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2016 | 63 | 4 | 709-716
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Selected small molecules as inducers of pluripotency

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The general idea of regenerative medicine is to fix or replace tissues or organs with live and patient-specific implants. Pluripotent stem cells are capable of indefinite self-renewal and differentiation into all cell types of the body. An easily accessible source of induced pluripotent stem cells (iPSCs) may allow obtaining and culturing tissues in vitro. Many approaches in the methods leading to obtain iPSCs have been tested in order to limit immunogenicity and tumorigenesis, and to increase efficiency. One of the approaches causing pluripotency is usage of small molecule compounds. It would be of great importance to assess their specific properties and reveal their new capacity to induce pluripotent stem cells and to improve reprogramming efficiency. Identification of the epigenetic changes during cellular reprogramming will extend our understanding of stem cell biology and many therapeutic applications. In this paper we discuss mainly the nucleotide derivatives, already proven or for now only putative inducers of the cells' pluripotency, that modulate the epigenetic status of the cell.
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  • Department of Epigenetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland
  • Department of Chemical Biology, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland
  • Department of Epigenetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznań, Poland
  • Andersen JB, Factor VM, Marquardt JU, Raggi C, Lee YH, Seo D, Conner EA, Thorgeirsson SS (2010) An integrated genomic and epigenomic approach predicts therapeutic response to zebularine in human liver cancer. Sci Transl Med 2: 54ra77.
  • Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, Zhang Y, Yang W, Gruber PJ, Epstein JA, Morrisey EE (2011) Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8: 376-388.
  • Baum C, von Kalle C, Staal FJ, Li Z, Fehse B, Schmidt M, Weerkamp F, Karlsson S, Wagemaker G, Williams DA (2004) Chance or necessity? Insertional mutagenesis in gene therapy and its consequences. Mol Ther 9: 5-13.
  • Bhutani K, Nazor KL, Williams R, Tran H, Dai H, Džakula Ž, Cho EH, Pang AW, Rao M, Cao H, Schork NJ, Loring JF (2016) Whole-genome mutational burden analysis of three pluripotency induction methods. Nat Commun 7: 10536.
  • Bray M, Driscoll J, Huggins JW (2000) Treatment of lethal Ebola virus infection in mice with a single dose of an S-adenosyl-L-homocysteine hydrolase inhibitor. Antiviral Res 45: 135-147. 10.1016/S0166-3542(00)00066-8.
  • Brevini TA, Pennarossa G, Rahman MM, Paffoni A, Antonini S, Ragni G, Deeguileor M, Tettamanti G, Gandolfi F (2014) Morphological and molecular changes of human granulosa cells exposed to 5-azacytidine and addressed toward muscular differentiation. Stem Cell Rev 10: 633-642.
  • Brivanlou AH, Gage FH, Jaenisch R, Jessell T, Melton D, Rossant J (2003) Stem cells. Setting standards for human embryonic stem cells. Science 300: 913-916.
  • Buta C, David R, Dressel R, Emgård M, Fuchs C, Gross U, Healy L, Hescheler J, Kolar R, Martin U, Mikkers H, Müller FJ, Schneider RK, Seiler AE, Spielmann H, Weitzer G (2013) Reconsidering pluripotency tests: do we still need teratoma assays? Stem Cell Res 11: 552-562. Epub 2013 Mar 26.
  • Chen L, MacMillan AM, Chang W, Ezaz-Nikpay K, Lane WS, Verdine GL (1991) Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase. Biochemistry 30: 11018-11025.
  • Cheng JC, Matsen CB, Gonzales FA, Ye W, Greer S, Marquez VE, Jones PA, Selker EU (2003) Inhibition of DNA methylation and reactivation of silenced genes by zebularine. J Natl Cancer Inst 95: 399-409.
  • Cheng JC, Weisenberger DJ, Gonzales FA, Liang G, Xu GL, Hu YG, Marquez VE, Jones PA (2004) Continuous zebularine treatment effectively sustains demethylation in human bladder cancer cells. Mol Cell Biol 24: 1270-1278.
  • Chiang PK (1998) Biological effects of inhibitors of S-adenosylhomocysteine hydrolase. Pharmacol Ther 77: 115-134.
  • Chin MH, Mason J, Xie W, Volinia S, Singer M, Peterson C, Ambartsumyan G, Aimiuwu O, Richter L, Zhang J, Khvorostov I, Ott V, Grunstein M, Lavon N, Benvenisty N, Croce CM, Clark AT, Baxter T, Pyle AD, Teitell MA, Pelegrini M, Plath K, Lowry WE (2009) Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. Cell Stem Cell 5: 111-123.
  • Dougherty TJ, Pucci MJ, Macielag M (2012) Chemical properties of antimicrobials and their uniqueness. Antibiotic Discovery and Development pp 793−820. Springer: New York.
  • Durupt F, Koppers-Lalic D, Balme B, Budel L, Terrier O, Lina B, Thomas L, Hoeben RC, Rosa-Calatrava M (2012) The chicken chorioallantoic membrane tumor assay as model for qualitative testing of oncolytic adenoviruses. Cancer Gene Ther 19: 58-68.
  • Efe JA, Ding S (2011) The evolving biology of small molecules: controlling cell fate and identity. Philos Trans R Soc Lond B Biol Sci 366: 2208-2221.
  • Elliott MJ, De Coppi P, Speggiorin S, Roebuck D, Butler CR, Samuel E, Crowley C, McLaren C, Fierens A, Vondrys D, Cochrane L, Jephson C, Janes S, Beaumont NJ, Cogan T, Bader A, Seifalian AM, Hsuan JJ, Lowdell MW, Birchall MA (2012) Stem-cell-based, tissue engineered tracheal replacement in a child: a 2-year follow-up study. Lancet 380: 994-1000.
  • Fandy TE (2009) Development of DNA methyltransferase inhibitors for the treatment of neoplastic diseases. Curr Med Chem 16: 2075-2085.
  • Fong H, Hohenstein KA, Donovan PJ (2008) Regulation of self-renewal and pluripotency by Sox2 in human embryonic stemcells. Stem Cells 26: 1931-1938.
  • Frye SV (2010) The art of the chemical probe. Nat Chem Biol 6: 159-161.
  • Gertow K, Przyborski S, Loring JF, Auerbach JM, Epifano O, Otonkoski T, Damjanov I, Ahrlund-Richter L (2007) Isolation of human embryonic stem cell-derived teratomas for the assessment of pluripotency. Curr Protoc Stem Cell Biol Chapter 1: Unit 1B.4.
  • Gordon RK, Ginalski K, Rudnicki WR, Rychlewski L, Pankaskie MC, Bujnicki JM, Chiang PK (2003) Anti-HIV-1 activity of 3-deaza-adenosine analogs. Inhibition of S-adenosylhomocysteine hydrolase and nucleotide congeners. Eur J Biochem 270: 3507-3517.
  • Hagedorn M, Javerzat S, Gilges D, Meyre A, de Lafarge B, Eichmann A, Bikfalvi A (2005) Accessing key steps of human tumor progression in vivo by using an avian embryo model. Proc Natl Acad Sci USA 102: 1643-1648.
  • Hanna J, Saha K, Pando B, van Zon J, Lengner CJ, Creyghton MP, van Oudenaarden A, Jaenisch R (2009) Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462: 595-601.
  • Hentze H, Soong PL, Wang ST, Phillips BW, Putti TC, Dunn NR (2009) Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies. Stem Cell Res 2: 198-210.
  • Hoebaus J, Heher P, Gottschamel T, Scheinast M, Auner H, Walder D, Wiedner M, Taubenschmid J, Miksch M, Sauer T, Schultheis M, Kuzmenkin A, Seiser C, Hescheler J, Weitzer G (2013) Embryonic stem cells facilitate the isolation of persistent clonal cardiovascular progenitor cell lines and leukemia inhibitor factor maintains their self-renewal and myocardial differentiation potential in vitro. Cells Tissues Organs 197: 249-268.
  • Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, Zhao T, Ye J, Yang W, Liu K, Ge J, Xu J, Zhang Q, Zhao Y, Deng H (2013) Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 341: 651-654.
  • Huangfu D, Maehr R, Guo W, Eijkelenboom A, Snitow M, Chen AE, Melton DA (2008a) Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nat Biotechnol 26: 795−797.
  • Huangfu D, Osafune K, Maehr R, Guo W, Eijkelenboom A, Chen S, Muhlestein W, Melton DA (2008b) Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol 26: 1269-1275.
  • Hurd PJ, Whitmarsh AJ, Baldwin GS, Kelly SM, Waltho JP, Price NC, Connolly BA, Hornby DP (1999) Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone. J Mol Biol 286: 389-401.
  • Jaenisch R (2012) Nuclear cloning and direct reprogramming: the long and the short path to Stockholm. Cell Stem Cell 11: 744-747.
  • Jia F, Wilson KD, Sun N, Gupta DM, Huang M, Li Z, Panetta NJ, Chen ZY, Robbins RC, Kay MA, Longaker MT, Wu JC (2010) A nonviral minicircle vector for deriving human iPS cells. Nat Methods 7: 197-199.
  • Jiang X, Lim CZ, Li Z, Lee PL, Yatim SM, Guan P, Li J, Zhou J, Pan J, Chng WJ, Chai CL, Yu Q (2015) Functional characterization of D9, a novel deazaneplanocin A (DZNep) analog, in targeting acute myeloid leukemia (AML). PLoS One 10: e0122983.
  • Jung DW, Kim WH, Williams DR (2014) Reprogram or reboot: small molecule approaches for the production of induced pluripotent stem cells and direct cell reprogramming. ACS Chem Biol 9: 80-95.
  • Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K (2009) Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature 458: 771-775.
  • Kim D, Kim CH, Moon JI, Chung YG, Chang MY, Han BS, Ko S, Yang E, Cha KY, Lanza R, Kim KS (2009) Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 4: 472-476.
  • Kim HO, Yoo SJ, Ahn HS, Choi WJ, Moon HR, Lee KM, Chun MW, Jeong LS (2004) Synthesis of fluorinated cyclopentenyladenine as potent inhibitor of S-adenosylhomocysteine hydrolase. Bioorg Med Chem Lett 14: 2091-2093.
  • Li LH, Olin EJ, Buskirk HH, Reineke LM (1970) Cytotoxicity and mode of action of 5-azacytidine on L1210 leukemia. Cancer Res 30: 2760-276.
  • Li Z, Yang CS, Nakashima K, Rana TM (2011) Small RNA-mediated regulation of generation iPS cell. EMBO J 30: 823-834.
  • Liao B, Bao X, Liu L, Feng S, Zovoilis A, Liu W, Xue Y, Cai J, Guo X, Qin B, Zhang R, Wu J, Lai L, Teng M, Niu L, Zhang B, Esteban MA, Pei D (2011) MicroRNA cluster 302-367 enhances somatic cell reprogramming by accelerating a mesenchymal-to-epithelial transition. J Biol Chem 286: 17359-17364.
  • Lipinski CA (2004) Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov Today Technol 1: 337-341. http://
  • Lyko F, Brown R (2005) DNA methyltransferase inhibitors and the development of epigenetic cancer therapies. J Natl Cancer Inst 97: 1498-1506.
  • Ma T, Xie M, Laurent T, Ding S (2013) Progress in the reprogramming of somatic cells. Circ Res 112: 562-574.
  • Maherali N, Hochedlinger K (2009) TGFbeta signal inhibition cooperates in the induction of iPSCs and replaces Sox2 and cMyc. Curr Biol 19: 1718-1723.
  • Mikkelsen TS, Hanna J, Zhang X, Ku M, Wernig M, Schorderet P, Bernstein BE, Jaenisch R, Lander ES, Meissner A (2008) Dissecting direct reprogramming through integrative genomic analysis. Nature 454: 49-55.
  • Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113: 631-642.
  • Miyoshi N, Ishii H, Nagano H, Haraguchi N, Dewi DL, Kano Y, Nishikawa S, Tanemura M, Mimori K, Tanaka F, Saito T, Nishimura J, Takemasa I, Mizushima T, Ikeda M, Yamamoto H, Sekimoto M, Doki Y, Mori M (2011) Reprogramming of mouse and human cells to pluripotency using mature microRNAs. Cell Stem Cell 8: 633-638.
  • Montgomery JA, Clayton SJ, Thomas HJ, Shannon WM, Arnett G, Bodner AJ, Kion IK, Cantoni GL, Chiang PK (1982) Carbocyclic analogue of 3-deazaadenosine: a novel antiviral agent using S-adenosylhomocysteine hydrolase as a pharmacological target. J Med Chem 25: 626-629.
  • Morange M (2002) The relations between genetics and epigenetics: a historical point of view. Ann N Y Acad Sci 981: 50-60.
  • Müller FJ, Laurent LC, Kostka D, Ulitsky I, Williams R, Lu C, Park IH, Rao MS, Shamir R, Schwartz PH, Schmidt NO, Loring JF (2008) Regulatory networks define phenotypic classes of human stem cell lines. Nature 455: 401-405.
  • Müller FJ, Schuldt BM, Williams R, Mason D, Altun G, Papapetrou E, Danner S, Goldman JE, Herbst A, Schmidt NO, Aldenhoff JB, Laurent LC, Loring JF (2011) A bioinformatic assay for pluripotency in human cells. Nat Methods 8: 315-317.
  • Mykhaylyk O, Sánchez-Antequera Y, Vlaskou D, Hammerschmid E, Anton M, Zelphati O, Plank C (2010) Liposomal magnetofection. Methods Mol Biol 605: 487-525.
  • Nakamura K, Aizawa K, Nakabayashi K, Kato N, Yamauchi J, Hata K, Tanoue A (2013) DNA methyltransferase inhibitor zebularine inhibits human hepatic carcinoma cells proliferation and induces apoptosis. PLoS ONE 8: e54036.
  • Nakamura K, Nakabayashi K, Htet Aung K, Aizawa K, Hori N, Yamauchi J, Hata K, Tanoue A (2015) DNA Methyltransferase inhibitor zebularine induces human cholangiocarcinoma cell death through alteration of DNA methylation status. PLoS ONE 10: e0120545.
  • Nguyen AT, Zhang Y (2011) The diverse functions of Dot1 and H3K79 methylation.Genes Dev 25: 1345-1358.
  • Nie B, Wang H, Laurent T, Ding S (2012) Cellular reprogramming: a small molecule perspective. Curr Opin Cell Biol 24: 784-792.
  • Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322: 949-953.
  • Onder TT, Kara N, Cherry A, Sinha AU, Zhu N, Bernt KM, Cahan P, Marcarci BO, Unternaehrer J, Gupta PB, Lander ES, Armstrong SA, Daley GQ (2012) Chromatin modifying enzymes as modulators of reprogramming. Nature 483: 598-602.
  • Ott HC, Taylor DA (2006) From cardiac repair to cardiac regeneration - ready to translate? Expert Opin Biol Ther 6: 867-878.
  • Park HY, Noh EH, Chung HM, Kang MJ, Kim EY, Park SP (2012) Efficient generation of virus-free iPS cells using liposomal magnetofection. PLoS One 7: e45812.
  • Paull D, Sevilla A, Zhou H, Hahn AK, Kim H, Napolitano C, Tsankov A, Shang L, Krumholz K, Jagadeesan P, Woodard CM, Sun B, Vilboux T, Zimmer M, Forero E, Moroziewicz DN, Martinez H, Malicdan MC, Weiss KA, Vensand LB, Dusenberry CR, Polus H, Sy KT, Kahler DJ, Gahl WA, Solomon SL, Chang S, Meissner A, Eggan K, Noggle SA (2015) Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells. Nat Methods 12: 885-892.
  • Pennarossa G, Maffei S, Campagnol M, Tarantini L, Gandolfi F, Brevini TA (2013) Brief demethylation step allows the conversion of adult human skin fibroblasts into insulin-secreting cells. Proc Natl Acad Sci USA 110: 8948-8953.
  • Pesce M, Scholer HR (2001) Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19: 271-278.
  • Polo JM, Anderssen E, Walsh RM, Schwarz BA, Nefzger CM, Lim SM, Borkent M, Apostolou E, Alaei S, Cloutier J, Bar-Nur O, Cheloufi S, Stadtfeld M, Figueroa ME, Robinton D, Natesan S, Melnick A, Zhu J, Ramaswamy S, Hochedlinger K (2012) A molecular roadmap of reprogramming somatic cells into iPS cells. Cell 151: 1617-1632.
  • Santi DV, Norment A, Garrett CE (1984) Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc Natl Acad Sci USA 81: 6993-6997.
  • Saunders PP, Tan MT, Robins RK (1985) Metabolism and action of neplanocin A in Chinese hamster ovary cells. Biochem Pharmacol 34: 2749-2754.
  • Shyh-Chang N, Locasale JW, Lyssiotis CA, Zheng Y, Teo RY, Ratanasirintrawoot S, Zhang J, Onder T, Unternaehrer JJ, Zhu H, Asara JM, Daley GQ, Cantley LC (2013) Influence of threonine metabolism on S-adenosylmethionine and histone methylation. Science 339: 222-226.
  • Soldner F, Hockemeyer D, Beard C, Gao Q, Bell GW, Cook EG, Hargus G, Blak A, Cooper O, Mitalipova M, Isacson O, Jaenisch R (2009) Parkinson's disease patient-derived induced pluripotent stem cells free of viral reprogramming factors. Cell 136: 964-977.
  • Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K (2008) Induced pluripotent stem cells generated without viral integration. Science 322: 945-949.
  • Stresemann C, Lyko F (2008) Modes of action of the DNA methyltransferase inhibitors azacytidine and decitabine. Int J Cancer 123: 8-13.
  • Subramanyam D, Lamouille S, Judson RL, Liu JY, Bucay N, Derynck R, Blelloch R (2011) Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nat Biotechnol 29: 443-448.
  • Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676.
  • Tam EK, Nguyen TM, Lim CZ, Lee PL, Li Z, Jiang X, Santhanakrishnan S, Tan TW, Goh YL, Wong SY, Yang H, Ong EH, Hill J, Yu Q, Chai CL (2015) 3-Deazaneplanocin A and neplanocin A analogues and their effects on apoptotic cell death. Chem Med Chem 10: 173-182.
  • Tamada H, Van Thuan N, Reed P, Nelson D, Katoku-Kikyo N, Wudel J, Wakayama T, Kikyo N (2006) Chromatin decondensation and nuclear reprogramming by nucleoplasmin. Mol Cell Biol 26: 1259--1271.
  • Tan J, Yang X, Zhuang L, Jiang X, Chen W, Lee PL, Karuturi RK, Tan PB, Liu ET, Yu Q (2007) Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells. Genes Dev 21: 1050-1063.
  • Tang S, Xie M, Cao N, Ding S (2016) Patient-specific induced pluripotent stem cells for disease modeling and phenotypic drug discovery. J Med Chem 59: 2-15.
  • Taubenschmid J, Weitzer G (2012) Mechanisms of cardiogenesis in cardiovascular progenitor cells. Int Rev Cell Mol Biol 293: 195-267.
  • Taylor SM, Jones PA (1979) Multiple new phenotypes induced in 10T1/2 and 3T3 cells treated with 5-azacytidine. Cell 17: 771-779.
  • Taylor SM, Jones PA (1982) Mechanism of action of eukaryotic DNA methyltransferase. Use of 5-azacytosine-containing DNA. J Mol Biol 162: 679-692.
  • Wang Y, Fu Q, Zhao RY, Deng CL (2014) Muscular tubes of urethra engineered from adipose-derived stem cells and polyglycolic acid mesh in a bioreactor. Biotechnol Lett 36: 1909-1916.
  • Wesselschmidt RL (2011) The teratoma assay: an in vivo assessment of pluripotency. Methods Mol Biol 767: 231-241.
  • Williams R, Schuldt B, Müller FJ (2011) A guide to stem cell identification: progress and challenges in system-wide predictive testing with complex biomarkers. Bioessays 33: 880-890.
  • Wolfe MS, Borchardt RT (1991) S-Adenosyl-L-homocysteine hydrolase as a target for antiviral chemotherapy. J Med Chem 34: 1521-1530.
  • Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hamalainen R (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458: 766-770.
  • Xiong X, Lan D, Li J, Zhong J, Zi X, Ma L, Wang Y (2013) Zebularine and scriptaid significantly improve epigenetic reprogramming of yak fibroblasts and cloning efficiency. Cell Reprogram 15: 293-300.
  • Yoo CB, Cheng JC, Jones PA (2004) Zebularine: a new drug for epigenetic therapy. Biochem Soc Trans 32: 910-912.
  • You BR, Park WH (2012) Zebularine inhibits the growth of HeLa cervical cancer cells via cell cycle arrest and caspase-dependent apoptosis. Mol Biol Rep 39: 9723-9731.
  • Yu J, Hu K, Smuga-Otto K, Tian S, Stewart R, Slukvin II, Thomson JA (2009) Human induced pluripotent stem cells free of vector and transgene sequences. Science 324: 797-801.
  • Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318: 1917-1920.
  • Yusa K, Rad R, Takeda J, Bradley A (2009) Generation of transgene-free induced pluripotent mouse stem cells by the PiggyBac transposon. Nat Methods 6: 363-369.
  • Zhao T, Zhang ZN, Rong Z, Xu Y (2011) Immunogenicity of induced pluripotent stem cells. Nature 474: 212-215.
  • Zhou L, Cheng X, Connolly BA, Dickman MJ, Hurd PJ, Hornby DP (2002) Zebularine: a novel DNA methylation inhibitor that forms a covalent complex with DNA methyltransferases. J Mol Biol 321: 591-599.
  • Zhou W, Freed CR (2009) Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells 27: 2667-2674.
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