Chapter1 Introduction 1.1 Embryonic stem cells 1.1.1 Derivation and definition of embryonic stem cells During the transition of an embryo from the morula to the blastocyst stage, a differentiation event partitions a developing embryo into its extraembryonic and embryonic components The outer layer of cells of the blastocyst is the extraembryonic components and forms an epithelium, the trophectoderm Descendents of the trophectoderm are restricted to the generation of the trophoblast components of the placenta The embryonic component, located on the interior is referred to as the inner cell mass (ICM) and this embryonic component comprises a stem cell population The ICM represents a unique transitory cellular structure that differentiates into epiblast (also known as embryonic ectoderm) derived cell types, namely the mesodermal, endodermal and ectodermal lineages in a regulated manner during the course of embryo development Alternatively, the ICM may differentiate into the primitive endoderm which gives rise to extraembryonic tissues instead of the pluripotent epiblast during this other differentiative event The three germ layers are the precursors to all tissues of the body Ectoderm develops into the epidermis, retina, brain and nervous system etc, while the mesoderm gives rise to the bones, muscles and most of the cardiac and circulatory system Endoderm gives rise to the respiratory organs and gastrointestinal tract Cells of the ICM also have the potential to develop into germ cells (Chambers and Smith, 2004) Of great significance is the fact that explant cultures of ICM can generate pluripotent embryonic stem (ES) cell lines The derivation of pluripotent cell lines from blastocysts was first achieved in the murine system in 1981 by Martin Evans and Matthew Kaufman and independently by Gail R Martin (Evans and Kaufman, 1981; Martin, 1981) Subsequently, successful derivation and propagation of rodent, rabbit, primate and human ES cell lines have been reported (Fan and Collodi, 2006; Iannaccone et al., 1994; Reubinoff et al., 2000; Thomson and Marshall, 1998; Vackova et al., 2007; Thomson et al., 1998) While ES cells are generally considered an in vitro phenomenon and are not true equivalents of the ICM, ES cells are at least ICM-like in terms of their ability to differentiate into cells of all three germ layers This pluripotent nature of mouse ES cells was first demonstrated by their ability to contribute to all tissues of adult mice, including the germ line following injection into host blastocysts Also, these cells can also form teratomas - benign tumours which consist of a mixture of differentiated cell types from the three germ layers following ectopic engraftment to immune-compromised mice (Reubinoff et al., 2000) In the in vitro scenario, ES cells likewise display a remarkable capacity to form a plethora of differentiated cell types of the three germ layers in culture Another defining feature of ES cells is their acquired capacity to proliferate indefinitely in vitro without undergoing senescence while retaining a normal karyotype This characteristic, coupled with the potential to differentiate into more than 200 unique cell types render ES cells a very attractive cell source for potential use in regenerative medicine The successful isolation and propagation of human ES cells in November 1998 by Thomson et al further brought this promise of ES cell based therapy one step closer to realization (Thomson et al., 1998) 1.1.2 Applications of ES cells ES cells can contribute to the formation of chimeric organisms following engraftment into a host ICM, and continue to give rise to all three germ lineages This potential for germline transmission in chimeras, coupled with the amenability of mouse ES cells to genetic manipulation enable the generation of knock-out mice As such, mouse ES cells have since found widespread applications in applied pharmacogenetics and basic functional genomics research (Pease and Williams, 1990; Tesar, 2005; Voss et al., 1997; Wolf et al., 1994) As for the potential therapeutic application of ES cells, ES cell based therapies and transplantation have been the most frequently discussed This has yet to be realized in clinic due to caveats such as less than optimal efficiency of in vitro differentiation, the danger of tumorigenecity of transplanted tissues and graft versus host response triggered in recipients upon recognition of non-autologous hES cells-derived cells as foreign Once these limitations can be overcome, it is likely that monocellular deficiency states such as Parkinson's disease and type I diabetes will be among the first examples of intractable diseases that can be rectified through ES cell based replacement therapies (Burns et al., 2006; Fukuda and Takahashi, 2005; Takagi et al., 2005) Apart from cellular therapy, ES cell technology can also prove to be very useful in many other aspects of medicine For example, the availability of ES cell lines, coupled with the recent development of various differentiative and purification regimes for the generation of a broad spectrum of lineages from ES cells, have opened up exciting opportunities to model mammalian embryonic development in vitro The study of events regulating the earliest stages of lineage induction and specification is tedious in mouse embryos and prohibited in the human embryo due to ethical concerns ES cell based models provide a convenient means around these limitations Understanding the events that occur at the first stages of development has potential clinical significance for preventing or treating birth defects, infertility and pregnancy loss A thorough knowledge of normal development could ultimately allow the prevention or treatment of abnormal human development For instance, testing drugs on cultured human embryonic stem cells could help reduce the risk of drug-related birth defects (Seiler et al., 2006) The understanding gained from the study of stem cell biology may also profoundly improve the treatment of cancer as mechanistic links between ES cell selfrenewal and cancer stem cell proliferation might make it possible to improve the treatment of cancer by targeting inappropriately activated self-renewal pathways Investigation of a number of human diseases is severely constrained by a lack of animal and cell culture models For instance, a number of pathogenic viruses including human immunodeficiency virus and hepatitis C virus grow only in human or chimpanzee cells Primate and human ES cells might provide cell and tissue types that will greatly accelerate investigation into some of these viral diseases (Yamamoto et al., 2003) In short, the prospect for medical application of ES cell technology is vast and promising One great impediment to the unleashing of the immense potential of ES cells is however the current incomplete understanding of the molecular mechanism underlying self renewal and cell fate determination of ES cells 1.2 Molecular basis underlying ES cell self renewal Despite the importance of stem cell self renewal, we are only beginning to understand how it is regulated ES cell self renewal is a complex process that involves both the proliferation and maintenance of pluripotency Self renewal is an intricate interplay instigated by instructive and permissive instructions provided by signaling molecules in the microenvironment and also by intracellular regulators such as transcriptional factors Multiple factors are required to act in concert to maintain the embryonic stem cell phenotype Some factors regulate only proliferation, while others regulate developmental potential and prevent differentiation Some determinants regulate proliferation and inhibit differentiation (Molofsky et al., 2004) To date, several key signaling pathways, such as the LIF/gp130/STAT3, bone morphogenetic protein (BMP) and wingless-type MMTV integration site (WNT) have been established as important pathways maintaining ES cell self renewal and preventing cell differentiation Intracellularly, the transcriptional regulatory circuit governed by OCT4, SOX2 and NANOG, have also been established as being crucial for maintaining ES cells in an undifferentiated state 1.2.1 Extrinsic regulators and signaling pathways governing ES cell self renewal 1.2.1.1 LIF/gp130/STAT3 pathway When mouse ES cells were first derived in the 1980s, they were propagated in coculture with a layer of fibroblast in the presence of serum An indication that fibroblasts act by secreting a signal that inhibits ES cell differentiation was substantiated by the ability of Buffalo rat liver cell line conditioned medium to replace the fibroblast requirement (Smith and Hooper, 1987) Leukeamia inhibitory factor (LIF) was subsequently identified to be the active component of the conditioned medium through fractionation (Smith et al., 1988) Fibroblasts carrying deletions in Lif were also found to have reduced capacity to support ES cells This further supports the fact that LIF is a major determinant of the ability of feeders to support ES cell self renewal Today, most investigators culture mouse ES cells feeder free in the presence of serum with supplementation of LIF LIF is a member of the IL6 family of cytokines that signals through the transmembrane receptor, gp130 LIF maintains ES cell self renewal through the activation of STAT3, a member of the signal transducer and activator of transcription (Stat) family (Raz et al., 1999) Evidence strongly indicates that STAT3 is the key transcription factor downstream of the LIF/gp130 pathway Forced expression of a dominant negative Stat3 mutant caused mouse ES cell differentiation even in the presence of LIF (Niwa et al., 1998) Also, point mutation of the tyrosine residue of gp130 responsible for STAT3 binding abrogated the ability of LIF to maintain self renewal It was also shown that mouse ES cells expressing a fusion molecule consisting of STAT3 and estrogen receptor could be maintained in the presence of estrogen derivative tamoxifen (Matsuda et al., 1999) which translocates the fusion LIF to the nucleus The first event involved in the LIF signaling cascade involves binding of LIF to the LIF receptor (LIFR) that contains a long cytoplasmic tail with a homology to the gp130 The LIF/LIFR complex then recruits gp130 to form a trimeric complex (Zhang et al., 1997) Heterodimer formation of the LIF receptor and gp130 receptor then results in the activation of the tyrosine kinase JAK The activated JAK phosphorylates tyrosine residues of gp130 which then serves as a docking site for STAT3 STAT3 is then activated and translocated into the nucleus to elicit transcriptional responses that prevent differentiation (Niwa et al., 1998) LIF and LIFR are expressed in blastocysts and expression of LIFR can be detected in the ICM However, mutant embryos deficient in LIF/gp130/STAT3 signaling forms normal ICM Lif deficient mice exhibit normal development (Stewart et al., 1992), while Lifr deficient mice showed perinatal lethality (Li et al., 1995; Ware et al., 1995) Embryos deficient in gp130 die progressively between dpc 12.5 and term Stat3 deficient embryos developed into egg cylinder stage until embryonic day 6.0 and rapidly degenerate between E6.5-7.5 Lack of phenotype pertaining to pluripotency in LIF/gp130/STAT3 suggests other mechanisms are normally responsible for maintenance of pluripotency in vivo In fact, a role for LIF/gp130 becomes only apparent for the prolonged survival of blastocysts during diapause Diapause is a physiological adaptation to the presence of suckling litter that allows embryos to persist for several weeks without implantation Upon cessation of suckling, the embryos implant in the uterus and development proceeds normally gp130 mutants however lose the epiblast component after days in delay and can no longer generate a foetus upon implantation (Nichols et al., 2001) This observation suggests LIF/gp130/STAT3 pathway is not fundamental for pluripotency, but instead functions primarily to extend the period of pluripotency in vivo The LIF/gp130/Stat3 pathway also appears to be dispensable for the maintenance of pluripotency and self- renewal of ES cells (Daheron et al., 2004; Ying et al., 2003) Even though Lifr-/- mouse ES cells are less pluripotent than wildtype, it was found that undifferentiated colony formation was not completely inhibited This indicates that there are LIFR-independent means by which fibroblast can support mouse ES cell self renewal Indeed, this may truly be the case as maintenance of several mouse ES cell lines does not require LIF Also, it has been reported that LIF is not necessary for human ES cell culture and that maintenance of pluripotency in human ES cells is STAT3 independent (Daheron et al., 2004; Thomson et al., 1998) Furthermore, STAT3 is expressed in a wide range of cell types, and in some cases drives differentiation (Hirano et al., 2000) Therefore, other core pathways and mechanisms that maintain pluripotency and ES cell self renewal may exist 1.2.1.2 Bone morphogenetic factor and BMP signaling In ES cell media without LIF, there is limited self renewal of mouse ES cells and the induction of neural differentiation It has recently been demonstrated by Ying et al that the requirement for serum can be replaced by Bone Morphogenetic Factors (BMP) BMP4 treatment suppresses neural differentiation and in combination with LIF, is sufficient to sustain ES cell self renewal without feeder or serum factors (Ying et al., 2003) This concurs with the fact that BMP signaling inhibits premature neural differentiation in the mouse embryo (Di-Gregorio A et al, 2007) The mechanistic action of BMP signaling has been attributed to the activation of inhibitor of differentiation (Id) genes by the downstream signal transducers, SMAD1/5/8 Forced Id1, Id2 and Id3 expression did not impair ES cell self-renewal nor block differentiation in the presence of serum ES cells transfected with Id1, Id2 and Id3 can however self-renew in serum-free culture containing LIF strongly, thereby suggesting that BMP/SMAD1/5/8 acts through Id1/2/3 proteins Id proteins exert a neuroectoderm lineage-specific block on ES cell differentiation by preventing precocious expression of proneural basic helix loop helix transcription factors as the Mash genes It could also be that IDs exert their effect by interaction with non bHLH proteins such as the PAXs Lefty in ES cells, it was then hypothesized that Lefty regulates ES cell fate by modulating cells’ responses to diverse differentiation factors such as Nodal or other signals that require EGF-CFC as a coreceptor Here, I report that Lefty prevents cell differentiation, at least in part through regulation of autocrine Nodal signaling in vitro 1.4 Nodal – A morphogen 1.4.1 Nodal signaling in embryo development and cancer Morphogens are defined as signaling molecules that activate target genes and induce cell fates in a concentration dependent manner in embryos (Hendrix et al., 2007) During embryogenesis, morphogens are secreted from a localized source and forms a concentration gradient as they spread by diffusion The concentration gradient provides spatial and temporal information to induce stem cell differentiation into different cell fates according to the position of the stem cells relative to the morphogen source during embryo development Morphogens are useful in regenerative medicine as they can potentially be used to promote differentiation of pluripotent cells into specific lineages Understanding the diverse mechanisms regulating morphogen action is hence important The identification of target genes regulated by morphogens, the delineation of the responses of target genes to graded morphogen signals and the elucidation of mechanism underlying the translation of different thresholds of morphogen-induced signaling into different cell fates are 26 fundamental questions that must be addressed before morphogens can be applied in stem cell therapies Nodal is a morphogen of the TGF superfamily Nodal was first isolated from a retroviral insertional mutagenesis screen in mouse ES cells A single Nodal ligand is found in mouse, chick and human while nodal related ligands exist in frogs (XNR1-6) and in zebrafish (cyclops and squint) Nodal ligands bind to type I serine-threonine kinase receptors (ALK4/5/7) and type II receptors ActRII (ActRIIA or ActRIIB), and signal through the receptor associated SMAD2/SMAD3 branch of the TGF pathway Nodal requires the EGF-CFC coreceptors for signaling EGF-CFC members are cysteine rich extracellular proteins that are attached to the plasma membrane through a glycosyl-phosphotidylinositol linkage In mammals, the EGF-CFC genes family has two members, Cripto and Cryptic Nodal ligands are generally expressed as homodimeric proproteins, and are secreted in a precursor form The precursor Nodal ligands are then cleaved by the subtilisin-like proprotein convertases Furin and PACE4 after secretion The inhibitory N-terminal pro domain stabilizes the precursor form while its cleavage leads to rapid turnover of the highly active mature protein Mature Nodal then bind to an EGF-CFC co-receptor in a complex with type I and II receptor Receptor activation leads to phosphorylation of the type I receptor kinase by the type II receptor kinase Activated type I receptors then phosphorylate cytoplasmic SMAD2 and/or SMAD3, leading to their interaction with the co-SMAD - SMAD4, and the subsequent translocation to the nucleus The receptor 27 complex undergoes internalization into endosomes and is a target by Dpr2 for lysosomal degradation Within the nucleus, activated SMAD2/3/4 complexes interact largely with the winged-helix transcription factors FOXH1 (also known as FAST2) or Mixer homeoproteins on target promoters This leads to transcription through interactions with ARC105 and the Mediator complex Examples of some current known target genes of the Nodal pathways are Nodal, Lefty1, Lefty2, Pitx2, Foxa2 and Lhx1 (Shen, 2007) Extracellular inhibitors that can modulate the activity of Nodal ligands includes Lefty proteins and Cerberus proteins Cerberus is a cysteine rich extracellular proteins that can block Nodal signaling through direct interaction with Nodal ligands As for the Lefty proteins, they antagonize Nodal signaling through their interactions with EGF-CFC co-recptor as well as Nodal ligands, thereby blocking receptor complex formation (Chen and Shen, 2004) A few membrane associated proteins, such as Tomoregulin-1 (TMEFF1) and Nicalin have also been proposed to represent pathway antagonists For example, Tomoregulin-1 (TMEFF1) was found to inhibit Nodal signaling through direct binding to the Nodal coreceptor, Cripto (Harms and Chang, 2003) Lineage tracing studies revealed the dynamic regulation of Nodal expression in space and time during development The earliest reported expression of Nodal during embryonic development was observed to be in the inner cell mass Nodal continues to be in the epiblast and the primitive endoderm The visceral endoderm that develops from the primitive endoderm also expresses Nodal Nodal expression in the epiblast is subsequently limited to the primitive streak during gastrulation After gastrulation, Nodal 28 expression is asymmetrically localized to the left side of the node, a structure that forms at the anterior end of the primitive streak Cells ingressing through the node give rise to mesendodermal tissues such as the prechordal plate and notochord which are the precursors of the head and central nervous system, as well as the anterior definitive endoderm that subsequently give rise to the esophagus, liver, lungs, pancreas, stomach and small intestines At 8.0dpc, Nodal is asymmetrically expressed more strongly in the left lateral plate mesoderm The splanchnic layer that forms the future circulatory system and gut wall originates from the lateral plate mesoderm During later development and adulthood, Nodal and components of its signaling such as Lefty are rarely expressed Numerous well conserved biological phenomena during early embryogenesis have been ascribed to the function of Nodal signaling A notable example is that of leftright specification (Brennan et al., 2002) Nodal is asymmetrically expressed on the left side of the developing embryo and activates other asymmetrically expressed genes such as Paired-like homeodomain transcription factor (Pitx2) Phenotypes of knockout mutants for various Nodal pathway components also suggest the importance of this pathway in left-right axis formation For example, mouse knockouts of type II receptor ActRIIB die of cardiac defects and display right pulmonary isomerism (Oh and Li, 1997) Right pulmonary isomerism and randomized embryonic turning was also observed for Cryptic mutant (co-receptor of Nodal) (Yan et al., 1999) Studies also show that Nodal signaling is important for the specification of the anterior-posterior (AP) axis (Brennan et al., 2001; Lu and Robertson, 2004) AP axis 29 specification is defined by two events: the formation and directional movement of the anterior visceral endoderm (AVE) In the absence of Nodal, Brennan et al and Norris et al observed no formation of the AVE and no apparent evidence of an AP axis In hypomorphic Nodal mutants or Cripto knockouts, the AVE forms, but is defective as it fails to migrate to the prospective anterior of the embryo GDF3, a ligand of the Nodal pathway is also critical for AVE formation and movement and both processes are affected in Gdf3 null mutants In addition to axis formation, Nodal signaling plays dual roles in neural patterning (Camus et al., 2006; Sampath et al., 1998) The generation of anterior neural tissues requires its inhibition, while the subsequent maintenance and patterning depends upon axial mesendoderm generated in response to nodal signals Nodal signaling is well known for its role in mesoderm and endoderm induction (Schier and Shen, 2000) Mouse Nodal knockout embryos are developmentally arrested prior to gastrulation Nodal mutants fail to develop a primitive streak and severely lack mesoderm (Conlon et al., 1994) Mouse mutants for Smad2, a transcriptional effector of Nodal signaling also failed to gastrulate or form mesoderm (Nomura and Li, 1998) Zebrafish double knockouts for Nodal related squint and cyclop lack endoderm (Feldman et al., 1998) A similar knockout phenotype is observed for Cripto mutant mouse embryos (co-receptor of Nodal) (Chu et al., 2005) These data taken together suggest that Nodal plays a central role in cell fate specification during development 30 Nodal signaling is also essential for induction of node formation The node is an organizer-like tissue that can induce surrounding tissues to develop a secondary axis when ectopically transplanted into embryos Foxh1 is a key transcriptional regulator of Nodal target genes and Foxh1 mouse mutant embryos fail to develop a node at the anterior primitive streak This leads to subsequent lack of prechordal plate mesoderm and definitive endoderm Such mutants also exhibit defects in AP patterning (Yamamoto et al., 2001) It is unclear how Nodal can achieve different functions in different tissues during the different stages of development This question was partially answered when Chen and Shier showed Nodal to be a morphogen in zebrafish (Chen and Schier, 2001) Injection of a single cell with different amount of squint RNA in zebrafish embryo triggered the activation of downstream targets in a concentration dependent manner Evidence also suggests that Nodal signaling induces different cell fate at different signaling threshold through activation of different genes in mouse Lowe et al previously demonstrated that distinct phenotypes results from Nodal hypomorphic mutants according to the severity of Nodal signaling attenuation A wildtype embryo develops a primitive streak that extends towards the distal tip where the node forms In a nodal hypomorphic mutant where Nodal function is partially suppressed, the primitive streak extends only half way down the posterior side and the node develops in a more proximal location instead In a Nodal knockout embryo where Nodal function is completely abolished, the embryo suffers from a pregastrulation block due to complete failure of primitive streak formation (Lowe et al., 2001) 31 Beyond its developmental role, Nodal has also been implicated in cancer (Postovit et al., 2007; Topczewska et al., 2006) Evidence suggests that Nodal pathway activity is upregulated in many cancers Pathway inhibition decreases tumourigeneity in xenograft assays while enhanced expression of Nodal in melanomas is correlated with malignancy Also, overexpression of Cripto has also been implicated in tumorigenesis Cripto is highly overexpressed in epithelial camcers including breast, pancreatic, colon and ovarian carcinomas, and overexpression of Cripto has been shown to transform mouse mammary epithelial cell line (Ciardiello et al., 1991a; Saloman et al., 2000) Depleting Cripto with antisense RNA also leads to loss of the transformed phenotype in human colon cancer cells (Ciardiello et al., 1991b) More functions of Nodal continue to emerge in recent years Some recent studies have suggested that Nodal/Activin signaling may even be required for the maintenance of pluripotency in pregastrulas Firstly, Nodal signaling in vivo appears to be necessary for the maintenance of pluripotency in epiblast Dunn et al has shown that loss of Smad2 and Smad3, the transcriptional effectors of Nodal signaling resulted in loss of mouse epiblast pluripotency by E7.5 as reflected by reduced Oct3/4 level This correlates well with the observation by Robertson et al that Nodal null mice have a substantial reduction in the epiblast cell population size and lacked Oct3/4 expression Genetic studies have also shown that embryos deficient in the Co-SMAD, SMAD4 display defective epiblast proliferation and retarded ICM outgrowths (Yang et al., 1998) James et al further investigated the role of Smad2/3 in mouse blastocysts First, they were able to detect the expression of phosphorylated SMAD2/3 and localization to the nucleus beginning at the four cell stage using immunofluorescence staining SMAD2/3 remains phosphorylated in 32 both the ICM and trophectoderm up to the blastocyst stage This confirms SMAD2/3 activation in pregastrula and concurs with the fact that Nodal related ligands and receptors are expressed very early in mouse preimplantation embryos They then went on to employ mouse blastocyst outgrowth assay to assess the requirement for SMAD2/3 in trophoblast and ICM, the two cell types that make up the blastocysts To assess the requirement for Smad2/3, they cultured blastocyst stage embryos ex vivo for days and assayed the presence of OCT3/4 in outgrowths in presence or absence of SB431542 SB431542 is a potent selective inhibitor of ALK4/5/7, the receptors mediating Nodal signals and it globally inhibits Nodal signaling Interestingly, they found that the OCT3/4 compartment is maintained in the controls, while the outgrowths cultured in the presence of SB431542 display loss of OCT3/4 staining They also suggested in their paper that mouse blastocysts outgrowths cultured ex vivo in presence of soluble receptors hrActRIB, hrActRIIB and hrCripto similarly lost OCT3/4 positive components, albeit less consistently Put together, all these data lead to the emergence of a paradigm in which Nodal signaling is necessary for the maintenance of pluripotent cell types in vivo and ex vivo (James et al., 2005) 1.4.2 Nodal signaling and ES cell fate decisions: What is known? Nodal signaling is also likely required for the maintenance of ES cell pluripotency in vitro Large scale transcriptome studies revealed that all key components of the Nodal pathway such as Nodal itself, Cripto, Lefty etc are highly expressed in both 33 undifferentiated mouse and human ES cells Also, chromatin immunoprecipitation coupled with DNA microarray (chIP on chip) studies revealed that Nodal related genes such as Skil, Lefty2 and Cripto are target genes co-bound by OCT4, NANOG and SOX2 in human ES cells while Lefty1 and Cripto are common targets of OCT4 and NANOG in mouse ES cells (Boyer et al., 2005; Loh et al., 2006) In fact, Cripto is one of the only 32 genes that are co-bound by OCT4 and NANOG in both mouse and human ES cells Apart from the abundant expression of Nodal related components, the undifferentiated state of human ES cells is also characterized by the activation of SMAD2/3 signaling James et al found that SMAD2/3 was phosphorylated and localized to the nucleus of undifferentiated human ES cells maintained in presence of MEF conditioned medium Vallier et al reported a similar finding that expression of SMAD2/3 could be detected in the nucleus of human ES cells expressing the pluripotency markers, Oct4 and SSEA-3 (Vallier et al., 2005) SMAD2/3 phosphorylation and nuclear localization were decreased in cells allowed to differentiate in non conditioned medium This indicates that the Nodal/Activin pathway is activated in the undifferentiated state Apart from the descriptive correlation of SMAD2/3 with “stemness”, SMAD2/3 activation has also been demonstrated to be necessary for maintenance of the undifferentiated state in human ES cells functionally James et al showed that upon withdrawal of MEF conditioned medium and subsequent reduction of SMAD2/3 phosphorylation, human ES cells exhibited reduced Oct3/4 and Nanog expression Even in presence of conditioned medium or BIO, inhibition of SMAD2/3 activation by SB431542 significantly reduced Oct3/4 and Nanog expression and promoted morphological changes of ES cells, indicative of differentiation James et al were able to 34 reproduce their findings by culturing human ES cells with a combination of human recombinant ActRIB, ActRIIB and Cripto, thereby strengthening the idea that intact Nodal/Activin signaling is important in maintaining pluripotent human ES cells Data gathered by several other groups are also consistent When Xiao et al compared an euploid H1 ES cell line vs an anueploid H1 ES cell line that demonstrated self renewal advantage over wildtype using microarray analysis, significant differential gene expression for Nodal, Lefty A and Lefty B was detected (Xiao et al., 2006) Vallier et al also demonstrated that Nodal is able to support prolonged feeder-free culture for more than 10 passages when the Nodal transgene is constitutively overexpressed in human ES cells They also found that inhibition of Nodal signaling by overexpression of Lefty or Cerberus short, both Nodal antagonists precipitated human ES cell differentiation Smith et al (2008) later also showed that inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm It is an established fact that mouse and human ES cells not differentiate in the presence of a mouse embryonic fibroblast (MEF) layer, be it in the presence or absence of LIF It is now known that MEF secretes a huge abundance of TGF-b ligands such as Activin A Activin A can utilize the core components of Nodal pathway and have been shown to be able to generate Nodal –like responses in vivo It is a possibility that the self renewal of ES cells grown on MEFs can in part, be mediated by the SMAD2/3 signaling triggered by paracrine Activin A signals from MEF It has recently been shown that 50ng/ml recombinant Activin A, in combination with 50ng/ml FGF7 and 10nM nicotinamide can maintain human ES cells cultured on laminin substrate in the absence of feeder layers long term (Beattie et al., 2005) Xiao et al has similarly also demonstrated the long term 35 maintenance of human ES cells cultured on matrigel in presence of serum replacement with just 5ng/ml Activin A under serum free, feeder free condition The crosstalk between the Nodal/Activin and FGF pathways was demonstrated by Vallier et al to be important for the long term maintenance of human ES cell pluripotency in a serum free chemically defined medium They showed that long term maintenance of ES cell pluripotency can be achieved with Nodal or Activin, with basic FGF as competence factor in absence of feeder cell layers, conditioned medium or serum replacement As for mouse, Miyazono et al have also shown that Nodal/Activin signaling is involved in the propagation of mouse embryonic stem cells in serum free condition (Ogawa et al., 2007) An in vitro assay that can be used to assess the pluripotency of ES cell is that of embryoid body formation Undifferentiated ES cells typically form embryoid bodies when cultured in suspension and these embryoid bodies contain cells from all three germ layers It was found however that when Nodal signaling was attenuated in the presence of SB431542, the efficiency of BGN2 human ES cells to form embryoid body was markedly reduced It was reported that the few embryoid bodies which did form in presence of SB431542 were of an atypical, multi-cystic morphology (James et al., 2005) This data suggest that inhibition of Nodal signaling abrogates ES cell pluripotency Vallier et al carried out the reciprocal experiment and found that overexpression of Nodal in human ES cells inhibits neuroectodermal differentiation within embryoid bodies, and maintains cells in the undifferentiated state, while simultaneously promoting visceral endoderm differentiation at the surface of embryoid bodies (Vallier et al., 2004) These observations 36 are consistent with findings that Nodal signaling in vivo is required to maintain epiblast pluripotency and prevent precocious neural differentiation It can be surmised from current lines of evidence that Nodal signaling mediated by SMAD2/3 indeed plays a role in self renewal and cell fate decisions of pluripotent cell populations in vivo and also that of embryonic stem cells in vitro An in-depth mechanistic understanding of how Nodal action actually translates to different cell fates at the molecular level is however lacking Nodal signaling has till far been predominantly examined in a binary on/off context It is unclear if ES cells can sense and interpret multilevel Nodal activity, as is the case with morphogens during embryo development This awaits further investigation 1.5 iPS cells- an alternative platform to ES cells for the study of pluripotency and pluripotent cell self renewal Scientists have recently reported the successful reprogramming of mouse and human somatic cells into pluripotent stem cells, designated as induced pluripotent stem (iPS) cells Mouse and human iPS cells could be induced by overexpressing the transcription factors quartet - Oct4, Sox2, c-Myc and Klf4 (Takahashi et al., 2006, Takahashi et al., 2007 and Yu et al., 2007) Such landmark discoveries not only open up avenues for the derivation of patient derived stem cells, but also provide an alternative platform to ES cells for the study of the molecular framework underlying pluripotency and pluripotent cell self renewal 37 Currently, very little is known about the mechanistic details of the reprogramming process The consensus is however that Oct4 is the key factor responsible for directing iPS cells towards pluripotency, since all current working protocols invariably require the application of Oct4 to the somatic cells As for Sox2, the current thinking is that Sox2 may not be necessary for the reprogramming process This is because iPS cells can be derived with just Oct4, Klf4 and c-Myc, without Sox2; and these cells are somewhat similar to ES cells in terms of morphology and proliferation rate (Takahashi et al., 2006) The only difference lies in the fact that iPS cells without Sox2 fail to differentiate and lack the property of pluripotency Hence, this implies that Sox2 may be important for the pluripotency and proper differentiation of the iPS cells, rather than the reprogramming process per se Klf4 and c-Myc have more to with the regulation of the growth and self renewal of iPS cells rather than the induction of pluripotency per se, as overexpression of Klf4 and c-Myc resulted in formation of tumour cells that are not pluripotent (Takahashi et al., 2006) It is now believed that common mechanisms underlie the induction of iPS self renewal and transformation, and that the balance of Klf4 and c-Myc is critical in regulating p21 and p53 dependent cell cycle programs Yu et al (2007) reported that forced expression of Nanog increased the survival rate of early reprogrammed cells, and this corroborates with the role of Klf4 and c-Myc in stimulating iPS cell proliferation and self renewal Also, recent work indicates that KLF4 and c-MYC can be substituted for by NANOG in the reprogramming of human somatic 38 cells (Yu et al., 2007) In mouse ES cells, Klf4 can activate Nanog enhancer, while Nanog has been found to be able to bind to Klf4 transcription regulatory regions (Jiang et al., 2008) Also, c-Myc is a target of Nanog in mouse ES cells (Loh et al., 2006) Together, these findings suggest that Nanog can regulate iPS self renewal by regulating Klf4, c-Myc and their respective target genes in reprogrammed cells Currently, most of the knowledge regarding the mechanistic details of reprogramming pertains to the role of transcription factors Nothing much is known about the signaling processes and molecules regulating the reprogramming process Such knowledge would however be critical for the medical application of iPS cells as ability to induce reprogramming through the use of exogenous factors circumvents the use of retroviral introduction of transcription factors, which is the only tried and tested mode of factor delivery available currently It is hence tempting to extrapolate the role of Lefty and Nodal signaling in ES cell self renewal and pluripotency to the reprogramming process One impetus for doing so would be that the Lefty genes are regulated by the key reprogramming factors - Oct4, Sox2, Nanog and Klf4 Also, Lefty has been shown to repress TGF- -induced expression of the p21 gene in pluripotent embryonal carcinoma cells, P19 Furthermore, Activin-Nodal signaling has been shown to be involved in propagation of mouse embryonic stem cells, and it can be speculated that this pathway can similarly support the self renewal of iPS cells In essence, Lefty and Nodal signaling may play very important roles in the iPS settings and this awaits further investigation 39 1.6 Goals and objectives The current dogma of ES cell self renewal and pluripotency revolves around the roles of LIF/STAT3, BMP and WNT signaling pathways, and that of the core transcriptional regulatory circuit governed by OCT4, SOX2 and NANOG The understanding of what constitutes the fundamental mechanics governing ES cell self renewal and pluripotency is however still incomplete The primary aim of this project is to identify additional factors and pathways that are required for the maintenance of ES cell self renewal and pluripotency This work will detail the functional dissection of one factor- Lefty2, which was identified using a candidate gene approach The question addressed here is if and what role(s) Lefty2 plays in the maintenance of ES cells Also, since Lefty2 is intricately linked to the Nodal signaling pathway during embryonic development, and that Nodal signaling has recently been implicated in ES cell fate determination, the second part of this thesis is also dedicated to clarifying how Nodal signaling influence ES cell fate decisions 40 ... the unleashing of the immense potential of ES cells is however the current incomplete understanding of the molecular mechanism underlying self renewal and cell fate determination of ES cells 1.2... pluripotent embryonic stem (ES) cell lines The derivation of pluripotent cell lines from blastocysts was first achieved in the murine system in 1981 by Martin Evans and Matthew Kaufman and independently... underlying ES cell self renewal Despite the importance of stem cell self renewal, we are only beginning to understand how it is regulated ES cell self renewal is a complex process that involves