7-pluripotent stem cells origin maintenance and induction

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Pluripotent Stem Cells: Origin, Maintenance and Induction

Maria P. De Miguel&

Sherezade Fuentes-Julián&

Yago Alcaina

Published online: 29 July 2010# Springer Science+Business Media, LLC 2010

Abstract Pluripotency is defined as the potential of a cellto differentiate into cells of the three germ layers:endoderm, mesoderm and ectoderm. In vivo, the presenceof pluripotent stem cells is transient during the very earlyembryo. However, immortal cell lines with the same properties can be obtained in vitro and grown indefinitelyin laboratories under specific conditions. These cells can beinduced to differentiate into all the cell types of theorganism through different assays, thereby proving their functional pluripotency. This review focuses on the plurip-otent stem cells of mammals, giving special attention to thecomparison between mouse and human. In particular,embryonic stem cells, epiblast-derived stem cells, primor-dial germ cells, embryonic germ cells, very smallembryonic-like cells and induced pluripotent stem cellswill be compared in terms of the following: in vivospecification and location; surface and intracellular markers; in vitro dependence on growth factors; signaltransduction pathways; epigenetic characteristics; and plu-ripotency genes and functional assays.

Keywords Embryonic stem cells . Cell therapy .Primordial germ cells . Embryonic germ cells .Very-small embryonic-like stem cells . Mesenchymal stemcells . Induced pluripotent stem cells

Pluripotency from the Beginning: From Fertilizationto Gastrulation

Mammalian Early Development

Pluripotency is defined as the potential of a cell todifferentiate into cells of the three germ layers: endoderm,mesoderm and ectoderm. This means that pluripotent stemcells can give rise to any fetal or adult cell type but not toextraembryonic tissues, such as the placenta. This is why pluripotent stem cells cannot alone develop a completeanimal, in contrast to totipotent cells like the zygote.Totipotent state is maintained from the zygote to earlyembryonic blastomeres until the 4-cell stage embryo [ 1].Progenitor cells capable of differentiating into a limitednumber of cell types are called multipotent. Precursor cellsthat have the capacity to differentiate into only one type of cell are unipotent and, finally, terminally differentiated cellsthat have lost the ability of self-renewal are known asnullipotent. All cells that can develop into another andmaintain the capacity to renew themselves are known asstem cells, even unipotent cells.

In vivo, the presence of pluripotent stem cells is transient during the very early embryo. However, immortal cell lineswith the same properties can be obtained in vitro and grownindefinitely in laboratories under specific conditions. Thesecells can be induced to differentiate into all the cell types of the organism through different assays, thereby proving their functional pluripotency. Also they can differentiate, both invitro and in vivo, into embryonic bodies (EB) andteratomas respectively, macroscopic structures that containcells representing the three germ layers. Generation of chimeras by cell aggregation with eight-cell embryos or cellinjection into blastocysts prove the potential of these cellsto contribute to all tissues of a completely developed

M. P. De Miguel ( * ) : S. Fuentes-Julián : Y. Alcaina Cell Engineering Laboratory, La Paz Hospital Research Institute,IdiPAZ, Paseo Castellana 261,Madrid 28046, Spaine-mail: [email protected]

Stem Cell Rev and Rep (2010) 6:633 – 649DOI 10.1007/s12015-010-9170-1



animal, but not the trophoblast derived lineages [ 2]. Finally,the tetraploid complementation assay, consisting of theaggregation of diploid pluripotent cells with tetraploidembryos, shows that the whole fetus can be obtained by pluripotent cells, as tetraploid host cells provide theextraembryonic tissues to support the embryonic develop-ment but do not contribute to embryonic lineages [ 3].

This review will focus on the pluripotent stem cells of mammals, giving special attention to the comparison between mouse and human (for a summary see Fig. 1).

Embryogenesis starts after fertilization, when the unionof the male sperm and the female oocyte takes place toform the zygote. As early as this phase there are differences between mouse and human, since fertilization in mice isdependent on maternal centrosomes whereas in humans thesperm centrosome is introduced into the oocyte, and is

therefore paternally derived. That is why fertilizationstudies were developed using primate models rather thanmice [4].

The zygote undergoes cleavage, dividing by mitosis.Early development relies on maternal transcripts and proteins that are progressively degraded while embryonicgenome transcription progressively increases. This is calledmaternal to embryonic transition (MET). The embryonicgenome activation (EGA) is a global activation of genesnecessary to establish the preimplantation developmental program. It occurs abruptly at the 2-cell stage in mouse, but in human embryo takes place over several cell cycles withweak transcriptional activity from the end of the 1-cellstage, and major transcriptional activation at the 4-cellstage. [ 5] In addition, the epigenetic changes associatedwith EGA vary between species. Active paternal DNA

Fig. 1 Schematic diagram

of the different pluripotent stemcells originated from the epiblast at different stages of development

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demethylation is less pronounced in human than in mouse:a partial asymmetrical demethylation has been reported inonly half of human embryos [ 6].

When the morula stage (16 cells) is reached, the embryoexperiences compaction which also establishes embryo polarity. The cells bind tightly to each other forming a compact sphere in which there are two different cell layers.The outermost layer becomes the trophoblast or trophoec-toderm (TE) that gives rise to the outer layers of the placenta; and the inner cells become the inner cell mass(ICM) that gives rise to the embryo and the remainingstructures, such as yolk sac, allantois and amnion.

After that, by embryonic day 3 in the mouse and days 5 –

6 in the human, cavitation occurs, when trophoblast cellssecrete fluid into the morula forming an inner cavity calledthe blastocoel. The zona pellucida begins to degenerate,allowing the embryo to increase its volume (a processcalled hatching). This stage is known as the blastocyst (Fig. 2). The time difference between mouse and humanwill obviously become greater throughout their develop-ment until birth, since embryonic and fetal development takes 18 – 20 days in mice and 9 months in humans. Thecells of the ICM are pluripotent and when cultured areknown as embryonic stem cells (ES) and maintain their pluripotency in vitro [ 7]. On the other hand, trophoblast cells in culture, known as trophoblast stem cells (TS),maintain the capacity to proliferate but contribute only tonormal trophoblast derived cell types, even when contrib-uting to chimeras [ 8]. At this point the embryo undergoes a remethylation that also varies between species since inhuman DNA methylation is higher in the TE than in theICM while in the mouse it is higher in the ICM [ 6].

The trophoblast then differentiates into two distinct layers: the inner is the cytotrophoblast consisting of cuboidal cells that are the source of dividing cells, and theouter is the syncytiotrophoblast that implants the blastocyst in the endometrium of the uterus at about day 4.5 in miceand day 8 – 9 in humans. In mice, trophoblast cells that overlie the ICM (polar trophoectoderm) form the ectopla-cental cone, a cap-like mass that penetrates the uterus [ 9].

As the syncytiotrophoblast starts to penetrate the wall of the uterus, the inner cell mass forms a bilaminar embryo(Fig. 3). The lower layer, closest to the blastocyst cavity, isthe hypoblast, or primitive endoderm, made of cuboidalcells that will generate only extraembryonic structures, suchas the lining of the primary yolk sac. The layer adjacent tothe trophoblast, made of columnar cells, is the epiblast or primitive ectoderm and produces all three germ layers of the embryo: ectoderm, mesoderm, and endoderm. Epiblast cells of early postimplantation mouse embryos have beenisolated to obtain another pluripotent cell type, the EpiSCs(epiblast-derived stem cells) [ 10].

Gastrulation takes place next in the epiblast. In mice a gastrula with a cup-shaped structure called the egg cylinder is formed (Fig. 4a ). Within it appears the proamniotic cavityon day 6 of gestation, which then extends to form the proamniotic canal and finally extends towards the ectopla-cental cone. In contrast, human gastrula has a planar morphology like most mammals and initiates on day 13of gestation (Fig. 4b). First a new cavity is formed, theamniotic cavity, by separation from the trophoblast. This islined with the amniotic membrane made up of cells that come from the epiblast (amnioblasts). The blastocoel becomes the primary yolk sac when it is lined by hypoblast cells that secrete extracellular matrix, forming the Heuser ’ smembrane.

Cytotrophoblast cells and cells of Heuser ’ s membranecontinue secreting extracellular matrix between them,which is called the extraembryonic reticulum. Epiblast cellsmigrate along the outer edges of this reticulum and form theextraembryonic mesoderm. In mice the extraembryonicmesoderm is derived exclusively from epiblast cells, but inhumans it is originally derived from hypoblast andeventually replaced by primitive streak-derived mesoderm.At this stage in humans the prechordal plate appears, a thickening of the anterior endoderm that represents the first indication of the anterior-posterior axis establishment. It is believed that this structure is homologous to the mouseanterior visceral endoderm, involved in the establishment of embryonic polarity [ 11].

Then the chorionic cavity forms in the reticulum.Another layer of cells leaves the hypoblast and migratesalong the inside of the primary yolk sac. The primary yolk sac is pushed to the opposite side of the embryo, while a new cavity forms, the secondary or definitive yolk sac. This

Fig. 2 Drawing showing a schematic mammalian embryo at the blastocyst stage. ICM: internal cellular mass

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one persists and functions throughout gestation in mouseembryo, but in humans it functions only in early embryo-genesis [ 12].

ES and EpiSC

As stated in the previous section, Embryonic stem cells(ES) are pluripotent cells derived from culturing the inner cell mass (ICM) of blastocysts. These cells prolongundifferentiated proliferation, and give rise to cells of allthree germ layers (endoderm, mesoderm and ectoderm),

and trophoectoderm [ 13]. The first ES lines were generatedfrom mouse blastocysts (mES) [ 7] using feeder layers of mouse embryonic fibroblasts or without feeders in the presence of leukemia inhibitory factor (LIF) [ 14]. Morerecently, human ES (hES) cells were isolated [ 15] andseveral differences between mES and hES cells were found.

m&h ES cells grow as colonies sharing most but not allcharacteristics with the pluripotent cells of the embryo.These include expression of cell surface antigens like theglycolipids stage specific embryonic antigens (SSEA1 inmES an SSEA3/4 in hES), the keratan sulfate antigens Tra-1-60, Tra-1-81, GCTM2 and GCT343, and the proteinantigens CD9, Thy1 and HLA1. Also, they expressstemness and development related genes like Oct4,Sox2, Nanog, Tdgf1, Dnmt3b, Gabrb3 and Gdf3 [ 16].They also share enzymatic activities such as tissue non-specific alkaline phosphatase (TNAP) and telomerase,which consists of the addition of TTAGGG repeats ontotelomeres. Telomeres shortening is related with chromo-somal instability and reduction of the cell life span bylimiting the number of cell divisions. Telomerase activity istherefore essential for continued self-renewal in any celltype, including stem, tumor and germ line cells [ 17]. Allthese markers and characteristics are rapidly lost upondifferentiation.

Oct4, Sox2 and Nanog are transcription factors that havean essential role in the stemness regulatory circuitry,maintaining an undifferentiated state and self-renewal.These genes regulate a wide number of target genes, manyof them transcription factors involved in development. It has been demonstrated that Oct4, Sox2 and Nanog share a great portion of their target genes, which indicates a

Fig. 3 Drawing showing a schematic mammalian embryo at the bilaminar stage

Fig. 4 Drawings showing sche-matic mammalian embryos at the gastrula stage. a Mouseembryo. b Human embryo




strongly autoregulated circuitry [ 18]. Other transcriptionfactors, like Klf4 [ 19], c-Myc, Stat3 [ 20] or Rest [21] arealso part of the mES transcriptional network, whichinvolves not only transcription factors and signal transduc-tion factors, but also microRNAs and epigenetic modifica-tions as well.

DNA and chromatin epigenetic processes, especially inregulatory and coding regions, seem to play an important role in maintaining stem cell properties. Epigenetic mod-ifications refer to heritable changes in gene expression that are not coded in the DNA sequence itself. DNA methyla-tion takes place in CpG dinucleotides and is related withtranscription repression. Histone modifications involve thecore histones H2A, H2B, H3, and H4 and consist of severaldifferent modifications, including acetylation, methylation,and phosphorylation. Therefore, there is a great diversity of histone markers, some involved in gene activation andothers in repression. Specific ES genes have lower DNAmethylation levels and are occupied by active chromatinmarkers such as H3K4me or H3K9ac that correlate withtheir active transcriptional state. On the other hand, genesthat are expressed in terminally differentiated cells aremethylated and occupied by the repressive chromatinmarker (H3K27me). However, those genes that areexpressed in early differentiating progenitors are associatedwith both active and repressive chromatin markers that define a bivalent histone state [ 22].

Mouse ES cell maintenance depends on leukemia inhibitory factor (LIF). The pathway by which LIFsignaling acts to promote mES cell self-renewal has beenwell studied. LIF signals via heterodimerization of the low-affinity LIF receptor (LIFR), and the common subunit,gp130 [ 23]. The cytoplasmic domain of gp130 associateswith JAK kinases after ligand-stimulated dimerization. JAK kinases activate the transcription factor STAT3, whoseactivation is sufficient for stem cell maintenance. Stimula-tion of gp130 signaling in mES cells also phosphorylatesSHP-2 and leads to activation of the mitogen-activated protein (MAP) kinases, ERK1 and ERK2. This pathwayseemed to promote self-renewal over differentiation. The invivo relevance of the LIF pathway for embryo development is not entirely clear. LIF is expressed in the trophoectodermof the blastocyst and the LIF receptor in the ICM, as would be expected. However, neither LIF mutants, nor mutants of the receptors LIFR and gp130, show any defects in thedevelopment of the ICM or early epiblast probably due toredundancy with other cytokines of the same family [ 8].

Recently, another pluripotent cell type has been isolatedfrom mice, the EpiSCs (epiblast-derived stem cells). Thesecells are derived from the epiblast of early postimplantation(embryonic day 5.5 – 6.5) mouse embryos. Like m&hEScells, mEpiSCs grow as colonies when explanted, expresstranscription factors associated with pluripotency (Oct4,

Nanog and SSEA-1) and proliferate indefinitely in vitrowhile maintaining their capacity to differentiate into thethree primary germ layers [ 10].

Strikingly, mEpiSC more closely resemble hES cellsthan mES cells. Mouse and human ES use different signaling pathways to maintain their pluripotent status.mES cells depend on LIF and bone morphogenetic protein(BMP) to maintain pluripotency, whereas hES and mEpiSCrely on Activin/Nodal and fibroblast growth factor 2 [ 24].Activin/Nodal signaling acts through Smad2/3 activation.Smad2/3 protein interacts directly with Nanog, a coretranscription factor of the pluripotency regulatory circuitry,modulating the transcriptional activity. Nanog plays a critical role in regulating cell fate of ICM during embryonicdevelopment, maintaining the pluripotency of the epiblast cells and preventing their differentiation [ 25]. Likewise, Nanog interacts with the transcription factors Sox2 andOct4 to establish ES cell identity. These three factorsregulate each other and a set of common target genes (suchas Esrrb, Rif1, BMP4 and Foxd3 among others) in a regulatory network involved in maintaining ES pluripo-tency [ 26, 27]. Accordingly, inhibition of Activin/Nodalsignaling resulted in a loss of Nanog expression whileinducing differentiation towards neuroectoderm. Neither Nodal nor Activin is sufficient to sustain long-term hES[24]. FGF2 can also maintain expression of pluripotencymarkers while suppressing cell death and apoptosis genes, but the molecular mechanisms by which FGF-2 promotesthe undifferentiated growth of hES cells are unclear. It is believed that FGF2 acts through activation of FGFreceptors (FGFRs), stimulating the mitogen-activated protein kinase (MAPK) pathway [ 28]. FGF2 also actsthrough indirect mechanisms, stimulating the feeder layer of mouse embryonic fibroblasts (MEFs) to release Activin[29 ]. Recently, it has been reported that the FGF2mechanisms of action in hES cells and EpiSCs are not thesame. In hES cells FGF2 cooperates with the Smad2/3 pathway to activate Nanog, whereas in EpiSCs Nanogremains unaltered [ 30].

In addition, the pattern of gene expression and epigeneticregulation and stability are more similar between hES andmEpiSC than with mES cells. The morphology of thecolonies is also different: the mEpiSC and hES colonieswere large and grew as a monolayer, whereas mES colonieswere usually smaller, more compact and domed [ 31]. Onthe other hand, mEpiSC and hES single cells behavior differs from mES cells in that they have a very lowefficiency when subcloned and go through widespread celldeath when passaging by single-cell dissociation methods.Furthermore, mEpiSC have a limited capacity to contributeto chimeras. The pre-implantation embryo does not repre-sent a compatible environment for mEpiSCs, possiblyowing to a developmental asynchrony. Sparse chimerism




could be explained by the limited capacity of mEpiSCs for development into early cell lineages, but the morula aggregation shows no better integration in the blastocyst [10]. Until recently, another difference was thought to bethat hES cells and mEpiSC differentiated into trophoblast-like cells in the presence of BMP4, whereas mouse ES cellshad little or no such capacity [ 31, 32]. However, Hayashi et al., [33] recently showed that in fact mES can indeeddifferentiate into trophoblast-like cells as well. The contra-dictory results of previous reports were due to mES cells being usually derived in media containing LIF, which prevents the trophoblast differentiation induced by BMP4.

The epigenetic stability of hES cells is greater than mEScells due to their extent of monoallelically imprinted geneexpression [ 34]. Observing the histone modification patternof some genes (Stella, Otx2 and Nanog) shows that EpiSCsand human ES cells ’ epigenetic regulation is also verysimilar, but distinct from mouse ES cells [ 10].

Despite the differences between mES and hES cell pathways, a core transcription factor circuit involving Oct4,Sox2 and Nanog is necessary for pluripotency in bothspecies. Indeed, constitutive expression of Nanog issufficient to prevent differentiation of mES and hES cells,and loss of function confirms that Nanog is necessary to block primitive endoderm differentiation. FGF2 was not strictly required during EpiSCs and hES cells derivation but improved the overall quality of the cultures, suggesting that it reinforces the efficiency of activin signaling. In hES cellsand mEpiSC Smad2/3 the downstream effectors of Activin/ Nodal signaling bind and directly control the activity of the Nanog gene [ 24].

Gene expression analysis shows that Oct-4, Nanog andSox2 are expressed at similar levels in mEpiSCs and mEScells. However, these genes are not regulated in the samemanner in both cell types. In mice there are two different regulatory sites that direct expression of the Oct-4 gene.The distal enhancer regulates the expression in mES cells,while the proximal enhancer does so in the mEpiSCs [ 35].

Downstream of the core transcription factor circuitryshared by mEpiSC, hES and mES cells, more differencesare found. Target genes for Oct-4 and Nanog are verydifferent. Despite the limited overlap of Oct-4 targets, hESshare with mEpiSC seven-fold more Oct-4 targets than withmES cells [ 10]. This fact correlates well with the geneexpression pattern: genes associated with the ICM such asRex1, Pecam1, Tbx3 and Gbx2 are expressed at higher levels in mES cells, whereas genes associated with theepiblast and early germ layers such as FGF5, Nodal, Otx2,Eomes, Foxa2, brachyury (T), Gata6, Sox17 and Cer1 areexpressed at higher levels in mEpiSCs and hES cells. Dax1(a central component of the protein interaction network downstream Oct4 [ 36] required for the maintenance of mEScells in culture) was strikingly underexpressed in mEpiSCs

and human ES cells [ 10]. This similar gene expression pattern in mEpiSCs and hES cells, along with the other common features mentioned before, suggests a moredifferentiated state of hES cells compared to mES cells. It is possible that in culture human blastocysts continue their development towards an epiblast stage and are the reasonwhy hES cells are more like mEpiSCs than like mES cells.Differences in gene expression between the ICM and hEScell support this idea, like the expression of the somaticmarker Ncam1 and the early germ cell marker Dazl in hESeven though they are not expressed in the ICM [ 37].

Human ES cells are not the only cells that undergochanges when cultured. For instance, there is a surprisingdecline in alkaline phosphatase activity of mEpiSCs inculture, in contrast to higher activity in both human andmouse ES cells, in early germ cell lineage and also in lateepiblast [ 31]. The real effect of the environment on stemcell culture is still not well known and could hold theanswer to many burning questions. Recently, it has beenshown that mEpiSC could revert to ES-cell-like cells (rES)in response to LIF-STAT3 signaling. This reversioninvolves the reactivation of the inactive X chromosome,DNA demethylation and expression of E-cadherin. Geneexpression pattern also changes resembling mES cells.Most notably, unlike mEpiSCs, rES cells could participatein chimeras and contribute to the germ line [ 38]. Thisfinding could lead to the discovery of a similar way toreverse hES cells to a state close to ICM and mES cells that could have more therapeutic potential. What was thought to be due to unavoidable species differences might be due todevelopmental state and could therefore be reversible.

Maintenance of Potency: Epiblast and the Germ Line

PGCs

Shortly before gastrulation, when the epiblast is about togive rise to all three germ layers (ectoderm, mesoderm, andendoderm), primordial germ cells (PGCs) become specifiedin the proximal part of the epiblast at 6.25 days post coitum(dpc) in mice [ 39, 40] and around the third week of gestation in humans. Thus, PGC precursors are the first population of stem cells that is specified in the embryo at the beginning of gastrulation. PGC are the founder cell population of the gametes. During gastrulation, the PGCsthat originate from the posterior epiblast ingress through the posterior primitive streak together with the allantoic,intermediate and lateral plate mesoderm [ 41 – 43] (Fig. 5a & b). PGCs can be identified in mice at about 7 dpc and inhumans around day 22 of gestation by expression of TNAPactivity. mPGCs also express SSEAs and mouse vasa homolog, Mvh, on the surface [ 44] and intracellular Oct-




4, Stella, Fragilis, Smad1, Nobox, Hdac68, Sox2, Esg1,Dazl, Nanos3, Nanog, Blimp1 and germ cell nuclear antigen, GCNA ([ 45], for a review see [ 46]). Epigeneticreprogramming commences immediately after specificationand results in global loss of histone H3 lysine 9 methylation(H3K9me2) and increased H3K27me3 between 7.5 and8.5 dpc mouse PGCs ([ 47, 48]; for a detailed comprehen-sive review see [ 49]).

PGC subsequently migrate to the base of the allantois,which is located in the extraembryonic mesoderm. After-wards they are incorporated into the epithelium of thehindgut, from which they start to move in mice at day9.5 dpc first into the dorsal mesentery (10.5 dpc) and at weeks 4 and 5 in human embryos. The mesoderm of theembryo gives rise to an anterior lateral region called the para-aorta-splanchnopleura, which later contributes tothe future aorta, gonads and mesonephros (hence, it istermed the AGM region). PGCs migrate from thehindgut, reaching the AGM region at 10.5 dpc in miceand around the 5th to 6th week in humans, then intothe genital ridges that lie on the dorsal body wall(11.5 dpc in mice and the 6th week in humans).

There are several molecular markers that distinguishearly germ cells from other pluripotent cells of the earlyembryo. Germ cell markers are expressed by ES cellsthemselves, including those, such as Stella (also know asDppa3 or PGC7), which help distinguish germ cells from primitive ectoderm [ 39, 50, 51]. Other markers, such asTNAP and GCNA are strongly expressed by m&hPGCs(and by m&hES cells as stated above, Table 1), but areweakly expressed by the epiblast and other surroundingembryonic cells [ 52]. In addition, early germ cells areFragilis (Ifitm3) positive [ 39]. Only the expression of moremature germ cell markers (such as Mvh) enable in vitro-

derived germ cells to be distinguished from ES cellsthemselves. Using immunocytochemistry, it was alsoshown that most individual human ES cells in a populationexpress the early germ cell markers Stella-related (StellaR)and deleted in azoospermia-like (Dazl), indicating that a minor subset of randomly differentiating cells in a mixed population is not responsible for the expression of germ cellmarkers in hES cell cultures. In a gene expressioncomparison, Elliott et al. [ 53] found 24 genes highlyexpressed in 10.5 dpc mPGCs in comparison to mES cells.The existing gene expression data are consistent with theidea that the closest in vivo equivalent to mES cells is not the ICM or even the epiblast, but an early germ cell, more precisely mPGC precursors set aside within epiblast cellsaround 6 dpc [ 46, 54]. However, some of the properties of mES cells suggest that they are not merely the equivalent of early germ cells. For example, the earliest mPGCs do not self-renew for prolonged periods of time, but instead followa series of differentiation steps, beginning with germ cellmigration and ending in the highly morphological special-ization of sperm or egg [ 55]. Also, although ES cells candifferentiate into more mature germ cells in vitro, they doso relatively inefficiently. Importantly, isolated PGCs arenot pluripotent (see below), so an exact equivalence to EScells is unlikely [ 54].

Clonal analysis of the lineage potency of epiblast cellsrevealed that some cells in the proximal epiblast of the pre-to early streak stage embryo can give rise to SSEA andTNAP-expressing cells that colonize the allantoic meso-derm and the hindgut endoderm. The location of PGC precursors in the proximal epiblast suggests that onlyepiblast cells in this region of the early gastrula may possess germ-line potency. However, cells that maycontribute descendants to the mPGC population are not

Fig. 5 Drawings showing sche-matic mammalian embryos at the stage of formation of theepiblast. a Mouse embryo. bHuman embryo




Table 1 Presence of specific markers comparison between the different pluripotent stem cells in mouse (m) and human (h). Human epiblast is not included due to lack of data. +: presence. −: absence. Low: Low expression. ND: not determined

TNAP TRAs SSEA1 SSEA4 Oct4 Dppa3 Mvh Blimp Sox2 Nanog

hICM ND + − + + + − + + +[152 ] [152 ] [152 ] [37] [37] [153 ] [154 ] [155 ] [156 ]

mICM + − + − + + − − + +[90] [152 ] [157 ] [152 ] [90] [158 ] [159 ] [61] [160 ] [18]

hES + + − + + Low − ND + +[15] [15] [15] [15] [18] [10] [37] [18] [18]

mES + − + − + + − − + +[90] [161 ] [162 ] [161 ] [90] [162 ] [159 ] [45] [162 ] [90]

mEpiblast + − + ND + − − − + +[163 ] [152 ] [162 ] [10] [90] [159 ] [61] [160 ] [10]

mEpiSC − ND + ND + − − − + +[31] [31] [10] [31] [63] [31] [10] [10]

hPGC + − + + + + + + − +[100 ] [164 , 165 ] [166 ] [166 ] [155 ] [37] [153 ] [154 ] [155 ] [155 ]

mPGC + − + ND + + + + + +[90] [161 ] [103 ] [90] [90] [159 ] [104 ] [104 ] [90]

hEG + + + + + ND ND ND − +[100 ] [100 ] [100 ] [100 ] [155 ] [155 ] [155 ]

mEG + − + − + + − − + +[90] [161 ] [103 ] [161 ] [90] [104 ] [159 ] [104 ] [104 ] [90]

hEC + + − + + + − − + +[167 ] [167 ] [15] [167 ] [167 ] [167 ] [168 ] [154 ] [167 ] [167 ]

mEC + − + − + ND − ND + +[169 ] [161 ] [157 ] [161 ] [170 ] [159 ] [171 ] [172 ]

hiPS + + − + + + ND + + +[173 ] [121 ] [115 ] [121 ] [121 ] [130 ] [130 ] [121 ] [121 ]

miPS + ND + ND + + ND ND + +[114 ] [114 ] [114 ] [174 ] [114 ] [114 ]

hVSEL ND ND ND + + ND ND ND ND +[78] [78] [78]

mVSEL + ND + ND + + ND ND + +[175 ] [75] [75] [75] [82] [75]




localized specifically to any area in the proximal epiblast.Oct-4 expression is widely localized in the epiblast of theearly gastrula and shows no regionalization to the proximalepiblast [ 56].

The lack of a pre-determined population of mPGC progenitors implies that germ-cell formation is unlikely to be restricted to subsets of epiblast cells. This was elegantlyverified by a series of experiments testing the ability of epiblast cells that are located outside the proximal epiblast to form PGCs. Distal epiblast cells that normally display a neuroectodermal fate were transplanted to the proximalregion of the epiblast and differentiated into cells showingthe typical PGC pattern of TNAP activity [ 57]. In thereciprocal transplantation, proximal epiblast colonized thehost neural tissue but did not form PGC-like cells after grafting to the distal epiblast [ 42 , 58]. These resultssuggested that cells in the distal and the proximal epiblast are equally competent of forming PGCs provided they are placed in an environment that is appropriate for germ cellspecification. This raised the intriguing possibility that PGCformation might be subject to local environmental influen-ces unique to the proximal epiblast [ 58]. Interestingly,descendants of these proximal epiblast cells do not onlycontribute to PGCs but also to somatic tissues [ 59].Therefore, cells that are destined to be germ cells need torepress their somatic program and it is thought that the keymolecular determinant for this specification is the transcrip-tional repressor Blimp1 [ 60 , 61 ]. Additional changesfollow, such as reexpression of pluripotency genes Nanogand Sox2 [ 45, 62]. Remarkably, it has also been shownrecently that mEpiSC have an infinite capacity for generating mPGCs under conditions that sustain their pluripotency and self-renewal [ 63].

m&h PGCs can be isolated and cultured for up to10 days while still maintaining their phenotype [ 64, 66].They depend on SCF (KL), LIF and BMP4 (for a recent useful review see 46). PGCs are not pluripotent, as they donot form teratomas when injected into immunodeficient mice. Moreover, mPGCs do not contribute to chimeraswhen injected into blastocysts [ 54]. However, PGCs areconsidered developmentally pluripotent, as they generatethe whole totipotent embryo after fertilization. It is knownthat the pluripotency of mPGC nuclei depends on themethylation status of genomic imprinted genes such asH19, Igf-2, Igf-2R and Snrpn [ 67 , 68]. Until 9.5 dpc, mousePGC display a somatic imprint (paternal and maternal pattern of methylation) which is crucial in maintaining their pluripotency. This imprint is erased by genome-widedemethylation during the migration of mPGCs, and at their arrival at the genital ridges at 10.5 dpc [ 69, 70]. The erasingof the imprint in early PGC could be envisioned as one of the mechanisms that shuts down PGC developmental pluripotency, making these cells resistant to potential

parthenogenesis and teratoma formation in humans [ 71].Soon afterwards a proper somatic imprint would bereestablished in sperm and oocytes, so that a fertilized eggexpressed a developmentally proper somatic imprint of these crucial genes. Re-establishment of a proper somaticimprint in PGCs is possible, as demonstrated by thederivation of pluripotent cells from PGCs both in vitroand in vivo (see below, EC and EG section).

In addition to PGCs, the mid-gestation embryo alsoharbors other types of at least multipotent stem cells,namely the hematopoietic stem cells (HSC) and themesenchymal stem cells (MSC) that will later colonize the bone marrow, and which are thought to arise at the AGMregion as well. These cell progenitors and their relationshipwith the epiblast are the subject of another recent review[72], and will not be discussed here.

VSELs

The presence of stem cells in peripheral blood (PB) wasfirst reported in 2004 [ 73]. mRNA for markers for earlymuscle, neural and liver cells were found at low percen-tages in PB and cell percentages were enhanced after administration of G-CSF using a mobilization protocol.Thus, they hypothesized that those stem cells resided in BMand could be mobilized to PB after tissue injury. These bone marrow derived cells were negative for CD45, whichmeans that they were not hematopoietic [ 74], although theyare Sca-1+ and Lin-. When Kucia et al. [ 75] characterizedthe morphology and molecular markers of these cells, theyrenamed them Very Small Embryonic-Like cells (VSELs).These cells were found to express CXCR4 receptor, theligand for SDF-1, which supports their chemotactic skills[75]. It was already known that CXCR4 positive cells were present in PB, but it was only in 2008 that this scientificgroup reported the presence of these CXCR4+ Sca-1+ Lin —

CD45-cells (VSELs) in murine PB under steady conditionsat approximately 160 cells/ml [ 76]. The percentage of mVSELs in PB showed a 5 fold increase by administrationof G-CSF, accompanied by an increase in the expression of mRNA for embryonic and mVSELs markers such as Oct-4, Nanog, Rex1, Rif-1 and Stella. The number of mVSELs present in PB in steady conditions decreased with aging.Furthermore, these cells were able to form spheres that resemble embryoid bodies when plated over C2C12 murinesarcoma cell feeder layer, but no spheres were observedfrom cells isolated from old mice [ 75]. During this research,the mobilization of these cells from BM to PB after liver and skeletal muscle damage was confirmed. Mobilizationof VSELs after acute myocardial infarction has been provedin mice and human beings with a maximum peak at 48 and24 h respectively [ 77, 78]. Since injured tissue upregulatesstromal derived factor-1 (SDF-1) expression, they are




thought to migrate towards damaged organs in a SDF-1chemotactic fashion.

Very Small Embryonic-like cells are tiny (2 – 4 μ m) pluripotent cells shown to reside in various organs (brain,kidney, muscle, BM, pancreas, lung, spleen) as a pool of stem cells in readiness for healing an injury [ 79]. They havea large nucleus surrounded by a narrow rim of cytoplasm,resembling ES cells. Likewise, mVSELs contain open-typechromatin and express mRNAs for pluripotent stem cellssuch as Oct-4, Nanog, Rex-1, Dppa3 and Rif1, at similar levels to those of ES cells. They also express ES cellmarkers such as SSEA-1, Oct-4 and Nanog at the proteinlevel. These cells are able to differentiate in vitro at least into cardiomyocytes (mesodermal), neurons (ectodermal)and pancreatic cells (endodermal) [ 75]. Taken together,these data support their pluripotency characteristics and theidea of a primitive origin of these cells. However, as theydo not confirm their pluripotency by more stringent analysis, like in vivo teratoma formation and chimera contribution, it is not clear if they are only multipotent.

The next question would be what is the VSELs origin?Since they show epiblast/germ cell markers and expressCXCR4, they are thought to migrate from the epiblast towards chemotactic signals, like PGCs and hematopoieticstem cells (HSCs), and to be deposited during organogen-esis in developing organs as a population of pluripotent stem cells. Hematopoietic progenitors migrate from theyolk sac to the umbilical cord to the AGM region andfinally to the fetal liver and thymus [ 80]. VSELs arethought to migrate in a similar fashion. This hypothesis issupported by the fact that mVSELs have been found tocolonize murine fetal liver with a maximum percentage of cells at 12.5 dpc and a decrease of up to a minimum at 17.5 dpc [ 81]. This is concurrent with the dynamic of HSCs, whose numbers increase and decrease in parallelwith VSELs in fetal liver, as they follow a SDF-1 gradient in order to colonize the developing BM. Therefore, VSELsare thought to follow the same chemotactic strategy for colonizing the BM as HSCs. Of note is that so far the invivo pluripotency of these cells has not been proved, sotheir pluripotency might only be in vitro. In addition, it hasyet to be proved that after mobilization to damaged organsthey differentiate into the lineage of the damaged tissue.

It is accepted that the epiblast contributes to the AGMregion cells that create embryonic precursors of mesenchy-mal stem cells (MSC) that will colonize the BM. SinceVSELs seem to follow the same pathway, they could bethought to be the same cell type, however they do not sharethe same morphology (MSC are spindle shaped) nor thesame markers (MSC are Stro-1+ and CD49a+). VSELshave not been able to contribute to embryonic development after blastocyst injection, so they are believed to undergo anerasure of their somatic imprinting during their migration,

as PGCs do [ 69]. This erasing consists of an epigeneticmodification of the somatic imprinted genes that protect PGCs from uncontrolled expansion and lead to a temporaryloss of pluripotency [ 82]. VSELs share several markerswith PGCs such as TNAP, Oct-4, SSEA-1, CXCR4, Mvh,Stella, Fragilis, Nobox and Hdac68 [ 83] and they are thought to be related to each other. But why then are VSELs not just extragonadal PGCs? Firstly, because when cultured in vitro,PGCs are mortal and undergo terminal differentiation [ 84].Secondly, because ectopic PGCs have been reported not toform functional germ cells and are thought to degenerate postnatally [ 40, 85]. And thirdly, because up to 3% of malignant pediatric tumors are caused by germ cells andmost of them and 18% of adult germ cell tumors arise out of the gonadal area [ 86]. Ectopic germ cells that failed to dieare thought to be the cause of these tumors [ 87]. Thus,ectopic PGCs could not spread out and remained in everyorgan as a population of VSELs without giving rise to germcell tumors or dying at a high percentage.

As an alternative hypothesis, VSELs might represent thetumor stem cells. Virchow proposed the “ Embryonal-rest hypothesis ” of tumor formation in 1855 [ 88], supported bythe fact that PGC-derived cells generate teratomas when placed in ectopic locations. This theory has been further demonstrated by a model of murine stomach cancer caused by a chronic helicobacter pylori infection. BM-derived cellswere identified as a source of the adenocarcinoma, and theSDF-1-CXCR4 axis was shown to be implicated at theinitial stages of tumor formation [ 89]. Whether these BM-derived cells are VSELs or not needs further research.

In conclusion, VSELs may represent a pool of epiblast pluripotent stem cells that are spread along the body duringontogenesis, follow a HSCs-like pathway, and are prone to be mobilized when an injury occurs. Their mechanism of pluripotency silencing might resemble that of the PGCs inorder to avoid tumorigenesis.

Induction of Potency in Differentiated Cells

EC and EG

Historically, it has been described that PGCs can give rise totwo types of pluripotent stem cells. In vivo, m&hPGCs cangive rise to embryonal carcinoma (EC) cells, the pluripotent stem cells of testicular tumors [ 90]. In fact, ES cell derivationwas based on studies of teratocarcinoma cells. The transplan-tation of mouse genital ridges or of egg-cylinder-stageembryos into ectopic sites, such as under the kidney capsuleof adult mice, gave rise to teratocarcinomasat a high frequencyin strains that did not spontaneously produce these tumors [ 91].The stem cell of these tumors is the embryonal carcinoma cell, which can be serially transplanted between adult mice




[92]. If the EC compartment disappears, the resulting tumor develops by differentiating into a benign teratoma.

Some of the genes that affect teratoma formation have been identified in mice and humans. In mice, the Teratoma (Ter) mutation is the most potent one that affects teratoma formation. Introduction of the Ter allele onto the 129/Svstrain background increases tumor incidence to 30% [ 91].Several other genes have been shown to affect teratoma formation in mice and include several tumor suppressor genes such as phosphatase and tensin homolog (PTEN) andthe Steel (stem cell factor) gene [ 93].

In humans it is accepted that an inappropriate germ cellnumber in the testis, brought about either by increasedtemperature, cryptorchidism or by other physical and geneticconditions, is linked to increased rate of testicular cancer (for review see 71). In humans, genetic studies have identifiedseveral chromosomal translocations associated with testicular tumors. Interestingly, one such frequently observed translo-cation, isochromosome 12p, contains some genes that could play important roles in germ cell and stem cell development [94]. These include the human homologs of the Stella and Nanog genes. In addition, a mutation in the Kit gene is alsofrequent in testicular tumors. The role of these genes intesticular tumor formation is unclear at present but obviouslyeither of these candidates could play a role in transformingPGCs into pluripotent stem cells in vivo.

The first mEC lines were established more than 40 yearsago [95], and are considered the malignant counterpart of ES cells, as they share the same pluripotency markers(Table 1) but are usually aneuploid. Most lines are derivedfrom mouse PGCs from 8.5 to 10.5 dpc. Notably, germcells rapidly lose the ability to give rise to cells that formteratomas after 12.5 dpc in mice suggesting that at this timethey lose developmental potential. The main difference between mES and mEC is that the latter are usuallyaneuploid. However, their malignancy is highly dependent on the microenvironment. Indeed, mEC cells injected intomouse blastocysts can contribute to either the normaltissues of the resulting chimera [ 96] or in some cases, totumors [ 97]. In vivo testicular non-seminomatous embryo-nal carcinoma tumors are invariably composed of pure ECs,and differentiation starts only after invasion [ 71].

In vitro, cultured m&hPGCs exposed to a specificcocktail of growth factors (KL, LIF and FGF2) generate pluripotent embryonic germ (m&hEG) cells [ 98 – 100]. Theconversion of PGCs into pluripotent stem cells is a remarkably similar process to nuclear reprogramming after somatic nuclear injection into the egg cytoplasm [ 90]. EGhave been derived from pre-migratory and postmigratoryPGCs (7.5 – 11.5 dpc) in mice and at different stages of development in humans (5 – 10 weeks of gestation) [ 98 – 100].The ability of mouse germ cells to form mEG cell lines invitro declines rapidly after 12.5 dpc in mice and coincides

nicely with the ability of mPGCs to give rise to mEC cells invivo. Thus, the developmental potential of mPGCs seems todecline rapidly after 12.5 dpc indicating that important differentiation events are occurring at this time. Of note isthat the potential to derive mEGs from mPGCs coincideswith high proliferation rates and with the presence of a specific chromatin signature, including enrichment of meth-ylated histone marks H3K27me3 and H3K4me3 [ 47].

m&hEG cells are pluripotent by embryoid body forma-tion [98, 100 , 101 ]. mEG have also demonstrated teratoma formation [ 98], although, interestingly, hEG have not [ 102 ].In addition, mEG cells, in contrast to PGCs, fully contributeto blastocyst complementation yielding all somatic lineagesand germ cells in the developing embryo (obviously onlymEG have been tested). The main inducer of thisdedifferentiation of m&hPGCs into m&hEG cells is FGF2[98, 99]. The exact role of this growth factor has recentlystarted to be elucidated [ 46, 103, 104], and is related to downregulation of the germ lineage specification transcriptionalregulator Blimp 1. The exposure of mPGCs to FGF2 alsoupregulates two key transcription factors, Klf4 and c-Myc[104].

Both m&hEC and m&hEG cells, like m&hES cells,share the specific markers SSEAs, Oct4, Nanog and TNAPamong others [ 65, 100 , 101 ], (Table 1). In contrast tom&hEC cells, m&hEG cells are euploid, making themmore similar to m&hES cells than to EC cells. AlthoughmEG cell lines are remarkably similar to mES cell lines,they have some differences [ 90]. Since during germ cellmigration and maturation the somatic status of imprintedgenes is progressively erased [ 68], mEG cells isolated at various stages of development retain some of these differ-ences, such as the reduced methylation of many imprintedgenes, including H19 and Snrpn. The analysis of mousePGCs at 10.5 dpc suggests that methylation erasure hasalready begun by this time [ 68]. Imprinted genes exhibit imprinted (somatic) expression patterns in 9.5 dpc mousePGCs, but by 10.5 dpc, they have switched to a bi-allelicmode of expression [ 68]. The relationship between theepigenetic state and PGC reprogramming has been recentlydemonstrated. Exposure of mouse PGCs to trichostatin A, a histone deacetylases inhibitor, induces PGC reprogramminginto EG cells, and can even substitute for FGF2 [ 104 ]. It appears that the epigenetic reprogramming observed inmouse PGCs also occurs in humans, as extensive DNAdemethylation is required to rest the imprints and for Xreactivation [ 49], and also because human EGs, unlikehuman ES cells, are dependent on LIF [ 100 ].

In addition to embryonic germ cells, neonatal and adult male germline stem cells can also be used to derive pluripotent cells similar to ES cells [ 105 – 108 ], however this topic is out of the scope of this review and has beenrecently reviewed elsewhere [ 109 ].




iPS Generation

Under specific conditions, differentiated cells can bereverted into a less differentiated state by nuclear reprog-ramming. The genetic and epigenetic changes needed tosucceed in this dedifferentiation can be achieved by severalmethods such as somatic cell nuclear transfer (for review see[110, 111 ]), cell fusion with pluripotent cells [ 112], incuba-tion with pluripotent cell extracts [ 113], or derivation of pluripotent embryonic germ (EG) cells from unipotent PGCs by the addition of a specific cocktail of growth factors (99,and see above section EC and EG). More recently, studies onthe different genes involved in reprogramming led to theidentification of a defined set of transcription factors that canconvert a mature cell into a pluripotent state, creating the so-called induced pluripotent stem (iPS) cells [ 114 , 115]. Thefour factors used were Oct4, Sox2, Klf4, and c-Myc reachingan efficiency of 0.1%.

These revolutionary cells created a great deal of enthusiasm in the scientific community, as previous ethicalconcerns involving the use and generation of ES were nolonger an issue. Also, a great deal of research already performed on ES-directed differentiation towards specificcell lineages could now be straightforwardly applied. Inaddition, the possibility of generating patient-derived iPScells represents a step forward in autologous transplantationand application of the technology to human beings.

Takahashi and Yamanaka [ 114 ] did not manage to obtainviable chimeras with the first miPS cells. Those cells werereported to be incompletely reprogrammed and when theselection method changed to an Oct4 or Nanog selectablesystem [ 116 – 118 ] they obtained miPS cells in a fullyreprogrammed state, with an efficiency of 0.1%.

Soon afterwards this technology was used to develophuman iPS [ 115 , 116 ]. hiPS cells always yielded anefficiency of one order of magnitude lower than that obtained with mouse cells, around 0.01% in human cells[115 ]. In parallel, Yu et al. [ 119 ] also created hiPS cellsusing another set of four genes (this time Oct4, Sox2, Nanog and Lin28) with an efficiency of 0.02%.

Over the last 3 years a lot of research has aimed not onlyto improve the efficiency of the technique but also toovercome its drawbacks. Some of the four primary factorsare known oncogenes that induce tumors in iPS cell-derivedmice and their offspring [ 117 ], also retroviruses can causethe same effect by insertional mutation [ 117 , 120 ], so thefollowing attempts were channeled into avoiding the use of oncogenes and transgenes retrovirally. C-Myc was shownto be dispensable for direct reprogramming of mouse andhuman fibroblasts, although the process is substantiallydelayed and overall efficiency was reduced by one to twoorders of magnitude [ 118 , 121 ]. Furthermore, using thisapproach, Yamanaka ’ s group was able to obtain miPS cells

that expressed ES-cell markers to more similar levels tothose in proper ES-cells, with fewer “ background ” non-iPScells. They also contributed to chimeras that did not die of tumors within 100 days of the experiment [ 121 ]. Recently,Esrrb, an orphan nuclear receptor proved capable of replacing Klf4 in the reprogramming of mouse cells [ 122 ]with 2 fold less efficiency than that obtained with the 4initial factors. When MEFs were transduced with Oct4 andKlf4, treated with valproic acid, with E-616452 added (aninhibitor of TGF- β ) the reprogramming efficiency obtainedwas 0.01 – 0.1% making Sox2 and c-Myc unnecessary[123 ]. Likewise, Oct4 seemed to be indispensable for reprogramming until 2010 when it could be replaced bythe orphan nuclear receptor Nr5a2. This receptor together with the 3 remaining factors showed an efficiency of 0.02%at reprogramming mouse cells [ 124 ].

New strategies aim to avoid multiple viral insertionevents, which have been proved unnecessary for inducing pluripotency [ 125 ]. Although retroviral vectors are silencedduring reprogramming [ 126 ], transgene reactivation mayoccur after completely induced pluripotency [ 125 ]. Somenon-integration approaches have been performed in mousecells involving the use of adenoviral vectors transientlyexpressing the four primary factors, or the use of expression plasmids carrying the same factors, [ 127 , 128 ] with a maximal efficiency of 0.001 and 0.003% respectively. Astrategy based on Epstein-Barr nuclear antigen-1-basedepisomal vectors has been used to generate hiPS cellssuccessfully [ 129, 130] at an efficiency rate of 0.1 – 1%.Likewise, excision approaches have been carried out in order to remove transgenes once the cell is reprogrammed. Firstly,using a DOX-inducible lentiviral that could be excised withCre-recombinase, Soldner et al. [ 131] generated hiPS cellsfree of reprogramming factors with an efficiency of 0.01%.Two similar approaches have been successfully performed inmice [132, 133] reaching an efficiency of 2,5%: a) a loxP site and vector DNA external to the loxP sites that remainsintegrated in the iPS cell genome, and b) the piggyBactransposon/transposase system has been used to generatemiPS cells, improving on the success of Cre/l oxP system,due to the fact that it is able to excise itself completely [ 132,134]. Finally, two studies have overcome the limitationscaused by DNA-based reprogramming methods by usingrecombinant reprogramming proteins both in human andmouse cells [ 135, 136].

In addition to fibroblasts, different cells have beenrecently reprogrammed to a pluripotent state, such asmurine liver and stomach cells [ 125 ] with a non determinedefficiency, pancreatic β cells [137 ] (0.001% efficiency) or lymphocytes [ 138 ]. Non-terminally differentiating mouse Bcells were able to be reprogrammed with the four factors, but terminally differentiated B cells could not be dediffer-entiated by the same method. These cells needed additional




help via transduction with CCAAT/enhancer-binding protein- α (C/EBP α ), which disrupts the function of Pax5,a transcription factor that regulates mature B cell develop-ment and function. The efficiency of reprogrammingterminally differentiated B cells was 0.03%. These resultsthrew light on the fact that different initial cell fates yielddifferent reprogramming efficiency. Cells in a more primitive state could more easily be dedifferentiated withfewer transcription factors as demonstrated by Eminli et al.[139 ] who reprogrammed mouse neural progenitor cells(NPCs) in the absence of exogenous Sox2 at an efficiencyrate of 0.002%. This could be achieved because NPCsexpress high levels of endogenous Sox 2. Furthermore,these mouse and human NPCs have been reprogrammedonly with Oct4 [ 130 , 140 ] (efficiency not determined and0.004% respectively).

iPS cells highly resemble ES cells despite not beingidentical. They show a similar global gene expression andchromatin configuration, as well as reactivation of Xchromosome [ 116 ] and acquisition of ES telomeres char-acteristics [ 141 ]. They even generate late gestation embryosthrough tetraploid complementation [ 118 ]. But when theyare analyzed in detail, they show several differences. hiPScells express not sufficiently induced genes important inearly embryonic fate such as Stella or Rex1, not sufficientlysuppressed genes related to specific lineage fate such asZic1, Olig2, En2 and Ptx3 and upregulated genes such asSnai2 that are thought to be downstream of the inductionfactors [ 130 ]. The most relevant differences between iPSand ES cells involve epigenetic events [ 142 – 144 ]. Severalapproaches have been used to overcome the limiting step by reverting the global epigenetic state in partiallyreprogrammed cells with small molecule compounds. Suchmolecules are 5-aza-cytidine (DNA methyltransferaseDnmt1 inhibitor) [ 145 ], Bix-01294 (G9a histone methyl-transferase inhibitor) [ 146 , 147 ], and valproic acid (histonedeacetylase inhibitor) [ 148 ]. When a single round of transduction with reprogramming factors is performed,mouse cells enter a pre-iPS state which can be promotedto ground state pluripotency by two small moleculeinhibitors of Mek/Erk pathway and of GSK3, plus LIF[149 ]. Unexpectedly, BayK, a calcium channel agonist which has effects upstream in cell signaling and does not directly cause epigenetic modification, has a positive effect on reprogramming mouse cells [ 147 ]. These moleculecompounds have managed to deem some of the reprogram-ming transcription factors, such as Sox2, Klf4 and c-Mycdispensable, bringing us closer to a therapeutic reality.Strikingly, Vitamin C, another molecule not related toepigenetics, showed a very positive effect in reprogrammingMEFs with the original factors without c-Myc, reaching anefficiency of up to 3.8% [ 150]. iPS cells rely on the Activin/ nodal pathway to maintain their pluripotency like hES cells.

hiPS cells grown with the addition of BMP4 in the absenceof activin and FGF2 differentiate into primitive endodermand trophoectoderm [ 151] in a similar way to hES cells andmEpiSCs. Therefore, hiPS cells can be differentiated intoextraembryonic tissues, sharing this ability with hES cellsand mEpiSCs, but not with mES cells.

Acknowledgements This work was supported in part by grants fromthe “ Fondo de Investigaciones Sanitarias ” , Ministry of Health, andAgencia Laín Entralgo,Madrid, Spain;SAF2008-03837 from the Ministryof Science and Innovation, Spain and from the Foundation Mutua Madrileña, Spain. The authors wish to acknowledge Fatima Dominguezfor excellent technical assistance, Jaime Posadas Fernandez for originaldrawing art and Gareth William Osborne for linguistic assistance.

Conflict of Interest The authors declare no potential conflicts of interest.

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