-2020, Lebanon
| | - Joseph Azar
- Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Beirut 1107-2020, Lebanon
| | - Mohamad Al-Fares
- Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Beirut 1107-2020, Lebanon
| | - Helene Boeuf
- Inserm, Biotis, U1026, University Bordeaux, Bordeaux F-33000, France
| | - Wassim Abou-Kheir
- Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Beirut 1107-2020, Lebanon
| | - Dana Zeineddine
- Rammal Rammal Lab, Physio-Toxicity Environmental Team, Faculty of Sciences, Lebanese University, Nabatieh 0000, Lebanon
| | - Ola Hadadeh
- Department of Anatomy, Cell Biology and Physiological Sciences, American University of Beirut, Beirut 1107-2020, Lebanon.
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Xin X, Xu Z, Wei J, Zhang Y. MiR-376a-3p increases cell apoptosis in acute myeloid leukemia by targeting MT1X. Cancer Biol Ther 2022; 23:234-242. [PMID: 35316152 PMCID: PMC8942499 DOI: 10.1080/15384047.2022.2054243] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022] Open
Abstract
Metallothioneins (MTs) are a group of low-molecular weight cysteine-rich proteins that play vital roles in oxidative stress, metal homeostasis, carcinogenesis and drug resistance. However, few studies have analyzed the roles of MTs in acute myeloid leukemia (AML). In this study, we revealed that the expression of metallothionein1X (MT1X), a main isoform of MTs, was highly expressed and acted as a candidate of prognostic indicator in AML patients. In vitro cell function experiments verified that silencing MT1X inhibited the proliferation of AML cells, sensitized cells to doxorubicin, and increased their apoptosis. We also showed that the downregulation of MT1X expression suppressed nuclear factor-κB (NF-κB) signaling by reducing p65, p-IκB-α, and downstream effectors. Elevated p65 and MT1X levels were indicators in AML. Moreover, we revealed that miR-376a-3p had binding sites with 3'-UTR of MT1X, suggesting that MT1X was negatively regulated by miR-376a-3p. Cell functional assay results indicated that miR-376a-3p overexpression significantly inhibited the proliferation, arrested the AML cells in the G0/G1 phase and induced cell apoptosis. The rescue experiments further confirmed that miR-376a-3p could reverse the promotion of MT1X overexpression on the progress of AML cells. Taken together, our results revealed that elevated MT1X expression might be involved in the mechanism underlying AML progression, indicating that the miR-376a/MT1X axis might serve as a promising novel target for the effective treatment of patients with AML.
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Affiliation(s)
- Xiangke Xin
- Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China.,Department of Hematology, Xinyang Hospital Affiliated to Zhengzhou University, Xinyang, Henan, China
| | - Zheng Xu
- Department of Hematology, Xinyang Hospital Affiliated to Zhengzhou University, Xinyang, Henan, China
| | - Jia Wei
- Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
| | - Yicheng Zhang
- Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
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Wei TH, Hsieh CL. Effect of Acupuncture on the p38 Signaling Pathway in Several Nervous System Diseases: A Systematic Review. Int J Mol Sci 2020; 21:E4693. [PMID: 32630156 PMCID: PMC7370084 DOI: 10.3390/ijms21134693] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2020] [Revised: 06/26/2020] [Accepted: 06/28/2020] [Indexed: 12/16/2022] Open
Abstract
Acupuncture is clinically used to treat various diseases and exerts positive local and systemic effects in several nervous system diseases. Advanced molecular and clinical studies have continually attempted to decipher the mechanisms underlying these effects of acupuncture. While a growing understanding of the pathophysiology underlying several nervous system diseases shows it to be related to inflammation and impair cell regeneration after ischemic events, the relationship between the therapeutic mechanism of acupuncture and the p38 MAPK signal pathway has yet to be elucidated. This review discusses the latest advancements in the identification of the effect of acupuncture on the p38 signaling pathway in several nervous system diseases. We electronically searched databases including PubMed, Embase, and the Cochrane Library from their inception to April 2020, using the following keywords alone or in various combinations: "acupuncture", "p38 MAPK pathway", "signaling", "stress response", "inflammation", "immune", "pain", "analgesic", "cerebral ischemic injury", "epilepsy", "Alzheimer's disease", "Parkinson's disease", "dementia", "degenerative", and "homeostasis". Manual acupuncture and electroacupuncture confer positive therapeutic effects by regulating proinflammatory cytokines, ion channels, scaffold proteins, and transcription factors including TRPV1/4, Nav, BDNF, and NADMR1; consequently, p38 regulates various phenomena including cell communication, remodeling, regeneration, and gene expression. In this review article, we found the most common acupoints for the relief of nervous system disorders including GV20, GV14, ST36, ST37, and LI4. Acupuncture exhibits dual regulatory functions of activating or inhibiting different p38 MAPK pathways, contributing to an overall improvement of clinical symptoms and function in several nervous system diseases.
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Affiliation(s)
- Tzu-Hsuan Wei
- Department of Chinese Medicine, China Medical University Hospital, Taichung 40447, Taiwan;
| | - Ching-Liang Hsieh
- Department of Chinese Medicine, China Medical University Hospital, Taichung 40447, Taiwan;
- Chinese Medicine Research Center, China Medical University, Taichung 40402, Taiwan
- Graduate Institute of Acupuncture Science, College of Chinese Medicine, China Medical University, Taichung 40402, Taiwan
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4
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Dokanehiifard S, Soltani BM, Ghiasi P, Baharvand H, Reza Ganjali M, Hosseinkhani S. hsa-miR-766-5p as a new regulator of mitochondrial apoptosis pathway for discriminating of cell death from cardiac differentiation. Gene 2020; 736:144448. [DOI: 10.1016/j.gene.2020.144448] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Revised: 01/26/2020] [Accepted: 02/04/2020] [Indexed: 12/19/2022]
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Isolation and Culture of Human Stem Cells from Apical Papilla under Low Oxygen Concentration Highlight Original Properties. Cells 2019; 8:cells8121485. [PMID: 31766521 PMCID: PMC6952825 DOI: 10.3390/cells8121485] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2019] [Revised: 11/07/2019] [Accepted: 11/14/2019] [Indexed: 12/12/2022] Open
Abstract
Stem cells isolated from the apical papilla of wisdom teeth (SCAPs) are an attractive model for tissue repair due to their availability, high proliferation rate and potential to differentiate in vitro towards mesodermal and neurogenic lineages. Adult stem cells, such as SCAPs, develop in stem cell niches in which the oxygen concentration [O2] is low (3–8% compared with 21% of ambient air). In this work, we evaluate the impact of low [O2] on the physiology of SCAPs isolated and processed in parallel at 21% or 3% O2 without any hyperoxic shock in ambient air during the experiment performed at 3% O2. We demonstrate that SCAPs display a higher proliferation capacity at 3% O2 than in ambient air with elevated expression levels of two cell surface antigens: the alpha-6 integrin subunit (CD49f) and the embryonic stem cell marker (SSEA4). We show that the mesodermal differentiation potential of SCAPs is conserved at early passage in both [O2], but is partly lost at late passage and low [O2], conditions in which SCAPs proliferate efficiently without any sign of apoptosis. Unexpectedly, we show that autophagic flux is active in SCAPs irrespective of [O2] and that this process remains high in cells even after prolonged exposure to 3% O2.
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Binó L, Veselá I, Papežíková I, Procházková J, Vašíček O, Štefková K, Kučera J, Hanáčková M, Kubala L, Pacherník J. The depletion of p38alpha kinase upregulates NADPH oxidase 2/NOX2/gp91 expression and the production of superoxide in mouse embryonic stem cells. Arch Biochem Biophys 2019; 671:18-26. [PMID: 31176685 DOI: 10.1016/j.abb.2019.06.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2018] [Revised: 05/31/2019] [Accepted: 06/05/2019] [Indexed: 01/04/2023]
Abstract
P38alpha kinase plays an important role in the regulation of both cell stress response and cell fate. In this study, we report that p38alpha kinase-deficient embryonic stem cells exhibit a higher production of reactive oxygen species (ROS) in contrast to their wild-type counterpart. Analysis of the expressions of NADPH oxidases (NOXs) and dual oxidases, crucial enzymes involved in intracellular ROS formation, shows NOX2/gp91phox is over-expressed in p38alpha deficient cells. The particular increase in superoxide formation was confirmed by the specific detection of hydroethidine derivate 2-hydroxyethidium. ROS formation decreased when the level of NOX2 was silenced by siRNA in p38alpha deficient cells. These data suggest the importance of p38alpha kinase in the regulation of ROS metabolism in embryonic stem cells and the significance of the observed phenomena of cancer cell-like phenotypes, which is discussed.
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Affiliation(s)
- Lucia Binó
- Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic; Department of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
| | - Iva Veselá
- Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
| | - Iva Papežíková
- Department of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
| | - Jiřina Procházková
- Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
| | - Ondřej Vašíček
- Department of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
| | - Kateřina Štefková
- Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
| | - Jan Kučera
- Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
| | - Markéta Hanáčková
- Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
| | - Lukáš Kubala
- Department of Free Radical Pathophysiology, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic.
| | - Jiří Pacherník
- Institute of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic.
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7
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Vlaski-Lafarge M, Loncaric D, Perez L, Labat V, Debeissat C, Brunet de la Grange P, Rossignol R, Ivanovic Z, Bœuf H. Bioenergetic Changes Underline Plasticity of Murine Embryonic Stem Cells. Stem Cells 2019; 37:463-475. [PMID: 30599083 DOI: 10.1002/stem.2965] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2018] [Revised: 11/13/2018] [Accepted: 12/03/2018] [Indexed: 12/19/2022]
Abstract
Murine embryonic stem cells (mESCs) are endowed by a time-dependent window of plasticity during their early commitment steps. Indeed, while mESCs deprived of leukemia inhibitory factor (LIF) for 24 hours revert to their naive pluripotent state after subsequent LIF readdition, cells deprived of LIF for 48 hours are no longer efficient in reverting, upon LIF addition, and undergo irreversible differentiation. We investigated undisclosed bioenergetic profiles of early mESC-derived committed cells versus their undifferentiated states in order to reveal specific bioenergetic changes associated with mESC plasticity. Multiparametric bioenergetic analysis revealed that pluripotent (+LIF) and reversibly committed cells (-LIF24h) are energetically flexible, depending on both oxidative phosphorylation (OXPHOS) and glycolysis. They exhibit high mitochondrial respiration in the presence of the main energetic substrates and can also rely on glycolysis in the presence of OXPHOS inhibitor. Inhibition of the glycolysis or mitochondrial respiration does not change drastically the expression of pluripotency genes, which remain well expressed. In addition, cells treated with these inhibitors keep their capacity to differentiate efficiently upon embryoid bodies formation. Transition from metabolically active mESCs to irreversibly committed cells is associated with a clear change in mitochondrial network morphology, to an increase of adenosine triphosphate (ATP) produced from glycolysis and a decline of ATP turnover and of the mitochondrial activity without change in the mitochondrial mass. Our study pointed that plasticity window of mESCs is associated with the bivalent energetic metabolism and potency to shift to glycolysis or OXPHOS on demand. LIF removal provokes glycolytic metabolic orientation and consecutive loss of the LIF-dependent reversion of cells to the pluripotent state. Stem Cells 2019;37:463-475.
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Affiliation(s)
- Marija Vlaski-Lafarge
- R&D Department, Etablissement Français du Sang Nouvelle-Aquitaine, Bordeaux, France.,Inserm/U1035, University of Bordeaux
| | - Darija Loncaric
- R&D Department, Etablissement Français du Sang Nouvelle-Aquitaine, Bordeaux, France.,Inserm/U1035, University of Bordeaux
| | - Laura Perez
- R&D Department, Etablissement Français du Sang Nouvelle-Aquitaine, Bordeaux, France
| | - Véronique Labat
- R&D Department, Etablissement Français du Sang Nouvelle-Aquitaine, Bordeaux, France.,Inserm/U1035, University of Bordeaux
| | - Christelle Debeissat
- R&D Department, Etablissement Français du Sang Nouvelle-Aquitaine, Bordeaux, France.,Inserm/U1035, University of Bordeaux
| | - Philippe Brunet de la Grange
- R&D Department, Etablissement Français du Sang Nouvelle-Aquitaine, Bordeaux, France.,Inserm/U1035, University of Bordeaux
| | | | - Zoran Ivanovic
- R&D Department, Etablissement Français du Sang Nouvelle-Aquitaine, Bordeaux, France.,Inserm/U1035, University of Bordeaux
| | - Hélène Bœuf
- Inserm/U1026, University of Bordeaux, Bordeaux, France
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8
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Qi YH, Mao FF, Zhou ZQ, Liu DC, Deng XY, Li JW, Mei FZ. The release of cytochrome c and the regulation of the programmed cell death progress in the endosperm of winter wheat (Triticum aestivum L.) under waterlogging. PROTOPLASMA 2018; 255:1651-1665. [PMID: 29717349 DOI: 10.1007/s00709-018-1256-7] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/28/2018] [Accepted: 04/16/2018] [Indexed: 06/08/2023]
Abstract
It has been shown in mammalian systems that the mitochondria can play a key role in the regulation of apoptosis by releasing intermembrane proteins (such as cytochrome c) into the cytosol. Cytochrome c released from the mitochondria to the cytoplasm activates proteolytic enzyme cascades, leading to specific nuclear DNA degradation and cell death. This pathway is considered to be one of the important regulatory mechanisms of apoptosis. Previous studies have shown that endosperm cell development in wheat undergoes specialized programmed cell death (PCD) and that waterlogging stress accelerates the PCD process; however, little is known regarding the associated molecular mechanism. In this study, changes in mitochondrial structure, the release of cytochrome c, and gene expression were studied in the endosperm cells of the wheat (Triticum aestivum L.) cultivar "huamai 8" during PCD under different waterlogging durations. The results showed that waterlogging aggravated the degradation of mitochondrial structure, increased the mitochondrial permeability transition (MPT), and decreased mitochondrial transmembrane potential (ΔΨm), resulting in the advancement of the endosperm PCD process. In situ localization and western blotting of cytochrome c indicated that with the development of the endosperm cell, cytochrome c was gradually released from the mitochondria to the cytoplasm, and waterlogging stress led to an advancement and increase in the release of cytochrome c. In addition, waterlogging stress resulted in the increased expression of the voltage-dependent anion channel (VDAC) and adenine nucleotide translocator (ANT), suggesting that the mitochondrial permeability transition pore (MPTP) may be involved in endosperm PCD under waterlogging stress. The MPTP inhibitor cyclosporine A effectively suppressed cell death and cytochrome c release during wheat endosperm PCD. Our results indicate that the mitochondria play important roles in the PCD of endosperm cells and that the increase in mitochondrial damage and corresponding release of cytochrome c may be one of the major causes of endosperm PCD advancement under waterlogging.
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Affiliation(s)
- Yuan-Hong Qi
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Fang-Fang Mao
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Zhu-Qing Zhou
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, China.
| | - Dong-Cheng Liu
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
| | - Xiang-Yi Deng
- College of Food and Biological Science and Technology, Wuhan Institute of Design and Sciences, Wuhan, 430070, Hubei, China
| | - Ji-Wei Li
- College of Food and Biological Science and Technology, Wuhan Institute of Design and Sciences, Wuhan, 430070, Hubei, China
| | - Fang-Zhu Mei
- Division of Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, China
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9
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Cho YS, Park HL. Exploitation of necroptosis for treatment of caspase-compromised cancers. Oncol Lett 2017; 14:1207-1214. [PMID: 28789335 PMCID: PMC5529905 DOI: 10.3892/ol.2017.6285] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2016] [Accepted: 06/30/2016] [Indexed: 11/05/2022] Open
Abstract
Programmed necrosis, or necroptosis, is a type of specialized cell death with necrotic characteristics, including the loss of membrane integrity and swollen organelles in dying cells. However, unlike simple necrosis, it may be induced as an alternative form of cell death when apoptosis is blocked and it is mediated in an orchestrated manner, similar to apoptosis, by a series of signaling molecules. Necroptosis-associated proteins and their specific small molecules have been extensively identified in order to illuminate the underlying mechanisms by which necroptosis is activated through a novel signaling pathway. However, the biological significance of necroptosis, which is known as a secondary route of apoptosis, remains under debate. Concurrent with these concerns, the clinical application of necroptosis has been cautiously proposed to treat necroptosis-associated diseases, and to overcome resistance to anticancer drugs. Accordingly, the present review will highlight the harnessing of necroptosis for anticancer therapy. To this end, the state-of-the art technique of necroptosis as a cancer therapy will be briefly described, and then its potential for clinical purposes will be delineated. For a further understanding of necroptosis, the present review begins with a basic introduction to necroptosis and its multifaceted physiological consequences.
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Affiliation(s)
- Young Sik Cho
- College of Pharmacy, Keimyung University, Daegu 704-701, Republic of Korea
| | - Hey Li Park
- College of Pharmacy, Keimyung University, Daegu 704-701, Republic of Korea
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10
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Ren L, Zhang C, Tao L, Hao J, Tan K, Miao K, Yu Y, Sui L, Wu Z, Tian J, An L. High-resolution profiles of gene expression and DNA methylation highlight mitochondrial modifications during early embryonic development. J Reprod Dev 2017; 63:247-261. [PMID: 28367907 PMCID: PMC5481627 DOI: 10.1262/jrd.2016-168] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Well-organized mitochondrial functions and dynamics are critical for early embryonic development and are operated via a large number of mitochondria-related genes (MtGs) encoded by both the nuclear and the mitochondrial genome.
However, the mechanisms underlying mitochondrial modifications during the critical window between blastocyst implantation and postimplantation organogenesis are poorly understood. Herein, we performed high-resolution dynamic
profiling of MtGs to acquire a more detailed understanding of mitochondrial modifications during early development. Our data suggest that the resumption of mitochondrial mass growth is not only facilitated by increased
mitochondrial biogenesis and mitochondrial DNA (mtDNA) replication, but also by the appropriate balance between mitochondrial fission and fusion. In addition, increased levels of reactive oxygen species (ROS) resulting from
enhanced mitochondrial functions may be the critical inducer for activating the glutathione (GSH)-based stress response system in early embryos. The appropriate balance between the mitochondrial stress response and apoptosis
appears to be significant for cell differentiation and early organogenesis. Furthermore, we found that most MtGs undergo de novo promoter methylation, which may have functional consequences on mitochondrial
functions and dynamics during early development. We also report that mtDNA methylation can be observed as early as soon after implantation. DNMT1, the predominant enzyme for maintaining DNA methylation, localized to the
mitochondria and bound to mtDNA by the implantation stage. Our study provides a new insight into the involvement of mitochondria in early mammalian embryogenesis. We also propose that the epigenetic modifications during early
development are significant for modulating mitochondrial functions and dynamics.
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Affiliation(s)
- Likun Ren
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Chao Zhang
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Li Tao
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Jing Hao
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Kun Tan
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Kai Miao
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Yong Yu
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Linlin Sui
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Zhonghong Wu
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Jianhui Tian
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
| | - Lei An
- Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, P. R. China
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Abstract
To generate new hypotheses, sometimes a "systems" approach is needed. In this review, I focus on the mitogen-activated kinase p38 because it has been recently shown to play an important role in the developmental programing and senescence of normal and stressed reproductive tissues. What follows is an overview of (i) pathways of p38 activation and their involvement in basic biological processes, (ii) evidence that p38 is involved in the homeostasis of reproductive tissues, (iii) how focus on p38 can be incorporated into investigation of normal and stressed pregnancies. Existence of excellent reviews will be mentioned as well as relevant animal models.
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Affiliation(s)
- Elizabeth A Bonney
- Department of Obstetrics, Gynecology and Reproductive Sciences, University of Vermont, Burlington, VT, USA
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12
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He H, Wang C, Dai Q, Li F, Bergholz J, Li Z, Li Q, Xiao ZX. p53 and p73 Regulate Apoptosis but Not Cell-Cycle Progression in Mouse Embryonic Stem Cells upon DNA Damage and Differentiation. Stem Cell Reports 2016; 7:1087-1098. [PMID: 27866875 PMCID: PMC5161534 DOI: 10.1016/j.stemcr.2016.10.008] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2016] [Revised: 10/21/2016] [Accepted: 10/24/2016] [Indexed: 01/23/2023] Open
Abstract
Embryonic stem cells (ESCs) are fast proliferating cells capable of differentiating into all somatic cell types. In somatic cells, it is well documented that p53 is rapidly activated upon DNA damage to arrest the cell cycle and induce apoptosis. In mouse ESCs, p53 can also be functionally activated, but the precise biological consequences are not well characterized. Here, we demonstrated that doxorubicin treatment initially led to cell-cycle arrest at G2/M in ESCs, followed by the occurrence of massive apoptosis. Neither p53 nor its target gene p73 was required for G2/M arrest. Instead, p53 and p73 were fully responsible for apoptosis. p53 and p73 were also required for differentiation-induced apoptosis in mouse ESCs. In addition, doxorubicin treatment induced the expression of retinoblastoma protein in a p53-dependent manner. Therefore, both p53 and p73 are critical in apoptosis induced by DNA damage and differentiation.
p53/p73 are key for DNA damage-induced apoptosis but not G2/M arrest in mESCs Both p53 and p73 are required for differentiation-induced apoptosis in mESCs Doxorubicin induces RB via p53-mediated suppression of miR-17-92 and miR-106a-363 p73 expression is induced upon differentiation in mESCs
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Affiliation(s)
- Hanbing He
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Cheng Wang
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Qian Dai
- Department of Pediatrics, West China Second University Hospital, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Sichuan University, Chengdu 610041, China
| | - Fengtian Li
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Johann Bergholz
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Zhonghan Li
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China
| | - Qintong Li
- Department of Pediatrics, West China Second University Hospital, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education, Sichuan University, Chengdu 610041, China.
| | - Zhi-Xiong Xiao
- Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment, Ministry of Education, College of Life Sciences, Sichuan University, 19 Wang Jang Road, Chengdu 610064, China.
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13
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Singh NK, Ernst M, Liebscher V, Fuellen G, Taher L. Revealing complex function, process and pathway interactions with high-throughput expression and biological annotation data. MOLECULAR BIOSYSTEMS 2016; 12:3196-208. [PMID: 27507577 DOI: 10.1039/c6mb00280c] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The biological relationships both between and within the functions, processes and pathways that operate within complex biological systems are only poorly characterized, making the interpretation of large scale gene expression datasets extremely challenging. Here, we present an approach that integrates gene expression and biological annotation data to identify and describe the interactions between biological functions, processes and pathways that govern a phenotype of interest. The product is a global, interconnected network, not of genes but of functions, processes and pathways, that represents the biological relationships within the system. We validated our approach on two high-throughput expression datasets describing organismal and organ development. Our findings are well supported by the available literature, confirming that developmental processes and apoptosis play key roles in cell differentiation. Furthermore, our results suggest that processes related to pluripotency and lineage commitment, which are known to be critical for development, interact mainly indirectly, through genes implicated in more general biological processes. Moreover, we provide evidence that supports the relevance of cell spatial organization in the developing liver for proper liver function. Our strategy can be viewed as an abstraction that is useful to interpret high-throughput data and devise further experiments.
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Affiliation(s)
- Nitesh Kumar Singh
- Institute for Biostatistics and Informatics in Medicine and Ageing Research, Rostock University Medical Center, Ernst-Heydemann-Str. 8, 18057 Rostock, Germany.
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14
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Chen IC, Hernandez C, Xu X, Cooney A, Wang Y, McCarrey JR. Dynamic Variations in Genetic Integrity Accompany Changes in Cell Fate. Stem Cells Dev 2016; 25:1698-1708. [PMID: 27627671 DOI: 10.1089/scd.2016.0221] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Pluripotent stem cells hold the potential to form the basis of novel approaches to treatment of disease in vivo as well as to facilitate the generation of models for human disease, providing powerful avenues to discovery of novel diagnostic biomarkers and/or innovative drug regimens in vitro. However, this will require extensive maintenance, expansion, and manipulation of these cells in culture, which raises a concern regarding the extent to which genetic integrity will be preserved throughout these manipulations. We used a mutation reporter (lacI) transgene approach to conduct direct comparisons of mutation frequencies in cell populations that shared a common origin and genetic identity, but were induced to undergo transitions in cell fate between pluripotent and differentiated states, or vice versa. We confirm that pluripotent cells normally maintain enhanced genetic integrity relative to that in differentiated cells, and we extend this finding to show that dynamic transformations in the relative stringency at which genetic integrity is maintained are associated with transitions between pluripotent and differentiated cellular states. These results provide insight into basic biological distinctions between pluripotent and differentiated cell types that impact genetic integrity in a manner that is directly relevant to the potential clinical use of these cell types.
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Affiliation(s)
- I-Chung Chen
- 1 Department of Biology, University of Texas at San Antonio , San Antonio, Texas
| | - Christine Hernandez
- 1 Department of Biology, University of Texas at San Antonio , San Antonio, Texas
| | - Xueping Xu
- 2 Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center , Houston, Texas
| | - Austin Cooney
- 2 Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center , Houston, Texas.,3 Department of Pediatrics, Dell Pediatric Research Institute, University of Texas at Austin Dell , Medical School, Austin, Texas
| | - Yufeng Wang
- 1 Department of Biology, University of Texas at San Antonio , San Antonio, Texas
| | - John R McCarrey
- 1 Department of Biology, University of Texas at San Antonio , San Antonio, Texas
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15
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Knocking down of heat-shock protein 27 directs differentiation of functional glutamatergic neurons from placenta-derived multipotent cells. Sci Rep 2016; 6:30314. [PMID: 27444754 PMCID: PMC4957209 DOI: 10.1038/srep30314] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2016] [Accepted: 07/04/2016] [Indexed: 01/06/2023] Open
Abstract
This study presents human placenta-derived multipotent cells (PDMCs) as a source from which functional glutamatergic neurons can be derived. We found that the small heat-shock protein 27 (HSP27) was downregulated during the neuronal differentiation process. The in vivo temporal and spatial profiles of HSP27 expression were determined and showed inverted distributions with neuronal proteins during mouse embryonic development. Overexpression of HSP27 in stem cells led to the arrest of neuronal differentiation; however, the knockdown of HSP27 yielded a substantially enhanced ability of PDMCs to differentiate into neurons. These neurons formed synaptic networks and showed positive staining for multiple neuronal markers. Additionally, cellular phenomena including the absence of apoptosis and rare proliferation in HSP27-silenced PDMCs, combined with molecular events such as cleaved caspase-3 and the loss of stemness with cleaved Nanog, indicated that HSP27 is located upstream of neuronal differentiation and constrains that process. Furthermore, the induced neurons showed increasing intracellular calcium concentrations upon glutamate treatment. These differentiated cells co-expressed the N-methyl-D-aspartate receptor, vesicular glutamate transporter, and synaptosomal-associated protein 25 but did not show expression of tyrosine hydroxylase, choline acetyltransferase or glutamate decarboxylase 67. Therefore, we concluded that HSP27-silenced PDMCs differentiated into neurons possessing the characteristics of functional glutamatergic neurons.
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16
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Tang Y, Hong YZ, Bai HJ, Wu Q, Chen CD, Lang JY, Boheler KR, Yang HT. Plant Homeo Domain Finger Protein 8 Regulates Mesodermal and Cardiac Differentiation of Embryonic Stem Cells Through Mediating the Histone Demethylation of pmaip1. Stem Cells 2016; 34:1527-40. [PMID: 26866517 DOI: 10.1002/stem.2333] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2015] [Accepted: 01/06/2016] [Indexed: 12/30/2022]
Abstract
Histone demethylases have emerged as key regulators of biological processes. The H3K9me2 demethylase plant homeo domain finger protein 8(PHF8), for example, is involved in neuronal differentiation, but its potential function in the differentiation of embryonic stem cells (ESCs) to cardiomyocytes is poorly understood. Here, we explored the role of PHF8 during mesodermal and cardiac lineage commitment of mouse ESCs (mESCs). Using a phf8 knockout (ph8(-/Y) ) model, we found that deletion of phf8 in ESCs did not affect self-renewal, proliferation or early ectodermal/endodermal differentiation, but it did promote the mesodermal lineage commitment with the enhanced cardiomyocyte differentiation. The effects were accompanied by a reduction in apoptosis through a caspase 3-independent pathway during early ESC differentiation, without significant differences between differentiating wide-type (ph8(+/Y) ) and ph8(-/Y) ESCs in cell cycle progression or proliferation. Functionally, PHF8 promoted the loss of a repressive mark H3K9me2 from the transcription start site of a proapoptotic gene pmaip1 and activated its transcription. Furthermore, knockdown of pmaip1 mimicked the phenotype of ph8(-/Y) by showing the decreased apoptosis during early differentiation of ESCs and promoted mesodermal and cardiac commitment, while overexpression of pmaip1 or phf8 rescued the phenotype of ph8(-/Y) ESCs by increasing the apoptosis and weakening the mesodermal and cardiac differentiation. These results reveal that the histone demethylase PHF8 regulates mesodermal lineage and cell fate decisions in differentiating mESCs through epigenetic control of the gene critical to programmed cell death pathways. Stem Cells 2016;34:1527-1540.
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Affiliation(s)
- Yan Tang
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) University of Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Ya-Zhen Hong
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) University of Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Hua-Jun Bai
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) University of Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Qiang Wu
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) University of Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Charlie Degui Chen
- State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS), Shanghai, China
| | - Jing-Yu Lang
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) University of Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Kenneth R Boheler
- LKS Faculty of Medicine, Department of Physiology and Stem Cell and Regenerative Medicine Consortium, School of Biomedical Sciences, Jockey Club Building for Interdisciplinary Research, University of Hong Kong, Hong Kong, SAR China
| | - Huang-Tian Yang
- Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) University of Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, China.,Second Affiliated Hospital, Zhejiang University, Hangzhou, China
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17
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Dziegiel P, Pula B, Kobierzycki C, Stasiolek M, Podhorska-Okolow M. The Role of Metallothioneins in Carcinogenesis. ADVANCES IN ANATOMY EMBRYOLOGY AND CELL BIOLOGY 2016. [DOI: 10.1007/978-3-319-27472-0_4] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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18
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Hammoud AA, Kirstein N, Mournetas V, Darracq A, Broc S, Blanchard C, Zeineddine D, Mortada M, Boeuf H. Murine Embryonic Stem Cell Plasticity Is Regulated through Klf5 and Maintained by Metalloproteinase MMP1 and Hypoxia. PLoS One 2016; 11:e0146281. [PMID: 26731538 PMCID: PMC4701481 DOI: 10.1371/journal.pone.0146281] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/20/2015] [Accepted: 12/15/2015] [Indexed: 12/12/2022] Open
Abstract
Mouse embryonic stem cells (mESCs) are expanded and maintained pluripotent in vitro in the presence of leukemia inhibitory factor (LIF), an IL6 cytokine family member which displays pleiotropic functions, depending on both cell maturity and cell type. LIF withdrawal leads to heterogeneous differentiation of mESCs with a proportion of the differentiated cells apoptosising. During LIF withdrawal, cells sequentially enter a reversible and irreversible phase of differentiation during which LIF addition induces different effects. However the regulators and effectors of LIF-mediated reprogramming are poorly understood. By employing a LIF-dependent 'plasticity' test, that we set up, we show that Klf5, but not JunB is a key LIF effector. Furthermore PI3K signaling, required for the maintenance of mESC pluripotency, has no effect on mESC plasticity while displaying a major role in committed cells by stimulating expression of the mesodermal marker Brachyury at the expense of endoderm and neuroectoderm lineage markers. We also show that the MMP1 metalloproteinase, which can replace LIF for maintenance of pluripotency, mimics LIF in the plasticity window, but less efficiently. Finally, we demonstrate that mESCs maintain plasticity and pluripotency potentials in vitro under hypoxic/physioxic growth conditions at 3% O2 despite lower levels of Pluri and Master gene expression in comparison to 20% O2.
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Affiliation(s)
- Aya Abou Hammoud
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
- Lebanese University, Beyrouth, Liban
| | - Nina Kirstein
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | - Virginie Mournetas
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | - Anais Darracq
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | - Sabine Broc
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | - Camille Blanchard
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
| | | | | | - Helene Boeuf
- Univ. Bordeaux, CIRID, UMR5164, F-33 000 Bordeaux, France
- CNRS, CIRID, UMR 5164, F-33 000 Bordeaux, France
- * E-mail:
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19
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McCarrey JR. Distinctions between transgenerational and non-transgenerational epimutations. Mol Cell Endocrinol 2014; 398:13-23. [PMID: 25079508 DOI: 10.1016/j.mce.2014.07.016] [Citation(s) in RCA: 38] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/30/2014] [Revised: 07/25/2014] [Accepted: 07/25/2014] [Indexed: 12/22/2022]
Abstract
Recent studies have described numerous environmentally-induced disruptions of the epigenome (epimutations) in mammals. While some of these appear to be corrected by normal germline-specific epigenetic reprogramming and are therefore not transmitted transgenerationally, others are not corrected and are transmitted over multiple subsequent generations. The mechanism(s) that distinguish transgenerational and non-transgenerational epimutations have not been delineated. This review examines several potential molecular and developmental distinctions between transgenerational and non-transgenerational epimutations in the context of the likelihood that any of these may or may not contribute to transgenerational inheritance of epimutations.
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Affiliation(s)
- John R McCarrey
- Department of Biology, University of Texas at San Antonio, San Antonio, USA.
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20
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Kempf H, Olmer R, Kropp C, Rückert M, Jara-Avaca M, Robles-Diaz D, Franke A, Elliott DA, Wojciechowski D, Fischer M, Roa Lara A, Kensah G, Gruh I, Haverich A, Martin U, Zweigerdt R. Controlling expansion and cardiomyogenic differentiation of human pluripotent stem cells in scalable suspension culture. Stem Cell Reports 2014; 3:1132-46. [PMID: 25454631 PMCID: PMC4264033 DOI: 10.1016/j.stemcr.2014.09.017] [Citation(s) in RCA: 166] [Impact Index Per Article: 15.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2014] [Revised: 09/24/2014] [Accepted: 09/25/2014] [Indexed: 12/13/2022] Open
Abstract
To harness the potential of human pluripotent stem cells (hPSCs), an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion). Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity) was enabled that were directly applicable to bioartificial cardiac tissue formation.
Efficient cardiac differentiation protocol in suspension by chemical Wnt modulators Differentiation is CHIR concentration dependent, but aggregate size independent Bioreactor-controlled hPSC expansion dictates subsequent lineage differentiation Metallothionein is a potentially stress-induced marker of hPSC culture
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Affiliation(s)
- Henning Kempf
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Ruth Olmer
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; Member of the Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Christina Kropp
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Michael Rückert
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Monica Jara-Avaca
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Diana Robles-Diaz
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Annika Franke
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - David A Elliott
- Murdoch Childrens Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC 3052, Australia
| | - Daniel Wojciechowski
- Institute for Neurophysiology, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Martin Fischer
- Institute for Neurophysiology, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Angelica Roa Lara
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - George Kensah
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Ina Gruh
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Axel Haverich
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany
| | - Ulrich Martin
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; Member of the Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), 30625 Hannover, Germany
| | - Robert Zweigerdt
- Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation, and Vascular Surgery (HTTG), Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany; REBIRTH-Cluster of Excellence, Hannover Medical School, Carl-Neuberg-Straβe 1, 30625 Hannover, Germany.
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21
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Cooper DJ, Walter CA, McCarrey JR. Co-regulation of pluripotency and genetic integrity at the genomic level. Stem Cell Res 2014; 13:508-19. [PMID: 25451711 DOI: 10.1016/j.scr.2014.09.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Revised: 09/12/2014] [Accepted: 09/20/2014] [Indexed: 12/20/2022] Open
Abstract
The Disposable Soma Theory holds that genetic integrity will be maintained at more pristine levels in germ cells than in somatic cells because of the unique role germ cells play in perpetuating the species. We tested the hypothesis that the same concept applies to pluripotent cells compared to differentiated cells. Analyses of transcriptome and cistrome databases, along with canonical pathway analysis and chromatin immunoprecipitation confirmed differential expression of DNA repair and cell death genes in embryonic stem cells and induced pluripotent stem cells relative to fibroblasts, and predicted extensive direct and indirect interactions between the pluripotency and genetic integrity gene networks in pluripotent cells. These data suggest that enhanced maintenance of genetic integrity is fundamentally linked to the epigenetic state of pluripotency at the genomic level. In addition, these findings demonstrate how a small number of key pluripotency factors can regulate large numbers of downstream genes in a pathway-specific manner.
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Affiliation(s)
- Daniel J Cooper
- Department of Biology, University of Texas at San Antonio, USA
| | - Christi A Walter
- Department of Cellular and Structural Biology, University of Texas Health Science Center at San Antonio, USA
| | - John R McCarrey
- Department of Biology, University of Texas at San Antonio, USA.
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22
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Nagahara Y, Morita M, Nakata T, Iba A, Shinomiya T. Loss of Bcl-2 expression correlates with increasing sensitivity to apoptosis in differentiating ES cells. Cell Biol Int 2013; 38:381-7. [DOI: 10.1002/cbin.10214] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2013] [Accepted: 10/21/2013] [Indexed: 01/25/2023]
Affiliation(s)
- Yukitoshi Nagahara
- Division of Life Science and Engineering, College of Science and Engineering; Tokyo Denki University; Hatoyama, Hiki-gun Saitama 350-0394 Japan
| | - Misa Morita
- Division of Life Science and Engineering, College of Science and Engineering; Tokyo Denki University; Hatoyama, Hiki-gun Saitama 350-0394 Japan
| | - Tsubasa Nakata
- Division of Life Science and Engineering, College of Science and Engineering; Tokyo Denki University; Hatoyama, Hiki-gun Saitama 350-0394 Japan
| | - Akitoshi Iba
- Division of Life Science and Engineering, College of Science and Engineering; Tokyo Denki University; Hatoyama, Hiki-gun Saitama 350-0394 Japan
| | - Takahisa Shinomiya
- Division of Life Science and Engineering, College of Science and Engineering; Tokyo Denki University; Hatoyama, Hiki-gun Saitama 350-0394 Japan
- Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences; Aomori University; 2-3-1 Koubata, Aomori Aomori 030-0943 Japan
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Siatkowski M, Liebscher V, Fuellen G. CellFateScout - a bioinformatics tool for elucidating small molecule signaling pathways that drive cells in a specific direction. Cell Commun Signal 2013; 11:85. [PMID: 24206562 PMCID: PMC3833265 DOI: 10.1186/1478-811x-11-85] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2013] [Accepted: 10/29/2013] [Indexed: 12/12/2022] Open
Abstract
Background Small molecule effects can be represented by active signaling pathways within functional networks. Identifying these can help to design new strategies to utilize known small molecules, e.g. to trigger specific cellular transformations or to reposition known drugs. Results We developed CellFateScout that uses the method of Latent Variables to turn differential high-throughput expression data and a functional network into a list of active signaling pathways. Applying it to Connectivity Map data, i.e., differential expression data describing small molecule effects, we then generated a Human Small Molecule Mechanisms Database. Finally, using a list of active signaling pathways as query, a similarity search can identify small molecules from the database that may trigger these pathways. We validated our approach systematically, using expression data of small molecule perturbations, yielding better predictions than popular bioinformatics tools. Conclusions CellFateScout can be used to select small molecules for their desired effects. The CellFateScout Cytoscape plugin, a tutorial and the Human Small Molecule Mechanisms Database are available at https://sourceforge.net/projects/cellfatescout/ under LGPLv2 license.
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Affiliation(s)
| | | | - Georg Fuellen
- Institute for Biostatistics and Informatics in Medicine and Ageing Research, University of Rostock, Ernst Heydemann Strasse 8, D-18057 Rostock, Germany.
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Fraser L, Taylor AH, Forrester LM. SCF/KIT inhibition has a cumulative but reversible effect on the self-renewal of embryonic stem cells and on the survival of differentiating cells. Cell Reprogram 2013; 15:259-68. [PMID: 23768117 DOI: 10.1089/cell.2013.0015] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
The receptor tyrosine kinase c-KIT is expressed in embryonic stem cells (ESCs) and adult stem cells, and many functional studies have demonstrated the importance of the SCF/KIT signaling pathway in adult stem cell maintenance. In this study, we show that a high level of KIT expression in wild-type ESCs correlates with an enhanced self-renewal and that inhibition of KIT signaling in ESCs for extended periods of time has a cumulative but reversible effect on self-renewal. Together these data suggest that continued KIT signaling in some cells within a self-renewing ESC population is required for optimal ESC function. Using a KIT blocking antibody, we recapitulated the phenotype we previously reported for genetically deficient KIT-null cells, demonstrating that SCF/KIT signaling is essential for the survival of differentiating ESCs. Here we show that this phenotype is also reversible. Pharmacological inhibition of JNK also had a cumulative but reversible detrimental effect on the survival of differentiating cells, thus recapitulating the Kit null phenotype and implicating JNK as a downstream mediator of KIT signaling. In contrast, the self-renewal of ESCs was unaffected by prolonged exposure to the JNK inhibitor, suggesting that JNK-independent downstream pathways are involved in KIT-mediated ESC self-renewal whereas KIT-mediated survival of differentiating ESC is likely to be JNK dependent.
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Affiliation(s)
- Lindsay Fraser
- MRC Centre for Regenerative Medicine, Edinburgh EH16 4UU, UK
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Sánchez-Alvarez R, Gayen S, Vadigepalli R, Anni H. Ethanol diverts early neuronal differentiation trajectory of embryonic stem cells by disrupting the balance of lineage specifiers. PLoS One 2013; 8:e63794. [PMID: 23724002 PMCID: PMC3665827 DOI: 10.1371/journal.pone.0063794] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2013] [Accepted: 04/04/2013] [Indexed: 02/07/2023] Open
Abstract
Background Ethanol is a toxin responsible for the neurodevelopmental deficits of Fetal Alcohol Spectrum Disorders (FASD). Recent evidence suggests that ethanol modulates the protein expression of lineage specifier transcription factors Oct4 (Pou5f1) and Sox2 in early stages of mouse embryonic stem (ES) cell differentiation. We hypothesized that ethanol induced an imbalance in the expression of Oct4 and Sox2 in early differentiation, that dysregulated the expression of associated and target genes and signaling molecules and diverted cells from neuroectodermal (NE) formation. Methodology/Principal Findings We showed modulation by ethanol of 33 genes during ES cell differentiation, using high throughput microfluidic dynamic array chips measuring 2,304 real time quantitative PCR assays. Based on the overall gene expression dynamics, ethanol drove cells along a differentiation trajectory away from NE fate. These ethanol-induced gene expression changes were observed as early as within 2 days of differentiation, and were independent of cell proliferation or apoptosis. Gene expression changes were correlated with fewer βIII-tubulin positive cells of an immature neural progenitor phenotype, as well as a disrupted actin cytoskeleton were observed. Moreover, Tuba1a and Gapdh housekeeping genes were modulated by ethanol during differentiation and were replaced by a set of ribosomal genes with stable expression. Conclusions/Significance These findings provided an ethanol-response gene signature and pointed to the transcriptional dynamics underlying lineage imbalance that may be relevant to FASD phenotype.
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Affiliation(s)
- Rosa Sánchez-Alvarez
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
| | - Saurabh Gayen
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
| | - Rajanikanth Vadigepalli
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
- Daniel Baugh Institute for Functional Genomics and Computational Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
- * E-mail: (RV); (HA)
| | - Helen Anni
- Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
- * E-mail: (RV); (HA)
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Ogony JW, Malahias E, Vadigepalli R, Anni H. Ethanol alters the balance of Sox2, Oct4, and Nanog expression in distinct subpopulations during differentiation of embryonic stem cells. Stem Cells Dev 2013; 22:2196-210. [PMID: 23470161 DOI: 10.1089/scd.2012.0513] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023] Open
Abstract
The transcription factors Sox2, Oct4, and Nanog regulate within a narrow dose-range embryonic stem (ES) cell pluripotency and cell lineage commitment. Excess of Oct4 relative to Sox2 guides cells to mesoendoderm (ME), while abundance of Sox2 promotes neuroectoderm (NE) formation. Literature does not address whether ethanol interferes with these regulatory interactions during neural development. We hypothesized that ethanol exposure of ES cells in early differentiation causes an imbalance of Oct4 and Sox2 that diverts cells away from NE to ME lineage, consistent with the teratogenesis effects caused by prenatal alcohol exposure. Mouse ES cells were exposed to ethanol (0, 25, 50, and 100 mM) during retinoic acid (10 nM)-directed differentiation to NE for 0-6 days, and the expression of Sox2, Oct4, and Nanog was measured in single live cells by multiparametric flow cytometry, and the cellular phenotype was characterized by immunocytochemistry. Our data showed an ethanol dose- and time-dependent asymmetric modulation of Oct4 and Sox2 expression, as early as after 2 days of exposure. Single-cell analysis of the correlated expression of Sox2, Oct4, and Nanog revealed that ethanol promoted distinct subpopulations with a high Oct4/Sox2 ratio. Ethanol-exposed cells differentiated to fewer β-III tubulin-immunoreactive cells with an immature neuronal phenotype by 4 days. We interpret these data as suggesting that ethanol diverted cells in early differentiation from the NE fate toward the ME lineage. Our results provide a novel insight into the mode of ethanol action and opportunities for discovery of prenatal biomarkers at early stages.
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Affiliation(s)
- Joshua W Ogony
- Daniel Baugh Institute for Functional Genomics and Computational Biology, Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA
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Bouchard F, Paquin J. Differential effects of retinoids and inhibitors of ERK and p38 signaling on adipogenic and myogenic differentiation of P19 stem cells. Stem Cells Dev 2013; 22:2003-16. [PMID: 23441952 DOI: 10.1089/scd.2012.0209] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
All-trans-retinoic acid (atRA) is an essential signaling molecule in embryonic development. It regulates cell differentiation by activating nuclear retinoic acid receptors (RAR) and retinoid-X receptors (RXR), which both control gene expression. In addition, atRA could act in the cytoplasm by modulating the activity of mitogen-activated protein kinases (MAPK) ERK and p38, which also have a role in cell differentiation. AtRA can induce the differentiation of P19 embryonic carcinoma stem cells into adipocytes, cardiomyocytes, and skeletal muscle cells, concurrently, in the same culture. We postulated that combinations of atRA, atRA analogs exhibiting selectivity for RAR or RXR, and inhibitors of ERK and p38 signaling (ERKi and p38i) could be used to favor one mesodermal fate over the others in the P19 model. In a first series of experiments, we replaced atRA by an agonist of RXR (LG100268) or RAR (TTNPB) to preferentially stimulate one group of receptors over the other. LG100268 was as adipogenic and myogenic as atRA, whereas TTNPB strongly induced adipogenesis, but not myogenesis. ERKi enhanced the myogenic action of atRA, and p38i increased both adipogenesis and myogenesis. In a second series of experiments, we combined atRA with an RAR or RXR antagonist (RARatg or RXRatg) to preferentially deactivate each receptor group in turn. The combinations atRA+RXRatg and atRA+RARatg, including or not ERKi, had similar mesodermal actions as atRA. In contrast, there was no myogenesis with atRA+RXRatg+p38i treatment, and there were no myogenesis and no adipogenesis with the atRA+RARatg+p38i combination. Overall, the results indicate that p38 has a role in mesodermal differentiation that depends on the retinoid context. Indeed, p38 in conjunction with RXR is important in myogenesis, and p38 and RAR in adipogenesis. Under the conditions tested, it was possible to stimulate adipogenesis with a block on myogenesis, whereas increased myogenesis was accompanied by adipogenesis.
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Affiliation(s)
- Frédéric Bouchard
- Département de chimie-biochimie and Centre BioMed, Université du Québec à Montréal, Montreal, Quebec, Canada
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Yang HT, Zhang M, Huang J, Liang H, Zhang P, Boheler KR. Cardiomyocytes derived from pluripotent stem cells: Progress and prospects from China. Exp Cell Res 2013; 319:120-5. [DOI: 10.1016/j.yexcr.2012.09.011] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2012] [Accepted: 09/18/2012] [Indexed: 10/27/2022]
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Jeon K, Lim H, Kim JH, Han D, Lee ER, Yang GM, Song MK, Kim JH, Cho SG. Bax inhibitor-1 enhances survival and neuronal differentiation of embryonic stem cells via differential regulation of mitogen-activated protein kinases activities. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2012; 1823:2190-200. [PMID: 22906541 DOI: 10.1016/j.bbamcr.2012.08.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/17/2012] [Revised: 07/23/2012] [Accepted: 08/04/2012] [Indexed: 01/05/2023]
Abstract
Bax inhibitor-1 (BI-1), a member of the BI-1 family of integral membrane proteins, was originally identified as an inhibitor of stress-induced cell death in mammalian cells. Previous studies have shown that the withdrawal of leukemia inhibitory factor (LIF) results in differentiation of the majority of mouse embryonic stem (mES) cells into various cell lineages, while some ES cells die within 3days. Thus, to investigate the function of BI-1 in ES cell survival and neuronal differentiation, we generated mES cell lines that overexpress BI-1 or a carboxy-terminal BI-1ΔC mutant. Overexpression of BI-1 in mES cells significantly increased cell viability and resistance to apoptosis induced by LIF withdrawal, while the control vector or BI-1ΔC-overexpressing mES cells had no effect. Moreover, overexpression of BI-1 produced significant inhibition of the p38 mitogen-activated protein kinases (MAPK) pathway in response to LIF withdrawal, while activity of the extracellular signal-regulated kinase (ERK)/c-Jun N-terminal kinase (JNK) MAPK pathway was increased. Interestingly, we found that BI-1-overexpressing cells showed higher expression levels of neuroectodermal markers (Otx1, Lmx1b, En1, Pax2, Wnt1, Sox1, and Nestin) and greater neuronal differentiation efficiency than control or BI-1ΔC-overexpressing mES cells did. Considering these findings, our results indicated that BI-1-modulated MAPK activity plays a key role in protecting mES cells from LIF-withdrawal-induced apoptosis and in promoting their differentiation toward neuronal lineages.
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Affiliation(s)
- Kilsoo Jeon
- Department of Animal Biotechnology (BK21), Animal Resources Research Center, and SMART-IABS, Konkuk University, Seoul 143-702, Republic of Korea
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Takahashi S. Molecular functions of metallothionein and its role in hematological malignancies. J Hematol Oncol 2012; 5:41. [PMID: 22839501 PMCID: PMC3419633 DOI: 10.1186/1756-8722-5-41] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2012] [Accepted: 07/27/2012] [Indexed: 11/10/2022] Open
Abstract
Metallothionein (MT) was reported to be a potential negative regulator of apoptosis, and various reports have suggested that it may play roles in carcinogenesis and drug resistance, in at least a portion of cancer cells. The author summarizes the current understanding of the molecular functions of MT for tumor cell growth and drug resistance. These activities are regulated through intracellular metal ion modulation and free radical scavenging. Compared with analyses of solid tumors, few studies have analyzed the roles of MT in hematological malignancies. This review mainly describes the functions of MT in hematopoietic cells. Furthermore, through expression analyses of leukemias and lymphomas, the roles of MT in the biology of these diseases are particularly focused upon.
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Affiliation(s)
- Shinichiro Takahashi
- Division of Hematology, Kitasato University School of Allied Health Sciences, 1-15-1 Kitasato, Minami-ku, Sagamihara, 252-0373, Japan.
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31
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Mathieu ME, Saucourt C, Mournetas V, Gauthereau X, Thézé N, Praloran V, Thiébaud P, Bœuf H. LIF-dependent signaling: new pieces in the Lego. Stem Cell Rev Rep 2012; 8:1-15. [PMID: 21537995 PMCID: PMC3285761 DOI: 10.1007/s12015-011-9261-7] [Citation(s) in RCA: 70] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
LIF, a member of the IL6 family of cytokine, displays pleiotropic effects on various cell types and organs. Its critical role in stem cell models (e.g.: murine ES, human mesenchymal cells) and its essential non redundant function during the implantation process of embryos, in eutherian mammals, put this cytokine at the core of many studies aiming to understand its mechanisms of action, which could benefit to medical applications. In addition, its conservation upon evolution raised the challenging question concerning the function of LIF in species in which there is no implantation. We present the recent knowledge about the established and potential functions of LIF in different stem cell models, (embryonic, hematopoietic, mesenchymal, muscle, neural stem cells and iPSC). We will also discuss EVO-DEVO aspects of this multifaceted cytokine.
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Affiliation(s)
- Marie-Emmanuelle Mathieu
- Univ. de Bordeaux, CIRID, UMR5164, F-33000 Bordeaux, France
- CNRS, CIRID, UMR5164, F-33000 Bordeaux, France
| | - Claire Saucourt
- Univ. de Bordeaux, CIRID, UMR5164, F-33000 Bordeaux, France
- CNRS, CIRID, UMR5164, F-33000 Bordeaux, France
| | - Virginie Mournetas
- Univ. de Bordeaux, CIRID, UMR5164, F-33000 Bordeaux, France
- CNRS, CIRID, UMR5164, F-33000 Bordeaux, France
| | - Xavier Gauthereau
- Univ. de Bordeaux, CIRID, UMR5164, F-33000 Bordeaux, France
- CNRS, CIRID, UMR5164, F-33000 Bordeaux, France
| | - Nadine Thézé
- Univ. de Bordeaux, CIRID, UMR5164, F-33000 Bordeaux, France
- CNRS, CIRID, UMR5164, F-33000 Bordeaux, France
| | - Vincent Praloran
- Univ. de Bordeaux, CIRID, UMR5164, F-33000 Bordeaux, France
- CNRS, CIRID, UMR5164, F-33000 Bordeaux, France
| | - Pierre Thiébaud
- Univ. de Bordeaux, CIRID, UMR5164, F-33000 Bordeaux, France
- CNRS, CIRID, UMR5164, F-33000 Bordeaux, France
| | - Hélène Bœuf
- Univ. de Bordeaux, CIRID, UMR5164, F-33000 Bordeaux, France
- CNRS, CIRID, UMR5164, F-33000 Bordeaux, France
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Plourde D, Vigneault C, Laflamme I, Blondin P, Robert C. Cellular and molecular characterization of the impact of laboratory setup on bovine in vitro embryo production. Theriogenology 2012; 77:1767-78.e1. [DOI: 10.1016/j.theriogenology.2011.12.021] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2011] [Revised: 11/22/2011] [Accepted: 12/17/2011] [Indexed: 11/26/2022]
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Valproic acid inhibits neural progenitor cell death by activation of NF-κB signaling pathway and up-regulation of Bcl-XL. J Biomed Sci 2011; 18:48. [PMID: 21722408 PMCID: PMC3158748 DOI: 10.1186/1423-0127-18-48] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2011] [Accepted: 07/04/2011] [Indexed: 01/24/2023] Open
Abstract
Background At the beginning of neurogenesis, massive brain cell death occurs and more than 50% of cells are eliminated by apoptosis along with neuronal differentiation. However, few studies were conducted so far regarding the regulation of neural progenitor cells (NPCs) death during development. Because of the physiological role of cell death during development, aberration of normal apoptotic cell death is detrimental to normal organogenesis. Apoptosis occurs in not only neuron but also in NPCs and neuroblast. When growth and survival signals such as EGF or LIF are removed, apoptosis is activated as well as the induction of differentiation. To investigate the regulation of cell death during developmental stage, it is essential to investigate the regulation of apoptosis of NPCs. Methods Neural progenitor cells were cultured from E14 embryonic brains of Sprague-Dawley rats. For in vivo VPA animal model, pregnant rats were treated with VPA (400 mg/kg S.C.) diluted with normal saline at E12. To analyze the cell death, we performed PI staining and PARP and caspase-3 cleavage assay. Expression level of proteins was investigated by Western blot and immunocytochemical assays. The level of mRNA expression was investigated by RT-PCR. Interaction of Bcl-XL gene promoter and NF-κB p65 was investigated by ChIP assay. Results In this study, FACS analysis, PI staining and PARP and caspase-3 cleavage assay showed that VPA protects cultured NPCs from cell death after growth factor withdrawal both in basal and staurosporine- or hydrogen peroxide-stimulated conditions. The protective effect of prenatally injected VPA was also observed in E16 embryonic brain. Treatment of VPA decreased the level of IκBα and increased the nuclear translocation of NF-κB, which subsequently enhanced expression of anti-apoptotic protein Bcl-XL. Conclusion To the best of our knowledge, this is the first report to indicate the reduced death of NPCs by VPA at developmentally critical periods through the degradation of IκBα and the activation of NF-κB signaling. The reduced NPCs death might underlie the neurodevelopmental defects collectively called fetal valproate syndrome, which shows symptoms such as mental retardation and autism-like behavior.
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El Ghazi I, Martin BL, Armitage IM. New proteins found interacting with brain metallothionein-3 are linked to secretion. Int J Alzheimers Dis 2010; 2011:208634. [PMID: 21234102 PMCID: PMC3014675 DOI: 10.4061/2011/208634] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2010] [Revised: 10/19/2010] [Accepted: 11/05/2010] [Indexed: 11/20/2022] Open
Abstract
Metallothionein 3 (MT-3), also known as growth inhibitory factor (GIF), exhibits a neuroinhibitory activity. Our lab and others have previously shown that this biological activity involves interacting protein partners in the brain. However, nothing specific is yet known about which of these interactions is responsible for the GIF activity. In this paper, we are reporting upon new proteins found interacting with MT-3 as determined through immunoaffinity chromatography and mass spectrometry. These new partner proteins-Exo84p, 14-3-3 Zeta, α and β Enolase, Aldolase C, Malate dehydrogenase, ATP synthase, and Pyruvate kinase-along with those previously identified have now been classified into three functional groups: transport and signaling, chaperoning and scaffolding, and glycolytic metabolism. When viewed together, these interactions support a proposed model for the regulation of the GIF activity of MT-3.
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Affiliation(s)
- Issam El Ghazi
- Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street, SE, Minneapolis, MN 55455, USA
| | - Bruce L. Martin
- Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street, SE, Minneapolis, MN 55455, USA
| | - Ian M. Armitage
- Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church Street, SE, Minneapolis, MN 55455, USA
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Cha Y, Moon BH, Lee MO, Ahn HJ, Lee HJ, Lee KA, Fornace AJ, Kim KS, Cha HJ, Park KS. Zap70 functions to maintain stemness of mouse embryonic stem cells by negatively regulating Jak1/Stat3/c-Myc signaling. Stem Cells 2010; 28:1476-86. [PMID: 20641039 DOI: 10.1002/stem.470] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Zeta-chain-associated protein kinase-70 (Zap70), a Syk family tyrosine kinase, has been reported to be present exclusively in normal T-cells, natural killer cells, and B cells, serving as a pivotal regulator of antigen-mediated receptor signaling and development. In this study, we report that Zap70 is expressed in undifferentiated mouse embryonic stem cells (mESCs) and may critically regulate self-renewal and pluripotency in mESCs. We found that Zap70 knocked-down mESCs (Zap70KD) show sustained self-renewal and defective differentiation. In addition, we present evidence that the sustained self-renewal in Zap70KD is associated with enhanced Jak/Stat3 signaling and c-Myc induction. These altered signaling appears to result from upregulated leukemia inhibitory factor receptor and downregulated src homology region 2 domain containing phosphatase 1 (SHP-1) phosphatase activity. On the basis of these results, we propose that in undifferentiated mESCs, Zap70 plays important roles in modulating the balance between self-renewal capacity and pluripotent differentiation ability as a key regulator of the Jak/Stat3/c-Myc signaling pathway.
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Affiliation(s)
- Young Cha
- Department of Biomedical Science, College of Life Science, CHA University, Pochon-si Gyeonggi-do, Korea
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Courtois ET, Castillo CG, Seiz EG, Ramos M, Bueno C, Liste I, Martínez-Serrano A. In vitro and in vivo enhanced generation of human A9 dopamine neurons from neural stem cells by Bcl-XL. J Biol Chem 2010; 285:9881-9897. [PMID: 20106970 DOI: 10.1074/jbc.m109.054312] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Human neural stem cells derived from the ventral mesencephalon (VM) are powerful research tools and candidates for cell therapies in Parkinson disease. Previous studies with VM dopaminergic neuron (DAn) precursors indicated poor growth potential and unstable phenotypical properties. Using the model cell line hVM1 (human ventral mesencephalic neural stem cell line 1; a new human fetal VM stem cell line), we have found that Bcl-X(L) enhances the generation of DAn from VM human neural stem cells. Mechanistically, Bcl-X(L) not only exerts the expected antiapoptotic effect but also induces proneural (NGN2 and NEUROD1) and dopamine-related transcription factors, resulting in a high yield of DAn with the correct phenotype of substantia nigra pars compacta (SNpc). The expression of key genes directly involved in VM/SNpc dopaminergic patterning, differentiation, and maturation (EN1, LMX1B, PITX3, NURR1, VMAT2, GIRK2, and dopamine transporter) is thus enhanced by Bcl-X(L). These effects on neurogenesis occur in parallel to a decrease in glia generation. These in vitro Bcl-X(L) effects are paralleled in vivo, after transplantation in hemiparkinsonian rats, where hVM1-Bcl-X(L) cells survive, integrate, and differentiate into DAn, alleviating behavioral motor asymmetry. Bcl-X(L) then allows for human fetal VM stem cells to stably generate mature SNpc DAn both in vitro and in vivo and is thus proposed as a helpful factor for the development of cell therapies for neurodegenerative conditions, Parkinson disease in particular.
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Affiliation(s)
- Elise T Courtois
- Center of Molecular Biology Severo Ochoa (Consejo Superior de Investigaciones Científicas-UAM), Department of Molecular Biology, Autonomous University of Madrid, 28049 Madrid, Spain
| | - Claudia G Castillo
- Center of Molecular Biology Severo Ochoa (Consejo Superior de Investigaciones Científicas-UAM), Department of Molecular Biology, Autonomous University of Madrid, 28049 Madrid, Spain; Department of Biochemistry, Faculty of Medicine, University of San Luis Potosí, 782 San Luis Potosí, México
| | - Emma G Seiz
- Center of Molecular Biology Severo Ochoa (Consejo Superior de Investigaciones Científicas-UAM), Department of Molecular Biology, Autonomous University of Madrid, 28049 Madrid, Spain
| | - Milagros Ramos
- Center of Molecular Biology Severo Ochoa (Consejo Superior de Investigaciones Científicas-UAM), Department of Molecular Biology, Autonomous University of Madrid, 28049 Madrid, Spain
| | - Carlos Bueno
- Institute of Neurosciences, University Miguel Hernandez of Elche, 03550 Alicante, Spain
| | - Isabel Liste
- Center of Molecular Biology Severo Ochoa (Consejo Superior de Investigaciones Científicas-UAM), Department of Molecular Biology, Autonomous University of Madrid, 28049 Madrid, Spain
| | - Alberto Martínez-Serrano
- Center of Molecular Biology Severo Ochoa (Consejo Superior de Investigaciones Científicas-UAM), Department of Molecular Biology, Autonomous University of Madrid, 28049 Madrid, Spain.
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Type 3 inositol 1,4,5-trisphosphate receptor negatively regulates apoptosis during mouse embryonic stem cell differentiation. Cell Death Differ 2010; 17:1141-54. [DOI: 10.1038/cdd.2009.209] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
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Trouillas M, Saucourt C, Guillotin B, Gauthereau X, Ding L, Buchholz F, Doss MX, Sachinidis A, Hescheler J, Hummel O, Huebner N, Kolde R, Vilo J, Schulz H, Boeuf H. Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives. BMC Genomics 2009; 10:73. [PMID: 19203379 PMCID: PMC2674464 DOI: 10.1186/1471-2164-10-73] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2008] [Accepted: 02/09/2009] [Indexed: 12/29/2022] Open
Abstract
Background Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. Results By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. Conclusion We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.
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Siu MKY, Wong ESY, Chan HY, Ngan HYS, Chan KYK, Cheung ANY. Overexpression of NANOG in gestational trophoblastic diseases: effect on apoptosis, cell invasion, and clinical outcome. THE AMERICAN JOURNAL OF PATHOLOGY 2008; 173:1165-72. [PMID: 18772339 DOI: 10.2353/ajpath.2008.080288] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
Abstract
Gestational trophoblastic disease includes choriocarcinoma, a frankly malignant tumor, and hydatidiform mole (HM), which often leads to the development of persistent gestational trophoblastic neoplasia and requires chemotherapy. NANOG is an important transcription factor that is crucial for maintaining embryonic stem cell self-renewal and pluripotency. We postulated that NANOG is involved in the pathogenesis of gestational trophoblastic disease. In this study, significantly higher NANOG mRNA and protein expression levels, by quantitative PCR and immunoblotting, respectively, were demonstrated in HMs, particularly those that developed persistent disease, when compared with normal placentas. In addition, significantly increased nuclear NANOG immunoreactivity was found by immunohistochemistry in HMs (P < 0.001) and choriocarcinoma (P = 0.002). Higher NANOG expression levels were demonstrated in HMs that developed persistent disease, as compared with those that regressed (P = 0.025). Nuclear localization of NANOG was confirmed by confocal microscopy and immunoblotting in choriocarcinoma cell lines. There was a significant inverse correlation between NANOG immunoreactivity and apoptotic index assessed by M30 CytoDeath antibody (P = 0.012). After stable knockdown of NANOG in the choriocarcinoma cell line JEG-3 by an shRNA approach, increased apoptosis was observed in relation to with enhanced caspases and poly(ADP-ribose) polymerase activities. NANOG knockdown was also associated with decreased mobility and invasion of JEG-3 and down-regulation of matrix metalloproteases 2 and 9. These findings suggest that NANOG is involved in the pathogenesis and clinical progress of gestational trophoblastic disease, likely through its effect on apoptosis, cell migration, and invasion.
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Affiliation(s)
- Michelle K Y Siu
- Department of Pathology, The University of Hong Kong Special Administrative Region, China
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Naujok O, Francini F, Jörns A, Lenzen S. An efficient experimental strategy for mouse embryonic stem cell differentiation and separation of a cytokeratin-19-positive population of insulin-producing cells. Cell Prolif 2008; 41:607-24. [PMID: 18616698 DOI: 10.1111/j.1365-2184.2008.00541.x] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
OBJECTIVES Embryonic stem cells are a potential source for insulin-producing cells, but existing differentiation protocols are of limited efficiency. Here, the aim has been to develop a new one, which drives development of embryonic stem cells towards insulin-producing cells rather than to neuronal cell types, and to combine this with a strategy for their separation from insulin-negative cells. MATERIALS AND METHODS The cytokeratin-19 (CK19) promoter was used to control the expression of enhanced yellow fluorescence protein in mouse embryonic stem cells during their differentiation towards insulin-producing cells, using a new optimized four-stage protocol. Two cell populations, CK19(+) and CK19(-) cells, were successfully fluorescence sorted and analysed. RESULTS The new method reduced neuronal progeny and suppressed differentiation into glucagon- and somatostatin-producing cells. Concomitantly, beta-cell like characteristics of insulin-producing cells were strengthened, as documented by high gene expression of the Glut2 glucose transporter and the transcription factor Pdx1. This novel protocol was combined with a cell-sorting technique. Through the combined procedure, a fraction of glucose-responsive insulin-secreting CK19(+) cells was obtained with 40-fold higher insulin gene expression and 50-fold higher insulin content than CK19(-) cells. CK19(+) cells were immunoreactive for C-peptide and had ultrastructural characteristics of an insulin-secretory cell. CONCLUSION Differentiated CK19(+) cells reflect an endocrine precursor cell type of ductal origin, potentially suitable for insulin replacement therapy in diabetes.
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Affiliation(s)
- O Naujok
- Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany
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Trouillas M, Saucourt C, Duval D, Gauthereau X, Thibault C, Dembele D, Feraud O, Menager J, Rallu M, Pradier L, Boeuf H. Bcl2, a transcriptional target of p38alpha, is critical for neuronal commitment of mouse embryonic stem cells. Cell Death Differ 2008; 15:1450-9. [PMID: 18437159 DOI: 10.1038/cdd.2008.63] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF) cytokine. LIF starvation leads to cell commitment, and part of the ES-derived differentiated cells die by apoptosis together with caspase3-cleavage and p38alpha activation. Inhibition of p38 activity by chemical compounds (PD169316 and SB203580), along with LIF withdrawal, leads to different outcomes on cell apoptosis, giving the opportunity to study the influence of apoptosis on cell differentiation. By gene profiling studies on ES-derived differentiated cells treated or not with these inhibitors, we have characterized the common and specific set of genes modulated by each inhibitor. We have also identified key genes that might account for their different survival effects. In addition, we have demonstrated that some genes, similarly regulated by both inhibitors (upregulated as Bcl2, Id2, Cd24a or downregulated as Nodal), are bona fide p38alpha targets involved in neurogenesis and found a correlation with their expression profiles and the onset of neuronal differentiation triggered upon retinoic acid treatment. We also showed, in an embryoid body differentiation protocol, that overexpression of EGFP (enhanced green fluorescent protein)-BCL2 fusion protein and repression of p38alpha are essential to increase formation of TUJ1-positive neuronal cell networks along with an increase in Map2-expressing cells.
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Aksoy I, Sakabedoyan C, Bourillot PY, Malashicheva AB, Mancip J, Knoblauch K, Afanassieff M, Savatier P. Self-renewal of murine embryonic stem cells is supported by the serine/threonine kinases Pim-1 and Pim-3. Stem Cells 2007; 25:2996-3004. [PMID: 17717068 DOI: 10.1634/stemcells.2007-0066] [Citation(s) in RCA: 60] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
pim-1 and pim-3 encode serine/threonine kinases involved in the regulation of cell proliferation and apoptosis in response to cytokine stimulation. We analyzed the regulation of pim-1 and pim-3 by the leukemia inhibitory factor (LIF)/gp130/signal transducer and activator of transcription-3 (STAT3) pathway and the role of Pim-1 and Pim-3 kinases in mouse embryonic stem (ES) cell self-renewal. Making use of ES cells expressing a granulocyte colony-stimulating factor:gp130 chimeric receptor and a hormone-dependent signal transducer and activator of transcription-3 estrogen receptor (STAT3-ER(T2)), we showed that expression of pim-1 and pim-3 was upregulated by LIF/gp130-dependent signaling and the STAT3 transcription factor. ES cells overexpressing pim-1 and pim-3 had a greater capacity to self-renew and displayed a greater resistance to LIF starvation based on a clonal assay. In contrast, knockdown of pim-1 and pim-3 increased the rate of spontaneous differentiation in a self-renewal assay. Knockdown of pim-1 and pim-3 was also detrimental to the growth of undifferentiated ES cell colonies and increased the rate of apoptosis. These findings provide a novel role of Pim-1 and Pim-3 kinases in the control of self-renewal of ES cells. Disclosure of potential conflicts of interest is found at the end of this article.
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Affiliation(s)
- Irène Aksoy
- Institut National de la Santé et de la Recherche Médicale INSERM U846, 18 avenue Doyen Lépine, 69500 Bron, France
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Cervera RP, Stojkovic M. Human embryonic stem cell derivation and nuclear transfer: impact on regenerative therapeutics and drug discovery. Clin Pharmacol Ther 2007; 82:310-5. [PMID: 17597709 DOI: 10.1038/sj.clpt.6100278] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
This review aims to introduce current and future uses of human embryonic stem cells derived from in vitro-fertilized embryos. These and stem cells derived from parthenogenetic and nuclear transfer embryos could be used for cell therapy, as in vitro cell models for drug discovery/screening, and for studying early human development and pathogenesis of human diseases. However, development of therapeutic and screening applications and products from embryonic stem cells is hampered by several barriers. Therefore, gaps in our current understanding of the basic science of stem cells need to be filled before either application can move forward with confidence.
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Affiliation(s)
- R P Cervera
- Cellular Reprogramming Laboratory, Centro de Investigación Príncipe Felipe, Valencia, Spain
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Sumi T, Tsuneyoshi N, Nakatsuji N, Suemori H. Apoptosis and differentiation of human embryonic stem cells induced by sustained activation of c-Myc. Oncogene 2007; 26:5564-76. [PMID: 17369859 DOI: 10.1038/sj.onc.1210353] [Citation(s) in RCA: 63] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Embryonic stem (ES) cells are self-renewing, pluripotent cell lines, characterized by their potential to differentiate into all cell types. The proto-oncogene product c-Myc has a crucial role in the self-renewal of mouse ES (mES) cells, but its role in human ES (hES) cells is unknown. To investigate c-Myc functions in hES cells, we expressed an inducible c-Myc fused to the hormone-binding domain of the estrogen receptor (c-MycER) protein that is activated by 4-hydroxy-tamoxifen. In contrast to its role in mES cells, activation of c-MycER in hES cells induced apoptosis and differentiation into extraembryonic endoderm and trophectoderm lineages concomitant with reduced expression of the pluripotent markers Oct4 and Nanog. Neither inhibition of caspase activity nor knockdown of p53 by RNA interference impaired the induction of differentiation markers induced by c-Myc activation. In addition, differentiation induced by c-Myc activation was associated with downregulation of alpha6 integrin expression, suggesting an important role for the integrin/extracellular matrix interaction in the regulation of ES cell behavior. None of these effects occurred with deletion of the c-Myc transactivation domain, indicating that c-Myc promotes both apoptosis and differentiation in a transcriptional activity-dependent manner. Together, our results provide new insights into the c-Myc functions regulating hES cell fate.
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Affiliation(s)
- T Sumi
- Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
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Mantel C, Guo Y, Lee MR, Kim MK, Han MK, Shibayama H, Fukuda S, Yoder MC, Pelus LM, Kim KS, Broxmeyer HE. Checkpoint-apoptosis uncoupling in human and mouse embryonic stem cells: a source of karyotpic instability. Blood 2007; 109:4518-27. [PMID: 17289813 PMCID: PMC1885509 DOI: 10.1182/blood-2006-10-054247] [Citation(s) in RCA: 100] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Karyotypic abnormalities in cultured embryonic stem cells (ESCs), especially near-diploid aneuploidy, are potential obstacles to ESC use in regenerative medicine. Events causing chromosomal abnormalities in ESCs may be related to events in tumor cells causing chromosomal instability (CIN) in human disease. However, the underlying mechanisms are unknown. Using multiparametric permeabilized-cell flow cytometric analysis, we found that the mitotic-spindle checkpoint, which helps maintain chromosomal integrity during all cell divisions, functions in human and mouse ESCs, but does not initiate apoptosis as it does in somatic cells. This allows an unusual tolerance to polyploidy resulting from failed mitosis, which is common in rapidly proliferating cell populations and which is reduced to near-diploid aneuploidy, which is also common in human neoplastic disease. Checkpoint activation in ESC-derived early-differentiated cells results in robust apoptosis without polyploidy/aneuploidy similar to that in somatic cells. Thus, the spindle checkpoint is "uncoupled" from apoptosis in ESCs and is a likely source of karyotypic abnormalities. This natural behavior of ESCs to tolerate/survive varying degrees of ploidy change could complicate genome-reprogramming studies and stem-cell plasticity studies, but could also reveal clues about the mechanisms of CIN in human tumors.
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Affiliation(s)
- Charlie Mantel
- Department of Microbiology & Immunology and the Walther Oncology Center, Indiana University School of Medicine, and the Walther Cancer Institute, Indianapolis, IN 46202, USA.
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Babaie Y, Herwig R, Greber B, Brink TC, Wruck W, Groth D, Lehrach H, Burdon T, Adjaye J. Analysis of Oct4-dependent transcriptional networks regulating self-renewal and pluripotency in human embryonic stem cells. Stem Cells 2006; 25:500-10. [PMID: 17068183 DOI: 10.1634/stemcells.2006-0426] [Citation(s) in RCA: 260] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The POU domain transcription factor OCT4 is a key regulator of pluripotency in the early mammalian embryo and is highly expressed in the inner cell mass of the blastocyst. Consistent with its essential role in maintaining pluripotency, Oct4 expression is rapidly downregulated during formation of the trophoblast lineage. To enhance our understanding of the molecular basis of this differentiation event in humans, we used a functional genomics approach involving RNA interference-mediated suppression of OCT4 function in a human ESC line and analysis of the resulting transcriptional profiles to identify OCT4-dependent genes in human cells. We detected altered expression of >1,000 genes, including targets regulated directly by OCT4 either positively (NANOG, SOX2, REX1, LEFTB, LEFTA/EBAF DPPA4, THY1, and TDGF1) or negatively (CDX2, EOMES, BMP4, TBX18, Brachyury [T], DKK1, HLX1, GATA6, ID2, and DLX5), as well as targets for the OCT4-associated stem cell regulators SOX2 and NANOG. Our data set includes regulators of ACTIVIN, BMP, fibroblast growth factor, and WNT signaling. These pathways are implicated in regulating human ESC differentiation and therefore further validate the results of our analysis. In addition, we identified a number of differentially expressed genes that are involved in epigenetics, chromatin remodeling, apoptosis, and metabolism that may point to underlying molecular mechanisms that regulate pluripotency and trophoblast differentiation in humans. Significant concordance between this data set and previous comparisons between inner cell mass and trophectoderm in human embryos indicates that the study of human ESC differentiation in vitro represents a useful model of early embryonic differentiation in humans.
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Affiliation(s)
- Yasmin Babaie
- Roslin Institute, Department of Gene Function and Development, Roslin, Midlothian, United Kingdom
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