EMBRYONICSTEMCELLSBASICBIOLOGYTOBIOENGINEERINGEditedbyMichaelS.Kallos Embryonic Stem Cells Basic Biology to Bioengineering Edited by Michael S. Kallos Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright 2011 InTech All chapters are Open Access articles distributed under the Creative CommonsNon Commercial Share Alike Attribution 3.0 license, which permits to copy,distribute, transmit, and adapt the work in any medium, so long as the originalwork is properly cited. After this work has been published by InTech, authorshave the right to republish it, in whole or part, in any publication of which theyare the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is acceptedfor the accuracy of information contained in the published articles. The publisherassumes no responsibility for any damage or injury to persons or property arising outof the use of any materials, instructions, methods or ideas contained in the book.

Publishing Process Manager Romina Krebel Technical Editor Teodora Smiljanic Cover Designer Jan Hyrat Image Copyright Knorre, 2011. Used under license from Shutterstock.com First published August, 2011 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from orders@intechweb.org Embryonic Stem Cells Basic Biology to Bioengineering, Edited by Michael S. Kallos p.cm.ISBN 978-953-307-278-4 free online editions of InTech Books and Journals can be found atwww.intechopen.com Contents PrefaceIX Part 1Challenges and Possibilities From New Cell Linesto Alternative Uses of Cryopreserved Embryos1 Chapter 1Embryonic Stem Cells for Therapies Challenges and Possibilities3 Ronne Wee Yeh Yeo and Sai Kiang Lim Chapter 2Derivation and Characterization of New hESC Linesfrom Supernumerary Embryos, Experience from Turkey19 Zafer Nihat Candan and Semra Kahraman Chapter 3Cryopreserved Embryos: A Catholic Alternativeto Embryonic Stem Cell Research and Adoption33 Peter A. Clark Part 2Methods, Tools and Technologies for Embryonic Stem Cell Culture, Manipulation and Clinical Application47 Chapter 4Bioprocess Developmentfor the Expansion of Embryonic Stem Cells49 Megan M. Hunt, Roz Alfred, Derrick E. Rancourt, Ian D. Gates and Michael S. Kallos Chapter 5Small-Scale Bioreactorsfor the Culture of Embryonic Stem Cells73 Allison Van Winkle, Ian D. Gates and Michael S. Kallos Chapter 6Synthetic Surfacesfor Human Embryonic Stem Cell Culture89 Andrei G. Fadeev and Zara Melkoumian Chapter 7Efficient Integration of Transgenes and Their Reliable Expression in Human Embryonic Stem Cells105 Kenji Sakurai, Miho Shimoji, Kazuhiro Aiba and Norio Nakatsuji VIContents Chapter 8Embryonic Stem Cells: Introducing ExogenousRegulators into Embryonic Stem Cells123 Yong-Pil Cheon Chapter 9Functional Control of Target Single Cells in ES Cell Clusters and Their Differentiated Cells by Femtoinjection149 Hideaki Matsuoka, Mikako Saito and Hisakage Funabashi Chapter 10From Pluripotency to Early Differentiationof Human Embryonic Stem Cell Cultures Evaluatedby Electron Microscopy and Immunohistochemistry171 Janus Valentin Jacobsen, Claus Yding Andersen,Poul Hyttel and Kjeld Mllgrd Part 3Applications of Embryonic Stem Cellsin Research and Development191 Chapter 11Methods to Generate Chimeric Micefrom Embryonic Stem Cells193 Kun-Hsiung Lee Chapter 12Embryonic Stem Cells in Toxicological Studies213 Carmen Estevan, Andrea C. Romero, David Pamies, Eugenio Vilanova and Miguel A. Sogorb Chapter 13Teratomas Derived from Embryonic Stem Cellsas Models for Embryonic Development,Disease, and Tumorigenesis231 John A. Ozolek and Carlos A. Castro Part 4Pluripotency and Molecular Biologyof Embryonic Stem Cells263 Chapter 14Illuminating Hidden Features of Stem Cells265 Gideon Grafi, Rivka Ofir, Vered Chalifa-Caspi and Inbar Plaschkes Chapter 15Signaling Pathways in Mouse Embryo Stem Cell Self-Renewal283 Leo Quinlan Chapter 16Building a Pluripotency Protein InteractionNetwork for Embryonic Stem Cells305 Patricia Ng and Thomas Lufkin Chapter 17Profile of Galanin in Embryonic Stem Cells and Tissues321 Maria-Elena Lautatzis and Maria Vrontakis Chapter 18Rho-GTPases in Embryonic Stem Cells333 Michael S. Samuel and Michael F. Olson ContentsVII Chapter 19Cripto-1: At the Crossroadsof Embryonic Stem Cells and Cancer347 Nadia Pereira Castro, Maria Cristina Rangel, Tadahiro Nagaoka, Hideaki Karasawa, David S. Salomon and Caterina Bianco Chapter 20Molecular Mechanisms Underlying Pluripotencyand Lineage Commitment The Role of GSK-3369 Bradley W. Doble, Kevin F. Kelly and James R. Woodgett Part 5Lessons from Development389 Chapter 21Embryonic Stem Cells and the Germ Cell Lineage391 Cyril Ramathal, Renee Reijo Pera and Paul Turek Chapter 22Techniques and Conditionsfor Embryonic Germ Cell Derivation and Culture425 Maria P De Miguel, Candace L Kerr,Pilar Lpez-Iglesias and Yago Alcaina Chapter 23Pluripotent Gametogenic Stem Cellsof Asexually Reproducing Invertebrates449 Valeria V. Isaeva Preface TheisolationandcultureofhumanembryonicstemcellsbyThomsoninthelate1990shasacceleratedaparadigmshiftinmedicinethatwasstartedmuchearlierbyTillandMcCulloch in the early 1960s with the discovery of the first stem cells in mice. Theburgeoning field of regenerative medicine will ultimately transform modern humanhealthcarefromamoleculebasedfocus,whichservestoalleviatesymptoms,toacellandtissuebasedfocuswhichhasthepromiseofactuallyrestoringfunction.Althoughthe potential is enormous, the road is long and there are certainly many milestonesalongtheway.Thisbook,EmbryonicStemCellsBasicBiologytoBioengineeringanditscompanion,EmbryonicStemCellsDifferentiationandPluripotentAlternatives,serveasasnapshotofmanyoftheactivitiescurrentlyunderwayonanumberofdifferentfronts.This book is divided into five parts and provides a foundation upon which futuretherapiesandusesofembryonicstemcellscanbebuilt.Part1:ChallengesandPossibilitiesFromNewCellLinestoAlternativeUsesofCryopreservedEmbryosChapters 13 offer a broad overview ofsome of the challenges in bringing embryonicstemcellbasedmedicinetotheclinic,aswellasacasestudyofthederivationofnewembryonicstemcelllines,andanalternativetotheuseofcryopreservedembryos.Part2:Methods,ToolsandTechnologiesforEmbryonicStemCellCulture,ManipulationandClinicalApplicationChapters 410 present a wide variety of tools and technologies ranging from largescale bioreactors to scaleddown bioreactor arrays and synthetic surfaces that can beused for embryonic stem cell culture. In addition, methods for introducing foreigngenesintoembryonicstemcellsandcontrollinggeneexpressionaredescribed.Lastly,the use of imaging is presented as a tool to measure pluripotency and earlydifferentiation.Part3:ApplicationsofEmbryonicStemCellsinResearchandDevelopmentChapters 1113 present methods to generatechimeric micefor use in research, andinaddition,describetheuseofembryonicstemcellsintoxicologicalstudiesandtheuseXPreface of teratomas derived from embryonic stem cells as models for early development,disease,andtumorigenesis.Part4:PluripotencyandMolecularBiologyofEmbryonicStemCellsChapters1420describeourunderstandingofpluripotencyaswellassomeofthekeymolecules involved in regulating not only pluripotency but cancer and earlyembryonictissues.Part5:LessonsfromDevelopmentChapters 2123 examine the knowledge we have gained from studying embryonicgerm cells and pluripotent gametogenic stem cells of asexually reproducinginvertebrates.In the book Embryonic Stem Cells Differentiation and Pluripotent Alternatives, the storycontinueswithasampleofsomeofthestudiescurrentlyunderwaytoderiveneural,cardiac,endothelial,hepaticandosteogeniclineages.Inaddition,inducedpluripotentstem cells are introduced and other unique sources of pluripotent stem cells areexplored.I would like to thank all of the authors for their valuable contributions. I would alsolike to thank Megan Hunt who provided me with much needed assistance and actedas a sounding board for early chapter selection, and the staff at InTech, particularlyRomina Krebel who answered all of my questions and kept me on track during theentireprocess.Calgary,Alberta,Canada,July2011MichaelS.KallosPharmaceuticalProductionResearchFacility(PPRF),SchulichSchoolofEngineering,UniversityofCalgary,AlbertaCanadaDepartmentofChemicalandPetroleumEngineering,SchulichSchoolofEngineering,UniversityofCalgary,Alberta,Canada Part 1 Challenges and Possibilities From New Cell Lines to Alternative Uses of Cryopreserved Embryos 1 Embryonic Stem Cells for Therapies Challenges and PossibilitiesRonne Wee Yeh Yeo and Sai Kiang Lim Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR); Yong Loo Lin School of Medicine, National University of Singapore Singapore 1. Introduction The successful establishment of human embryonic stem cells (hESCs) in culture (Thomson et al.,1998)hasraisedunprecedentedpublicinterestandexpectationoftreatingintractable diseasessuchasdiabetes,spinalcordinjuries,neurodegenerativeandcardiovascular diseases. Much of this enthusiasm was predicated on the unlimited self-renewal capacity of hESCsandtheirremarkableplasticityindifferentiatingintoeverycelltypeinourbody. Thesefeaturespresentedthetantalizingpossibilityofanunlimitedcellsourcein regenerativemedicinetogenerateanytissuestoreplaceinjuredordiseasedtissues. However,translatingthepotentialofhESCintotherapieshasbeenchallenging.Although translation of hESC has been severely impeded by social and political constraints placed on hESCresearchthroughethicalandreligiousconcernsoverthedestructionofviable blastocystsduringhESCisolation,themainchallengeshavebeensafetyandtechnical issues.2. Challenges in ESC therapy 2.1 Overcoming tumor formation ThetwodefiningcharacteristicsofESCsare:1)theirpluripotency,orthepotentialto differentiateintoallcelltypesintheadultbody;and2)theirunlimitedself-renewal capacity,ortheabilitytoremaininanundifferentiatedstateanddivideindefinitely.For mESCs,pluripotencyisoftendemonstratedbytheproductionofmESC-derivedanimals throughgermlinetransmissionbychimerasresultingfrominjectionofthecellsinto blastocystsorthroughtetraploidcomplementation.InhESCs,proofofpluripotencyhas been limited to formation of teratomas or teratocarcinomas, which are tumors composed of randomlydistributedtissuesfromthethreeprimordialgermlayersinimmunologically incompetentmice(Lenschetal.,2007).Karyotypicallynormal,lowpassagehESCsform benign teratomas that do not contain undifferentiated tissues and are less invasive (Blum et al., 2009; Reubinoff et al., 2000; Thomson et al., 1998) while high passage hESCs which have becomekaryotypicallyabnormalgiverisetohighlyinvasive,malignantteratocarcinomas (Herszfeld et al., 2006; Plaia et al., 2006; Werbowetski-Ogilvie et al., 2009; Yang et al., 2008).Pluripotencycoupledwithunlimitedself-renewalnotonlydefineESCs,theyarealsothe main appeal of ESC as the cell source for regenerative medicine but at the same time,pose Embryonic Stem Cells Basic Biology to Bioengineering 4 significantchallengestothetransplantationofdifferentiatedESCstoreplaceinjuredor diseased tissues. The propensity of ESC to differentiate into teratomas necessitates the need to eliminate any residual ESCs in the differentiated cell preparation. There have been many strategiestoeliminateresidualESCsorenhancethepurityofdifferentiatedESC preparations.Theuseofheterologousselectablegenemarkerssuchasantibioticresistance geneorfluorescentproteinmarkers(Klugetal.,1996;M.Lietal.,1998; Mulleretal.,2000; Soriaetal.,2000)isgenerallynotastrategyofchoiceasthiscouldintroducepotentially deleteriousgenemutations.Mostofthestrategiescenteredaroundtheuseofendogenous markersthatareuniqueorhighlyexpressedonESCsandnotontheirdifferentiated progeny.Forexample,SSEA-4andTRA-1-60whicharehighlyexpressedonhESCshave showntobehighlyefficientinphysicallyremovingcontaminatingESCsbymagneticor fluorescence-activatedcellsorters(MACSorFACS)(Fongetal.,2009b).Anotherstrategy exploittheflotationdensityofcellondiscontinuousdensitygradientssuchasPuresperm- or Percoll-based gradients (Fong et al., 2009a). Using a relatively novel strategy, Choo et al. hasraisedantibodiesagainstundifferentiatedhESCs(Chooetal.,2008)andidentifiedan antibodythatwascytotoxicagainsthESCsbyoncosis.ThisantibodywasanIgMthat recognizespodocalyxin-likeprotein-1(PODXL).hESCsthatweretreatedwithmAB84did notformteratomawhentransplantedintoSCIDmiceevenafter18-24weeks.Therefore, thereareviabletechnologiestoremoveorreduceresidualhESCsindifferentiatedhESC preparationandmitigatetheriskofteratomaformationinpatientsreceivinghESC-based cell therapy.2.2 Overcoming immunorejection Likealltissuetransplants,hESC-basedcelltherapywillhavetocircumventhostimmune rejectiontoengraftintherecipients.OneproposedstrategywastoestablishESC repositorieswithlinesexpressingthecombinationsofHLAmoleculesthatarecompatible withHLAhaplotypespresentinthepopulation(Nakajimaetal.,2007;Tayloretal.,2005). Alternatively,thehostsimmunesystemcouldbemanipulatedtoinducetoleranceto foreign tissues by ablation of donor-reactive T cell in the thymus, generation of tolerogenic dendritic cells and induction of Treg cells [reviewed in (Chidgey et al., 2008)]. However, with thedevelopmentofinducedpluripotentstemcelltechnologythatmakesthecreationof patient-specificpluripotentcellscontainingthesamegeneticmaterialastherecipienta highly viable and practical option, the issue of host rejection has become a non issue.Thequesttocreatepatient-specificpluripotentcellsbeganwiththerapeuticcloningor somatic cell nuclear transfer (SCNT) where the diploid nucleus of a somatic cell was injected into a haploid enucleated egg to be reprogrammed by soluble factors in the host cell. Upon stimulation, the re-programmed cell divides to form a blastocyst with an inner cell mass that has identical nuclear genetic composition as the nucleus donor. Although this approach has workedtogenerateESCsfromdifferentanimalssuchasmice,rabbits,cats,sheep,cattle, pigs,goats[reviewedin(Wilmutetal.,2002)]andevenprimates(Byrneetal.,2007),no hESChasbeengeneratedthroughthisapproachasitremainsahighlyinefficientprocess and the use of human oocytes is ethically controversial (French et al., 2008; J. Li et al., 2009b). ESCs generated through SCNT are in principle, heterogeneous in their genetic composition as they contain nuclear DNA of the nucleus donor and mitochondrial DNA of the egg donor (Evans et al., 1999). This raises the possibility that SCNT-derived ESCs could be rejected by the innate immune systemof the host with which the ESCs share the same nuclear but not mitochondrial genetic material (Ishikawa et al., 2010). Embryonic Stem Cells for Therapies Challenges and Possibilities 5 Fig. 1. Mitigating tumor formation and immune rejection. Two of the major challenges to the translation of ESCs into clinical applications are teratoma formation by residual undifferentiated ESCs in the cell preparation and immune rejection of ESC-derived cells or tissues due to incompatible HLA profiles of ESC and recipient. To mitigate the risk of teratoma formation, several methods to remove residual hESCs have been developed using either physical or biological methods. Some of the physical separation methods are based on magnetic- or fluorescence-activated cell sorters (MACS or FACS) that sort against cells with ESC-associated surface markers, SSEA-4 and TRA-1-60 or on cellular density using discontinuous gradients of Percoll or PureSperm. Alternatively, residual ESCs can be destroyed using a cytotoxic antibody (mAb 84) specific for undifferentiated hESCs. To prevent immune rejection, one strategy proposed the establishment of ESC repositories to carry lines expressing HLA combinations compatible with all possible haplotypes in the Embryonic Stem Cells Basic Biology to Bioengineering 6 population. Alternatively, donor cell tolerance can be induced by manipulating host immune defenses, such as eliminating donor-reactive T cells in the thymus, generating tolerogenic dendritic cells and inducing Treg cells. An ideal approach would be to generate patient-specific ESCs. Some of early efforts include the use of somatic cell nuclear transfer (SCNT). More recently, induced pluripotent stem cell (iPSC) technology has enabled with great ease the generation of self pluripotent stem cells without the destruction of oocytes or embryos, hence bypassing ethical controversies. Thebreakthroughincreatingpatient-specificpluripotentcellswasachievedwhen YamanakademonstratedthattheintroductionoftranscriptionfactorswhichregulateESC self-renewal,includingOct3/4andSox2wassufficienttoreprogramsomaticcellsintoES-likecells(Takahashi&Yamanaka,2006).Theseinducedpluripotentstemcells(iPSCs)are karyotypicallynormalwithgeneexpressionprofileshighlysimilartoESCsandcan differentiate into cells of all three germ layers (Takahashi et al., 2007; Yu et al., 2007). Apart frombeingpatient-specific,themajorattractionofiPSCsliesintheirderivationfrom somatictissuesandnotfromethicallycontentioustissuessuchashumanoocytesor embryos.However,retroviralandlentiviralvectorswererequiredtoexpressthe transcriptionfactorsforreprogrammingofthesomaticcellsandthiscarriesariskof insertionalmutagenesis.Tocircumventtheneedforviralvectors,non-viralgenetic modificationapproachesweredeveloped(Okitaetal.,2008;Soldneret al.,2009;Woltjenet al., 2009). Recently iPSCs were obtained via a direct delivery of reprogramming factors into cells using poly-arginine protein transduction domains (Zhou et al., 2009) or mRNA (Plews et al., 2010), thereby circumventing any form of genetic manipulation. These improvements haveessentiallyabrogatedtheissueofhost/donorcellimmunecompatibilityand considerablyenhancedtheprospectsofgeneratingpatient-specificiPSCsforregenerative medicine.However,arecentstudydemonstratedthatsomehiPSCderivativesexhibit limited expansion capability, increased apoptosis and early cellular senescence as compared totheirhESC-derivedcounterparts,raisingdoubtsabouttheclinicalvalueofthis reprogrammingtechnology(Fengetal.,2010).Also,itremainstobedeterminedifthe progeny of these cells, which are genetically identical to the reprogrammed cell, will trigger any immune response when reintegrated into the donor. 2.3 ESC differentiationESCowesitsallureasthesourceofstemcellsforregenerativemedicinetotwoimportant potentials: 1) unlimited self-renewal potential and 2) the potential to differentiate into all the celltypesinanadult.Unfortunately,therecenttechnologicaladvancestocircumventthe risksassociatedwithtransplantationofESC-derivedcells,namelyteratomaformationand hostimmunerejection,werenotmatchedbysimilarprogressindifferentiatinghESCsinto cells suitable for regenerative medicine. In contrast to adult stem cells where hundreds of clinicaltrialshavebeenconductedtoevaluatetheirclinicalefficacy,thefirsttestingofa hESC-basedtherapeuticcandidatehasonlyjustbeeninitiated.InOct2010,GeronCorp announced the enrollment of the first patient to test the safety of human embryonic stem cell (hESC)-derived oligodendrocyte progenitor cells, GRNOPC1, in treating spinal cord injury. WiththeprogressmadeinreducingtheriskofteratomaformationbyresidualESCin differentiatedESCpreparationsandthegenerationofpatient-specificiPSC,themajor impedimenttothedevelopmentofhESC-basedcelltherapiesremainsthegenerallackof progress in developing protocols for efficient and reproducible differentiation of hESCs into Embryonic Stem Cells for Therapies Challenges and Possibilities 7 clinicallyrelevantcelltypesinsufficientquantityandpuritysuitablefortransplantation studies in clinically relevant large animal models.ThepluripotentdifferentiationpotentialofhESCshasalwaysbeenpredicatedontheir ability to form teratomas in immune-compromised animals and embryoid bodies consisting of tissues from the three germ layers. This ability suggest that differentiation of ESC into the variouscelltypesintheadultanimalwasnotcontingentonthepresenceofanembryonic microenvironment.Instead,itreliesonaratherminimalenvironmentthatdidnotsupport thepluripotentandself-renewingstateoftheESCsandborelittleresemblancetothe dynamically evolving microenvironment of a developing embryo. Nevertheless, much effort to direct differentiation of hESCs into potentially therapeutic cell types have focused on the recapitulation of the embryonic microenvironment based on a yet to be tested rationale that the embryonic microenvironment represents the optimal micro-environment for directed in vitro differentiation ofESC. 2.3.1 Recapitulating embryonic development to induce lineage commitmentEmbryogenesisisahighlydynamiccomplexprocessthatisstillbeingunraveleddespite yearsofintensiveresearchandmuchprogressinelucidatingthemolecularandcellular processes involved in formation of an embryo. From a developmental perspective, the ESC represents cells that were frozen in the developmental state of a late-stage embryo just prior to differentiation and lineage commitment. The ability of ESC to re-enter the developmental processanddifferentiatewhenreturnedtothemicro-environmentofablastocysthas providedcompellingimpetustousethedevelopingembryotoguideanddirectinvitro differentiationofESCtoaspecificcelltype.Muchefforthasthereforebeendevotedto identifyingthemolecularcuesthatwereinvolvedinthedifferentiationofpluripotentcells intheblastocystintospecificterminallydifferentiatedcells.Theunderlyingrationalehas alwaysbeenthatatemporalandspatialrecapitulationofthesecuesinvitrowilldirect differentiation of ESC towards a specific cell type. An early and critical phase of embryogenesis is gastrulation. During this process, the mono-layeredblastulaundergoesaseriesoftransformationtoformthetri-layeredgastrula.The formationofthesethreegermlayers(endoderm,mesodermandectoderm)marksthefirst stage of cell fate determination. This is followed by organogenesis when tissues and organs areformedfromfurtherdifferentiationofthegermlayers.Theendodermgivesrisetothe epitheliaofthegutandrespiratorysystem,andorganssuchasliverandpancreas;the mesoderm gives rise to muscles, the circulatory system, bone and connective tissues; and the ectodermgivesrisetothenervoussystemandtheepidermis.Similarly,theinitialstep towardsderivingfunctionalcellsandtissuesfromESCsmayinvolvegermlayerinduction in vitro. Thefirstvisiblesignofgastrulationistheformationofthesymmetry-breakingstructure calledtheprimitivestreak(PS).Epiblastcells,whicharederivedfromtheinnercellmass, ingressthroughthePStoformthemesodermanddefinitiveendoderm.Theremaining epiblastcellsthatdonotingressformtheectoderm.Manymolecularfactorshavebeen implicated in this process and they include members of the large transforming growth factor (TGF)andWntsignalingfamilies(Conlonetal.,1994;Hogan,1996;Schier,2003; Yamaguchi,2001).Painstakingresearchhasrevealedsomeofthetemporalandspatial effectsofthesefactorsduringembryogenesisandmanyofthesefactorsexertedsimilar effectsonthedifferentiationofESCcells.AsreviewedbyMurryandKeller(Murry& Embryonic Stem Cells Basic Biology to Bioengineering 8 Keller, 2008)], differentiation of ESCs into each of the three germ layers could be induced by thesamefactorsknowntoinducethemduringgastrulation.Forexample,Wnt,Nodalor BMP4 which have been shown to be important in the formation of epiblast cells in the PS of adevelopingembryo(Kispert&Herrmann,1994)couldsimilarlyinducetheformationof PS-like cells from ESC (Kubo et al., 2004; Lindsley et al., 2006; Ng et al., 2005; Nostro et al., 2008).Asingastrulation,exposureofthePS-likecellstohighlevelsofNodalfurther differentiate these cells to a Foxa2hi cells that are comparable to cells in the anterior PS that formsthedefinitiveendoderm(D'Amouretal.,2005;Kuboetal.,2004).Incontrast, exposure to Wnt, low level of activin (which activates Nodal) and BMP4 causes the PS-like cellstodifferentiateintoaFlk-1+posteriorPS-equivalentpopulationthatformsthe mesoderm (Nostro et al., 2008). Therefore, the three germ layers can be induced in ESCs by exposingthecellstofactorsknowntobeimportantintheformationofthesethreegerm layersduringembryogenesis.Further,bymodulatingthesefactorsinaconcentrationand temporalmannerthatrecapitulatesearlyembryonicdevelopment,commitmentofESCsto one of the germ layers could be enhanced.2.3.2 Enhancing lineage commitment TheintensiveresearcheffortstoinduceabiasindifferentiatingpluripotentESCstowards oneofthegermlayerswould,inprinciple,enhancethesubsequentproductionofspecific tissuecelltypesofthisgermlayere.g.musclesfrommesoderm.However,enhancing commitmentofdifferentiatingESCtooneofthethreegermlayersmaynotbethelimiting factoringeneratingclinicallyusefulcelltypesinsufficientnumberandpurityfor therapeutic or screening applications. For example, the most efficient derivation of clinically useful cell types from ESC is neural cell types and not surprisingly, the first ESC-derived cell type to be clinically tested is oligodendrocytes. The relative efficiency of generating neurons, astrocytes and oligodendrocytes from ESC probably lies not in the ease of generating neural progenitorcellsbutintherelativelyhighexpansioncapacityofESC-derivedneural progenitor cells (Dottori & Pera, 2008; Studer, 2009). The high expansion capacity of neural progenitorcellswouldeasilycircumventalimitingsupplyofrareneuralprogenitorcells formedduringESCdifferentiationandobviatestheneedtofirstbiasdifferentiationof pluripotentESCstowardsanectodermalgermlineage.Therefore,therationaleunderlying theintensiveresearcheffortstobiasdifferentiatingpluripotentESCstowardsoneofthe germlayersmayberedundantatleastforthederivationofneuralcelltypes.Unlike ectodermal differentiation which is generally considered the default differentiation pathway forESC,thederivationofmesodermalorendodermalcelltypesfromESCcouldstillbe enhancedbytherecapitulationofearlyembryonicdevelopmentprocessestoenhance mesodermal or endodermal commitment. 2.3.3 Terminal differentiation of ESC In 2005, D'Amour et al reported the use of a multi-stage protocol that attempts to temporally recapitulateembryonicdevelopmentforthedifferentiationofhESCintoinsulin-producing pancreatic cells for diabetes treatment. During this differentiation regime, they observed the formationofsequentialtransientcellpopulationswithmarkersthatmappedontothe developmentalpathwayofpancreaticendoderm.Thefinalcellpopulationrepresenting pancreaticendodermwastransplantedinmiceforfurtherdifferentiationandmaturation. Whenthesetransplantedanimalsweretreatedwithstreptozotocin,theinductionof Embryonic Stem Cells for Therapies Challenges and Possibilities 9 hyperglycemiawasattenuated.Itwasobservedthatsomeofthetransplantedmice developedteratomas,suggestingthecellpreparationwasheterogenousandcontaminated withESCsthatcoulddifferentiateintoallcelltypes.Incontrast,differentiationofmouse ESCstoinsulin-producingcells isoftenathree-stepprotocolconsistingoftheformationof embryoid bodies, spontaneous differentiation into ecto-, endo- and mesoderm lineages and finallyinductionofpancreaticdifferentiation(Schroederetal.,2006).However,thecell populations generated using this protocol have low insulin content. Using an approach that combineselementsfromthisprotocolandthatusedinneuraldifferentiation,wefirst derivedhighlyexpansibleE-RoSHcelllineswithmeso-endodermpotentialfrom spontaneously differentiating EBs (Lian et al., 2006; Yin et al., 2004). Like neural stem cells, theseE-RoSHcelllinesarehighlyproliferativeandprovideunlimitedsupplyofcellsfor differentiation.Serumstarvationandnicotinamidesupplementationinducedifferentiation of E-RoSH cells to form a heterogenous, insulin-producing culture. Limiting dilution of such culturesyieldedindependentlyderivedclonalinsulin-producingERoSHKcelllines.These cellscontainequimolarofinsulinandC-peptidethatwasstablymaintainedover30 passages at a high concentration of 300-500 pmol/106 cells. The insulin-producing ERoSHK cellsresemblepancreaticcellsanddisplaythedefiningfunctionalpropertiesofbonafide pancreaticbetacells(G.Lietal.,2009a).Theysynthesizeandstoreinsulinintypical intracellular vesicles. Under stimulation by secretagogues such as glucose, tolbutamide and glibenclamide,thesecellsclosetheirATP-sensitiveK+channels,leadingtomembrane depolarization,openingofCa2+channelsandthesubsequentreleaseofinsulinandC-peptideinequimolarratio,amechanismresemblingthatofprimarybetacells.Most importantly, these cells can reverse hyperglycemia when grafted into streptozotocin-treated mice. Relative to their progenitor E-RoSH cells, ERoSHK cells also exhibit enhanced activity in biochemical pathways that are also highly characteristic of beta cells such as the pentose phosphatepathway,clathrin-mediatedendocytosisandPPARsignaling(T.S.Chenetal., 2010).Importantly,transplantationofERoSHKcellsinhyperglycemicstreptozotocin-treatedmicereversesthehyperglycemiaandremovalofthetransplantedcellsrestoresthe hyperglycemia.Thetransplantedcellsdonotformteratomas.Together,thesestudies illustrated the diversity of approaches that have been taken to differentiate ESCs to insulin-producingcellsandtherelativepotentialofeachapproachingeneratingthedesiredend product on a scale to support potential therapeutic application. They also prompted doubts on the need to recapitulate the precise developmental pathway when differentiating ESC. ThisquestionwaspreviouslyraisedbyBurnsetal(Burnsetal.,2004).Fromtheir perspective,developmentaleventsdirectingduodenalendodermtowardsaninsulin-expressing-cellphenotypearetheresultofmillionsofyearsofevolutionaryselection, drivenbyenvironmentalpressuresratherthanbyconsciousdesign.Therefore,insteadof mappingexperimentalprotocolsontotheknowndevelopmentalpathwaysofpancreatic endocrine cells, they proposed that conscious design may be a less circuitous route to arrive atthesameend-point.However,inlieuofknowndevelopmentalpathways,thereisno obvious source to guide and rationalize such a design. In essence, a conscious design would inevitably have to be an empirical approach of careful observation, trial and error, and high throughput screens.2.3.4 Empirical differentiation of ESC Despiteapervasivebeliefthatahighfidelityrecapitulationofdevelopmentalprocess representsthebeststrategyforefficientdifferentiationofpluripotentstemcellsto Embryonic Stem Cells Basic Biology to Bioengineering 10therapeutically useful cell types , the two human ESC-derived cell types ready for testing in manwerederivedbyempiricallyformulatedprotocols.Fortuitously,someelementsin theseprotocolsweresubsequentlyfoundtomapontosimilarpathwaysinembryonic development. In the basic protocol for deriving Geron Corporations GRNOPC1 which is already in Phase Iclinicaltrial,oneofthekeyelementsininducingneuralcommitmentinESCstoform neurospheresisretinoicacid(RA)(Nistoretal.,2005).RAwasfirstobservedtobean inducer of neural differentiation in embryonal carcinoma cells (ECs) (Jones-Villeneuve et al., 1982)beforethefirstretinoicacidreceptor(nowknownasRARa1)wasclonedin1987 (Giguereetal.,1987;Petkovichetal.,1987).BasedontheempiricalobservationthatRA inducedneuraldifferentiationinP19tetracarcinomacells,RAwasusedtoenhanceneural lineagecommitmentinESCs(Bainetal.,1995).Today,RAisoftenusedtoenhanceneural lineagecommitmentinESCtogenerateneurospherescontainingneuralstemcellsandfor thesubsequentterminaldifferentiationofneurospherestoproduceneurons, oligodendrocytes and astrocytes. Therefore, the use of RA to induce neural differentiation in ESCs was rationalized on empirical observation of their effects on EC cells and this preceded the cloning of RA receptors and our understanding of its role in embryonic development. Todate,thereislittleevidencethatRAplaysasignificantroleinneuraldifferentiation duringgastrulation.ThefirstRAsignalinginthegastrulatingvertebrateembryooccursin theposteriormesodermalcellswhenRAisfirstsynthesizedbyretinaldehyde dehydrogenase2(RALDH2)(Niederreitheretal.,1997).ThereishowevernoRAsignaling in the anterior regions of the embryo due to the presence of RA metabolizing enzymes such as CYP26A1 and CYP26C1 (Hernandez et al., 2007; Ribes et al., 2007; Uehara et al., 2007). In fact,RAreceptorsintheprospectiveheadregionoftheXenopusgastrulafunctionas transcriptionalrepressorstopreventinappropriateactivationofgenesactingasposterior determinants.Also,theabsenceofendogenousRAsynthesisinmiceaffectprimarily forebraindevelopmentbutdidnotcompromisetheearlyneurallineagecommitmentor differentiation (Natalia Molotkova et al., 2007; N. Molotkova et al., 2005; Niederreither et al., 2000;Sirbuetal.,2005).Infact,thepathwayforneuraldifferentiationduringembryonic developmentcouldnothaveinformedontheusefulnessofinsulin,triiodothyroidin,EGF andFGFinenhancingtheinvitroproliferationanddifferentiationofESC-derived oligodendrocyte precursors and increase oligodendrocyte survival.ThesecondESC-derivedcelltypemostlikelytobetestedinmanisAdvancedCell Technologysretinalpigmentepithelial(RPE)cellswhichhasbeengivenFDAclearanceto initiate a Phase I/II multicenter clinical trial to treat patients with Dry AMD. The derivation of these RPE cells relies primarily on spontaneous differentiation of hESCs (Klimanskaya et al.,2004).RPEcellsareformedascoloniesofpigmentedcellswhenhESCsundergo spontaneousdifferentiationbyFGF2withdrawalorembryoidbodyformation.These coloniesofpigmentedcellswerethenpickedandexpandedusingveryunremarkable culture medium.2.3.5 Strategizing differentiation of ESC for therapeutic applications The progress of ESC-derived oligodendrocytes and RPE cells to clinical testing attests to the robustnessandefficiencyoftheempirically-drivendifferentiationprotocols.Incontrast, differentiation of ESCs by meticulous mapping on embryonic development pathway has not yielded cells that are ready for clinical testing. Despite this dichotomy in outcomes, there is Embryonic Stem Cells for Therapies Challenges and Possibilities 11 still a prevalent belief that high fidelity recapitulation of embryonic development process is theroutetogeneratethemostphysiologicallyrelevantcells.Embryonicdevelopmentisa timetestedsuccesswithdefinedmilestones.Incontrast,anempiricallydriven differentiation strategy is an inherently inefficient chance event.The success of a differentiation strategy based on embryonic development is predicated not onlyontheelucidationbutalsotherecapitulationofthehighlydynamictemporaland spatialchangesintheembryonicmicroenvironmentthatisinfluencedbybothintra-and Fig. 2. Strategies for differentiation of ESCs into therapeutically useful cell types. The strategies currently being used could be broadly classified into a developmental or an empirical approach. The developmental approach (upper panel) to produce a desired cell type (green stars) relies on the recapitulation of the developmental pathway (blue arrows) during embryogenesis that produces that desired cell type. The general expectation is that identifying the cues that direct the developmental process during embryogenesis and recapitulating these cues spatially and temporally in vitro will be most optimal in yielding physiologically functional cell type (e.g. pancreatic insulin-producing cells). The empirical approach involves the differentiation of ESCs either spontaneously, or using novel factors identified empirically, such as through high-throughput molecular screening or in vitro cell studies (e.g. neural induction of EC cells by RA). These factors may or may not play a role in development. Embryonic Stem Cells Basic Biology to Bioengineering 12extra-embryonicfactors.Notwithstandingthis,translatingsuchacomplexdifferentiation strategytoascalablecommerciallyviablemanufacturingprocesswillbeanequally confoundingunknown.Ontheotherhand,developingadifferentiationstrategyusingan empiricalapproachisachanceprocessoftrialanderrorandfortuitousobservation.This inherentinefficiencycanbecircumventedbyhighthroughputscreenstoidentifyinducing moleculesorcombinationsofmolecules.Thereisalsoalikelihoodthatsuchastrategy wouldprovideforapotentiallyscalablemanufacturingprocessthatwillsupportclinical applications. 3. ESC therapeutics: cell versus biologic AmuchoverlookedformofESC-derivedtherapeuticsisbiologicalproductsorbiologics fromESC.Todate,thepredominantoronlyformsofESC-derivedtherapeuticsthatare beingevaluatedareprimarilycell-based.ThecapacityofESCtoundergospontaneous differentiationinaminimalculturemediumtoformtissuesfromallthreegermlayers suggestthatdifferentiatingESCscanproduceaninductiveandsustaining microenvironmentforthevariouscelltypesthatarebeingformed.Itisconceivablethat someofthismicroenvironmentmayalsoinduceorsustainsometissueregenerationand repairinadult.Howevercapturingthismicroenvironmentandtranslatingittoascalable manufacturing process would be a challenge. Biologic-basedtherapeuticshaveseveraladvantagesovercell-basedtherapies.Biologics eliminatestheneedtopreserveviabilityduringmanufacture,storageandtransport,and administrationtothepatient.Thissubstantiallyreducesthecostandcomplexityof productionanddelivery.Maintainingcellviabilitybeforeandaftertransplantationhas alwaysbeenanimportantconsiderationincell-basedtherapy.Althoughpreservingthe activityofbiologicsisnotaminorconsideration,itis,neverthelessmoretractablethan preservingcellviability.Celltherapyisgenerallyapermanentorlongtermtherapeutic sustainedbythereplicativecapacityofthetransplantedcellswithlittlerecoursefor termination of therapy except when removal of the graft is possible. In contrast to biologics, cell therapy presents increased risks of tumor formation and acute immunological rejections. All things considered, ESC-based biologics is an attractive alternative to develop ESC-based therapeutics.AsanillustrationofapotentialESC-derivedbiologic,wehavedemonstratedthat mesenchymalstemcellsderivedfromhESCs(Lianetal.,2007)secretefactors(Szeetal., 2007)thatarecardioprotectiveinpigandmousemodelsofmyocardial ischemia/reperfusioninjury(Timmersetal.,2008).Theactivecomponentinthissecretion wassmalllipidvesiclesof50-100nmknownas exosomes (Laietal.,2010).Immortalization ofthesemesenchymalstemcellsdidnotcompromisetheproductionoractivityofthe exosomes(T.S.Chenetal.,2011).Thesestudiesprovidedforthedevelopmentofa sustainable scalable manufacturing process to produce potentially therapeutic exosomes for testing in the clinic.4. Conclusion ESCisaversatilecellthathasexertedsignificantimpactonourunderstandingand investigationofcellbiology,differentiationanddevelopment.Ithasprovidedexciting possibilitiesforthetreatmentofhighlyintractablediseases.AsthefirstESC-derivedcell Embryonic Stem Cells for Therapies Challenges and Possibilities 13 type makes its way into clinical testing, there is an apprehensive hope that ESC will justify itshypenotonlyasatherapeuticagentbutonethatwilltreatamultitudeofintractable diseasesaswiderangingasitsdifferentiationpotential.TheestablishmentofiPSC technology by Shinya Yamanaka (Takahashi & Yamanaka, 2006) represents a paradigm shift innotonlyourunderstandingofstemcellbiologybutalsoinovercomingtheethicaland immunechallengesthathadstymiedthetranslationofESCintoclinicalapplications.His approachofre-programmingterminallydifferentiatedcellsintopluripotentstemcells contradictedfundamentalprinciplesindevelopmentalbiology.Thisapproachofexploring beyondtheobviousandlogicalusingempiricalandexperimentalstrategiesmaybe necessary to transcend this current bottleneck in generating the quantity and quality of ESC-derived cells for therapy. 5. 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Zhou, H., Wu, S., Joo, J.Y., Zhu, S., Han, D.W., Lin, T., Trauger, S., Bien, G., Yao, S., Zhu, Y., Siuzdak,G.,Scholer,H.R.,Duan,L.,&Ding,S.(2009).Generationofinduced pluripotentstemcellsusingrecombinantproteins.CellStemCell,Vol.4,No.5, (May 8), pp. (381-384). 2 Derivation and Characterization of New hESC Lines from Supernumerary Embryos, Experience from Turkey Zafer Nihat Candan and Semra Kahraman Memorial Hospital ART & Reproductive Genetics Unit, Istanbul, Turkey 1. IntroductionHuman embryonic stem cell (hESC) lines, which are derived from inner cell mass (ICM) of supernumeraryblastocysts-stageembryos,havewellknownuniqueproperties;long-term self-renewing ability with maintenance of an undifferentiated state and pluripotent capacity todifferentiateintoallderivativesofthreeembryonicgermlayers(Hoffman&Carpenter, 2005;Semb,2005;Trounson,2006).Sinceitsfirstderivationandcharacterizationby Thomson et al in 1998, these intrinsic properties have made hESC very popular worldwide and, thereby, many studies describing isolation and characterization of new hESC lines have beenreported(Findiklietal.,2005;Simonetal.,2005;Thomson,1998).HESCshavebeen consideredveryvaluableandpromisingcellsourceforresearchinvolvingmainlyhuman embryogenesis,oncology,drugtoxicologyanddevelopmentalbiologyaswellasforcell based regenerative therapies (Edwards, 2004).Obviously,studiesonhESCsmostlyfocusontheirpotentialusefortreatmentof degenerativehumandiseases.However,duetothelargelyunknowncharacteristicsof establishedlinesandtheuseofanimalbasedmaterialintheircultures,mostofthelines could not be suitable for prospective transplantation studies (Findikli et al., 2006; Rodriguez etal.,2006).Therefore,registrationofexistinghESClineswiththeircharacteristicsinstem cellbankswouldprovidedatabaseofcelllines,cooperationandco-regulationfor researchers.Inthischapter,itwasaimedtoreportthemethodstoderive18hESClineswhichwere establishedandcharacterizeduntilthedeclarationofprohibitiononhESCresearchin Turkey by Health Ministry in 2005. Additionally, it was discussed the current legal situation of hESC research and perspectives to that issue in Turkey. 2. Material and methods for derivation and characterization of hESC lines DerivationandcharacterizationofallhESClineswereundertakeninMemorialHospital ART&ReproductiveGeneticsCentre,R&DLaboratory,Istanbul,TurkeybetweenJanuary 2003andSeptember2005.Alldonatedsupernumeraryembryoswereusedafterobtaining writteninformedconsentsfromcouples.AllhESClineswereestablishedonlyforresearch purposeratherthanforanyfinancialinterest.Thisstudywasapprovedandcontrolledbythe local ethic committee/Internal Review Board of Istanbul Memorial Hospital. Embryonic Stem Cells Basic Biology to Bioengineering 202.1 Source of supernumerary human embryos SupernumeraryhumanembryosusedforderivationofhESClineswereobtainedafterin vitrofertilization(IVF)orintracytoplasmicsperminjection(ICSI)andpreimplantation geneticdiagnosis(PGD)cycles.Mostoftheseembryoshadpoorqualityandthereby,were considered insufficient for replacement or cryopreservation. Preimplantationgeneticdiagnosisisappliedtothreegroupsofpatientswithvarietyof indicationsinourclinic.ThefirstgroupofPGDincludespatientswhohaveahighriskof transmitting their single gene disorder to their offspring. After diagnosis, abnormal embryos were used to derive hESC lines with specific single gene mutation.In the second group of PGD cycles, embryos belonging to couples with advanced maternal age,ahistoryofrecurrentmiscarriagesandrepeatedimplantationfailuresaredestinedto chromosomal screening (Kahraman et al., 2000, 2006; Lavon et al., 2008; Munne et al., 2005; Verlinsky et al., 2005). Following PGD, chromosomally abnormal embryos were subjected to derivation of hESC lines having chromosomal aneuploidies.Inthethirdgroup,PGDisusedforidentificationofembryosforhumanleukocyteantigen (HLA)matchingtoanaffectedoldersiblingwhorequireshematopoieticstemcell transplantation. Furthermore, the HLA matching can be combined with mutational analysis for genetic diseases in cases where the sibling is affected with this monogenic disorder and waiting for stem cell transplantation. Therefore, in those cases, embryos having mismatched HLA type or carrying genetic disorder were used for derivation of hESC lines. 2.2 Isolation and preparation of feeder cells As feeder cells both mouse embryonic fibroblast (MEF) and human foreskin fibroblast (HFF) wereusedduringisolationandlongtermcultureofhESClines.MEFswereisolatedfrom embryosofthe12-to14-daypregnantBALb/cmice(Conner,2000).Toisolatesinglecell suspension of MEF mouse embryos isolated from the sacrificed mice by cervical dislocation were dissociated into small pieces with scissor. Then dissociated tissues were trypsinized in 0.25% trypsin-EDTA (Gibco BRL; Invitrogen, Gaithersburg, MD, USA) for 15min to produce single cell suspension.Humanforeskinfibroblastswereisolatedfromcircumcisedtissuesof0-1yearoldmales. Cellisolationwasperformedasdescribedpreviously(Hovattaetal.,2003;Richardetal., 2002).Inabrief,followingtheisolationofdermisfromtheepidermisbyscissororrazor blade,tissuewasdissectedintosmallpiecesandthentrypsinizedin0.05%trypsin-EDTA (Gibco BRL) for approximately 1 h to dissociate into single cells.Both 25 cm2 and 75 cm2 culture flasks were used to culture HFF and MEF lines. These lines weregrowninfeedercellculturemedium,consistingof85%highglucoseDMEM(Gibco BRL), 10%FBS (Gibco BRL), 1% penicillin streptomycin-amphotericin (Biological Industries) and 2mM L-glutamine (Gibco BRL) at 37C with 5% CO2. Supportive medium was changed ineverythreedays.MEFlinescouldbeusedincultureofhESClinesupto6passages, whereas HFFs had supportive potential up to 15 passages.Mitoticinactivationoffeedercellswasperformedafterexposingfeedercellstoculture mediumcontaining10g/mlmitomycinC(Sigma-Aldrich,Poole,Dorset,UK)for2.5-3h. Inactivated cells were seeded on a 0.1% gelatin-coated at a concentration of 1.5 105 cells / ml. After 2 days incubation organ culture dishes were ready to use as feeder plates. Feeder plates could be used for the following 7 d.Derivation and Characterization of New hESC Linesfrom Supernumerary Embryos, Experience from Turkey 21 2.3 Isolation and long-term culture of hESC lines Blastocyst stage embryos, that were graded according to the Gardners scoring criteria, were processedforhESCisolationbyeitherimmunosurgeryordirectculture(Gardneretal., 2000;Findiklietal.,2005).Priortotheimmunosurgeryordirectculture,zonapellucidaof embryowasremovedbytheshort-termexposuretothe5IU/ml(finalconc.)pronase (Sigma)containingembryoculturemediumforupto5min.Immunosurgerywasapplied basedonthepreviouslypublishedprotocol(SolterandKnowles,1975).Afterlysisof trophoblastic cells, the resulted intact inner cell mass clumps were placed on feeder cells and cultured until to observe the primary hESC colonies.Inthedirectculturemethod,zonafreeblastocystsweredirectlyplacedonfeedercellsand cultured until the appearance of outgrowth, which lasted about 6 to 8 days. Then compacted outgrowthsincludingcellsofhESC-likemorphologyweremechanicallysplitintosmall clumps. The cell clumps were transferred on new feeder plates. The primary colonies were generally observed after about 5 to 7 days (Figure 1A-B).HESClineswereculturedat37Cin5%CO2inthecompletestemcell medium(CSCM) withthecompositionof85%Ko-DMEM(GibcoBRL),15%FBS(Hyclone,South,Logan, UT,USA),1penicillin/streptomycin/amphotericinB(BiologicalIndustries,Haemek, Israel), 1 non essential amino acid stock solution (Sigma), 0.1%2mM L Glutamine (Gibco A)B) C) D) Fig. 1. Establishment of hESC line on HFF and MEF feeder cells. Phase contrast microscopy of NS-10 line at different stages of development. A) The formation of outgrowth from the inner cell mass of blastocyst after direct culture of zona-free blastocyst on HFF. B) Cell clump, which was formed after mechanically dissociation of outgrowth, included primary hESC like cells. C) Circular primary NS-10 colony on MEF. D) Polarized colony morphology of NS-10 on HFF. Original magnifications: (A-D) X 200 Embryonic Stem Cells Basic Biology to Bioengineering 22BRL),4ng/mlbasicfibroblastgrowthfactor(bFGF)(Chemicon,Temecula,CA,USA),-mercaptoethanol (0.1 mmol/l; Sigma), and 0.1% insulin transferrin selenium complex (Gibco BRL). Culture media was changed daily.UndifferentiatedhESCcoloniesweresplitintosmallcolonypiecesmechanicallywiththe flame-drawnglasseveryafter7-8daysofculture.EnzymaticdissociationofhESCwasnot preferred in this study. Each hESC line was cultured at least up to fifteen passages and was cryopreservedbyvitrificationtechniqueaccordingtothepreviouslyreportedprotocols (Reubinof et al., 2001; Vanderzwalmen et al., 2003). Briefly, colonies were first mechanically splitintosmallpiecesandweresequentiallyvitrifiedintwosolutionsincludingdifferent concentration of DMSO and ethylene glycol.Cryopreserved hESC lines were warmed sequentially in solutions including 0.5M and 0.25M sucrose to control efficiency of vitrification and following warming techniques (Reubinoff et al., 2001).2.4 Karyotyping and immunocytochemistry of hESC lines G-banding technique was used to karyotype hESC lines. HESC colonies were first incubated with culture medium including 0.1 g / ml Colcemid (Biological Industries) for 2 h at 37 C ina%5CO2.Thencoloniesweresplitintosmallpiecesmechanicallyandincubatedin 0.075M KCI hypotonic solution for 17 minutes at 37C. Colonies were fixed with methanol-aceticacidsolution(3:1)andprocessedforG-bandinganalysis.Foreachlineatleast20 metaphases were analyzed for confirmation.Karyotypingofeachlinewasperformedseveraltimestoassesswhetherkaryotypeswere stable during their long term culture. Confirmation of genetic mutation in hESC line derived from affected embryo was performed by the same procedures applied for single blastomere mutational analysis.Surfaceexpressionmarkers(SSEA-3,SSEA-4,TRA-1-60andTRA-1-81),whichareunique forundifferentiatedhESClines,wereimmunochytoemicallyanalyzedaccordingtothe manufacturersinstructions(Chemicon)byusingflouresceinisothiocyanate(FITC)-conjugatedgoatanti-mousesecondaryantibody(anti-IgG)(SantaCruzBiotechnology, California, USA). Negative controls were performed by addition of phosphate buffer saline instead of the primary antibody. All the other reagents were the same as in the slides run for specificantibodies,exceptnucleusofcellswerestainedby4',6-diamidino-2-phenylindole (DAPI) for visualization.Alkalinephosphataseactivity(Chemicon)ofhESClineswasdetectedbyusingthe ChemiconAlkalinePhophataseDetectionkit(Chemicon).ExpressionsofOCT-4and housekeepinggene,glyceraldehyde-3phosphatedehydrogenase(GADPH),geneswere detectedbyreversetranscriptasePCR(RT-PCR).Briefly,extractionofRNAfrom undifferentiatedhESCsandsynthesisofcDNAwerecarriedoutbyusingRneasyMiniKit (Qiagen Gmbh, Strasse, Germany) and Sensiscript RT Kit (Qiagen), respectively. Then, PCR and following analysis were performed according to the protocol of Amit et al (Amit et al., 2002). 2.5 Differentiation potential of hESC lines ThedifferentiationabilityofhESClineswereanalyzedonlybyinvitro.Forinvitro differentiation,embryoidbodies(EBs)werefirstgeneratedaccordingtopreviously published protocol (Itskovits-Eldor et al., 2000; Carpenter et al., 2003). In a brief, small pieces Derivation and Characterization of New hESC Linesfrom Supernumerary Embryos, Experience from Turkey 23 ofundifferentiatedhESCcoloniesweretransferredintothenonadherentbacterialpetri dishesandwereculturedinCSCMwithoutbFGFfor8-10days.TheresultingEB,which theoreticallycomprisethreeembryonicgermlayers,werethenplatedonto0.1%gelatin coated plastic petri dishes and cultured for long-term for spontaneous differentiation.DifferentiationfeaturesofhESClineswasexaminedunderphasecontrastmicroscopeand differentiatedcellswereanalyzedbyimmunocytochemicalstainingwithmarkersfor endoderm(cytokeratin18,specificmarkerforepithelialcells,Chemicon),mesoderm (troponin I, specific for cardiac muscle; Chemicon) and ectoderm (Nestin, specific marker for progenitorofneuron,andMAP2AB,specificmarkerformatureneuron;Chemicon) according to manufacturers instruction.Additionally,intheselines(OZ,OZ-1andOZ-2),rhythmicallybeatingofcardiomyocytes withinspontaneouslydifferentiatingembryonicstemcellswerefurtheranalyzedby transmission electron microscopy (TEM). Briefly, cell clumps with spontaneous contractions weregentlyremovedfromthecultureplateandfixedin2%glutaraldehydein0.1 mol/sodiumcacodylatebuffer(pH7.4)for2hours.Secondaryfixationwasperformedin 1%OsO4inthesamebufferfor1.5h.Thegridsweredehydratedingradedethanoland embedded in Epon 812. The very thin sections about 80 nm were cut and stained with lead citrate for 8 min in order to identify the cellular structures of cardiac muscle cells. 3. Results of hESC study ExperienceofhESCfromMemorialHospitalcomprisethreephrases;derivationoffirst hESC lines in Turkey, which was reported previously (Findikli et al., 2005), using HFF as a feedercellinsteadofMEFtoderivenewhESClinesandderivationofhESClinesfrom donated embryos from PGD cycles (Candan & Kahraman, 2010).In the first phase, nine hESC lines, which were named NS-1, NS-2, NS-3, NS-4, NS-5, NS-6, NS-7,NS-8andMINE,werederivedfrom26donatedblastocystsstagehumanembryos with a 34.6% success rate (Table 1). Twenty blastocysts were spare IVF/ICSI embryos and 5 hESClines(NS-1,NS-2,NS-3,NS-4andMINE)werederivedfromtheseembryos.The remaining6embryoshadmismatchedHLAtypeand,thereforewerenoteligiblefor transferringinPGDcycle.Fromtheseembryos,4hESClines(NS-5,NS-6,NS-7andNS-8) No.of blastocysts No. of stem cell line, n, (%) Names of derived hESC lines IVF/ICSI embryos 308, (27) NS-1,2,3,8, MINE, and OZ, OZ-1, OZ-2 PGDfor single gene disorder81, (13)OZ-8 PGDfor single gene disorder & HLA typing 64, (67)*NS-4, 5, 6, and 7 PGD for chromosomal screening427, (17) OZ-3, OZ-4, OZ-5, OZ-6,OZ-7, NS-9, NS-10 Immunosurgery method154, (27)NS-1,2,3,4 Direct culture method7616, (21) Table 1. A total number of embryos used for hESC lines and overall outcomes Embryonic Stem Cells Basic Biology to Bioengineering 24werederived.Fourlines(NS-1,NS-2,NS-3,NS-4)outof9hESClineswereobtainedby immunosurgeyandremaining5lineswerederivedbydirectculture(Table1).Twocell lines(NS-1andNS-2)werespontaneousdifferentiatedduringtheirfirstdaysofinvitro culture. InthesecondphaseofhESCresearchinIstanbulMemorialHospital,asanalternativeto MEF,HFFwasusedasafeedercellforestablishmentandlong-termcultureofnewhESC lines.ThreehESClines(OZ,OZ-1andOZ-2)werederivedfrom10blastocyststagespare IVF/ICSI embryos by the direct culture technique with a 30% success rate (Table 1). Unlike to circular colony morphology of hESC colonies on MEF, hESC colonies on HFF had angular shaped morphology, due to the polarity of HFF cells (Figure 1 C-D).Inthefinalphase,followingPGD,embryosdiagnosedashavingchromosomal abnormalities andsinglegenemutationswereusedtoestablishhESC lines(Table1). Forty twoblastocystswithdifferentchromosomalaneuploidiesweredirectlyplacedeitheron MEF or on HFF. From those embryos 7 hESC lines (OZ-3, OZ-4, OZ-5, OZ-6, OZ-7, NS-9 and NS-10)werederived(Table1and2).Ofthese7hESClines,oneline(OZ-3)wasderived frombiopsiedembryowhosediagnosiswassuspicious.Althoughchromosomalcontentof biopsiedblastomerefromthisembryowasidentifiedasabnormal(trisomy15)byFISH, because of the fragmentations in nuclear structure of blastomere, we could not interpret the result exactly and thereby assumed it as an abnormal (Candan & Kahraman, 2010). Threeembryosdiagnosedascarryingcysticfibrosisand5embryoswithbeta-thalassemia wereusedtoisolatehESClineswithgeneticdisorder.However,onlyonehESC(OZ-8), which had a single gene mutation causing beta-thalassemia, was isolated successfully. Only 4hESClineswereisolatedbyimmunosurgeryandtheremaininghESClineswerederived after direct culture of blastocysts on feeder cells (Table 2). Following to either direct culture orimmunosurgery,thedevelopingthreedimensionaloutgrowthsfromICMweresplit mechanicallyintosmallclumpsandtransferredontonewfeederplate.Durationfor successful derivation of first primary hESC colonies was ranged 15 to 20 days, based on the quality of inner cell mass of blastocysts, and application of isolation techniques properly. FollowingthefirstseveralpassagesofprimaryhESCcolonies,flatcoloniesofcellswitha distinguishablecompactedcolonystructure,welldefinedcolonyborderandcellular morphologywithhighernucleustocytoplasmratioandprominentnucleoliwereobtained (Figure 1C-D).Regardless of type of feeder cells, these unique colony features were similar in all hESC. Each of hESC lines were passaged mechanically every after about 7-8d for more than15passages(Table2).WhilepassaginghESCcolonies,spontaneouslydifferentiated cells,whichwereobservedfrequentlyinthecentralorintheperipherypartofcolonies, were always removed mechanically to maintain undifferentiation state of colonies.3.1 Unique features of hESC lines AllhESClineswerecharacterizedforcellsurfaceexpressionmarkers,whichareuniqueto undifferentiatedhumanembryonicstemcells.AsshowninFigure2A,establishedhESC linesrepresentedahighlevelofalkalinephosphatesactivity.Furthermore, immunocytochemical staining revealed that derived 18 hESC lines were positive for SSEA-3, SSEA-4,TRA-1-60andTRA-1-81(Figure2B).Negativecontrolslidesof immunocytochemicalstainingshowedthatprimaryantibodiesspecificallyboundtothe certain surface antigens (Figure 2C).Derivation and Characterization of New hESC Linesfrom Supernumerary Embryos, Experience from Turkey 25 NS-3NS-4NS-5NS-6NS-7NS-8NS-9NS-10MINE Embryo source IVF/ICSI IVF/ICSIPGDPGDPGDPGDPGSPGSIVF/ICSI Embryo grades 4BB3BA4AA4AA4AB3AB4BB4BA4BB PGD/PGS results -- HLA mismatched HLA mismatched HLA mismatched HLA mismatched Tetraploid Monosomy-13 - Feeder cellMEFMEFMEFMEFMEFMEFMEFMEFMEF Alkaline phosphatase +++++++++ Immuno-staining +++++++++ SSEA-3+++++++++ SSEA-4+++++++++ TRA-1-60+++++++++ TRA-1-81+++++++++ OCT-4+++++++++ In vitro differentiation +++++++++ Max. passage no. of lines 356039202222212849 Karyotype46 XY46 XX46 XX46 XY46 XX46 XX46 XY46 XX46XX OZOZ-1OZ-2OZ-3OZ-4OZ-5OZ-6OZ-7OZ-8 Embryo source IVF/ICSI IVF/ICSIIVF/ICSIPGSPGSPGSPGSPGSPGD Embryo grades 4AA3AA4BB4BB4AB3BB4AC4BB4AB PGD/PGS results --- Undiagnosed embryo Trisomy 21 Monosomy15+Trisomy 21 Monosomy 16+ Trisomy 16 Monosomy 16 Beta-Thalassemia Type of feeder cell HFFHFFHFFHFFHFFHFFMEFMEFMEF Alkaline phosphatase +++++++++ Immuno-staining +++++++++ SSEA-3+++++++++ SSEA-4+++++++++ TRA-1-60+++++++++ TRA-1-81+++++++++ OCT-4+++++++++ In vitro differentiation +++++++++ Max. passage no. of lines 191515221720221818 Karyotype46 XY46 XX46 XX46 XX46 XX46 XY46 XX46 XY46 XX Table 2. Unique features of 18 hESC lines. (+) represents that HESC line is shown to be positive for those expression markers. Embryonic Stem Cells Basic Biology to Bioengineering 26 A)B)C) D)E) Fig. 2. Unique features and karyotyping of OZ-6 hESC line. A) alkaline phosphatase staining, B) SSEA-3, SSEA-4, TRA-1-60, TRA-1-81and C) negative control for immunostaining ofhESC lines (cells were stained by DAPI for visualization). Expression of OCT-4 and housekeeping gene GADPH in hESC lines (OZ, OZ-1-7). E) Karyotype analyze of OZ-6 line in passage 5, 46XX. Original magnifications: (A-C) X 200. Abbreviations: OCT-4, octamer-4 and GADPH, glyceraldehyde-3 phosphate dehydrogenase. OurhESClineswerenotanalyzedforSSEA-1expression,aspecificmarkerformouse embryonic stem cells. In consistent with the previous reports, expression intensity of SSEA-3 among hESC lines was variable and comparably weaker than SSEA-4 which was consistent andexpressedhigherinallhESClines(Ohetal.,2005).Additionally,OCT-4expressions werehigherinallundifferentiatedhESClineswhencomparedtoexpressionlevelof housekeeping gene GADPH (Figure 2D).TestingdifferentiationcapacityofeachhESClinebyembryoidbodyformationinvitro revealed that these lines were capable of differentiating into various cell types derived from thethreeembryonicgermlayers(Figure3).Spontaneouscontractingcellclumps,neural rosettestructures,neural-likecells,epitheliallikecellswereobservedunderphase microscopeandthesedifferentiatingcellswerediscriminatedbyimmunocytochemically (Figure3B).However,celllinesshowedarelativelydifferentcapacityortendencyto differentiate into certain type of cell lineage. Rhythmicallycontractionsincellclusterswerestartedapproximatelyaftertendaysfrom platingEBsonbacterialcultureplatesandkeptcontinuinguptosixweeks.Duringthis Derivation and Characterization of New hESC Linesfrom Supernumerary Embryos, Experience from Turkey 27 period,beatingcellclusters,belongingtoOZ,OZ-1andOZ-2hESClines,werefurther analyzedbytransmissionelectronmicroscopy.Thereby,sarcomere,intercalateddiscand myofibril structures were well defined in cardiac muscle cells by TEM analysis (Figure 3E).Followingvitrificationandthawingprocedures,allcelllinescouldretaintheirunique properties; long-term extension and pluripotency capacity in vitro. A)B)C) D)E) Fig. 3. In vitro differentiation of NS-10 hESC line by embryoid body formation A) Phase contrast microscopy of 10-day EBs. Spontaneously differentiating cells B) neuron-like cells, positive for neuron specific nestin, C) epithelial-like cells, positive for cytokeratin 18 (green) and D) cardiac muscle cells, positive for troponin I. E) TEM photographs of beating cardiomyocytes. (A) X 100, (B-D) X200, (E) X12000. 3.2 Karyotyping and genetic analysis of hESC lines KaryotypinganalysisofallhESClineswereperformedatthe5thpassages.HESClines, whichwerederivedfromsupernumeraryembryosafterIVF/ICSIcyclesandfromPGD embryos, having genetic disorder and/or having mismatched HLA, had normal karyotypes (Figure 2E and Table 2).HESC lines derived from chromosomally abnormal embryos were first analyzed by FISH at 1stpassagewhethertheyhaddetectedchromosomalabnormality.Surprisingly, chromosomalabnormalitieswerenotconfirmedintheselines.Contrarily,analyzed chromosomeswereeuploidinnumber.Furtherconfirmationwasperformedby Embryonic Stem Cells Basic Biology to Bioengineering 28karyotyping at 5th passages of these lines. In consistent with results after FISH at 1st passage, karyotypinganalysisrevealedthatincontrasttothediagnosisafterPGDtheirkaryotypes were normal (Table 2).KaryotypingofallhESClineswereperformedfurtherpassageswhethercelllinesretained normal karyotypes. All hESC lines had stable karyotypes.MutationinOZ-8hESCcelllinewithbetathalassemiadisorderwasconfirmedbyPCRand subsequent sequencing procedures at passage 6. The homozygote single nucleotide transition (guanine to adenine nucleotide transition) in second exon of beta-globin gene was detected. 4. Discussion Humanembryonicstemcellisoneofthemostcontradictoryscientificissuessinceitwas first reported by Thomson in 1998. Obviously, this ongoing dispute has been arisen from the use of human embryo for derivation of hESC. In regard to that concern, alternative methods havebeenproposedbyseveralresearches.However,spareembryosgeneratedfor reproductiveandtherapeutictreatmentsstillremainasa mainsourceforhESCderivation. Therefore,registeringallexistinglinesinadatabase,likestemcellbank,maydecreasethe necessitiestoderivenewhESClinesworldwideandeventuallyethicalconcernmaybe alleviated among the public. In that regard, all these hESC lines, which had been established andcharacterizedinIstanbulMemorialHospitaluntilrulingonbanonhESCresearchby Turkish Health Ministry, were registered to European hESCreg in 2008.During the hESC derivation study, 86 donated embryos, which were considered insufficient fortransferandcryopreservationafterIVF/ICSIcyclesandwerediagnosedashaving genetic disorder or chromosomal aneuploidies and having mismatched HLA type after PGD cycles, were used. ThederivationefficiencyofhESClineswas 20%andsuccessratewasdirectlyrelatedwith thequalityofblastocysts.Four(NS-1,NS-2,NS-3andNS-4)outof20hESClineswere successfully derived from 15 blastocysts after immunosurgery. However, two of these lines werespontaneouslydifferentiatedattheearlynumberofpassages.Remaining16hESC lineswereisolatedthroughdirectcultureofwholeblastocystsonfeedercells.Thesetwo methods had comparable success rates (27% vs 21%, p>0.05).Alllinesdescribedinthischapterhadsimilarcolonyandcellularmorphology.Theselines expressed unique cell surface expression markers, including SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81.TheyalsohadhighlevelofalkalinephosphataseactivityandexpressedOCT-4 gene,whichkeepspluripotencyofhESClinesduringlong-termculture(Figure2). Moreover,these18hESClineswereproventohavepluripotentcapacityinvitrobyEBs formation(Figure3).Therefore,theselineshavesimilaruniquepropertiesaspreviously reported existing hESC lines.Although all lines were capable of differentiating into derivatives of three embryonic germ layers,differentiatingcharacteristicwasvariedamonglines.Theprevalenceof differentiating cardiomyocytes was higher in EBs generated from OZ-3 line, whereas higher percentage of neuron-like cells were observed in EBs generated from OZ line. The presence of specific cell lines at a various degree in differentiating cell cultures of hESC lines may be attributedtothedevelopmentalstageofembryousedforderivation,genomicand epigenetic differences. Derivation and Characterization of New hESC Linesfrom Supernumerary Embryos, Experience from Turkey 29 InPGDforchromosomalscreeningcycles,embryosdiagnosedashavingchromosomal abnormalitieswerelikelytobediscardedandnottobeconsideredeligiblefortransfer.In consistentwiththepreviousresults,inwhichitwasaimedtoderivechromosomally abnormalhESClinesforinvestigationofvariousaspectsofearlyembryonicdevelopment, all our hESC lines derived from those embryos had normal karyotypes (Munne et al., 2005; Lavon et al., 2008). As stated in these studies, this unexpected result has been suggested to self correction of embryos during long-term in vitro culture (Hazan et al., 2008; Lavon et al., 2008).As a result of proposed mosaicism and trisomy rescue mechanisms in developing embryos, chromosomal self correction may be occurred. In the mosaic embryos, it was suggested that euploidblastomerescangrowpreferentiallytoabnormalcellsduringearlyembryogenesis oralternatively,thesenormalcellswerepreferentiallyallocatedtotheinnercellmass. Therefore,embryomaybesubjecttoselfcorrectionmechanismandmayevolvetoafetus witheuploidchromosomalnumber.Intrisomicrescuemechanism,ithasbeenspeculated thatembryoscanbecorrectedintermsofchromosomalnumberthroughanaphase-lag, nondisjunction,orchromosomaldemolition.However,thesemechanismsarestill assumptionsandexactsmechanismsshouldbeprovenbyfurtherstudies(Hazanetal., 2008).Furthermore,wespeculatedthatthelong-termculturemaynotonlyinducethechangein chromosomalcontentbutalsomayresultinvariousgeneticorepigeneticmodificationsin hESClineswhichcouldeventuallyimpairtheirpluripotentandselfrenewalcapacity. However,duetoinuredrestrictiononhESCresearch,thesederivedlinescouldnotbe analyzed regarding to these aspects (Candan et al 2010).Embryos,havingsinglegenemutations,areofgreatimportance,whileconsideringasa potentialsourceingeneticbasedresearchesonunderstandingthemechanismsofdisease anddevelopingnewdrugs(Pickeringetal.,2003;Verlinskyetal.,2005).Wederivedone hESClinehavingmutation,thatcausebetathalassemiadisorder,from5donatedembryos afterPGD.Thiscelllinecanprovideasacellsourcetostudythepathologyofbeta thalassemia and its effects on different cell types.5. Conclusions HESCresearcheshavebeenincreasingcontinuouslysinceitwasfirstisolatedand characterized.AlthoughseveralproblemsincludingderivationclinicalgradehESClines, risk for teratoma formation, HLA incompatibility of cells and establishment of well defined differentiationprotocolsofhESClinestocertaincelltypeshavebeennotbeensolved efficiently, there is still hope for coming day in which hESC lines will be used effectively in cellreplacementtherapiestotreatsuchdevastatinghumandiseases;myocardialinfarcts, spinalcordinjuriesanddiabetes.Therefore,hESClinesreportedinthischaptercouldbe potentialcellsourceforin-vitrostudiesinvolvingbasicmolecularandstemcellbiology.. Moreover,asinourstudy,derivationofhESCfromPGDembryoswithintrinsicgenetic contentandthediseaseprofilecouldbeanextremelyvaluablesourceforresearchon genetic diseases. Inthischapter,itisaimedtosummarizedestablishmentprotocolsandfeaturesoffirst reported hESC lines in Turkey. Turkey is among the countries in which earlier hESC studies Embryonic Stem Cells Basic Biology to Bioengineering 30wereestablishedfirst.Today,however,TurkeyisoneofthecountriesinwhichhESC researcheswereprohibitedbyGovernment.Unfortunately,sofarnewlegislationor regulation has not been declared. However, establishment of new guideline is on the agenda ofgovernmentalinstitutionsnowadays.Recentliftingofbanonthefederalfundingfor hESC research in US and given permission on first clinical trial of hESC may, in fact, affect thepoliticalopinionandhopefullymayinureregulations,whichresumehESCresearches under the control of authority and new guidelines in Turkey.6. Acknowledgments We would like to specifically thank to Necati Findikli, Oya Akcin and Ayla Eker Sariboyaci for their previous contributions on the derivation and characterization of hESC lines and all IVFstafffortheirkindlyhelpinhumanembryocultureandsupport.Additionally,weare verythankfultoallGeneticlaboratorystaffforPGDanalysisandkaryotypingallhESC lines. Study was supported and funded by Istanbul Memorial Hospital.7. References AmitM,MarguletsV,SegevH.(2002).Humanfeederlayersforhumanembryonicstem cells. Biology of Reproduction, 68, 2150-2156 Candan NZ & Kahraman S. (2010). Establishment and characterization of human embryonic stem cell lines, Turkey perspectives. In Vitro Cell.Dev.Biol.Animal, 46, 345355 CarpenterMK,RoslerE,RaoMS.(2003).Characterizationanddifferentiationofhuman embryonic stem cells. Cloning and Stem Cells, 5, 79-88 Conner DA. (2000) Mouse embryo fibroblast (MEF) feeder cell preparation. 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