Bright/ARID3A was initially discovered for its role in immunoglobulin heavy chain transcription in the mouse. Bright has also been implicated as a target of p53 and as an E2F binding partner. We have previously shown that Bright is necessary for hematopoietic stem cell development in the embryo. In this work, we show that Bright has a much broader role in development than previously appreciated. Loss of Bright in mice usually results in embryonic lethality due to lack of hematopoietic stem cells. Rare survivor mice initially appear smaller in size than either wildtype or heterozygous littermates, but as they age, these differences diminish. We show that adult Bright null mice have age-dependent kidney defects. Previous work in the adult mouse has not indicated a role for Bright in kidney function. We observed an increase in cellular proliferation in Bright null kidneys, indicating a possible mechanism behind our observation. Loss of Bright has recently been implicated in causing developmental plasticity in somatic cells. Our data indicate that loss of Bright is sufficient to fully reprogram mouse embryonic fibroblasts (MEFs) back to a pluripotent state. We term these cells Bright repression induced pluripotent stem cells (BriPS). BriPS derived from Bright knockout MEFs can be stably maintained in standard embryonic stem cell culture conditions: they express pluripotency markers and can form teratomas in vivo. We further viii show that Bright is active during embryonic stem cell differentiation. Bright represses key pluripotency genes, suggesting the mechanism of reprogramming may be Bright’s direct repression of key pluripotency factors in somatic cells. Recent advances in inducing pluripotency in somatic cells (iPS cells) have created a new field of disease modeling, increased our knowledge of how pluripotency is regulated, and introduced the hope that they can be adapted to treat disease. However, current methods for producing iPS involve overexpression of potentially oncogenic transcription factors, leaving a large gap between the lab and the clinic. Our results mark the first demonstration of an alternative method to reprograming somatic cells that is not mediated by overexpression of pluripotency factors. / text

No description available.

Differentiation and cell cycle regulation in stem cell have a key function for embryonic development, organ homeostasis and tissue repair. Recent results have shown that these two mechanisms are intrinsically connected. Indeed, cell cycle machinery directly controls maintenance of pluripotency and initiation of differentiation. More precisely, the cell cycle regulator Cyclin D appears to control the transcriptional activity of Activin/Nodal signalling during progression of the cell cycle in human Embryonic Stem Cells (hESCs). As a consequence, hESCs can only differentiate into endoderm in the Early G1 phase when Cyclin Ds are expressed at low levels. These results show the mechanisms by which the cell cycle defines differentiation propensity of stem cells. However, these observations also imply the existence of interplays coordinating extra cellular signalling pathways with the epigenetic state, chromatin structure and transcriptional networks during cell cycle progression and these mechanisms remain to be fully uncovered. Here, I have utilised the FUCCI reporter system combined with ATAC-Seq to analyse chromatin dynamics during cell cycle progression in hESCs. Furthermore, I performed ChIP-Seq analyses to define the genomic location of transcriptional regulators during cell cycle progression as well as RNA-Seq to confirm variation in gene expression pattern. Integration of these data shows that the chromatin status in hESCs is highly dynamic and the core pluripotency transcription factors and epigenetic modifiers change genomic location during cell cycle progression. I also showed that hESCs in the Late G1 phase accumulate transcripts that are important for differentiation and development; therefore, indicating this phase represents a unique portion of the cell cycle for cell fate decisions. Taken together, these results uncover that transcriptional networks are unexpectedly dynamic during the progression of cell cycle in stem cells. I hypothesise that these modifications are necessary to prime hESCs for different cell fate choices allowing a diversity of differentiation that is otherwise impossible. Overall these mechanisms underline the need to study transcriptional and epigenetic mechanisms in the dynamic context of the cell cycle and have major implications for adult tissue homeostasis and disease.

In embryonic stem cells (ESCs), the SWI/SNF, CHD, and INO80 families of ATP-dependent chromatin remodellers have been implicated in maintaining pluripotency-associated gene expression, however the involvement of ISWI family remodellers has yet to be defined. Here, we explore the importance of the mammalian ISWI homologue SNF2H (Smarca5) by deriving a conditional knockout mouse ESC line and observing the consequences of SNF2H depletion on the pluripotent state. Cre-mediated deletion of Snf2h disrupts hallmark characteristics of pluripotency, resulting in distinct morphological changes; reduced expression of the master transcription factors Oct4, Sox2, and Nanog; and reduced alkaline phosphatase activity. To understand the mechanisms of SNF2H-mediated regulation, we mapped SNF2H-bound nucleosomes genome-wide. SNF2H is broadly distributed across the genome, but is preferentially enriched at active regulatory regions and transcription factor binding sites. These findings demonstrate the importance of SNF2H in ESCs and shed light on genome-wide mechanisms of transcriptional regulation.

LSD1 is the first described histone demethylase which demethylates H3K4me1/2 (Shi et el., 2004), thus, causing transcriptional repression. Alternatively, LSD1 was demonstrated to have H3K9me1/2 demethylase activity when bound by androgen receptor, hence, causing transcriptional activation (Schule et al., 2005). LSD1 is commonly recruited by the so called CoREST core complex including: RCOR1, HDAC1 and HDAC2 among others and therefore is coupled with histone deacetylation and transcriptional repression (Foster et al., 2010). It is an important regulator of pluripotency in early development and it occupies, along with pluripotency factors NANOG and OCT4, the promoters of major lineage determining genes that are poised for activation in the pluripotent state, (Adamo et al., 2011). There are four described isoforms for LSD1: LSD1, LSD1-E2a, LSD1-8a and LSD1-E2a/E8a (Zibetti et al., 2010). While the Cterminus of LSD1 is extensively studied and the function of the isoforms LSD1-E8a and LSD1-E8aE2a is described, there is scarce knowledge on LSD1 N-terminus unstructured region and the SWIRM domain. In this project I examined the role of the differently spliced exon 2a on the function of the SWIRM domain through generation of eight constructs coding for the N-terminal portion of LSD1 SV1 and SV2 fused with a C- or N-terminus FLAG tag. I then performed an immunoprecipitation experiment followed by mass spectrometry and proteomics analysis that led to the identification of previously unknown binding partners to the LSD1 SWIRM domain: NONO and IGF2B3.

DNA methylation is one of the principal epigenetic modifications that plays an essential role in transcriptional regulation. After fertilization, mammalian embryos undergo dynamic changes in genome-wide DNA methylation patterns and the changes are essential for normal embryo development. Ten-eleven translocation (TET) methylcytosine dioxygenases are implicated in DNA demethylation by catalyzing the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The three members of TET protein family, TET1, TET2, and TET3, are highly expressed in preimplantation embryos in a stage-specific manner. Previous studies demonstrated that TET proteins are involved in diverse biological processes such as gene regulation, pluripotency maintenance, and cell differentiation by mediating 5mC oxidation. My dissertation research was conducted to elucidate the mechanistic roles of TET proteins in epigenetic reprogramming of mammalian embryos using porcine embryos as a model. The first set of studies focused on the relationship between TET proteins and pluripotency. To understand the role of TET proteins in establishing pluripotency in preimplantation embryos, CRISPR/Cas9 technology and TET-specific inhibitors were applied. TET1 depletion unexpectedly resulted in an increased expression of NANOG and ESRRB genes in blastocysts, although the DNA methylation levels of NANOG promoter were not changed. Interestingly, transcript abundance of TET3 was increased in blastocysts carrying inactivated TET1, which might have had an effect on the increase of NANOG and ESRRB. When the activity of TET enzymes was inhibited to eliminate the compensatory increase of TET3 under the absence of functional TET1, the expression levels of NANOG and ESRRB were decreased and methylation level of NANOG promoter was increased. In addition, ICM specification was impaired by the inhibition of TET enzymes. These results suggest that the TET family is a critical component of the pluripotency network of porcine embryos by regulating expression of genes involved in pluripotency and early lineage specification. In the next set of studies, the presence of TET3 isoforms in porcine oocytes and cumulus cells was investigated to dissect the gene structure of TET3 that could assist in understanding mechanistic actions of TET3 in the DNA demethylation process. Among the three TET3 isoforms identified in cumulus cells, only the pTET3L isoform, which contains CXXC domain that carry DNA binding property, was verified in mature porcine oocytes. Expression level of the pTET3L isoform was much higher in mature oocytes compared to that in somatic cells and tissues. In addition, the transcript level of this isoform was significantly increased during oocyte maturation. These results suggest that pTET3L isoform is predominantly present in mature porcine oocytes and that CXXC domain may play an important role in DNA demethylation in zygotes. In a follow-up study, the role of the TET3 CXXC domain in controlling post-fertilization demethylation in porcine embryos was investigated by injecting TET3 GFP-CXXC into mature porcine oocytes. The injected CXXC was exclusively localized in the pronuclei, indicating that the CXXC domain may localize TET3 to the nucleus. The CXXC overexpression reduced the 5mC level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, the transcript abundance of NANOG and ESRRB was increased in blastocysts derived from GFP-CXXC overexpressing zygotes. These results provide an evidence that the CXXC domain of TET3 is critical for post-fertilization demethylation of porcine embryos and proper expression of pluripotency related genes in blastocysts. In the last set of studies, the impact of MBD proteins on porcine embryo development was examined under the hypothesis that competitive binding of MBD and TET proteins to 5mC contributes to the proper maintenance of DNA methylation levels in embryos. Cloning of porcine MBD1, MBD3, and MBD4 from mature oocytes indicates that the genes are highly conserved among different species, implying the involvement of porcine MBD proteins in the maintenance of DNA methylation. MBD1 overexpression in oocytes impaired preimplantation development of porcine embryos, suggesting that the MBD1 overexpression may have negatively affected porcine embryo development because proper DNA methylation levels were not preserved under the high level of MBD1. Collectively, the studies in my dissertation demonstrate that TET family proteins are important epigenetic players involved in the regulation of pluripotency and reprogramming of DNA methylation, and are thus crucial for normal embryo development. The findings in the dissertation will improve our understanding of epigenetic events occurring in mammalian embryos, and have the potential to overcome epigenetic defects that are observed in pluripotent stem cells and in-vitro derived embryos. / Doctor of Philosophy / Epigenetic modifications are heritable changes affecting the level of gene expression without changing the sequence of the genome. DNA methylation, one of the biggest epigenetic marks in mammalian genome, is often correlated to gene repression. In mammals, DNA methylation patterns are dramatically changed during preimplantation development to acquire embryonic developmental potential. Understanding of the epigenetic changes occurring in preimplantation embryos is necessary for producing healthy domestic animals in agriculture and for developing strategies for the treatment of epigenetic defects in human. Ten-eleven translocation (TET) family enzymes, TET1, TET2, and TET3, are known to function as a DNA methylation modifier by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). My dissertation research was performed to elucidate the role of TET family during preimplantation development using porcine embryos as a model. Pluripotency refers to the ability of cells to differentiate into all cell types of a mature organism. Pluripotent cells emerge in embryos as embryonic cells acquire lineage-specific characteristics. The first set of studies focused on the role of TET enzymes in regulating the pluripotency of porcine embryos. The impacts of inhibited activities of TET enzymes on the expression of pluripotency related genes were examined. We found that the inhibition of all TET enzymes leads to a decreased expression of pluripotency related genes, an altered DNA methylation level on a gene segment controlling pluripotency, and the impaired formation of pluripotent cell lineage in porcine embryos. This study demonstrates that the TET family is critical for the acquisition of pluripotency in porcine embryos. In the following sets of studies, the function of TET3 protein in the demethylation process occurring in preimplantation embryos was investigated. Fertilized mammalian embryos undergo genome-wide demethylation process to reset germ cell specific epigenetic marks into the embryonic epigenome. Previous studies indicate that TET3 is responsible for the demethylation process in mammalian embryos, although detailed mechanistic action of TET3 is still elusive. Here, we identified a predominant expression of a specific TET3 gene in porcine oocytes. The TET3 gene contained a CXXC domain, a potential DNA binding module, suggesting that TET3 may mediate DNA demethylation through its DNA binding property. To examine the function of the CXXC domain in TET3-mediated DNA demethylation, isolated CXXC domain was injected into porcine oocytes. The injection of CXXC domain facilitated DNA demethylation in embryos, demonstrating that the DNA binding property of TET3 is important for its functionality. In the last study, we investigated the importance of genes known to interact with TET enzymes in porcine embryos. Methyl-CpG-binding domain proteins (MBDs) have the ability to bind methylated region on the genome and play a critical role in mediating DNA methylation and gene repression. Our hypothesis was that a competitive binding of MBD and TET proteins to methylated regions was critical for proper DNA methylation levels in embryos. We identified that porcine MBD sequences were very similar to other species in terms of gene structure, indicating that the genes could also possess gene repressing activity by competing with TET enzymes during porcine embryo development. Injection of MBD1 mRNA to oocytes impaired normal embryo development, suggesting that the injected MBD1 may have negatively affected early embryo development in pigs by disrupting the proper maintenance of DNA methylation levels. My dissertation researches demonstrate that maintaining proper DNA methylation levels through the TET family is critical for normal embryo development in pigs. This research assists in improving our understating of epigenetic dynamics occurring in mammalian embryos and offers a potential solution to the epigenetic defects frequently observed in mammalian embryos produced through artificial reproductive technologies and pluripotent stem cells reprogrammed from somatic cells.

Human pluripotent stem cells (hPSCs) exist in multiple states of pluripotency, broadly categorised as naïve and primed states. These provide an important model to investigate the earliest stages of human embryonic development. Naïve cells can be obtained through primed-to-naïve reprogramming; however, there are no reliable methods to prospectively isolate unmodified naïve cells during this process. Moreover, the current isolation strategies are incompatible for enrichment of naïve hPSCs early during reprogramming. Consequently, we know very little about the temporal dynamics of transcriptional changes and remodelling of the epigenetic landscape that occurs during the reprogramming process. To address this knowledge gap, I sought to develop an isolation strategy capable of identifying nascent naïve hPSCs early during reprogramming. Comprehensive profiling of cell-surface markers by flow cytometry in naïve and primed hPSCs revealed pluripotent state-specific antibodies. By compiling the identified state-specific markers into a multiplexed antibody panel, I was able to distinguish naïve and primed hPSCs. Moreover, the antibody panel was able to track the dynamics of primed-to-naïve reprogramming, as the state-specific surface markers collectively reflect the change in pluripotent states. Through using the newly identified surface markers, I found that naïve cells are formed at a much earlier time point than previously realised, and could be subsequently isolated from a heterogeneous cell population early during reprogramming. This allowed me to perform the first molecular characterisation of nascent naïve hPSCs, which revealed distinct transcriptional changes associated with early and late stage naïve cell formation. Analysis of the DNA methylation landscape showed that nascent naïve cells are globally hypomethylated, whilst imprint methylation is largely preserved. Moreover, the loss of DNA methylation precedes X-chromosome reactivation, which occurs primarily during the late-stage of primed-to-naïve reprogramming, and is therefore a hallmark of mature naïve cells. Using the antibody panel at discrete time points throughout reprogramming has allowed an unprecedented insight into the early molecular events leading to naïve cell formation, and permits the direct comparison between different naïve reprogramming methods. Taken together, the identified state-specific surface markers provide a robust and straightforward method to unambiguously define human PSC states, and reveal for the first time the order of transcriptional and epigenetic changes associated with primed to naïve reprogramming.

A produção de células-tronco induzidas (iPSC) a partir de fibroblasto fetal canino abre caminhos para a obtenção de células pluripotentes e o estudo de sua aplicabilidade para terapias alternativas na medicina veterinária. Neste contexto, este trabalho investigou metodologias adequadas avaliando a eficiência destas, para a produção de células-tronco pluripotentes no modelo canino in vitro (CTE-like), uma vez que a produção de células-tronco embrionárias verdadeiras, cultivadas a partir da MCI de blastocistos, ainda não foi completamente caracterizada em animais domésticos. Os experimentos visaram o aumento do conhecimento de fatores envolvidos no processo de reprogramação em cães, bem como a produção de tais linhagens e sua completa caracterização. No primeiro experimento, foi comparada a infecção retroviral, já padronizada por diversos grupos, com a reprogramação epissomal, inédita para a espécie, na tentativa de induzir células à pluripotência sem a integração viral, e ainda, estratégias para o aumento da eficiência de reprogramação, onde o plasmídeo epissomal foi somado a fatores de transcrição. A reprogramação epissomal gerou colônias quando acrescida do fator c-MYC, que provavelmente, aumentou a proliferação destas células produzindo colônias iPS com morfologia típica e positivas para o teste da fosfatase alcalina. Tais resultados, ainda preliminares pra conclusões, são essenciais para o processo de obtenção de linhagens sem a integração viral, aumentando a aplicabilidade na terapia celular. No segundo experimento objetivou-se avaliar os fatores OCT4 e SOX2 associados a proteínas repórteres. Os fibroblastos que receberam estes fatores, foram analisados por citometria de fluxo, permitindo a avaliação da influência de cada fator no processo de reprogramação, além de permitir a separação (sorting) das células que integraram o gene, aumentando a eficiência de reprogramação e o conhecimento biológico dos mecanismos de integração rastreados por uma proteína repórter. A análise por microscopia de fluorescência revelou que a distribuição de proteínas repórteres foi semelhante entre as duas diferentes construções proteicas e que não se restringe a uma região da célula em particular. OCT4 e SOX2 mostraram uma elevada expressão exógena de cada gene alvo, bem como células dupla positivas. No entanto, nenhuma interação foi observada pelo menos 6 dias após a transdução. O último capítulo experimental descreveu o mecanismo de reprogramação por integração lentiviral para indução da pluripotência em fibroblastos fetais de cão. As linhagens obtidas e completamente caracterizadas neste estudo foram independentes de LIF ou qualquer outra suplementação com inibidores, resistentes ao repique enzimático (Tryple Express), sendo apenas bFGF dependentes. Foram obtidas 66 linhagens clonais, das quais 10 (7 h+mOSKM e 3hOSKM) se mantiveram por 15 ou mais passagens e foram utilizadas para todos os testes de caracterização in vitro, com eficiência máxima de reprogramação de 0,001%. Todas as colônias foram positivas para o teste da fosfatase alcalina, bem como formaram corpos embrióides e se diferenciaram de forma espontânea, além de expressarem altos níveis dos fatores endógenos OCT4 e SOX2. In vivo, as colônias foram capazes de desenvolver tumor 120 dias após a inoculação (confirmado por análise histopatológica) comprovando sua origem predominantemente mesodérmica. A integridade cromossomal das linhagens foi avaliada por hidridização FISH, que não evidenciou qualquer tipo de anomalia. A completa caracterização de tais linhagens, bem como os experimentos não integrativos e com fatores associados a proteínas repórteres, aumentam o conhecimento da tecnologia de reprogramação, estabelecendo novas estratégias para indução da pluripotência de forma mais eficaz e segura para seu uso em testes clínicos e terapia celular / The production of induced pluripotent stem cells (iPSC) from canine fetal fibroblast opens new ways for obtaining pluripotent cells and study its applicability for alternative therapies in veterinary medicine. In this context, this study investigated appropriate methods for producing pluripotent stem cells using a in vitro canine model (ESC-like), so far the production of true embryonic stem cells from ICM cultured blastocysts has not been fully characterized in domestic animals. The experiments aimed at increasing knowledge of the factors involved in reprogramming process in dogs, as well as the production of such strains and complete characterization. In the first experiment, a retroviral infection was compared to episomal reprogramming (never done for this specie) in an attempt to induce cells to pluripotency state without viral integration, also to observe the development of cells receiving separately the episomal plasmid plus transcription factors. The generation of colonies was possible only in the episomal plus c-MYC factor group, leading to increased cell proliferation producing iPS colonies with typical morphology and positive for the alkaline phosphatase detection. These results, so far as preliminary conclusions, are essential to obtaining strains without viral integration, increasing its applicability for clinical cell therapy. In the second experiment, we aimed to evaluate the OCT4 and SOX2 factors associated with fluorescent reporter proteins. These were analyzed by flow cytometry allowing the performance evaluation of each factor on the reprogramming process the fluorescence activated separation of cells containing the integrated gene, increasing the enriching the efficiency of reprogramming. Fluorescence microscopy analysis showed that the distribution of reporter protein was similar between the two different protein structures and not restricted to a particular cell region. OCT4 and SOX2 showed a high exogenous expression of each target gene, and double positive cells. However, no colony formation was observed at least 6 days after transduction. The last experimental chapter aimed to described the reprogramming mechanism of lentiviral integration to induce pluripotency in dog fetal fibroblasts. The lines obtained were fully characterized in this study, showing independency of LIF or any other supplemental inhibitors, resistance to enzymatic process (Tryple Express) and bFGF dependency only. A total of 66 clonal strains were obtained (hOSKM and h+mOSKM) while 10 (7 h+m and 3h) were maintained for 15 or more passages and used for in vitro characterization tests, with maximum efficiency of reprogramming 0.001% . All colonies were positive for the alkaline phosphatase detection, embryoid bodies formation, spontaneously differentiated and expressed high levels of endogenous OCT4 and SOX2. In vivo, the colonies were able to developed tumors 120 days after inoculation (confirmed via histopathology analysis), with predominantly mesodermal tissues. Chromosomal evaluations were made by FISH hybridization showing no chromosomal abnormality in iPSCs canine lines. The fully characterization of such lines as well as non-integrated experiments and factors associated via reporter proteins increases the knowledge of the iPSCs technology, establishing new strategies for more efficient and safe induction of pluripotency for potential use in cell therapy and clinical trials

Células somáticas podem ser reprogramadas para um estádio pluripotente (iPS) adquirindo propriedades semelhantes às células-tronco embrionárias (CTE). O interesse nas células pluripotentes reside em sua capacidade de originar todos os tipos de células somáticas e germinativas, podendo ser aplicadas no tratamento de diversas doenças crônico-degenerativas. Desde sua primeira descrição, diferentes combinações de moléculas já foram utilizadas com sucesso para a geração de iPS. Entretanto, os mecanismos pelos quais a transdução de fatores específicos atuam na reprogramação celular não estão esclarecidos. Este trabalho teve como objetivo induzir a expressão do gene TCL1 em fibroblastos humanos e avaliar a ação deste gene no processo de reprogramação celular. Para tal, foram estabelecidas linhagens celulares de fibroblastos humanos com a expressão estável de TCL1 e essas células foram cultivadas em condições de pluripotência. Após a modificação, as células adquiriram morfologia sugestiva de colônias de células-tronco pluripotentes com marcação positiva para a proteína intracelular NANOG e com níveis de expressão gênica elevados de SOX2, MYC, NANOG, LIN28, TP53, CDH1 e reduzidos de SLUG, quando comparados com fibroblastos virgens. Com intuito de avaliar as alterações transcricionais decorrentes da inserção de TCL1 e do cultivo em condições favorecedoras da pluripotência, foram comparados os perfis de expressão gênica obtidos por microarray de diferentes bibliotecas, incluindo as células modificadas com TCL1, fibroblastos, CTE e iPS. A análise exploratória dos dados mostrou que a introdução de TCL1 modificou o perfil de expressão dos fibroblastos e as células resultantes adquiriram um perfil transcricional que se assemelhou mais com o perfil de células pluripotentes do que com o perfil das células somáticas de origem. A análise diferencial dos dados revelou que vias importantes para a reprogramação celular foram moduladas pela inserção de TCL1, como: Pluripotência de células-tronco embrionárias humanas, Sinalização Wnt/?-catenina e Regulação da transição epitelial-mesenquimal. Os resultados deste trabalho propõem que TCL1 interage com AKT1, aumentando sua atividade, que por sua vez ativa NANOG, acionando a maquinaria de pluripotência e, contribuindo assim, para a reprogramação celular / Somatic cells can be reprogrammed into pluripotent stage (iPS) acquiring properties similar to embryonic stem cells (ESC). The interest in pluripotent stem cells lies in their ability to originate all types of somatic and germ cells, and in their possible application in the treatment of various chronic and degenerative diseases. Since its first description, different combinations of molecules have been successfully used for the generation of iPS. However, the mechanisms by which the transduction of specific factors act on cell reprogramming remain unclear. This study aimed to induce the TCL1 gene expression in human fibroblasts and to evaluate its effect on the cell reprogramming process. We established human fibroblast cell lines with stable expression of TCL1 and cultured these cells under pluripotency conditions. After modification, the cells acquired a pluripotent stem cells-like morphology, stained positive for intracellular protein NANOG, expressed high levels of SOX2, MYC, NANOG, LIN28, TP53, CDH1, and reduced levels of SLUG, as compared to nontransduced fibroblasts. In order to evaluate the transcriptional changes resulting from the insertion of TCL1 and from the culture conditions favoring the pluripotency, we compared the gene expression profiles obtained by microarray among different libraries, including the TCL1 modified cells, fibroblasts, ESC and iPS. Exploratory data analysis showed that the introduction of TCL1 gene modified the expression profile of cells and the resulting fibroblasts acquired a transcriptional profile that resembled more to the profile of pluripotent cells than with the profile of the somatic cells. Differential data analysis revealed that pathways important for cell reprogramming were modulated by TCL1 insertion such as: Human embryonic pluripotent stem cell pathway, Wnt / ?-catenin signaling pathway, and Regulation of epithelial-mesenchymal transition. The results of this study suggest that TCL1 interacts with AKT1, increasing its activity, which in turn activates NANOG, triggering the machinery of pluripotency and thus contribute to cellular reprogramming.

Pluripotency is defined as the capacity to differentiate into cells from each of the three primary germ layers, the ectoderm, mesoderm and endoderm. This is a property of cells located in the inner cell mass (ICM) of preimplantation blastocysts and in the epiblast layer of postimplantation, presomite embryos. Preimplantation and postimplantation pluripotency can be captured indefinitely in cultured embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively. Preimplantation pluripotency in ES cells is regulated by a network of genes centred on three transcription factors (TFs) Oct4, Sox2 and Nanog. Oct4 and Sox2 form a mutually-reinforcing circuit and cooperatively stimulate transcription of downstream genes, including Nanog. All three TFs are expressed in EpiSCs and in the postimplantation epiblast. Functional studies established a role for Oct4 and Nanog in the specification of ICM cell identity, and a role for Oct4 in the maintenance of postimplantation pluripotency. Although the role of Sox2 in preimplantation ICM cells is unclear, it is critical for the establishment of egg cylinder following implantation and indispensable for ES cell pluripotency. However, despite the presence of Sox2 in postimplantation pluripotent cells the role of Sox2 in postimplantation pluripotency is unknown. In this thesis the role of Sox2 in the regulation of postimplantation pluripotency was examined. In contrast to the situation in the preimplantation ICM, Sox2 and Nanog are expressed in opposing gradients in the gastrulation-stage postimplantation epiblast, with Sox2 highest anteriorly and Nanog highest posteriorly. Interestingly the posterior epiblast of neural-plate (NP)-staged embryos was shown not to be pluripotent. Furthermore, forced expression of Sox2 but not Oct4 in this region rescued pluripotency. The ability of Oct4 to reinstate pluripotency in the somitogenesis-stage embryo is limited to Sox2-positive tissues. This strongly suggests that coexpression of Sox2 and Oct4 is important for establishing postimplantation pluripotent identity. Sox2HIGH cultured EpiSCs were not positively correlated with NanogHIGH cells. This reciprocal relationship emerged during the transition from ES cells to EpiSCs in culture. Using mutant cells with reduced levels of Sox2 or Nanog, Sox2 positively influences Nanog but Nanog negatively influences Sox2 expression post-transcriptionally. The negative influence of Nanog on Sox2 protein level was confirmed using doxycycline-inducible Nanog overexpressing EpiSCs. This negative relationship indicates that the regulation of Sox2 expression is different in postimplantation pluripotency and that Nanog may negatively regulate Sox2 on the protein level in the posterior epiblast. Sox2 is expressed at a lower level in EpiSCs than ES cells and the significance of this was further investigated by microarray transcription profiling using cells in which a fluorescent reporter (tdTomato) was knocked in to the Sox2 gene. Sox2- tdTomatoHIGH cells cultured in LIF/FCS/GMEMβ correlate with an undifferentiated cell identity and Sox2-tdTomatoLOW cells are associated with non-neural differentiation. Interestingly the global profile of ES cells and EpiSCs that share similar Sox2-tdTomato signal are non-identical. This suggests that Sox2 has different roles in different pluripotent states. ES cells with enforced Sox2 expression were unable to enter the EpiSC state, while ES cells with lowered Sox2 levels were inefficient in neural differentiation. Therefore, levels of Sox2 are critical for cell fate decisions. Strikingly, given the apparent requirement for Sox2 during Oct4-induced reinstatement of post-implantation pluripotency, deletion of Sox2 had no effect on the maintenance of EpiSC pluripotency. This is likely due to the presence of redundant Sox factors and indeed Sox3 is able to rescue the Sox2-null phenotype in ES cells. Taken together, these results suggest the hypothesis that postimplantation pluripotency is maintained by multiple Sox factors, while Nanog negatively regulates Sox2 post-transcriptionally to repress neural specification in the posterior epbilast. The positive influence of Sox2 on Nanog protein level suggests a possible negative feedback loop to balance the proneural and pluripotent properties of Sox2 in postimplantation pluripotency.