SpringerBriefs in Stem Cells For furthervolumes: http://www.springer.com/series/10206 Sibel Yildirim Induced Pluripotent Stem Cells 123 Sibel YildirimPhD Department of PediatricDentistry Faculty ofDentistry SelcukUniversity Konya Turkey e-mail: [email protected] ISSN 2192-8118 e-ISSN2192-8126 ISBN 978-1-4614-2205-1 e-ISBN978-1-4614-2206-8 DOI 10.1007/978-1-4614-2206-8 SpringerNewYorkDordrechtHeidelbergLondon LibraryofCongressControlNumber:2011942210 (cid:2)TheAuthor(s)2012 Allrightsreserved.Thisworkmaynotbetranslatedorcopiedinwholeorinpartwithoutthewritten permissionofthepublisher(SpringerScience+BusinessMedia,LLC,233SpringStreet,NewYork,NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software,orbysimilarordissimilarmethodologynowknownorhereafterdevelopedisforbidden. Theuseinthispublicationoftradenames,trademarks,servicemarks,andsimilarterms,eveniftheyare notidentifiedassuch,isnottobetakenasanexpressionofopinionastowhetherornottheyaresubject toproprietaryrights. Printedonacid-freepaper SpringerispartofSpringerScience+BusinessMedia(www.springer.com) Preface ‘‘The world is changing’’ is a classical concern for people over forties. However, the era we live is a little bit different than the previous generations. We are more likelyinbetweenclassicalandcontemporaryorbetweenfictionsandfacts.Change is unbearably fast and the story of biology is still far from to be completed. It is only a few weeks ago (September 22, 2011) particle physicists on the particle detect or named ‘‘Oscillation Project with Emulsion-tRacking Apparatus (OPERA)’’experimentdetectedneutrinostravelingfasterthanlight.Althoughitis too early to declare Einstein’s theory of special relativity is wrong, results would be said being so revolutionary at least. The same has happened in cell biology in 2006.ShinyaYamanakaandhisteamdiscoveredthatthecompletelydifferentiated somatic cell could exert its embryonic stem cell state potential with the available conditions. The method for turning a somatic cell to a pluripotent one was rela- tively easy, at least easier than one could imagine till Yamanaka’s paper. They named those cells as induced pluripotent stem cells (iPSCs). Science is used to have such sudden pulses. However, there is always a resistancetounexpectedchanges.Itgenerallytakes longer time thannecessaryto interpret the discoveries having results that are applicable to many situations. Some scientists want to directly apply those results to a daily life as quickly as possible, while the others, whom being fascinated by reaching one more level of endless mystery, display a tendency to beware there is actually more. However, some with a special consciousness embark through explorations of unknown realities. The discovery of iPSCs broughtabout all. Now many researchers are trying to refinethetechniquetoservethosecellstorestorehumanhealth.Someofthemare trying to reach the furthest point in the dark corridors of cell biology, while they areawarethatthebatteryoftheirtorchispushingitslimits.However,veryfewof them opened a completely renewed era in biology by mathematical biology. This manuscriptis trying toexplainthe fundamentals behindthe iPSCsandits applications. Most importantly, it attempts to show why we have to use mathe- maticstogofurtherwithiPSCsoranotheryetundiscoveredcells.Thetheoriesof Stuart Kauffman and Sui Huang pointed out the ways to solve many problems in v vi Preface cell biologyandarebeingpored overbymanypeopletoquench theirintellectual thirst.Dr.Huangisshowingbravelyhowimpossibletofathombycommon sense ofalldatafloodingfrom‘omics’worksofbiology.Fortunately,heisusinggeneral concepts or principles of physics and mathematics to establish a firm theoretical foundation. Researchers from my team started from tooth regeneration and inescapably ended up with stem cell biology. I individually had begun to be involved in microbialaspectsofdentaldiseases.NowIambeingcaptivatedbycomplexityand system biology, because it is hard not being exposed to the emergent patterns of every system that has a common connection: mathematics. Obviously iPSCs are providing great tools to study every aspects of biology, fundamentals through applications. In conclusion, iPSCs opened minds of scientist by showing that we should compel our imagination limits to see more. I am grateful to Sui Huang for his generous and humble guidance. Thanks to KursatTurksentofindmesufficienttowritethismanuscript.ThankstoSpringer’s publishing team, especially to Renata Hutter and Aleta Kalkstein for their kind assistance. Thanks also to Kamil Can Akçalı who encouraged me to follow my instincts.LastbutnotleastthankstoMuammerSaglamforhisunconditionallove that led me to feel the light. Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 Pluripotent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.1 Different Pluripotent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.2 Transcriptional Networks and Signaling Pathways of Pluripotency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 2.2.1 Transcriptional Network of Pluripotency . . . . . . . . . . . . 8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3 Induced Pluripotent Stem Cells (iPSCs) . . . . . . . . . . . . . . . . . . . . 11 3.1 Generation the First iPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3.2 Reprogramming. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 3.2.1 Factor Delivery into Target Cells . . . . . . . . . . . . . . . . . 12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 4 Molecular Mechanisms of Pluripotency. . . . . . . . . . . . . . . . . . . . . 21 4.1 Steps in Reprogramming . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 4.1.1 Increase in the Cell Cycle Rate . . . . . . . . . . . . . . . . . . 21 4.1.2 Morphological Changes. . . . . . . . . . . . . . . . . . . . . . . . 22 4.1.3 Late Events Toward Pluripotency. . . . . . . . . . . . . . . . . 22 4.2 Mechanisms in Reprogramming . . . . . . . . . . . . . . . . . . . . . . . 23 4.2.1 Genetic Factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.2.2 Signaling Pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . 24 4.3 Dynamics of Direct Reprogramming . . . . . . . . . . . . . . . . . . . . 25 4.4 Epigenetic Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 4.5 Similarities and Differences Between iPSCs and ESCs . . . . . . . 27 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 vii viii Contents 5 Modeling Disease in a Dish. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 5.1 Disease-Specific iPSCs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 5.2 Choosing Cell Sources. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 5.3 Identification of iPSC Colonies. . . . . . . . . . . . . . . . . . . . . . . . 40 5.4 Characterization of Genetic Mutation. . . . . . . . . . . . . . . . . . . . 42 5.5 iPSCs Differentiation into Desired Cell Types . . . . . . . . . . . . . 42 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 6 Challenges to Therapeutic Potential of hiPSCs . . . . . . . . . . . . . . . 51 6.1 Is Reprogramming Necessary for Regenerative Therapies?. . . . . 53 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 7 New Approach to Understand the Biology of Stem Cells. . . . . . . . 57 7.1 Health Versus Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 7.2 Changed Paradigm: Reprogramming as Rare But Robust Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 7.3 From Reductionism to Wholeness. . . . . . . . . . . . . . . . . . . . . . 62 7.4 More Future Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . 66 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 8 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 About the Author . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 Chapter 1 Introduction Although cell fates during development are neither restrictive nor irreversible, interestingly enough the deeply rooted attitude in cell biology has been that the terminallydifferentiated cellshave lost thepotential ofproducing othercell types (Huang 2009). The first study showed that the nuclei offrog blastula cells could produce complete embryos when transplanted into enclosed embryos (Briggs and King 1952). Only a few years later Gurdon et al. (1958) reprogrammed fully differentiated intestinal cells from Xenopus by frog oocytes. It took 20 years of incubation for Evans and Kaufman to publish the successful isolation of embry- onic stem cells (ESCs) in 1981 (Evans and Kaufman 1981). Although changing a somatic cell fate was achieved using frog oocytes, somatic cell nuclear transfer (NT)couldnotbesucceeded inotherspecies untillate 1990s. ThenWilmutet al. (1997) cloned sheep Dolly. The first successful derivation of human ESCs was reported a year after (Thomson et al. 1998). However, ethical complications and paucity of human egg cells for research purposes, extremely low efficiency, high technical difficulty levels and aberrant ploidy caused researches on ESCs much more controversial than expected (Walia et al. 2011). Earlier studies showed that the ‘terminally differentiated’ state of human cells was not fixed, but could be altered, and the expression of previously silent genes typical of other differentiated states could be induced (Blau and Baltimore 1991; Bhutanietal.2010;YamanakaandBlau2010).Accordingly,pluripotentstemcell lines can be generated directly from completely differentiated adult somatic cells using alternative approaches, such as nuclear transfer, cell fusion and direct reprogramming.Whenanucleusfromadifferentiatedsomaticcellistransplanted into an enucleated oocyte, nuclear reprogramming is initiated, leading to cloning of the original somatic cell. These experiments clearly showed that cell special- izationneedsonlychangeingeneexpression,notingenecontentandthisprocess ofdifferentiationcanbefullyreversed(YamanakaandBlau2010).Heterokaryons constitute another complementary approach to nuclear reprogramming and it involves cellfusion,in which two ormore cell types fuse toformsingle entity. It has been shown that reprogramming in heterokaryons was influenced by DNA S.Yildirim,InducedPluripotentStemCells,SpringerBriefsinStemCells 1 DOI:10.1007/978-1-4614-2206-8_1,(cid:2)TheAuthor(s)2012 2 1 Introduction methylationstatus,tissueoforiginandtherelativeratioofnucleithatdictatesthe balance of regulators (Blau and Baltimore 1991; Zhang et al. 2007). By using interspeciesheterokaryons(mouseESCs,humanfibroblasts)Bhutanietal.(2010) have showed that mammalian AIDis required for active DNA demethylation and initiation of nuclear reprogramming toward pluripotency in human somatic cells (Bhutani et al. 2010). Intenseefforttoidentifythemastertranscriptionfactorofcellphenotypeshave beenexertedtosupporttheideathatasmallnumberofmastertranscriptionfactors can control cell state in various cell types including reprogramming and trans- differentiation (Young 2011). Only in 2006, attempts to identify the main regu- lators of the ESC state was reached to a plateau with the study of Shinya Yamanaka and colleagues who showed that a combination of only four tran- scription factors could generate ESC-like pluripotentcells frommousefibroblasts (Takahashi and Yamanaka 2006). These generated cells are called induced plu- ripotentstemcells(iPSCs).Thediscoveryoffactor-directedreprogramminghada seismic effect on stem cell biology and its potential application (Wilmut et al. 2011). Today many fundamental systems in biology are changing to accept that maturebodycellscouldberevertedtoanembryonicstatewithoutthehelpofeggs or embryos. Thence, reprogramming not only sidesteps the necessity of using embryos to collectandcultureESCs,butalsocomeswiththeadditionalexpectedadvantageof circumventing the immunological problems associated with engraftment, which includes transplant rejection and graft versus host disease. On the other hand the efficacy of the technique is just a wide place in the road. There are still many technical roadblocks in the process. This review will focus on the story of iPSCs that opened a new era in cell biology only in the very beginning of 2000. References Bhutani N et al (2010) Reprogramming towards pluripotency requires AID-dependent DNA demethylation.Nature463(7284):1042–1047 Blau HM, Baltimore D (1991) Differentiation requires continuous regulation. J Cell Biol 112(5):781–783 BriggsR,KingTJ(1952)TransplantationofLivingNucleiFromBlastulaCellsintoEnucleated Frogs’Eggs.ProcNatlAcadSciUSA38(5):455–463 Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos.Nature292(5819):154–156 GurdonJBetal(1958)SexuallymatureindividualsofXenopuslaevisfromthetransplantationof singlesomaticnuclei.Nature182(4627):64–65 Huang S (2009) Reprogramming cell fates: reconciling rarity with robustness. Bioessays 31(5):546–560 TakahashiK,YamanakaS(2006)Inductionofpluripotentstemcellsfrommouseembryonicand adultfibroblastculturesbydefinedfactors.Cell126(4):663–676 Thomson JA et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145–1147 References 3 Walia B et al (2011) Induced pluripotent stem cells: fundamentals and applications of the reprogrammingprocessanditsramificationsonregenerativemedicine.StemCellRevJun14. [Epubaheadofprint] Wilmut I et al (1997) Viable offspring derived from fetal and adult mammalian cells. Nature 385(6619):810–813 WilmutIetal(2011)Theevolvingbiologyofcellreprogramming.PhilosTransRSocLondB BiolSci366(1575):2183–2197 YamanakaS,BlauHM(2010)Nuclearreprogrammingtoapluripotentstatebythreeapproaches. Nature465(7299):704–712 YoungRA(2011)Controloftheembryonicstemcellstate.Cell144(6):940–954 ZhangFetal(2007)Activetissue-specificDNAdemethylationconferredbysomaticcellnuclei instableheterokaryons.ProcNatlAcadSciUSA104(11):4395–4400