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Stem Cells

Edited byRobert A. Meyers

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Stem Cells

From Biology to Therapy

Advances in Molecular Biology and Medicine

Edited byRobert A. Meyers

Volume 1

Editor

Dr. Robert A. MeyersEditor in ChiefRAMTECH LIMITED122, Escalle LaneLarkspur, CA 94939USA

Cover

Image of human iPSCs in feeder-free culture con-ditions. For more details see figure 1 in chapter2 ‘‘Induced Pluripotent Stem Cells’’ authored byKazutoshi Takahashi and Shinya Yamanaka.

Limit of Liability/Disclaimer of Warranty: Whilethe publisher and author have used their best ef-forts in preparing this book, they make no repre-sentations or warranties with respect to the accu-racy or completeness of the contents of this bookand specifically disclaim any implied warrantiesof merchantability or fitness for a particular pur-pose. No warranty can be created or extended bysales representatives or written sales materials.The Advice and strategies contained herein maynot be suitable for your situation. You should con-sult with a professional where appropriate. Nei-ther the publisher nor authors shall be liable forany loss of profit or any other commercial dam-ages, including but not limited to special, inciden-tal, consequential, or other damages.

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V

Contents

Preface ix

Volume 1

Part I Basic Biology 1

1 Epigenetic Regulation in Pluripotent Stem Cells 3Lin Liu and Lingyi Chen

2 Induced Pluripotent Stem Cells 41Kazutoshi Takahashi and Shinya Yamanaka

3 Naturally Occurring Adult Pluripotent Stem Cells 63Henry E. Young and Asa C. Black Jr

4 Spermatogonial stem cell (SSCs) system 95G. Ian Gallicano and Shenglin Chen

5 Stem Cell Dormancy: Maintaining a Reserved Population 119John M. Perry, Xi C. He, Ryohichi Sugimura and Linheng Li

6 Stem Cells in the Adult Brain: Neurogenesis 133Michael A. Bonaguidi, Guo-li Ming, and Hongjun Song

7 Embryonic Stem Cells 151Mahendra Rao

Part II Laboratory Methods 175

8 Cardiomyocytes from Human Embryonic Stem Cells 177Xiu Qin Xu, Manasi Nandihalli, Kar Tong Tan, and William Sun

9 Cloned Mice from Adult Stem Cells 209Haruko Obokata and Teruhiko Wakayama

VI Contents

10 Cloned Mice from Embryonic Stem Cells 233Chong Li and Teruhiko Wakayama

11 Haploid Embryonic Stem Cells 255Ni Hong and Yunhan Hong

12 Muscle Stem Cells: Their Discovery, Properties, and In-Vitro Manipulation 273Sean McFarland, Ioanna Pagani, and Irina Conboy

13 Nuclear Transfer for Cloning Animals 299Andras Dinnyes, Xiuchun Cindy Tian, and Bjorn Oback

14 Induction of Pluripotent Stem Cells from Umbilical Cord Blood 345Kejin Hu and Igor Slukvin

15 Development and Renewal of Ventricular Heart Muscle from IntrinsicProgenitor Cells 371William C.W. Chen and Bruno Peault

Volume 2

Part III Stem Cell Therapy 389

16 Gene Therapy of Genetic Diseases of Blood Cells 391Gabriela Kuftinec, Jennifer Wherley and Donald B. Kohn

17 Mesenchymal Stem Cells Characteristics, Niches, and Applicationsfor Cell Therapy 429Joni H. Ylostalo and Thomas J. Bartosh

18 Stem Cells and Parkinson’s Disease 471Emma Lane, Maria Sundberg, and Jan Pruszak

19 Stem Cell-Based Approaches to Spinal Cord Injury 503Alexa L. Reeves and Hans Keirstead

20 Therapeutics against Cancer Stem Cells: Targeting the Root of Cancer 521Kristen M. Smith and Catriona H. M. Jamieson

21 Translating Stem Cells to the Clinic: From Modeling Disease to CellularProducts 573Emmanuel Nivet, Ignacio Sancho-Martinez, and Juan Carlos Izpisua Belmonte

Part IV Stem Cells and Disease 601

22 Cancer Stem Cells 603Mei Zhang and Jeffrey M. Rosen

Contents VII

23 Normal and Neoplastic Stem Cells 627Axel Schulenburg and Brigitte Marian

24 Prostate Tissue Stem Cells and Prostate Cancer Progression 655Collene R. Jeter and Dean G. Tang

25 The Stem Cell Niche and Its Role in Self-Renewal, Aging, and Malignancy 677Peter Breslin S.J., Andrew Volk and Jiwang Zhang

26 Stem Cells and Colon Cancer 727Simone Di Franco, Antonina Benfante, Flora Iovino, Sebastiano Bonventre,Francesco Dieli, Giorgio Stassi and Matilde Todaro

Index 751

IX

Preface

Five Nobel Laureates are associated with this book: the Encyclopedia of MolecularBiology and Molecular Medicine Board members, Sir Martin Evans, who won a Nobel inPhysiology or Medicine in 2007 for isolating embryonic stem cells and then growingthem in culture; as well as David Baltimore, Gunter Blobel, and Phil Sharp; andnow contributing author Shinya Yamanaka, whose 2012 Nobel Prize for Physiology orMedicine was awarded for reprogramming mature cells to become pluripotent stemcells. Professor Yamanaka’s chapter on Induced Pluripotent Stem Cells, written for ourbook, forms a central component, tying together all aspects of stem cells biology andapplications.

In his chapter, Professor Yamanaka points out the central issues associated withclinical application of stem cells. ‘‘Because pluripotent stem cells can theoreticallydifferentiate into all cell types in the body, applications for cell therapy are expected.However, it is unclear when ES and/or iPS cells would be effective for cell therapy. Themost common issue preventing the clinical use of ES and iPS cells is the risk of teratomaformation after transplantation. Residual undifferentiated cells in differentiated cellcultures used for a transplant can cause a teratoma, and should be removed before use.Both effective methods for the removal of undifferentiated cell contamination, such asthe use of flow cytometry, and more efficient procedures for differentiation are beingdeveloped’’. Beyond these, there are additional important potential hurdles to clinicalapplications, including: the need for xeno-free stem cell lines, epigenetic memory andaberrant genetic errors which may be higher for iPS cells as compared with ES cells. Allof these factors are covered in detail in our chapters.

The 26 detailed chapters, prepared by leaders in the field, cover the basic biology ofstem cells, laboratory methods, stem cells and disease and stem cell therapy approachesand translation to the clinic for treatment of many diseases including Parkinson’sdisease, spinal cord trauma, diseases of blood cells, and many types of cancer as wellas regeneration of cardiac and other muscle tissue. The chapter on ‘‘Translating StemCells to the Clinic: from modeling disease to cellular products’’ by Juan Carlos IzpisuaBelmonteand his team at the Salk Institute presents the state and future of stem cellclinical applications including 1) ‘‘disease in a dish’’ laboratory substrates providingpatient-specific iPS cells which can be employed for disease modeling and drugdevelopment; 2) the possibility to generate every desired cell type in vitro for restorationof any injury from lost tissue by cell replacement and gene-editing technologies that

Stem Cells: From Biology to Therapy, Advances in Molecular Biology and Medicine, First Edition. Edited by Robert A. Meyers.© 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

X Preface

efficiently target both and 3) pluripotent cells as well as adult stem cells giving rise tothe possibility for gene-correction followed by autologous transplantation which couldbe employed for the actual cure of monogenic inherited diseases in patients.

Our team hopes that you, the reader, will benefit from our hard work, finding thecontent useful in your research and educational. We wish to thank our Managing Editor,Sarah Mellor as well as our Executive Editor, Gregor Cicchetti for both their advice andhard work in the course of this project.

Larkspur, California, March 2013

Robert A. MeyersEditor-in-Chief

RAMTECH LIMITED

1

Part IBasic Biology


3

1Epigenetic Regulationin Pluripotent StemCells*

Lin Liu and Lingyi ChenNankai University, The Ministry of Education, Key Laboratory of BioactiveMaterials, Laboratory of Stem Cells and Developmental Biology, College of LifeSciences, 94 Weijin Road, Tianjin 300071, China

1 Introduction 6

2 DNA Methylation 7

3 Histone Modifications and Histone Variants 11

4 Higher-Order Structure of Chromatin 16

5 X-Chromosome Inactivation 18

6 Regulation of ESC Pluripotency and Differentiation by miRNAs 19

7 Telomere Function and Genomic Stability in ESCs 21

8 Imprinting and ESC Stability 23

9 Epigenetic Interconversion among Mouse ESCs, EpiSCs, and Human ESCs 24

10 Summary 26

References 29

∗This chapter has previously been published in: Meyers, R.A. (Ed.) Epigenetic Regulation and Epigenomics,2013, ISBN 978-3-527-32682-2.


4 Epigenetic Regulation in Pluripotent Stem Cells

Keywords

Embryonic stem cells (ESCs)Pluripotent cells derived and cultured from the inner cell mass of blastocysts orfrom blastomeres of early embryos. These cells are able to proliferate and self-renewindefinitely, and to maintain undifferentiated states under correct culture conditions,while retaining the potential to differentiate into all types of cell in the body.

Induced pluripotent stem cells (iPSCs)By ectopic expression of a few transcription factors (e.g., Oct4, Sox2, Klf4, and c-Myc),differentiated cells are reprogrammed and give rise to ESC-like cells. The latter are alsopluripotent and able to self-renew; hence, they are termed iPS cells (iPSCs).

TotipotencyCells sufficient to form an entire organism by themselves. Examples are zygotes andfew cells in early-cleavage embryos in mammals.

PluripotencyThe developmental potential of a cell to differentiate into all types of cell in the body.The most stringent test for developmental pluripotency is the generation of offspringcompletely from ESCs/iPSCs by tetraploid embryo complementation, or by four- toeight-cell embryo injection. A less stringent test is the production of germline-competentchimeras by either diploid blastocyst or four- to eight-cell embryo-injection methods.

ReprogrammingAn increase in the developmental potency from a differentiated to an undifferentiatedstage; also referred to as dedifferentiation in some instances.

EpigeneticsChanges in gene function that are mitotically and/or mitotically inheritable, and thatdo not entail a change in DNA sequences. Epigenetic information includes changesin gene expression by DNA methylation, microRNAs, histone modifications, histonevariants, nucleosome positioning, and higher-order chromatin structure.

DNA methylationThe addition of methyl groups to DNA, mostly at CpG sites, to convert cytosine to5-methylcytosine. DNA methylation usually represses gene expression.

HistoneProteins enriched in positively charged amino acid residuals, found in eukaryoticcell nuclei. These proteins package and order the DNA into structural units callednucleosomes.

Epigenetic Regulation in Pluripotent Stem Cells 5

NucleosomeThe basic unit of chromatin. In a nucleosome, a DNA fragment of 147 bp is wrappedaround spools of histone proteins.

Histone modificationModification in the entire sequence of histones, particularly at the unstructuredN-termini (‘‘histone tails’’), including acetylation, methylation, ubiquitylation, phospho-rylation, and SUMOylation. Histone acetylation or the inhibition of histone deacetylationis generally linked to transcriptional activation.

ImprintingThe allele-specific expression of a small subset of mammalian genes in a parent-of-originmanner (either the paternal or maternal is monoallelically expressed). The establishmentof genomic imprinting is controlled mostly by DNA methylation, and also by histonemodifications, noncoding RNAs, and specialized chromatin structures. Aberrantimprinting disrupts fetal development, and is associated with genetic diseases, somecancers, and a number of neurological disorders.

X chromosome inactivationIn each mammalian female cell, one of the two X chromosomes is transcriptionallyinactivated to compensate any X-linked gene dosage effect between male (XY) andfemale (XX).

TelomereRepeated DNA sequences (TTAGGG)n and associated protein complexes that cap theend of chromosomes to maintain genomic stability. Telomere shortening is associatedwith cell senescence and organism aging, and also cancer.

TelomeraseAn enzyme that specifically adds telomeric repeats de novo during each cell division,and is composed of two major components: a telomerase RNA template component(Terc); and Tert, a reverse transcriptase as a catalytic unit. ESCs acquire high telomeraseactivity to maintain telomere length.

Epigenetic stability is tightly controlled in embryonic stem cells (ESCs) forself-renewal and pluripotency, but is changed during the differentiation ofESCs to various cell lineages. The derivation and culture of ESCs also induceepigenetic alterations, which could have long-term effects on gene expressionand the developmental and differentiation potential of ESCs. Developmentaland cancer-related genes, and also imprinted genes, are particularly susceptible


to changes in epigenetic remodeling, particularly DNA methylation, microRNA(miRNA), and histone modification. In recognition of the tremendous potential ofESC/induced pluripotent stem cells (iPSCs) in regenerative medicine, the epigeneticinstability must be closely monitored when considering human ESCs/iPSCs fortherapeutic and technological applications.

1Introduction

Embryonic stem cells (ESCs) are de-rived from the inner cell mass (ICM)of blastocysts [1–3]. Under the correctconditions, ESCs are able to prolifer-ate indefinitely. A group of genes isrequired for ESC self-renewal. Thesepluripotency-associated genes, which arehighly expressed in ESCs, are mostlydownregulated upon ESC differentiation[4–6]. Among these genes, three transcrip-tion factors – Oct4, Sox2, and Nanog – playpivotal roles in the maintenance of pluripo-tency [7–10]. These three transcriptionfactors regulate themselves and crossreg-ulate each other, thus, forming a coreregulatory circuitry for pluripotency [11].Moreover, Oct4, Sox2, and Nanog acti-vate many pluripotency-associated genes,while suppressing the expression of genesthat encode developmental regulators[11–14].

Importantly, ESCs also have the po-tential to differentiate into all types ofcell in the organism. Typically, ESCsform embryoid bodies (EBs) in vitro,which resemble early embryogenesis [1,2]. Following the subcutaneous injectionof ESCs into immunodeficient mice, thecells develop into a benign tumor (a ter-atoma), which consists of cells from threegerm layers [2]. When injected into blas-tocysts, ESCs contribute to embryonicdevelopment and give rise to chimericanimals; subsequently, through germlinetransmission in chimera, the genetic

information from the ESCs can be passedto the progeny [15]. Most importantly, livepups composed totally of ESCs can bederived by the tetraploid complementationor four- to eight-cell embryo injection[16–18].

These unique properties of ESCs –notably, self-renewal and differentiationpotential – are referred to as pluripotency.The self-renewal of ESCs can provide anunlimited supply of cells, whereas thedifferentiation potential of ESCs allowsany desired type of cell to be derived.Consequently, ESCs hold great promisefor the future development of regenerativemedicine.

Epigenetic events are defined as changesin gene function that are mitoticallyand/or miotically inheritable and thatdo not entail a change in DNA se-quences. Epigenetic information includesDNA methylation, histone modifications,histone variants, nucleosome position-ing, and higher-order chromatin structure.The activities of many enzymes, includ-ing DNA methyltransferases (DNMTs),histone demethylases, histone methyl-transferases (HMTs), histone deacety-lases (HDACs), histone acetyltransferases(HATs), and chromatin-remodeling en-zymes, are involved in the regulation ofepigenetics [19]. Moreover, as ESCs anddifferentiated cells share the same ge-netic materials, the pluripotency of ESCs ismainly attributed to the unique epigeneticregulation within ESCs.


2DNA Methylation

DNA methylation, which serves as a keyepigenetic event in the regulation of geneexpression, is in dynamic mode during de-velopment. Typically, the paternal genomeis actively demethylated in the male pronu-cleus shortly after fertilization, and this isfollowed by a passive DNA demethylationof the maternal genome [20]. Global denovo methylation increases rapidly in theblastocysts, the earliest stage of differen-tiation into trophectoderm cells, and alsoin the ICM, from which the ESCs areisolated. The reprogramming of promotermethylation represents one of the key de-terminants of the epigenetic regulationof pluripotency genes [21]. The methy-lation of DNA occurs on the cytosine inmost cytosine-guanine dinucleotide (CpG)islands in mammalian genomes, andis carried out by various DNMTs. Forexample, DNMT1 prefers hemimethylatedCpGs as a substrate, and maintains thepre-existing DNA methylation pattern dur-ing DNA replication. In contrast, DNMT3aand DNMT3b, which are known as denovo methyltransferases, prefer unmethy-lated CpGs as substrate and are respon-sible for the de novo methylation of DNA.The hypermethylation of DNA usually re-sults in repression of gene transcription.Many CpG islands through the genomeare hypomethylated and are actively tran-scribed in undifferentiated ESCs, but sub-sequently become methylated and silencedduring differentiation. Those genes thatare repressed in ESCs but required forlater differentiation are marked by biva-lent H3K4me3 and H3K27me3 domains,that render them poised for activation [22,23]. Approximately one-third of genes thatare not marked by histone H3 lysine 4trimethylation (H3K4me3) or H3K27me3,

but are mostly repressed in ESCs, aremarked by DNA methylation, comple-mentary to histone modifications [21, 22,24]. The DNA methylation patterns arebetter correlated with histone methyla-tion patterns than with the underlyinggenome sequence context. DNA methy-lation and histone modification pathwaysmay be interdependent, with any crosstalkbeing mediated by biochemical interac-tions between the SET domain histonemethyltransferases and the DNMTs [25].Moreover, the polycomb group (PcG) pro-tein Enhancer of Zeste homolog 2 (EZH2)is a histone methyltransferase that is as-sociated with transcriptional repression,interacts (within the context of the Poly-comb repressive complexes (PRC) 2 and3) with DNMTs, and also exerts a directcontrol over DNA methylation [26].

Undifferentiated ESCs express high lev-els of the de novo DNA methyltrans-ferases DNMT3a and DNMT3b, whichmay repress differentiation-related genes,thereby maintaining the ESCs in un-differentiated states. Both DNMT3a andDNMT3b are directly regulated by thecore pluripotency transcription factorsOct4, Sox2, Nanog, and Tcf3. In addi-tion, they are also indirectly regulatedby the miR-290 cluster that repressesretinoblastoma-like 2 (Rbl2) [27], whichin turn downregulates DNMT3a andDNMT3b [28]. The inactivation of bothDNMT3a and DNMT3b in mouse ESCswas shown to cause a progressive lossof methylation in various repetitive se-quences and single-copy genes. Typically,DNMT3a and 3b are both stably associ-ated with each other in ESCs [29], withthe two enzymes interacting to methylatethe promoters of Oct4 and Nanog genesin differentiating ESCs. The methylationof key regulatory genes Oct4 and Nanog


plays an important role in the differen-tiation of ESCs [30]. Generally, DNMT3aand 3b are required for remethylation inpost-implantation mouse embryos and ingerm cells [31]. ESCs which are deficient inDNMT1 are viable, but undergo cell deathwhen induced to differentiate [32], whereasfibroblasts die within a few cell divisionsafter the conditional deletion of DNMT1[33]. DNA methylation is also involved inchromatin structure regulation [34] and,in ESCs, also requires the lysine methyl-transferase G9a [35]. Whilst, together, theactivities of DNMTs and DNA methyla-tion are not essential for the self-renewalof ESCs, they are rather critical in orderfor the pluripotent cells to differentiateinto various types of specialized cell [36,37] (Table 1).

A comprehensive map of DNA methy-lation in 11 201 proximal promoters inmouse embryonic stem cells (mESCs),using methyl-DNA immunoprecipitation(MeDIP) in combination with microar-rays, showed that approximately 40% ofthe interrogated promoter regions aremethylated, 32% are unmethylated, and28% are indeterminate [59]. The methy-lated promoter regions are located pri-marily outside of the CpG islands, ofwhich only about 3% are methylated tosome degree in mESCs [59]. DNA methy-lation maps, created by high-throughputreduced representation bisulfite sequenc-ing and single-molecule-based sequenc-ing, have shown that the methylation ofCpGs undergoes extensive changes duringcell differentiation, particularly in regula-tory regions outside of core promoters.Any ‘‘weak’’ CpG islands that are associ-ated with a specific set of developmentallyregulated genes undergo aberrant hyper-methylation during extended proliferationin vitro [24]. Furthermore, genome-wide,

single-base-resolution maps of methy-lated cytosines in a mammalian genome,from both human embryonic stem cells(hESC) and fetal fibroblasts have shownwidespread differences in the compositionand patterning of cytosine methylationbetween the two genomes [60]. Almostone-quarter of all methylations identifiedin ESCs were in a non-CG context, whichsuggested that ESCs might also use dif-ferent methylation mechanisms to affectgene regulation. Non-CG methylation hasbeen shown to disappear upon the induceddifferentiation of ESCs, but to be restoredin induced pluripotent stem cells (iPSCs)[60].

Whilst ESC lines may differ in their DNAmethylation profiles, methylation changeshave been shown to accumulate duringprolonged culture [61]. Such epigeneticchanges are thought to reduce the develop-mental potential of high-passage ESC lines[62, 63]. Many female ESC lines rapidlylose their global DNA methylation follow-ing their derivation, and are associatedwith the activation of both X chromosomes[62, 64]. However, female ESCs are diffi-cult to maintain in culture, and often tendto lose one of their two X chromosomesand thus to exhibit genetic instability [64].Methylation changes are observed at theimprinting control regions (ICRs), includ-ing those at the growth-related imprintedIgf2 and Igf2r loci [62, 63]. In addition,a prolonged culture period and varyingculture conditions can affect the methyla-tion patterns of undifferentiated hESCs[65, 66]. The DNA methylation profileclearly distinguishes hESCs from all othercell types, including somatic stem cells.Yet, different hESC lines exhibit differentchanges randomly with time in culture,and the degree of overall change in methy-lation is related to the number of passages[67]. It should also be noted that some

EpigeneticR

egulationin

PluripotentStem

Cells

9

Tab. 1 Epigenetic modifying enzymes involved in the maintenance of cell pluripotency.

Epigenetic modification Enzyme Knockout/knockdown phenotypein ESCs

Knockout phenotype in mouse Reference(s)

DNA methylation DNMT1 Normal proliferation, but defectsin differentiation

Embryos die at E9.5 [38, 39]

DNMT3a/3b Normal proliferation, but defectsin differentiation

Embryos die at E11.5 [31, 40]

DNMT1/3a/3b Normal proliferation, but defectsin differentiation

NA [41]

Eed Reduction in mono-, di-, andtrimethylation of H3K27.Upregulation of PcG targetgenes. Strong tendency todifferentiate

Gastrulation failure. Defect inembryonic mesodermdevelopment. Embryos die at∼E8.5

[42–44]

His

ton

em

odifi

cati

ons

H3K

27m

eth

ylat

ion

Suz12 Global loss of H3K27me2 andH3K27me3. Impaireddifferentiation, failing torepress ESC markers and toactivate differentiation-specificgenes

Early developmental defect.Embryos die at ∼E7.5–E8.5

[45]

Ezh2 Reduction in H3K27me2 andH3K27me3, only negligibleeffect on H3K27me1. Impaireddifferentiation, yet less severethan Eed null ESCs

Lethal around gastrulation.Embryos die at ∼E7.5–E8.5

[46]

PR

C2 Jarid2 Unaffected global H3K27me3.

Impaired ESC differentiationDefect in neurulation. Embryos

die before E15.5[47–49]

(continued overleaf )

10Epigenetic

Regulation

inPluripotent

StemC

ells

Tab. 1 (continued)

Epigenetic modification Enzyme Knockout/knockdownphenotype in ESCs

Knockout phenotype in mouse Reference(s)H

3K9

met

hyl

atio

n SetDB1a Differentiation towardtrophectoderm lineage

Peri-implantation lethal.Embryos die at ∼E3.5–E5.5

[50–52]

Jmjd1aa Increased level of H3K9me2.ESC differentiation

Viable mice. Defect inspermatogenesis andbecome obese in adult mice

[53–55]

Jmjd2ca Increased level of H3K9me3.ESC differentiation

NA [53]

His

ton

eac

etyl

atio

n

Tip

60-p

400 Tip60a Flattened colony morphology.

AP activity, EB formation,and teratoma formation arecompromised.Upregulation of manydevelopmental genes

Embryo dies beforeimplantation

[56, 57]

Trrapa Flattened colony morphology Peri-implantation lethality [56, 58]

aThe phenotypes described here are knockdown ESCs.NA, data not available.


genes which frequently gain aberrant DNAmethylation are related to tumorigenesis[68].

In contrast, DNA demethylation is im-portant for the full reprogramming ofsomatic cells into iPSCs, by an en-forced expression of defined sets of tran-scription factors in somatic cells. Stablepartially reprogrammed cell lines showthe reactivation of a distinctive subsetof stem cell-related genes, an incom-plete repression of lineage-specifying tran-scription factors, and DNA hypermethy-lation at pluripotency-related loci [69].Thus, DNA demethylation might rep-resent an inefficient step in the tran-sition to pluripotency. Down-regulationof lineage-specifying transcription factorscan facilitate reprogramming, and treat-ment with DNMT inhibitors can improvethe overall efficiency of the reprogram-ming process [69]. Activation-induced cy-tidine deaminase (AID; also referred toas AICDA) is also required for promoteractive demethylation and the induction ofOCT4 and NANOG gene expression toinitiate nuclear reprogramming towardspluripotency in human somatic cells [70].Small molecules that modulate DNA andhistone methylation have also been shownuseful for facilitating the epigenetic mod-ification and reprogramming of somaticcells to iPSCs [71, 72].

3Histone Modifications and HistoneVariants

In eukaryotic cells, DNA is organized intochromatin, the basic unit of which is thenucleosome. In a nucleosome, a DNA seg-ment of approximately 147 bp is wrappedaround a histone octamer, which is it-self composed of two copies each of four

histones (H2A, H2B, H3, and H4) [73].The histones in nucleosomes are subjectedto many types of modification, includ-ing methylation, acetylation, ubiquitina-tion, phosphorylation, and SUMOylation.Many of these histone modifications re-side on the amino- and carboxy-terminalhistone tails, including the methylationof Lys4, Lys9, and Lys27 in histone H3(H3K4, H3K9, and H3K27), the acetyla-tion of H3K9 and H3K14, the acetylationof H4K5, H4K8, H4K13 and H4K16, andthe ubiquitination of H2BK123 (in yeast),and H2BK120 (in mammals) [19]. Histonemodifications can be classified broadlyinto two types – repressing and activat-ing. H3K4me3 and histone acetylationare frequently associated with active tran-scription, while H3K9me3 and H3K27me3belong to the repressive histone marks.The language of histone modification isnot always ‘‘black and white’’, for example,an active histone modification H3K4me3does not always mark actively transcribedgenes, and in many cases genes markedwith H3K4me3 are neither expressed, norstably bound, by RNA polymerase II (RNAPol II) [74]. H3K9me3, a repressive his-tone modification, is found at the codingregions of active genes [75]. Moreover,at some specific genomic loci, both ac-tive and inactive histone modifications arepresent simultaneously. Such a combina-tion of H3K4me3 (active modification) andH3K9me3 (repressive modification) is de-tected within open reading frames (ORFs),which indicate a dynamic transcriptionalactivity [76]. By contrast the so-called‘‘bivalent domain,’’ which harbors bothH3K4me3 (active) and H3K27me3 (repres-sive) modifications, maintains genes at apoised stage ready for transcription [23].

More recently, several studies havebeen conducted to characterize the


genome-wide profiles of histone mod-ifications in ESCs [23, 75–78]. Ingeneral, these profiles have revealed therelationships between various histonemodifications and gene expression; forexample, H3K4me3 and H3K27me3 caneffectively discriminate between genesthat are expressed, poised for expression,or stably repressed [77].

These genome-wide profiles of histonemodifications have also revealed somenovel regulation mechanisms for tran-scription. Although most promoters inhESCs have nucleosomes marked withH3K4me3 [75, 76, 78], only a small subsetof these genes will express full-length tran-scripts. Genes with H3K4me3, but not pro-ducing detectable full-length transcripts,actually experience a transcriptional ini-tiation, as evidenced by the presence ofH3K9,14 acetylation, and RNA Pol II attheir promoters. Yet, the fact that no elon-gation marker H3K36me3 is detected at

these genes suggests that they are regu-lated at post-initiation steps. The meansby which transcription is suppressed fol-lowing transcriptional initiation remainselusive; however. it should be noted thatthis regulation mechanism is not limitedto ESCs, as the same phenomenon is alsoobserved in differentiated cells [78].

These genome-wide analyses of his-tone modifications have revealed a spe-cific modification pattern, consisting ofa large region of H3K27me3 harboringa smaller region of H4K4me3 (Fig. 1).As this modification pattern has bothrepressive and activating histone modifi-cations, it is termed ‘‘bivalent domain.’’In ESCs, genes marked with bivalentdomains are normally expressed at lowlevels, and are enriched in developmentalfunction. Such genes also become eitheractivated or suppressed upon differenti-ation [23], which leads to the intriguinghypothesis that bivalent domains main-tain developmental genes at a status which

K27K4 K27K27K27K27K27K27K27 K4

Poised

K27K27K27K27K27K27K27K27 K27 K27 K4 K4 K4K4 K4 K4 K4 K4 K4 K4K4 K4

ActiveRepressed

ESCs cells:

Differentiated cells:

Fig. 1 Bivalent domain facilitates rapidgene activation in ESCs. Bivalent domainsare chromatin regions marked with both ac-tive H3K4me3 and repressive H3K27me3.Genes associated with bivalent domains are

not expressed or expressed at low levels inESCs. Upon differentiation, bivalent domainsbecome either H3K27me3 or H3K4me3, re-sulting in gene repression or gene activation,respectively.


is transcriptionally inactive, but capableof being activated; clearly, bivalent do-mains may play a critical role in themaintenance of pluripotency. Althoughthe inactivation of developmental genesallows ESCs to be self-renewed, mainte-nance of the ‘‘activatability’’ of these genesis essential to maintain the differentia-tion potential of ESCs. Again, bivalentdomains are not restricted to ESCs. Somepluripotency-associated genes – notablySOX2, OCT4, and NANOG – which aremarked with H3K4me3 alone in ESCs,become associated with bivalent domainsduring differentiation. Moreover, in a hu-man lung fibroblast cell line, IMR90, biva-lent domains were also detected at someES-specific and lineage-specific genes [65].It appeared that these ES-specific andlineage-specific genes were not ready to beactivated in IMR90 cells. Therefore, in ad-dition to the bivalent domain, there mightbe other mechanism(s) available to sup-press these genes in differentiated cells.Alternative, bivalent domains might func-tion in unison with other mechanism(s) tomaintain developmental genes poised fortranscriptional activation in ESCs.

The importance of histone modifica-tions in the maintenance of pluripotencyhas been further elucidated by studies ofhistone-modifying enzymes [42, 45–48,50, 53, 56–58, 79–81]. The PcG proteinshave essential roles in early embryoge-nesis, thus implying their functions inESC pluripotency. PcG proteins func-tion in two distinct Polycomb repressivecomplexes, PRC1 and PRC2, with thePRC2-mediated methylation of H3K27having been implicated in the mainte-nance of pluripotency. The core of PRC2is composed of three PcG proteins, Ezh2,Suz12, and Eed. Mouse ESCs lackingthe individual PRC2 core subunit can beestablished from respective homozygous

knockout blastocysts. Although these nullESCs retain a normal self-renewal capac-ity, they display defects in differentiation[45, 46, 81]. For example, Eed−/− ESCslack di- and trimethylation on H3K27,show significantly reduced H3K27me1,and also upregulate PcG target genes[42, 43]; consequently, Eed−/− ESCs havea strong propensity to differentiate [42].Similar phenotypes have been observed inSuz12−/− ESCs [45], whereas the knock-out of Ezh2 results in reductions ofH3K27me2 and H3K27me3, but has a neg-ligible effect on H3K27me1. A less-severedifferentiation defect is also observed inEzh2−/− ESCs than in Eed−/− ESCs.The residual HMT activity in Ezh2−/−

ESCs is provided by Ezh1, since cellslacking Ezh2 and depleted of Ezh1 re-semble Eed−/− ESCs [46]. Mapping thegenome-wide binding sites of PCR2 hasshown that PRC2 occupies many of thegenes that encode developmental regula-tors in ESCs. These genes are associatedwith H3K27me3-modified nucleosomes,which suggests their transcriptional in-active state, and they are preferentiallyactivated during ESC differentiation [42,82]. In addition, PRC1 co-occupies manyPRC2 target genes, which implies thatPRC1 might also be involved in suppress-ing developmental regulators [42]. More-over, both Eed−/− and Ezh2−/− ESCs failto completely silence a set of ES-specificgenes following a six-day differentiation;this suggests that PRC2 is also required forthe suppression of pluripotency-associatedgenes during differentiation [46]. Takentogether, PRC2 is capable of maintainingESC pluripotency by suppressing the ex-pression of developmental regulators inESCs, and also contributes to ESC differ-entiation by suppressing the expression ofpluripotency genes upon differentiation.


In addition to the core of PRC2, a found-ing member of the Jumonji family, Jarid2,is associated with PRC2 complex. Jarid2and PRC2 co-occupy the same genomic re-gions and, indeed, the occupancy of Jarid2and PRC2 at target genes is mutually de-pendent [47, 48, 80]. Interestingly, Jarid2modulates the HMT activity of PRC2, andfine-tunes the H3K27me3 in vivo. Simi-lar to null mutations of the core subunitsof PRC2, knockout of Jarid2 does not af-fect ESC self-renewal, but impairs ESCdifferentiation [48, 80].

Histone H3K9 also plays a role in ESCpluripotency. In a high-throughput shorthairpin RNA (shRNA) screen for novelchromatin regulators that influence theESC state, a group of H3K9 methyl-transferases was identified as essentialchromatin regulators for the maintenanceof pluripotency. In particular, the lossof SetDB1 (also named ESET), whichis an H3K9 HMT, had the most pro-found effect on the ESC state [79]. ASetDB1-null mutation was shown to leadto peri-implantation lethality between 3.5and 5.5 days post coitus (dpc), and no ESClines were obtained from the SetDB1-nullblastocysts [50]. SetDB1 knockdown wasshown to reduce both SetDB1 and Oct4expression levels, whereas the expressionlevels of differentiation markers were en-hanced [51, 52, 79]. Taken together, thesedata suggest a role for SetDB1 in themaintenance of ESC pluripotency. The re-sults of recent studies have shown thatthe knockdown of SetDB1 results in thedifferentiation of ESCs into a trophec-toderm lineage. In this case, SetDB1and Oct4 interact with each other, andco-occupy the Cdx2 promoter to inactivatetranscription. Hence, SetDB1 is requiredfor the maintenance of ESC pluripotency

by suppressing trophectoderm differen-tiation [51, 52]. Chromatin immunopre-cipitation, coupled with massively par-allel DNA sequencing (ChIP-Seq), hasrevealed that SetDB1 binds to both the ac-tive and repressed genes. The repressedgenes, which were bound by SetDB1,were significantly enriched for develop-mental regulators, whereas the activegenes were enriched for gene expressionand metabolism. About one-third of thegenes occupied and repressed by SetDB1were also targets of PRC2. Consequently,the ChIP-Seq result suggests a broaderfunction of SetDB1 in maintaining ESCpluripotency, by suppressing the genesthat encode the developmental regulators[79].

H3K9 methylation is regulated byboth HMTs and histone demethylases.H3K9 demethylases also play importantroles in the maintenance of pluripo-tency. Two JmjC domain-containing hi-stone demethylases, Jmjd1a and Jmjd2c,are involved in the regulation of ESCself-renewal, such that the depletion ofeither Jmjd1a or Jmjd2c causes ESC differ-entiation. Jmjd1a demethylates H3K9me2at the promoter regions of pluripotencygenes, such as Tcl1, Tcfcp2l1, and Zfp57,and activates the expression of these genes.Jmjd2c promote ESC self-renewal by pos-itively regulating a key pluripotency factorNanog. Jmjd2c removes the H3K9me3marks at the Nanog promoter, and conse-quently prevents binding of the transcrip-tional repressors heterochromatin protein1 (HP1) and KAP1 [53].

The Tip60-p400 HAT and nucleosomeremodeling complex is essential for ESCmaintenance. The deletion of Tip60 or Tr-rap, which are two components of theTip60-p400 complex, results in preim-plantation embryonic lethality [57, 58].The colonies of ESCs depleted Tip60-p400


complex exhibit a flattened and elongatedmorphology, which is different from thatof typical ESC colonies. Moreover, de-pletion of the Tip60-p400 complex com-promises three features of ESCs, namelyalkaline phosphatase (AP) activity, EB for-mation, and teratoma formation. Knock-down of the Tip60-p400 complex alsoleads to an upregulation of many devel-opmental genes, despite the expressionlevels of the ESC markers not beingsignificantly affected. Interestingly, theTip60-P400 knockdown expression profileoverlaps with that of Nanog, while thelatter promotes Tip60-p400 binding to itstarget sites. Since Tip60-p400 binding alsorequires H3K4me3 at the binding sites, ithas been suggested that Tip60-p400 reg-ulates gene expression in ESCs throughintegrating the signals from Nanog andH3K4me3 [56].

In summary, many histone-modifyingenzymes are essential to maintain thepluripotency of ESCs, by catalyzinghistone modification reactions to repressor activate target gene expression, andto maintain the unique transcriptionalprofile in ESCs. For example, PRC2 andSetBD1 methylate H3K27 and H3K9,respectively, thereby repressing manydevelopmental genes. In contrast, Jmjd1aand Jmjd2c remove the methylationfrom H3K9, and positively regulatepluripotency-associated genes, such asNanog. The ablation of these enzymaticactivities leads to changes in the epigeneticprofile, in association with a compromisedESC pluripotency (Table 1).

It is not only the canonical histones(H2A, H2B, H3, and H4) but also non-canonical histone variants that contributeto the formation of nucleosomes. The hi-stone variants, which add another layerof complexity to the regulation of nucleo-some dynamics and chromatin structure,

may be classified as two types: universaland lineage-specific variants. The univer-sal variants, such as centromeric histonevariant H3 (CenH3), H3.3, H2A.Z, andH2A.X, are found in almost all eukary-otes, whereas lineage-specific variants,with their unique biological functions,are only found in certain organisms. Forexample, in animal sperm the DNA istightly packaged with histone variants,protamines, and protamine-like proteins.Another example is the mammal-specificH2A Barr body-deficient (H2A.Bbd) whichlacks a complete docking domain at theC terminus. Typically, H2A.Bbd appearsto contribute to active chromatin, be-ing absent on inactive X chromosomesin fibroblasts and coinciding with acety-lated H4. Moreover, H2A.Bbd-GFP (greenfluorescent protein) undergoes a quickerexchange in the nucleosome than doesH2A-GFP [83].

These noncanonical histone variants areinvolved in a wide range of biologicalprocesses, including DNA repair, meioticrecombination, chromosome segregation,transcription initiation and termination,sex chromosome condensation, and spermchromatin packaging. The histone vari-ants also contribute to the maintenanceof pluripotency in ESCs. For example,H2AZ has been shown recently to beessential for ESC differentiation, sinceH2AZ-depleted ESCs could not supportnormal development in vivo by tetraploidcomplementation and chimeric analysis.Upon the withdrawal of leukemia in-hibitory factor (LIF) under non-adherentconditions, the H2AZ-depleted ESCs wereseen to differentiate into EBs. However,the H2AZ-depleted EBs proved to bemore disorganized than egg cylinder-stageembryos, and failed to form typicalstructures representing differentiated celltypes. The differentiation deficiency of


the H2AZ-depleted ESCs was similar tothat of Suz12 (a component of the PRC2core)-null ESCs. Consistent with these ob-servations, genome-wide binding profileanalysis revealed that H2AZ would mainlyoccupy the promoter regions, while H2AZand Suz12 shared a highly similar set ofgenes in ESCs. Most importantly, the oc-cupancy of H2AZ and Suz12 at promoterswas shown to be mutually dependent inESCs [84]. Taken together, H2AZ mightcooperate with PRC2 to regulate the ex-pression of key developmental regulatorsin ESCs, and also during ES differentia-tion.

4Higher-Order Structure of Chromatin

Nucleosome organization is the first-orderstructure of chromatin, resulting in a‘‘beads-on-a-string’’ fiber structure on thebasis of which chromatin is further foldedto form higher-order structures. This leadsto the formation of two distinct typesof chromatin, namely euchromatin andheterochromatin. Although the way inwhich chromatin is further packed isa controversial topic, the higher-orderorganization of chromatin represents animportant mechanism with regards togene regulation.

The chromatin in ESCs is maintainedin a unique state compared to otherdifferentiated cells:

1) The staining of HP1α, H3K9me3, orDNA reflects a large, poorly definedheterochromatin region in undifferen-tiated ESCs. In neural progenitor cells(NPCs) which have been differentiatedfrom ESCs, the heterochromatin is or-ganized into small, discrete foci withwell-defined borders. And the number

of heterochromatin foci per nucleusincreasing as the cells differentiate.

2) By using the technique of fluorescencerecovery after photobleaching (FRAP),the exchange dynamics of architecturalchromatin proteins, including HP1,H2B, H3, and the linker histone H1,have been shown to be faster in undif-ferentiated ESCs than in NPCs. Theserapid exchange dynamics of architec-tural chromatin proteins in ESCs mightbe due to an increased loosely bound orsoluble pool of these molecules. Con-sistently, biochemical studies have alsoshown that both endogenous H1 andHP1 are released more easily fromESC chromatin than from NPC chro-matin.

3) By regulating the hyperdynamic plastic-ity of chromatin proteins it is possibleto affect the differentiation of the ESCs.The deletion of HirA (a nucleosomeassembly factor) leads to a dramaticincrease in the rapid exchange of theunbound and loosely bound fractionsof both H3 and H3.3. As a resultof these enhanced exchange dynam-ics, HirA−/− ESCs show an accelerateddifferentiation. Conversely, when theexchange dynamics is reduced by theexpression of H1cc (an H1 mutant withan increased binding affinity to chro-matin), the ESCs do not differentiatenormally into neuroblasts [85].

Taken together, these data suggest thatESCs maintain their chromatin in anopen state, and the hyperdynamic bindingof chromatin proteins promotes an earlydifferentiation of ESCs.

Chd1, a chromatin-remodeling enzyme,has been shown to be an essential regu-lator of open chromatin in ESCs. Chd1extensively colocalizes with Pol II and


H3K4me3, which suggests that Chd1 asso-ciates globally with euchromatin in ESCs.When Chd1 is knocked down in ESCs,the number of heterochromatin foci is in-creased, even in the undifferentiated ESCsexpressing Oct4. Moreover, the depletionof Chd1 compromises the rapid exchangeof H1 in heterochromatin, indicating thatthe chromatin is condensed. As a conse-quence, Chd1 RNA interference (RNAi)results in a decreased expansion of ESCs.The differentiation potential of ESCs isalso compromised by Chd1 RNAi. Theloss of primitive endoderm and cardiacmesoderm differentiation, as well as an en-hanced neural differentiation, is observedin EBs from Chd1 RNAi ESCs [86]. Thesedata suggest that Chd1 is required for themaintenance of open chromatin in ESCs,while open chromatin is essential to main-tain ESC pluripotency.

Another factor involved in the regula-tion of open chromatin is the SWI/SNFchromatin-remodeling complex (alsoknown as the Brg/brahma-associatedfactors; BAFs). The BAF complexes utilizeenergy derived from ATP hydrolysisto alter the DNA–nucleosome contactand to modulate chromatin structure;thus, they have a critical role in generegulation. The BAF complexes consistof 11 core subunits, several of whichare encoded by gene families. Thecombinatorial assembly of alternativefamily members diversifies the BAFcomplexes with different functionalspecificities. The ESCs have been shownto possess a distinct subunit compositionof BAF complexes which differs fromthose in fibroblasts, brain, and somemammalian cell lines [87, 88]. Thedistinctive BAF complexes in ESCs(esBAF) are defined by the presenceof Brg, BAF155, and BAF60A, and theabsence of Brm, BAF170, and BAF60C

[88]. The esBAF complexes are criticalfor pluripotency. Null mutations ofBrg, BAF155, and BAF47 all causeperi-implantation death. Neither theICM nor trophectoderm of these mutantblastocysts can give rise to outgrowthin vitro [89–91]. The inactivation ordownregulation of subunits in the BAFcomplexes, such as BAF250, Brg, BAF47,BAF155, and BAF57, compromisesESC self-renewal and differentiation[56, 88, 92–94]. In undifferentiatedESCs, the esBAF complexes colocalizeextensively with the key pluripotencyfactors Oct4, Nanog, and Sox2. Inaddition, the esBAF complexes occupya large number of Smad1 and Stat3target genes. Both, Smad1 and Stat3are transcription factors downstream ofthe bone morphogenetic protein (BMP)and LIF signaling pathways, respectively.Thus, esBAF complexes participate inESC maintenance by cooperating withnot only key pluripotency factors butalso with transcription factors involvedin the signaling pathways [95]. Duringdifferentiation, esBAF complexes arerequired for the repression of Nanogand other self-renewal genes. Mostimportantly, BAF155 is necessary forheterochromatin formation during theretinoic acid-induced differentiation ofESCs [92].

The important role of open chromatinin pluripotency is further elucidated bythe derivation of iPSCs. The latter are es-tablished from differentiated cells by theectopic expression of certain transcriptionfactors, such as Oct4, Sox2, Klf4, andcMyc. The knockdown of Chd1 compro-mises the efficiency of iPSC derivation[86], and two components of esBAF com-plex, Brg1, and BAF155, synergisticallypromote the reprogramming efficiency[96].


5X-Chromosome Inactivation

In each mammalian female cell, one of thetwo X chromosomes is transcriptionallyinactivated to compensate for the X-linkedgene dosage effect between male (XY) andfemale (XX). This phenomenon, whichis referred to as X-chromosome inacti-vation (XCI) [97], is a critical epigeneticevent in the establishment of pluripo-tency and in differentiation. Two typesof XCI have been identified during em-bryogenesis, namely imprinted XCI andrandom XCI (Fig. 2). Imprinted XCI initi-ates at the two-cell stage, and preferentially

silences the paternal X chromosome [98,99]. Subsequently, at the morula stage,the paternal X chromosomes in all blas-tomeres are inactivated. As the embryosfurther develop into blastocysts, however,the inactivated paternal X chromosomeis reactivated in the epiblast, while im-printed XCI is maintained in the tro-phectoderm and the primitive endoderm[98, 100]. Following implantation, the epi-blast cells undergo another round of XCI,in which one of the two X chromo-somes is randomly silenced, regardlessof their parental origin [101]. Hence,this round of XCI is known as randomXCI.

PGCs(Xp

aXma)

or

or

Fetus(Xp

iXm

a orXp

aXmi)

Placenta(Xp

iXm

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TE and PE(Xp

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Acquisition of paternalX chromosome imprint

Male PGCs(XY) Imprinted

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aXma)

Blastocyst

RandomXCI

X reactivationin PGCs

Mai

nten

ance

of i

mpr

inte

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Maintenance of imprinted XCI

Fig. 2 X-chromosome inactivation (XCI) andreactivation cycle in mouse development. Sev-eral key events in XCI and inactivation, includ-ing imprinted XCI from two-cell to morulastage, X-reactivation in epiblast, and randomXCI as epiblast further develops, are illustratedin the diagram. The paternal and maternalX chromosomes are shown in blue and red

rectangles, respectively. The blue shading onthe paternal X chromosomes symbolizes pa-ternal imprints. Rectangles marked with twoblack ‘‘X’’ are inactive X chromosomes. TE,trophectoderm; PE, primitive endoderm; PGCs,primordial germ cells; Xp, paternal X; Xm, ma-ternal X; Xi, inactive X; Xa, active X.

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