Fibroblast growth factor (FGF) signalling plays many fundamentally important roles during the development of the mammalian embryo. However, its effects on pluripotent stem cells derived from mouse and human embryos appear to be markedly different. FGF2 is routinely added to culture medium for propagating undifferentiated human (hES) cells, whereas in mouse (mES) cell cultures FGFs have been described as regulators of their differentiated progeny. To assess the effect of FGF signalling on undifferentiated mES cells, the effects of FGF2 and 4 were analysed in the presence of saturating and sub-saturating levels of the inhibitor of differentiation, leukaemia inhibitory factor (LIF). Mouse ES cell self-renewal was quantified by measuring the expression of the stem cell specific reporter Oct4-LacZ in biochemical and fluorometric assays. Treatment with FGF reduced the expression of the OCT4-LacZ reporter, even under saturating concentrations of LIF and this was mirrored by decreased levels of OCT4 protein. Furthermore, treatment with FGF leads to upregulation of the ectodermal differentiation marker Pax6. These results suggest that FGF signalling has a direct impact on undifferentiated mES cells, and actively promotes their differentiation. To asses the effect of FGF signalling on hES cells without the influence of undefined factors, a feeder and serum free system was developed. Cells growing in this conditions for >20 passages maintained expression of surface (SSEA3 and TRA1-60 and 81) and internal (OCT4) markers specific for undifferentiated hES cells. Expression of these markers was dependant on the continuous presence of FGF2. Indeed, withdrawal of FGF2 resulted in a rapid decrease of in hES cell growth and of the emergence of cell flattened morphology and of the surface marker SSEA1, changes typically associated with differentiation. Two important signals activated by FGF in hES cells are the ERK/MAPK and PI3K pathways. To assess their functional relevance, hES cell cultures were treated with the drugs UO126 and LY294002, inhibitors of the MAPK and PI3K pathways respectively. Drug mediated suppression of the phosphorylation of these pathways, correlated with a reduction in cell growth, flattening of the colonies and reduction in SSEA4 expression. Use of SB431542, specific inhibitor of TGFβ/activin type I receptor kinase (Alk5) also resulted in the flattening of the colonies and the appearance of dispersed cells. Therefore, inhibition of MAPK and PI3K appears to impair growth and self-renewal in hES cells and this may be happening in conjunction with TGFβ/Activin pathway. Taken together, these results suggest that FGF signalling has opposite effects in mouse and human ES cells: inducing differentiation in mES and sustaining self-renewal in hES.

Optimizing culture conditions that reduce genomic instability in human pluripotent stem cells (hPSCs) is an unmet challenge in the field. Results from our lab and numerous research groups demonstrate that hPSCs are prone to genomic aberrations and single-cell passaging increases the rate of genomic alterations. However, single-cell based passaging maintains advantages for scale-up and standardizing differentiation protocols. In this study, we investigated the problem of genomic instability in hPSC cultures with the goal towards identifying and characterizing candidate genes that could contribute to generation and survival of abnormal hPSCs. Based on microarray analysis, we identify ARHGDIA, located on 17q25, as a candidate gene conferring selective advantage to trisomy 17 hPSCs. Using lentiviral approaches to overexpress ARHGDIA in hPSCs, [hPSC (Arg)], we functionally validate that in enzymatically passaged co-cultures, hPSC (Arg) lines exhibit competitive advantage against wild type hPSCs, [hPSC (WT)]. Additionally, hPSC (Arg) lines exhibit increased single-cell survival at low density plating. In co-cultures with hPSC (WT), ROCKi exposure attenuated the competitive advantage of hPSC (Arg) subpopulations. For the first time, this work demonstrates that increased expression of a gene on 17q25 confers selective advantage to hPSCs. In parallel studies, using medium devoid of bFGF containing LIF plus two inhibitors, MEK inhibitor (PD0325901) and p38 inhibitor (SB203580), we demonstrate that hPSCs are LIF responsive and can be stably maintained in naive pluripotent culture conditions. Based on their clonal viability, we propose that naive hPSCs are a more genetically stable population than primed hPSCs, when passaged as single- cells. These studies will aid the long-term goal of hPSC scale-up while promoting stable propagation of genomically normal hPSCs.

Central to the progression of all organisms is the maintenance of a stable genome despite continuous insults arising from genotoxic and environmental stresses. Embryonic stem cells show promise for treatment of a variety of diseases as well as for providing normal human tissue to conduct scientific research. A major obstacle for their application is that genomic instability arises in stem cells after prolonged cell culture. The most detrimental form of DNA damage is the DNA double-strand break (DSB), which is managed by cells through complex mechanisms, designated the DNA damage response. There are two major types of DSB repair; homologous recombination repair (HRR) and non-homologous end joining (NHEJ), both of which are regulated by members of the phosphatidyl-inositol-3’-kinase-related kinase (PIKK) family, including Ataxia Telangiectasia Mutated (ATM), Ataxia Telangiectasia Mutated and Rad3-related (ATR) and the DNA dependent protein kinase (DNA-PK). The aim of this study was to define the mechanisms and important proteins involved in repair of human embryonic stem cells. Here we have also described a system to differentiate hESCs into neural progenitors and astrocytes and were able to examine their DNA damage response. In both examining DNA repair markers and using a DNA repair reporter assay, this work shows that ATR is involved in DSB repair early in development, whereas ATM is essential in DSB repair in differentiated cells. We also show that HRR, a high fidelity form of repair, is used extensively by embryonic stem cells and HRR diminishes as cells differentiate. We also further defined the extent of NHEJ and the role of high fidelity NHEJ from the embryonic to differentiated state. These findings further the basic knowledge of repair fidelity in embryonic and mature human tissue. The data gives insight into what proteins maintain stem cell genomic stability and may be important to develop safe technologies for tissue engineering. Specifically, we have defined what DNA damage signaling pathways are used as embryologic cells progress to a mature, functional state.

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) plays an important role in cell cycle regulation, by inhibiting CDK2 and by targeting it for proteolysis. It is also known to bind the DNA polymerase alpha-primase complex and regulate the initiation step of DNA synthesis. Its overexpression has been shown to inhibit growth, reduce invasion and increase apoptosis in a number of cancer cell lines. In studies in which mouse embryonic stem cells (mESCs) with targeted deletion of the Cdk2ap1 gene were used, Cdk2ap1 was shown to be required for epigenetic silencing of Oct4 during differentiation. The goal of this thesis was to examine the role of CDK2AP1 in somatic cells (primary human dermal fibroblasts (HDFs)) and human embryonic stem cells (hESCs) and specifically assess its impact on proliferation, self-renewal and differentiation. In the first part of this study, using a short-hairpin RNA (shRNA) approach, we investigated the effect of CDK2AP1 downregulation in HDFs. Outcomes indicated: (a) reduced proliferation, (b) premature senescence, (c) cell cycle alterations, (d) DNA damage, and (e) an increase in p53, p21, and the p53-responsive apoptotic genes BAX and PUMA. Simultaneous downregulation of p53 and CDK2AP1 in HDFs confirmed that observed phenotype was p53 dependent. In the second part of this study, using a shRNA approach, we investigated the role of CDK2AP1 on hESC fate associated with self-renewal and differentiation. We found that CDK2AP1 knockdown in hESCs resulted in: (a) reduced self-renewal (b) enhanced differentiation (c) cell cycle alterations and (d) increase in p53 expression. Results indicate that the knockdown of CDK2AP1 in hESCs enhances differentiation and favors it over a self-renewal fate. Thus, this study has successfully identified novel functions for CDK2AP1, as its knockdown has a significant impact on self-renewal, differentiation and senescence. Results obtained from this study could contribute to development of directed differentiation strategies for generating uniform populations of differentiated phenotypes from hESCs for clinical applications.

Les cellules souches embryonnaires (cellules ES) sont capables de se multiplier de façon autonome en l’absence de facteurs de croissance et de cytokines, un état appelé “état fondamental de pluripotence”. Le cycle cellulaire des cellules ES se caractérise : (i) par une expression élevée et uniforme de la cycline E et des complexes Cycline E-CDK2 au cours de la progression dans le cycle cellulaire et (ii) par une phase G1 très courte (1 heure) dont la traversée ne dépend ni des MAPK ni des points de contrôles régulés par la protéine du rétinoblastome (RB) et p53. Ces observations soulèvent la question de l’existence d’un lien de cause à effet entre ce phénomène de réplication autonome et la pluripotence. Mon projet de thèse se construit autour de trois axes qui montrent que : 1/ la phase G1 des cellules ES de souris est une phase de sensibilité accrue aux inducteurs de différenciation. 2/ la balance entre autorenouvellement et différenciation est perturbée, (i) quand l’expression de la cycline E est altérée, ou (ii) quand l’association de la cycline E avec la kinase CDK2 et le centrosome est bloquée. 3/ La signalisation par le LIF contrôle la formation et l’activation des complexes Cycline E/CDK2. Dans les cellules ES naïves Rex1+, l’allongement de la durée de la phase G1 induit par la privation de LIF précède, ou est concomitante, à la diminution de l’expression de marqueurs de pluripotence et à l’activation des marqueurs les plus précoces de la différenciation. Finalement, nous proposons un modèle dans lequel la signalisation par le LIF régule la transition G1/S et permet le maintien de l’autorenouvellement des cellules ES murines / Pluripotency can be captured and propagated in vitro from the epiblast of the pre-implantation blastocysts in the form of embryonic stem cells (ESCs). ESCs are capable of unlimited proliferation in an undifferentiated state while maintain the potential to differentiate into cells of all three germ layers in the embryo, including the germline. Two key features the ES cell mitotic cycle are (i) a vastly elevated and uniform expression of Cyclin E and Cyclin E/CDK2 complexes throughout the cell cycle and (ii) a short G1 phase characterized by the lack of RB- and p53-dependent checkpoints, and reduced dependency on MAPK signalling. During my PhD project, we explored whether and how the regulation of the cell cycle actively sustains self-renewal of mouse ESCs (mESCs). We demonstrated that: 1/ the G1 phase of mESCs is a phase of increased susceptibility to differentiation inducers. Thus shortening of G1 might shield undifferentiated cells from differentiation inducers and help ESCs to self-renew in the pluripotent state. 2/ Cyclin E opposes differentiation and supports self-renewal of mESCs by two independent mechanisms, one of which being independent of CDK2 activation. 3/ LIF signalling regulates Cyclin E/CDK2 kinase activity therefore accelerating the G1 to S phase transition. Finally, we propose a model in which LIF signalling stimulates the G1 to S phase transition to shield mESCs from undesired differentiation signals and help them to self-renew in the pluripotent state

The sex determining region Y-box 2 (sox2) gene is one of the most important transcription factors during development, particularly the development of the central nervous system (CNS). It is expressed in embryonic stem cells and later in neural stem cells, where it modulates their maintenance and differentiation. In humans, heterozygous mutations are associated with eye malformations, including anophthalmia and severe microphthalmia. Also, a subset of patients has extra-ocular phenotypes, such as hearing loss, seizures and pituitary hypoplasia. Although the roles of sox2 in embryonic stem cells and eye development are well studied, the function of sox2 in brain development and disease is still elusive. The aim of this project was to characterize a novel role for sox2 in the development of zebrafish epithalamus, which was identified from an in silico screen previously performed in our laboratory. The zebrafish epithalamus, located in the dorsal diencephalon, consists of three main structures: the pineal gland, the parapineal organ and the habenular nuclei. The pineal gland, also known as epiphysis, is a photoreceptive (in zebrafish) and neuroendocrine organ that detects light and rhythmically produces melatonin in order to regulate the circadian rhythms. The parapineal organ is located to the left side of the pineal gland and is important for the elaboration of the asymmetries observed between the left and right habenular nuclei. Finally, the bilateral habenulae are part of the dorsal diencephalic conduction system that links the forebrain with the mid- and hindbrain. The left and right habenulae show both molecular and neuroanatomical asymmetries, including differences in neuropil organization, in levels of gene expression and in the morphology and connectivity of their neurons’ projections. The relatively simple architecture of the pineal gland and the asymmetric character of the habenulae provide a useful tool for studying cell-fate determination, cell migration and establishment of brain asymmetries. In this study, we used zebrafish as a model to dissect the novel functions of sox2 in the development of the epithalamus. We showed that sox2 works synergistically with Notch pathway to negatively regulate neurogenesis within the pineal gland. The pineal gland consists of only two cell types: the photoreceptors and the projection neurons. Previous studies showed that the Notch and BMP pathways are important for the proper specification of these cells. Here, we show that sox2 normally inhibits the photoreceptor cell fate, whereas it has no effect on the number of projection neurons. Therefore, sox2 complements Notch and BMP pathways in cell-fate determination within the pineal gland. In addition, downregulation of sox2 results in abnormal parapineal organ development and disruption of the asymmetric architecture of the habenulae. A subset of sox2 morphant embryos develops right-sided parapineal organs, which is consistent with abnormal bilateral expression of the Nodal gene, pitx2 (paired-like homeodomain transcription factor 2). Also, timelapse experiments showed that migration of the parapineal cells is defective, resulting in scattered cells. The aberrant parapineal development leads to disorganization of the habenular nuclei, as shown by the abnormal neuropil arrangement and the expression of the asymmetric marker kctd12.1 (potassium channel tetramerisation domain containing 12.1).

The thymus is the major site for T-cell generation and thus is important for the adaptive immune system. Development of a properly selected, functional T-cell repertoire relies on interactions between developing T cells and a series of functionally distinct thymic stroma cell types including the cortical and medullary thymic epithelial cells (TECs). The thymus is one of the first organs to degenerate in normal healthy ageing. Related to this, there is strong interest in developing protocols for improving thymus function in patients by cell replacement or regenerative therapies. Thymic epithelial progenitor cells (TEPCs) represent a potential source of cells for thymus transplantation. However, the only source of these cells for transplantation is currently fetal thymus tissue. If TEPCs could be generated from pluripotent cells, this could provide an alternative source of cells for transplantation. The work described in this thesis therefore had two central aims (i) to test the stability of thymic epithelial progenitor cells in vivo and (ii) to investigate the possibility of generating TEPCs or TECs from mouse embryonic stem (ES) cells. The forkhead transcription factor, Foxn1, is essential for the development of a functionally mature thymic epithelium, but is not necessary for formation of the thymic primordium or for medullary thymic epithelial sub-lineage specification. By reactivating Foxn1 expression postnatally in mice carrying a revertible hypomorphic allele of Foxn1, Foxn1R, I herein demonstrate that TEPCs that can express only low levels of Foxn1 mRNA can persist postnatally in the thymic rudiment in mice until at least 6 months of age, and retain the potential to give rise to both cortical and medullary thymic epithelial cells (cTECs and mTECs). These data demonstrate that the TEPC-state is remarkably stable in vivo under conditions of low Foxn1 expression. In parallel with this work, I confirmed the possibility of generating Foxn1-expressing cells from mouse ES cells by using a Foxn1 reporter cell line. As the thymic epithelium has a single origin in the third pharyngeal pouch (3pp) endoderm, I then tested whether or not TEPCs and /or TECs were generated during ES cell differentiation via existing protocols for generating anterior definitive endoderm differentiation cells from mouse ES cells. From this work, I showed that genes expressed in the 3pp and/or TEPC,-including Plet-1, Tbx1, Hoxa3 and Pax9, were induced by differentiation of ES cells using these protocols. I further showed that cells expressing both Plet-1, a marker of foregut endoderm and 3pp, and EpCAM, a marker of proliferating epithelial cells, were induced using a novel protocol (2i ADE) for generating ES cells from ADE. However, gene expression analysis and functional testing suggested that the majority of these cells were non-thymus lineage. I subsequently developed a novel protocol which combined this 2i ADE protocol with co-culturing of the differentiating ES cells with fetal thymic lobes, and demonstrated that this further induced 3pp and TEPC related genes. Finally, I modified the culture conditions in this protocol to conditions predicted to better support TEPC/TEC, and showed that in these conditions, the TEPC-specific markers Foxn1 and IL-7 were induced more strongly than in any other conditions tested. The data presented in this thesis therefore represent an advance towards an optimized protocol for successfully generating TEPCs from ES cells in vitro.

Embryonic stem (ES) cell pluripotency is sustained by a network of transcription factors centred on Oct4, Sox2 and Nanog. Whilst Oct4 and Sox2 expression is relatively uniform, ES cells fluctuate between states of high Nanog expression possessing high self-renewal efficiency, and low Nanog expression exhibiting increased differentiation propensity. Moreover, modulation in the level of Nanog expression determines the efficiency of ES cell self-renewal. To identify genes regulated by Nanog, genome-wide transcriptional profiling was performed on ES cells expressing different Nanog levels and Nanog-null ES cells expressing a Nanog-ERT2 fusion protein in which nuclear Nanog activity can be regulated by tamoxifen. Surprisingly, only a minor fraction of the genes to which Nanog binds showed significant changes in response to Nanog induction. Prominent amongst Nanog-responsive genes is Estrogen-related receptor b (Esrrb). Nanog binds directly to Esrrb, enhances binding and pause-release of RNAPolII from the Esrrb promoter and stimulates Esrrb transcription. Consistent with these findings, elevation of Nanog produces a cell population that expresses uniformly high Esrrb levels. Moreover, double fluorescent reporter lines show that Esrrb and Nanog levels are strongly correlated in individual cells. Loss of Nanog is required for downregulation of Esrrb, which coincides with commitment to differentiate. Esrrb overexpression results in LIF independent self-renewal, and blocks neural differentiation, even in the absence of Nanog. Cell fusion experiments between ES and neural stem (NS) cells show that elevated Esrrb levels allow the reprogramming of the NS cell genome in the absence of Nanog. Esrrb can rescue stalled reprogramming during the derivation of Nanog-/- induced pluripotent stem (iPS) cells. Moreover, targeted knock-in of Esrrb at the Nanog locus rescues the ability of Nanog null ES cells to maintain germ cell development beyond E12. Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent selfrenewal to ES cells. Together these data identify Esrrb as a critical downstream mediator of Nanog function.

No description available.

Em fêmeas de mamíferos, um dos cromossomos X é inativado proporcionando compensação de dose entre os produtos gênicos de machos e fêmeas. A inativação do cromossomo X (ICX) ocorre no embrião em desenvolvimento, e se caracteriza pela aquisição de marcas heterocromáticas no cromossomo X inativado (Xi), que são mantidas nas células somáticas ao longo das divisões celulares. O melhor modelo para estudo do início da ICX são as células-tronco embrionárias femininas. Provenientes da massa celular interna de blastocistos, elas representam um embrião em desenvolvimento e possuem os dois X ativos; a diferenciação das células promove a ICX in vitro, o que permite a identificação dos fatores e mecanismos moleculares envolvidos. A derivação de linhagens de célulastronco embrionárias humanas (human embryonic stem cells - hESCs) em 1998 permitiu novas possibilidades de estudo da ICX, pois a maioria dos trabalhos procurou esclarecer o mecanismo da ICX no modelo murino. Tradicionalmente, a manutenção da ICX em humanos tem sido investigada em células somáticas híbridas ou transformadas; porém, sabe-se que estas não representam um contexto celular natural. Assim, o presente trabalho teve como objetivos principais explorar a potencialidade de hESCs no estudo do início da ICX, e ainda investigar a função de três fatores na manutenção da ICX em células humanas imortalizadas: DNMT1 (enzima responsável pela manutenção da metilação do DNA), SMCHD1 (proteína da família de coesinas/condensinas), e XIST (um RNA não-codificador que inicia o processo de heterocromatinização do futuro Xi) foram selecionados para este estudo, uma vez que todos participam da manutenção da ICX em camundongos. Até o momento foram derivadas em nosso laboratório quatro linhagens de hESCs, as primeiras da América Latina. A caracterização das linhagens mostrou que, apesar de se manterem indiferenciadas, as hESCs femininas encontram-se em estágio pós-ICX, pois mesmo indiferenciadas já apresentam um dos X inativado. Nossos dados indicam que, submetidas às atuais condições de cultivo, as hESCs não são bons modelos para o estudo do início da ICX, e é possível que a inativação de um cromossomo X durante o cultivo confira alguma vantagem seletiva às células. A estratégia utilizada no estudo da manutenção da ICX foi o silenciamento dos três genes por interferência de RNA (RNAi). Não foi possível diminuir significativamente a expressão dos genes XIST e SMCHD1. Porém, o silenciamento de DNMT1 foi expressivo, e em resposta foi observada reativação do gene MAOA, localizado no cromossomo X e submetido à inativação. Apesar de nossas análises mostrarem que os efeitos da diminuição de DNMT1 foram restritos ao gene MAOA, estes resultados sugerem a existência de diferentes hierarquias de controle epigenético dos genes submetidos à ICX em células humanas / In female mammals, one of the X chromosomes is inactivated to achieve dosage compensation between males and females. The X chromosome inactivation (XCI) occurs early during embryogenesis and is characterized by the acquisition of heterochromatic features on the inactive X (Xi), which are maintained during all the subsequent cell divisions. Embryonic stem cells are the most suitable cells to study the establishment of XCI. They are obtained from the inner cell mass (ICM) of blastocysts, and can represent a developing female embryo, possessing two active X-chromosomes; when differentiated, these cells recapitulate XCI in vitro, and thus one can identify XCI regulators and factors involved. The derivation of human embryonic stem cells (hESCs) in 1998 offered new possibilities to study XCI, since most of the mechanistic studies of XCI have so far been investigated in the mouse model system. Traditionally, maintenance of XCI in humans has been addressed in somatic cell hybrids or transformed cells; however, they do not represent a natural cellular context. The main goals of the present work were to verify the potential of hESCs as models of XCI, and also to study the function of three important factors in XCI maintenance in immortalized human cells. DNMT1 (DNA-methyltransferase 1), SMCHD1 (a cohesin/condensin protein family member) and the XIST gene (a non-coding RNA which triggers XCI and promotes X heterochromatin formation on the future Xi) were selected, as they are key factors in XCI maintenance in the mouse. Until now four hESCs lines were derived in our lab. Their characterization showed that, in spite of been undifferentiated, the female hESCs have already undergone XCI. Our data suggest that, under the actual culture conditions, hESCs are not good models to study XCI, and it is also possible that X inactivation confers selective advantage to hESCs. Knockdown by RNA interference was used to study the roles of three genes in XCI maintenance. We could not efficiently knockdown XIST or SMCHD1. However, the DNMT1 silencing was substantial, and led to the reactivation of MAOA, an X-linked gene subjected to XCI. Although the effect of DNMT1 silencing was restricted to MAOA, our data suggest that there are different epigenetic hierarchies to control the expression of the genes subjected to XCI in human cells.