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Volumn 5, Issue 8, 2010, Pages

A combined epigenetic and non-genetic approach for reprogramming human somatic cells

Author keywords

[No Author keywords available]

Indexed keywords

BETA ACTIN; LAMIN A; LAMIN C; OCTAMER TRANSCRIPTION FACTOR 4; PROTEIN P21; PROTEIN P53; TRANSCRIPTION FACTOR NANOG; BIOLOGICAL MARKER; CDKN1A PROTEIN, HUMAN; CYCLIN DEPENDENT KINASE INHIBITOR 1A; DNA (CYTOSINE 5) METHYLTRANSFERASE; DOPAMINE; HISTONE DEACETYLASE INHIBITOR; HOMEODOMAIN PROTEIN; NANOG PROTEIN, HUMAN; POU5F1 PROTEIN, HUMAN;

EID: 77957895936     PISSN: None     EISSN: 19326203     Source Type: Journal    
DOI: 10.1371/journal.pone.0012297     Document Type: Article
Times cited : (45)

References (50)
  • 1
    • 36549033276 scopus 로고    scopus 로고
    • Producing primate embryonic stem cells by somatic cell nuclear transfer
    • Byrne JA, Pedersen DA, Clepper LL, Nelson M, Sanger WG, et al. (2007) Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 450: 497-502.
    • (2007) Nature , vol.450 , pp. 497-502
    • Byrne, J.A.1    Pedersen, D.A.2    Clepper, L.L.3    Nelson, M.4    Sanger, W.G.5
  • 2
    • 36248966518 scopus 로고    scopus 로고
    • Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors
    • Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, et al. (2007) Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. Cell 131: 861-872.
    • (2007) Cell , vol.131 , pp. 861-872
    • Takahashi, K.1    Tanabe, K.2    Ohnuki, M.3    Narita, M.4    Ichisaka, T.5
  • 3
    • 23944457397 scopus 로고    scopus 로고
    • Nuclear Reprogramming of Somatic Cells After Fusion with Human Embryonic Stem Cells
    • Cowan CA, Atienza J, Melton DA, Eggan K (2005) Nuclear Reprogramming of Somatic Cells After Fusion with Human Embryonic Stem Cells. Science 309: 1369-1373.
    • (2005) Science , vol.309 , pp. 1369-1373
    • Cowan, C.A.1    Atienza, J.2    Melton, D.A.3    Eggan, K.4
  • 4
    • 28644443829 scopus 로고    scopus 로고
    • Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells
    • Taranger CK, Noer A, Sørensen AL, Håkelien AM, Boquest AC, et al. (2005) Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells. Molecular Biology of the Cell 16: 5719-5735.
    • (2005) Molecular Biology of the Cell , vol.16 , pp. 5719-5735
    • Taranger, C.K.1    Noer, A.2    Sørensen, A.L.3    Håkelien, A.M.4    Boquest, A.C.5
  • 5
    • 47749105737 scopus 로고    scopus 로고
    • Current concepts in reprogramming somatic cells to pluripotent state
    • Han J, Sidhu KS (2008) Current concepts in reprogramming somatic cells to pluripotent state. Current Stem Cell Research and Therapy 3: 66-74.
    • (2008) Current Stem Cell Research and Therapy , vol.3 , pp. 66-74
    • Han, J.1    Sidhu, K.S.2
  • 7
    • 4744372577 scopus 로고    scopus 로고
    • Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells
    • Jackson M, Krassowska A, Gilbert N, Chevassut T, Forrester L, et al. (2004) Severe global DNA hypomethylation blocks differentiation and induces histone hyperacetylation in embryonic stem cells. Molecular and Cellular Biology 24: 8862-8871.
    • (2004) Molecular and Cellular Biology , vol.24 , pp. 8862-8871
    • Jackson, M.1    Krassowska, A.2    Gilbert, N.3    Chevassut, T.4    Forrester, L.5
  • 10
    • 2942718698 scopus 로고    scopus 로고
    • Histone Code Modifications on Pluripotential Nuclei of Reprogrammed Somatic Cells
    • Kimura H, Tada M, Nakatsuji N, Tada T (2004) Histone Code Modifications on Pluripotential Nuclei of Reprogrammed Somatic Cells. Mol Cell Biol 24: 5710-5720.
    • (2004) Mol Cell Biol , vol.24 , pp. 5710-5720
    • Kimura, H.1    Tada, M.2    Nakatsuji, N.3    Tada, T.4
  • 12
    • 19944430797 scopus 로고    scopus 로고
    • Genomic maps and comparative analysis of histone modifications in human and mouse
    • Bernstein BE, Kamal M, Lindblad-Toh K, Bekiranov S, Bailey DK, et al. (2005) Genomic maps and comparative analysis of histone modifications in human and mouse. Cell 120: 169-181.
    • (2005) Cell , vol.120 , pp. 169-181
    • Bernstein, B.E.1    Kamal, M.2    Lindblad-Toh, K.3    Bekiranov, S.4    Bailey, D.K.5
  • 13
    • 31544456085 scopus 로고    scopus 로고
    • The role of mammalian DNA methyltransferases in the regulation of gene expression
    • Turek-Plewa J, Jagodziński PP (2005) The role of mammalian DNA methyltransferases in the regulation of gene expression. Cellular and Molecular Biology Letters 10: 631-647.
    • (2005) Cellular and Molecular Biology Letters , vol.10 , pp. 631-647
    • Turek-Plewa, J.1    Jagodziński, P.P.2
  • 15
    • 0035937729 scopus 로고    scopus 로고
    • DNA Demethylation Reactivates a Subset of Imprinted Genes in Uniparental Mouse Embryonic Fibroblasts
    • El Kharroubi A, Piras G, Stewart CL (2001) DNA Demethylation Reactivates a Subset of Imprinted Genes in Uniparental Mouse Embryonic Fibroblasts. Journal of Biological Chemistry 276: 8674-8680.
    • (2001) Journal of Biological Chemistry , vol.276 , pp. 8674-8680
    • El Kharroubi, A.1    Piras, G.2    Stewart, C.L.3
  • 16
    • 0041861033 scopus 로고    scopus 로고
    • Epigenetic Characteristics and Development of Embryos Cloned from Donor Cells Treated by Trichostatin A or 5-aza-29-deoxycytidine
    • Enright BP, Kubota C, Yang X, Tian XC (2003) Epigenetic Characteristics and Development of Embryos Cloned from Donor Cells Treated by Trichostatin A or 5-aza-29-deoxycytidine. Biol Reprod 69: 896-901.
    • (2003) Biol Reprod , vol.69 , pp. 896-901
    • Enright, B.P.1    Kubota, C.2    Yang, X.3    Tian, X.C.4
  • 17
    • 42649130264 scopus 로고    scopus 로고
    • Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease
    • Wernig M, Zhao J-P, Pruszak J, Hedlund E, Fu D, et al. (2008) Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proceedings of the National Academy of Sciences 105: 5856-5861.
    • (2008) Proceedings of the National Academy of Sciences , vol.105 , pp. 5856-5861
    • Wernig, M.1    Zhao, J.-P.2    Pruszak, J.3    Hedlund, E.4    Fu, D.5
  • 18
    • 33748305875 scopus 로고    scopus 로고
    • Role of Histone Acetylation in Reprogramming of Somatic Nuclei Following Nuclear Transfer
    • Rybouchkin A, Kato Y, Tsunoda Y (2006) Role of Histone Acetylation in Reprogramming of Somatic Nuclei Following Nuclear Transfer. Biol Reprod 74: 1083-1089.
    • (2006) Biol Reprod , vol.74 , pp. 1083-1089
    • Rybouchkin, A.1    Kato, Y.2    Tsunoda, Y.3
  • 19
    • 46949085597 scopus 로고    scopus 로고
    • Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds
    • Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, et al. (2008) Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotech 26: 795-797.
    • (2008) Nat Biotech , vol.26 , pp. 795-797
    • Huangfu, D.1    Maehr, R.2    Guo, W.3    Eijkelenboom, A.4    Snitow, M.5
  • 20
    • 31544436888 scopus 로고    scopus 로고
    • Dietary HDAC inhibitors: Time to rethink weak ligands in cancer chemoprevention?
    • Dashwood RH, Myzak MC, Ho E (2006) Dietary HDAC inhibitors: time to rethink weak ligands in cancer chemoprevention? Carcinogenesis 27: 344-349.
    • (2006) Carcinogenesis , vol.27 , pp. 344-349
    • Dashwood, R.H.1    Myzak, M.C.2    Ho, E.3
  • 21
    • 33846783210 scopus 로고    scopus 로고
    • Targeting cellular memory to reprogram the epigenome, restore potential, and improve somatic cell nuclear transfer
    • Eilertsen KJ, Power RA, Harkins LL, Misica P (2007) Targeting cellular memory to reprogram the epigenome, restore potential, and improve somatic cell nuclear transfer. Animal Reproduction Science 98: 129-146.
    • (2007) Animal Reproduction Science , vol.98 , pp. 129-146
    • Eilertsen, K.J.1    Power, R.A.2    Harkins, L.L.3    Misica, P.4
  • 22
    • 48849110380 scopus 로고    scopus 로고
    • Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts
    • Bru T, Clarke C, McGrew MJ, Sang HM, Wilmut I, et al. (2008) Rapid induction of pluripotency genes after exposure of human somatic cells to mouse ES cell extracts. Experimental Cell Research August 15: 2634-2642.
    • (2008) Experimental Cell Research August , vol.15 , pp. 2634-2642
    • Bru, T.1    Clarke, C.2    McGrew, M.J.3    Sang, H.M.4    Wilmut, I.5
  • 23
    • 34248141152 scopus 로고    scopus 로고
    • Epigenetic reprogramming of OCT4 and NANOG regulatory regions by embryonal carcinoma cell extract
    • Freberg CT, Dahl JA, Timoskainen S, Collas P (2007) Epigenetic reprogramming of OCT4 and NANOG regulatory regions by embryonal carcinoma cell extract. Molecular Biology of the Cell 18: 1543-1553.
    • (2007) Molecular Biology of the Cell , vol.18 , pp. 1543-1553
    • Freberg, C.T.1    Dahl, J.A.2    Timoskainen, S.3    Collas, P.4
  • 24
    • 44949128225 scopus 로고    scopus 로고
    • Cell-Free Embryonic Stem Cell Extract-Mediated Derivation of Multipotent Stem Cells From NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair
    • Rajasingh J, Lambers E, Hamada H, Bord E, Thorne T, et al. (2008) Cell-Free Embryonic Stem Cell Extract-Mediated Derivation of Multipotent Stem Cells From NIH3T3 Fibroblasts for Functional and Anatomical Ischemic Tissue Repair. Circ Res 102: e107-117.
    • (2008) Circ Res , vol.102
    • Rajasingh, J.1    Lambers, E.2    Hamada, H.3    Bord, E.4    Thorne, T.5
  • 25
    • 38049086046 scopus 로고    scopus 로고
    • Mouse fibroblasts are reprogrammed to Oct-4 and Rex-1 gene expression and alkaline phosphatase activity by embryonic stem cell extracts
    • Neri T, Monti M, Rebuzzini P, Merico V, Garagna S, et al. (2007) Mouse fibroblasts are reprogrammed to Oct-4 and Rex-1 gene expression and alkaline phosphatase activity by embryonic stem cell extracts. Cloning and stem cells 9: 394-406.
    • (2007) Cloning and Stem Cells , vol.9 , pp. 394-406
    • Neri, T.1    Monti, M.2    Rebuzzini, P.3    Merico, V.4    Garagna, S.5
  • 26
    • 69349100455 scopus 로고    scopus 로고
    • Linking the p53 tumour suppressor pathway to somatic cell reprogramming
    • Kawamura T, Suzuki J, Wang YV, Menendez S, Morera LB, et al. (2009) Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460: 1140-1144.
    • (2009) Nature , vol.460 , pp. 1140-1144
    • Kawamura, T.1    Suzuki, J.2    Wang, Y.V.3    Menendez, S.4    Morera, L.B.5
  • 27
    • 19544366589 scopus 로고    scopus 로고
    • Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm
    • Alberio R, Johnson AD, Stick R, Campbell KHS (2005) Differential nuclear remodeling of mammalian somatic cells by Xenopus laevis oocyte and egg cytoplasm. Experimental Cell Research 307: 131-141.
    • (2005) Experimental Cell Research , vol.307 , pp. 131-141
    • Alberio, R.1    Johnson, A.D.2    Stick, R.3    Campbell, K.H.S.4
  • 28
    • 66449109143 scopus 로고    scopus 로고
    • Epigenetic marks in somatic chromatin are remodelled to resemble pluripotent nuclei by amphibian oocyte extracts
    • Bian Y, Alberio R, Allegrucci C, Campbell KH, Johnson AD (2009) Epigenetic marks in somatic chromatin are remodelled to resemble pluripotent nuclei by amphibian oocyte extracts. Epigenetics 4: 194-202.
    • (2009) Epigenetics , vol.4 , pp. 194-202
    • Bian, Y.1    Alberio, R.2    Allegrucci, C.3    Campbell, K.H.4    Johnson, A.D.5
  • 29
    • 65949095201 scopus 로고    scopus 로고
    • Cell-free extracts from mammalian oocytes partially induce nuclear reprogramming in somatic cells
    • Miyamoto K, Tsukiyama T, Yang Y, Li N, Minami N, et al. (2009) Cell-free extracts from mammalian oocytes partially induce nuclear reprogramming in somatic cells. Biology of Reproduction 80: 935-943.
    • (2009) Biology of Reproduction , vol.80 , pp. 935-943
    • Miyamoto, K.1    Tsukiyama, T.2    Yang, Y.3    Li, N.4    Minami, N.5
  • 31
    • 33747195353 scopus 로고    scopus 로고
    • Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors
    • Takahashi K, Yamanaka S (2006) Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell 126: 663-676.
    • (2006) Cell , vol.126 , pp. 663-676
    • Takahashi, K.1    Yamanaka, S.2
  • 32
    • 0034028901 scopus 로고    scopus 로고
    • Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells
    • Niwa H, Miyazaki JI, Smith AG (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genetics 24: 372-376.
    • (2000) Nature Genetics , vol.24 , pp. 372-376
    • Niwa, H.1    Miyazaki, J.I.2    Smith, A.G.3
  • 34
    • 2342437315 scopus 로고    scopus 로고
    • Epigenetic Control of Mouse Oct-4 Gene Expression in Embryonic Stem Cells and Trophoblast Stem Cells
    • Hattori N, Nishino K, Ko Y-G, Hattori N, Ohgane J, et al. (2004) Epigenetic Control of Mouse Oct-4 Gene Expression in Embryonic Stem Cells and Trophoblast Stem Cells. J Biol Chem 279: 17063-17069.
    • (2004) J Biol Chem , vol.279 , pp. 17063-17069
    • Hattori, N.1    Nishino, K.2    Ko, Y.-G.3    Hattori, N.4    Ohgane, J.5
  • 35
    • 33847663874 scopus 로고    scopus 로고
    • Epigenetic regulation of Nanog gene in embryonic stem and trophoblast stem cells
    • Hattori N, Imao Y, Nishino K, Hattori N, Ohgane J, et al. (2007) Epigenetic regulation of Nanog gene in embryonic stem and trophoblast stem cells. Genes to Cells 12: 387-396.
    • (2007) Genes to Cells , vol.12 , pp. 387-396
    • Hattori, N.1    Imao, Y.2    Nishino, K.3    Hattori, N.4    Ohgane, J.5
  • 36
    • 48349122654 scopus 로고    scopus 로고
    • Pluripotency Associated Genes Are Reactivated by Chromatin- Modifying Agents in Neurosphere Cells
    • Ruau D, Ensenat-Waser R, Dinger TC, Vallabhapurapu DS, Rolletschek A, et al. (2008) Pluripotency Associated Genes Are Reactivated by Chromatin- Modifying Agents in Neurosphere Cells. Stem Cells 26: 920-926.
    • (2008) Stem Cells , vol.26 , pp. 920-926
    • Ruau, D.1    Ensenat-Waser, R.2    Dinger, T.C.3    Vallabhapurapu, D.S.4    Rolletschek, A.5
  • 37
    • 0034669082 scopus 로고    scopus 로고
    • Clonally Derived Human Embryonic Stem Cell Lines Maintain Pluripotency and Proliferative Potential for Prolonged Periods of Culture
    • Amit M, Carpenter MK, Inokuma MS, Chiu C-P, Harris CP, et al. (2000) Clonally Derived Human Embryonic Stem Cell Lines Maintain Pluripotency and Proliferative Potential for Prolonged Periods of Culture. Developmental Biology 227: 271-278.
    • (2000) Developmental Biology , vol.227 , pp. 271-278
    • Amit, M.1    Carpenter, M.K.2    Inokuma, M.S.3    Chiu, C.-P.4    Harris, C.P.5
  • 38
    • 77955494512 scopus 로고    scopus 로고
    • Production of mice using iPS cells and tetraploid complementation
    • Zhao X-Y, Lv Z, Li W, Zeng F, Zhou Q (2010) Production of mice using iPS cells and tetraploid complementation. Nat Protocols 5: 963-971.
    • (2010) Nat Protocols , vol.5 , pp. 963-971
    • Zhao, X.-Y.1    Lv, Z.2    Li, W.3    Zeng, F.4    Zhou, Q.5
  • 39
    • 3142655794 scopus 로고    scopus 로고
    • Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media
    • Cheng J, Dutra A, Takesono A, Garrett-Beal L, Schwartzberg PL (2004) Improved generation of C57BL/6J mouse embryonic stem cells in a defined serum-free media. Genesis 39: 100-104.
    • (2004) Genesis , vol.39 , pp. 100-104
    • Cheng, J.1    Dutra, A.2    Takesono, A.3    Garrett-Beal, L.4    Schwartzberg, P.L.5
  • 40
    • 69349100179 scopus 로고    scopus 로고
    • Suppression of induced pluripotent stem cell generation by the p53-p21 pathway
    • Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, et al. (2009) Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460: 1132-1135.
    • (2009) Nature , vol.460 , pp. 1132-1135
    • Hong, H.1    Takahashi, K.2    Ichisaka, T.3    Aoi, T.4    Kanagawa, O.5
  • 41
    • 69349103956 scopus 로고    scopus 로고
    • The Ink4/Arf locus is a barrier for iPS cell reprogramming
    • Li H, Collado M, Villasante A, Strati K, Ortega S, et al. (2009) The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460: 1136-1139.
    • (2009) Nature , vol.460 , pp. 1136-1139
    • Li, H.1    Collado, M.2    Villasante, A.3    Strati, K.4    Ortega, S.5
  • 42
    • 69349094006 scopus 로고    scopus 로고
    • A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity
    • Marion RM, Strati K, Li H, Murga M, Blanco R, et al. (2009) A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460: 1149-1153.
    • (2009) Nature , vol.460 , pp. 1149-1153
    • Marion, R.M.1    Strati, K.2    Li, H.3    Murga, M.4    Blanco, R.5
  • 43
    • 0035206383 scopus 로고    scopus 로고
    • Mechanisms of p53-dependent apoptosis
    • Schuler M, Green DR (2001) Mechanisms of p53-dependent apoptosis. Biochem Soc Trans 29: 684-688.
    • (2001) Biochem Soc Trans , vol.29 , pp. 684-688
    • Schuler, M.1    Green, D.R.2
  • 45
    • 57449115567 scopus 로고    scopus 로고
    • The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation
    • Li X, Meng G, Krawetz R, Liu S, Rancourt DE (2008) The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells and Development 17: 1079-1085.
    • (2008) Stem Cells and Development , vol.17 , pp. 1079-1085
    • Li, X.1    Meng, G.2    Krawetz, R.3    Liu, S.4    Rancourt, D.E.5
  • 46
    • 80055113510 scopus 로고    scopus 로고
    • Generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human TF lentivirus set
    • doi: 10.3791/1553
    • Wu D, Hamilton B, Martin C, Gao Y, Ye M, et al. (2009) Generation of induced pluripotent stem cells by reprogramming human fibroblasts with the stemgent human TF lentivirus set. Journal of visualized experiments: JoVE;doi: 10.3791/1553.
    • (2009) Journal of Visualized Experiments: JoVE
    • Wu, D.1    Hamilton, B.2    Martin, C.3    Gao, Y.4    Ye, M.5
  • 47
    • 70349206185 scopus 로고    scopus 로고
    • Induction of stem cell gene expression in adult human fibroblasts without transgenes
    • Page RL, Ambady S, Holmes WF, Vilner L, Kole D, et al. (2009) Induction of stem cell gene expression in adult human fibroblasts without transgenes. Cloning and Stem Cells 11: 417-426.
    • (2009) Cloning and Stem Cells , vol.11 , pp. 417-426
    • Page, R.L.1    Ambady, S.2    Holmes, W.F.3    Vilner, L.4    Kole, D.5
  • 48
    • 73349124768 scopus 로고    scopus 로고
    • Non cell-autonomous reprogramming of adult ocular progenitors: Generation of pluripotent stem cells without exogenous transcription factors
    • Balasubramanian S, Babai N, Chaudhuri A, Qiu F, Bhattacharya S, et al. (2009) Non Cell-Autonomous Reprogramming of Adult Ocular Progenitors: Generation of Pluripotent Stem Cells without Exogenous Transcription Factors. Stem Cells 27: 3053-3062.
    • (2009) Stem Cells , vol.27 , pp. 3053-3062
    • Balasubramanian, S.1    Babai, N.2    Chaudhuri, A.3    Qiu, F.4    Bhattacharya, S.5
  • 50
    • 54049144605 scopus 로고    scopus 로고
    • PA6- induced human embryonic stem cell-derived neurospheres: A new source of human peripheral sensory neurons and neural crest cells
    • Pomp O, Brokhman I, Ziegler L, Almog M, Korngreen A, et al. (2008) PA6- induced human embryonic stem cell-derived neurospheres: a new source of human peripheral sensory neurons and neural crest cells. Brain Research 1230: 50-60.
    • (2008) Brain Research , vol.1230 , pp. 50-60
    • Pomp, O.1    Brokhman, I.2    Ziegler, L.3    Almog, M.4    Korngreen, A.5


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