Dissertations / Theses on the topic 'Pluripotencia'

The adaptive response of cells to external stimuli is an intriguing mechanism at the basis of the existence of life itself. For this purpose, signalling pathways and gene regulatory networks elegantly evolved translating extracellular signals into finely tuned cellular responses. Among them, the Wnt/ß-catenin signalling pathway converges on the regulation of ß-catenin protein, which, in turn regulates target gene expression. In particular the Wnt/ß-catenin pathway plays a pivotal role in sustaining pluripotency and somatic cell reprogramming. Here we identified a temporal of Wnt/ß-catenin activity during somatic cell reprogramming, controlling the expression levels of mesenchymal-to-epithelial transition and senescence-associated genes through TCF1. We demonstrated that the “Wnt-OFF” state is an early reprogramming marker and that dynamic modulation can be effectively used to increase the reprogramming efficiency. Furthermore the Wnt/ß-catenin pathway is a key regulator of pluripotency and self-renewal of mouse embryonic stem cells (mESCs) and a small-molecule activator of the Wnt pathway is widely used to maintain embryonic stem cells in a ground state of pluripotency. The role of ß-catenin in mESCs is however still controversial. We noticed available ß-catenin knock-out models are flawed by the production of N-terminally truncated proteins with unknown functions. We therefore generated a novel ß-catenin knock-out using CRISPR/Cas9 technology, hoping to have clearer insight of ß-catenin functions in mESCs. We have also found that ground state pluripotency promoted by sustained Wnt pathway activation cannot be maintained indefinitely, resulting in a “lapsed” ground state possibly due, among other factors, to regulatory negative feedback loops that impair Wnt/ß-catenin activity.
La respuesta adaptativa de las células a estímulos externos es un mecanismo fundamental de la existencia de la vida en sí misma. Para este fin, rutas de señalización y redes de regulación génica evolucionaron elegantemente, traduciendo señales extracelulares en respuestas celulares calibradas con precisión. Entre ellas, la ruta de señalización de Wnt/ß-catenin converge en la regulación de la proteína ß-catenin, que a su vez regula la expresión de genes diana. En particular, la ruta de Wnt/ß-catenin tiene un rol fundamental en el mantenimiento de la pluripotencia y la reprogramación de células somáticas. En esta tesis hemos identificado un papel temporal de actividad de Wnt/ß-catenin durante la reprogramación de células somáticas, lo que controla los niveles de expresión de genes asociados a transición mesenquima-epitelial y senescencia a través de TCF1. Además, la ruta de Wnt/ß-catenin es un regulador clave de la pluripotencia y la auto-renovación de células madre embrionarias de ratón (mESCs). Una pequeña molécula, activadora de la ruta Wnt es usada comúnmente para mantener las células madre embrionarias en “ground state” de pluripotencia. Sin embargo, el rol de la ß-catenin en las mESCs es aún controvertido. Observamos que los modelos disponibles de Knock-Out de ß-catenin producen proteínas truncadas en N-terminal con funciones desconocidas. Por ello, generamos un nuevo Knock-Out usando CRISPR/Cas9, al fin de clarificar funciones de ß-catenina en mESCs. Hemos encontrado también que el “ground state” de pluripotencia promovido por la activación sostenida de Wnt no puede ser mantenido indefinidamente, resultando esto en un“ lapsed ground state”; debido posiblemente, entre otros factores, a feedback-loop negativos que afectan negativamente la actividad de Wnt/ß-catenin.

X-Chromosome Reactivation (XCR) occurs in the epiblast cells of the blastocyst and in germ cells, thereby coupling XCR with pluripotency. We performed a screen in iPS cells by knocking down the expression of candidate genes picked from a single cell microarray expression profile in blastocysts. We thereby identified candidates which had an effect on both pluripotency and X-Reactivation. However, we also identified factors with a specific role in XCR. This suggests that XCR is not an absolute requirement for iPSC reprogramming and that the two processes can be uncoupled. Among these factors, there was the cohesin complex member Smc1a. In experiments based on Super resolution microscopy (STORM), we observed a preferential enrichment of Smc1a on the active compared to inactive X, suggesting a role in shaping the Xa structure. Therefore, we conclude that cohesin-mediated changes in X-chromosome structure are a key step during the XCR process.
La reactivación del cromosoma X (XCR) ocurre en las células epiblásticas del blastocisto y en las células germinales, acoplando XCR con la pluripotencia. Se realizó un cribaje durante la reprogramación de iPSC reduciendo la expresión de genes candidatos, seleccionados a partir de un microarray de expresión en blastocitos. Se identificaron factores con un efecto tanto en la pluripotencia como en la XCR y factores con un rol específico en la XCR. Esto sugiere que la XCR no es un requisito absoluto para la reprogramación de las iPSC, y que los dos procesos se pueden desacoplar. Se identificó el miembro Smc1a del complejo de cohesina. Mediante microscopía de súper resolución (STORM) se observó un enriquecimiento preferencial de Smc1a en el cromosoma X activo en comparación con el X inactivo, lo que sugiere un papel en la configuración de la estructura del X activo. Por lo tanto, concluimos que los cambios mediados por cohesina en la estructura del cromosoma X son un paso clave durante el proceso de XCR.

Embryonic stem cells (ESC) are derived from the inner cell mass (ICM) of the blastocyst and have the capacity for unlimited proliferation while retaining their potential to differentiate into a wide variety of cell types when cultured in vitro. These properties have made of human embryonic stem cells (hESC) an excellent model on which to study the conditions required for differentiation into specific cell lineages, and consequently the possibility of transplanting specific cell types into damaged tissues. The continued turn over of ESC while maintaining an undifferentiated state is dependent on unusual cell cycle properties. These unusual proliferative properties are responsible for the generation of tumours when these cells are injected into adult animals. Thus, the study of the unusual proliferative properties of hESC needs to be addressed if their potential is to be realized. To date, most studies of the cell cycle in hESC have been descriptive, lacking functional studies that reveal the mechanisms of how the cell cycle maintains pluripotency and self- renewal of hESC. In this thesis we sought to understand the mechanisms of cell cycle control of hESC. We asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC using a gain and loss of gene function strategy. We have identified that the protein expression of the p27Kip1 cell cycle inhibitor was low in human pluripotent cells, but its expression increased during differentiation together with changes in the cell cycle structure of pluripotent cells. By adopting a gain and loss of function strategy we increased or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency of Hesc, using undifferentiation conditions, overexpression of p27Kip1 in hESC lead to a G1 phase arrest with an enlarged and flattened hESC morphology and consequently loss of self-renewal ability. Loss of p27Kip1 caused an increase of self-renewal while maintaining an undifferentiated phenotype. Moreover, we have shown that a change in the balance of p27Kip1 levels in undifferentiated hESC affects expression of the mesoderm markers: BRACHYURY and TWIST. We have found that expression changes of TWIST are associated with the presence of p27Kip1 protein in the TWIST1 gene promoter. The results presented in this thesis have interesting implications in stem cell biology. Firstly, these results define that the maintenance of p27Kip1 protein levels at a certain level is essential for self-renewal and pluripotency of hESC. Secondly, p27Kip1 is involved in the regulation of TWIST which is upregulated in several types of tumours and induces an epithelial-mesenchymal transition to facilitate tumor metastasis.
Las células madre embrionarias humanas (conocidas como hESC por sus siglas en inglés de human embryonic stem cells) son derivadas de la masa celular interna de los blastocistos y poseen la capacidad para auto-renovarse ilimitadamente, reteniendo su potencial para diferenciarse hace una amplia variedad de tipos celulares (pluripotencia), cuando son cultivadas in vitro. Estas propiedades permiten el estudio de las condiciones requeridas para la diferenciación hacia linajes específicos y la posibilidad de trasplantar tipos celulares específicos en tejidos dañados. El continuo recambio de las hESC al mismo tiempo que mantienen un estado de indiferenciación es dependiente de sus inusuales propiedades proliferativas. El objetivo de esta tesis doctoral fue el estudio de los mecanismos de control del ciclo celular de las hESC. Nos preguntamos si una única proteína del ciclo celular podría regular las propiedades de auto-renovación o pluripotencia de las hESC. En esta tesis doctoral identificamos que la expresión proteica del inhibidor del ciclo celular p27Kip1 era baja en diversas líneas celulares humanas pluripotentes pero aumentó durante la diferenciación, al mismo tiempo que la estructura del ciclo celular cambió. Mediante una estrategia de ganancia y pérdida de función, aumentamos o reducimos la expresión de p27Kip1 a fin de definir su función en la auto-renovación y la pluripotencia de las hESC. En condiciones de indiferenciación, la sobreexpresión de p27Kip1 en las hESC resultó en un arresto del ciclo celular en fase G1 y un cambio hacia una morfología más grande y aplanada, y consiguiente pérdida de la propiedad de auto-renovación. La pérdida de p27Kip1 causó un aumento de la auto-renovación manteniendo un fenotipo indiferenciado. También, hemos demostrado que un cambio en la expresión de p27Kip1 en hESC indiferenciadas afecta la expresión de los reguladores de mesodermo: BRACHYURY y TWIST. Además, hemos descubierto que los cambios en la expresión de TWIST están asociados con la presencia de la proteína p27Kip1 en el promotor de TWIST1. Estos resultados definen que los niveles de expresión de p27Kip1 son críticos para la auto-renovación y la pluripotencia de las hESC y sugieren una función para p27Kip1 en el control de la transición de epitelio a mesénquima.

El periodonto está formado por varios tejidos: la encía, el ligamento periodontal, el cemento radicular y el hueso alveolar. El periodonto tiene dos funciones fundamentales: protección e inserción. La función de protección la realizan la encía y el epitelio de unión, mientras que la función de inserción se desempeña a través del ligamento periodontal, el cemento radicular y el hueso alveolar.- El ligamento periodontal es un tejido celular altamente vascularizado que rodea la raíz del diente. Se compone por haces de fibras de tejido conectivo fibroso que unen el cemento radicular y la pared del alveolo. Presenta una mayor anchura en el extremo cervical, apical y en dientes funcionales, siendo más estrecho en la parte central y en dientes no funcionales. – Las enfermedades periodontales son un conjunto de entidades patológicas de causa infecciosa y naturaleza inflamatoria que afectan a los tejidos que rodean los dientes. Su etiología es multifactorial. La enfermedad periodontal es un importante problema de salud pública y el desarrollo de terapias efectivas para tratar esta enfermedad debería ser un objetivo principal de la comunidad científica. Dependiendo del grado de afectación y de la susceptibilidad del individuo se distinguen dos tipos de enfermedad periodontal: la gingivitis y la periodontitis. La gingivitis se define como la inflamación de los tejidos gingivales sin que exista pérdida ósea y se trata de la enfermedad periodontal más habitual. Sin embargo, si no se trata a tiempo, la gingivitis puede derivar a periodontitis, una enfermedad menos habitual pero con peores consecuencias, ya que afecta al 90% de la población por encima de los 35 años y es la primera causa de perdida de dientes en todo el mundo. La periodontitis se caracteriza por la destrucción del tejido periodontal, incluyendo el ligamento periodontal, el cemento, el hueso alveolar y la encía. La importancia de la periodontitis no está en su prevalencia sino en la morbilidad asociada que conlleva la pérdida de dientes, consecuencia final de la enfermedad periodontal destructiva. En general, se estima que la periodontitis provoca el 30-35% de todas las extracciones dentales. – El objetivo principal del tratamiento periodontal es de prevenir la pérdida de inserción y de regenerar los tejidos del soporte periodontal. En los últimos años se han utilizado diferentes técnicas de tratamiento periodontal, métodos destinados a reproducir o reconstituir una parte perdida o dañada de los tejidos de soporte dentario con el objetivo de restaurar la arquitectura y función de estos tejidos. Las técnicas no-quirúrgicas como la terapia mecánica convencional y los procedimientos quirúrgicos y materiales regenerativos, como por ejemplo la regeneración tisular guiada, el uso de injertos óseos, la aplicación de factores de crecimiento y la modulación de factores del huésped, así como la combinación de varias de estas técnicas. En la mayoría de los casos se consigue una curación a través de un epitelio largo de unión, pero no se consigue una reparación del cemento o el hueso. – Por esta razón se plantea la terapia celular como una posibilidad futura para regenerar el periodonto, la cual requiere la implicación de: células madre, moléculas de señalización y la matriz extracelular tridimensional. – Por definición, una célula madre es una célula indiferenciada que es capaz de autorenovarse y de diferenciarse hacia múltiples tipos celulares. Estas dos propiedades, en conjunto, permiten a las células madre proliferar y regenerar los tejidos perdidos o dañados. Las células madre se han podido aislar de una amplia variedad de tejidos y se pueden clasificar en 3 categorías: células madre embrionarias, células madre pluripotentes inducidas y células madre adultas. – Las células madre embrionarias (ESC) son células pluripotentes, que se aíslan de la masa celular interna del blastocito. Las ESC tienen la capacidad de proliferar extensamente manteniéndose en un estadio indiferenciado pero, bajo unas condiciones adecuadas, son capaces de diferenciarse en células con características de las tres capas germinales (endodermo, mesodermo y ectodermo). Aunque presentan un gran potencial de proliferación y se pueden mantener indefinidamente en cultivo en un estado indiferenciado, su uso en terapias regenerativas está limitado por cuestiones legales y éticas, ya que para su obtención es necesaria la utilización de embriones. Debido a ésto, se intentaron conseguir poblaciones de células madre pluripotentes a partir de células somáticas mediante modificación genética, llamadas células madre pluripotentes inducidas (iPSC). Estas células tienen la ventaja de ser autólogas, al provenir del propio individuo, lo que evitaría los problemas de rechazo. Sin embargo, las manipulaciones genéticas pueden alterar el crecimiento y desarrollo de las células reprogramadas, lo que dificulta la previsibilidad de su comportamiento y, como tal, limita su aplicación terapéutica en la regeneración de tejidos. Como alternativa se aislaron las células madre adultas de prácticamente todo el cuerpo humano: médula ósea, sangre periférica, músculo esquelético, hígado, páncreas, epitelio de la piel y del intestino, pulpa dental, córnea, retina del ojo, sistema nervioso central y corazón. Sin embargo, la obtención de estas células madre resulta laboriosa, en algunos casos y, en otros, las células obtenidas tienen una capacidad de diferenciación limitada. – Nuestro grupo de investigación del Regenerative Medicine Research Institute de la UIC Barcelona ha conseguido aislar una nueva población de células madre adultas a partir de la pulpa dental de los terceros molares. El tercer molar es el último diente en desarrollarse y contiene una cantidad óptima de tejido pulpar para la extracción de células madre. Estas características de los terceros molares, junto con la utilización de un protocolo de cultivo específico, permitieron el aislamiento de una nueva población de células madre de la pulpa dental, las DPPSC (dental pulp pluripotent-like stem cells). Estas células, a diferencia de otras células madre adultas, tienen una capacidad regenerativa similar a la de las células madre embrionarias, al expresar marcadores de pluripotencia (Oct4, Nanog, Sox2 y Lin28) y tener la capacidad de diferenciarse a tejidos de las tres capas embrionarias, lo cual las convierte en una fuente muy prometedora de células madre para los estudios de medicina regenerativa. – El objetivo de esta tesis doctoral ha sido investigar el potencial regenerativo de las DPPSC para formar tejido periodontal nuevo in vitro usando dientes sanos y estériles como soporte. Nuestro estudio pretende reproducir la topografía de la superficie de la raíz natural del diente para favorecer la elongación celular perpendicular, condición óptima para la regeneración de las fibras periodontales. – Resultados – Se aislaron las células DPPSC del tercer molar y se cultivaron hasta pase 4. Se caracterizaron estas células a través de microscopia óptica y de microscopia electrónica de transmisión (TEM) donde se observaron que las células presentaban un tamaño pequeño, morfología triangular con un núcleo prominente y escaso citoplasma, característica especifica de las células embrionarias. – Para comprobar el fenotipo de las DPPSC indiferenciadas del cultivo primario se analizaron las células mediante citometría de flujo. Los resultados evidenciaron expresiones positivas para los marcadores de membrana: CD105 (92,15%), CD29 (99,63%), CD146 (15,54%), CD45 (0,02%) y para los marcadores de pluripotencialidad: OCT3/4 (70,72%) y NANOG (30,18%). Los resultados de la RT-PCR también revelaron la expresión genética positiva para los marcadores de pluripotencia OCT 3/4, NANOG y SOX-2. – Mediante el análisis de inmunofluorescencia se confirmó el fenotipo de las DPPSC indiferenciadas y los resultados mostraron la expresión del marcador de células adultas CD 13 y el marcador de pluripotencia SSEA4. – Para comprobar la estabilidad genética del cultivo antes de realizar la diferenciación periodontal se realizó la hibridación genética comparativa corta. El perfil genómico analizado en la muestra 8DD fue equilibrado y no se observó ninguna anormalidad cromosómica. – Después de la caracterización de nuestras células DPPSC se realizó la diferenciación de las DPPSC a tejido periodontal. El experimento consistía en sembrar las células DPPSC encima de los dientes sanos y estériles, tratados previamente con FN, y mantenerlas en cultivo durante 21 días en medio osteogénico. En total se sembraron 5 placas, 3 de las cuales (a razón de una por semana) fueron utilizadas para aislamiento de RNA. De las 2 restantes, una se destinó al análisis del SEM y la otra al examen histológico. – Con las muestras obtenidas cada semana, se realizaron las pertinentes RT-PCRs. Se analizó la expresión de diferentes marcadores (ALP, OC, PLAP-1, COL I, COL III, BGN, POSTN, S100A4) entre los 5 grupos (DPPSC, DPPSC diferenciadas en 2D, DPPSC diferenciadas en 3D en la primera semana, segunda semana y tercera semana de diferenciación). Los resultados mostraron altos niveles de expresión para los marcadores óseos y periodontales desde la primera semana a la tercera semana de diferenciación. La expresión de estos marcadores fue mayor que en la diferenciación 2D. – Después de cultivar las DPPSC in vitro durante 21 días, las matrices se analizaron al microscopio electrónico de barrido (SEM). Las imágenes de las raíces a diferentes aumentos (5000X, 15000X y 30000X) mostraron una gran densidad de masa celular encima de las superficies dentales para todas las muestras de DPPSC, indicando la capacidad de las células para adherirse y crecer sobre esa superficie. Después de 3 semanas de diferenciación, el análisis de SEM permitió observar la formación de fibras de colágeno de las matrices dentales. Se observaron algunos vasos sanguíneos y algunos cementoblastos en las muestras del grupo de DPPSC, al igual que en el grupo control positivo, que consistía en el ligamento periodontal de un diente erupcionado. Por el contrario, en el control negativo, que consistía en un diente estéril en el que previamente se había eliminado el ligamento periodontal, no se observó la presencia de células u otro tejido. – Con el fin de visualizar la formación de nuevos tejidos se realizaron las tinciones: tricrómica; Azul alcián y Hematoxilina-eosina (H&E). En los cortes histológicos del grupo de las DPPSC se observó la formación de fibras de colágeno insertadas perpendicular al cemento, pareciéndose a las fibras de Sharpey en comparación al grupo control negativo, donde no se observan estas fibras. A mayor aumento se reveló la homogeneidad de las fibras nuevas de colágeno unidas a las superficies a los 21 días de la diferenciación. – Discusión -- Las células utilizadas en este estudio se aislaron de la pulpa dental de los terceros molares. El desarrollo tardío del tercer molar, permite la extracción de una gran cantidad de células progenitoras. El aislamiento y cultivo de las DPPSC a partir de la pulpa de los terceros molares requiere de unas condiciones de cultivos específicos y bastante estrictos, por lo que es importante caracterizar esta subpoblación antes de realizar los experimentos. – Las DPPSC poseen grandes núcleos alargados, lo que indica su alta capacidad replicativa, característica típica de las células embrionarias. Sin embargo, la expresión del marcador CD13 observado en la inmunofluorescencia nos indica el origen adulto de estas células, por lo tanto, se trata de células adultas con características pluripotentes. La elección de estas células para la realización de esta tesis doctoral se debe a su alta capacidad de diferenciación y su alta proliferación, lo que va ligado a la expresión de marcadores de pluripotencia, como Oct-4, Sox-2, SSEA4 o Nanog, en su etapa indiferenciada. Estas características podrían favorecer la regeneración del ligamento periodontal en las raíces de los dientes. Además, la estabilidad genética observada en estas células es requisito indispensable para su futura aplicación clínica. – En este estudio se ha investigado la capacidad de los dientes humanos para actuar como una matriz óptima in vitro para la regeneración del tejido periodontal humano. Los resultados obtenidos en este estudio han demostrado que los cortes de dientes humanos tratados, junto con células madre de la pulpa dental (DPPSC), parecen constituir una buena combinación para la regeneración completa del tejido periodontal, ya que no solamente se observa la adhesión de estas células a la superficie de la raíz de los dientes, sino que, además, se consiguen generar in vitro gran parte de los componentes de este tejido, como fibras, células y vasos sanguíneos. No obstante el tratamiento de los dientes con la FN mejoró la adhesión de las células a los dientes. – Los estudios demostraron que los tejidos periodontales de ligamentos de diferentes pacientes pueden tener diferentes proteínas o genes. Debido que hasta la fecha, no se ha encontrado un marcador específico para identificar los fibroblastos del ligamento periodontal y diferenciarlos de otras células circundantes en nuestro estudio hemos utilizado más de un marcador periodontal, incluyendo PLAP-1, BGN, POSTN, S100A4, COL I, COL III; y algunos marcadores óseos, incluyendo la fosfatasa alcalina (ALP) y la osteocalcina (OC). – El perfil de expresión del ligamento periodontal mostró que el colágeno tipo I y tipo III fueron los genes más abundantes, lo que les hace ser considerados en nuestro estudio. El colágeno I es la principal proteína de membrana extracelular (ECM) del ligamento periodontal y proporciona un microambiente ideal para que las PDLPs (células madre del ligamento periodontal) se adhieran, proliferen y formen un tejido periodontal. Los ensayos de RT-PCR muestran un aumento de la expresión de este tipo de colágeno durante las dos primeras semanas de la diferenciación y descendiendo en la tercera semana. Este descenso podría estar asociado a un aumento en la mineralización del tejido diferenciado durante la tercera semana, como indica el incremento en la expresión de marcadores óseos como la osteocalcina (OC) o la fosfatasa alcalina (ALP). – La expresión de proteína PLAP-1 en células del ligamento periodontal in vitro está demostrada en algunos artículos y se observó el aumento durante el curso de la citodiferenciación de las células del ligamento periodontal en células formadoras de tejido mineralizado, tales como osteoblastos y cementoblastos. Estas observaciones sugirieron que PLAP-1 está implicada en la formación de la matriz mineralizada en los tejidos periodontales. Los resultados de este estudio muestran un aumento en la expresión de PLAP-1 a lo largo de la diferenciación en 3D, mientras que no se observa expresión de la misma durante la diferenciación en 2D. Este hecho podría estar indicando el papel del diente en la inducción de la diferenciación de las DPPSC hacia tejido periodontal y la mineralización de las mismas, como ya se ha observado que ocurre con otras matrices y otros tipos celulares. – Finalmente, POSTN (Periostin) es una proteína de membrana extracelular (ECM) muy importante para la homeostasis periodontal y mantenimiento del espacio periodontal. Su expresión cambia dinámicamente en respuesta a la tensión y compresión del ligamento periodontal. POSTN juega un papel importante en la respuesta característica de las células periodontales a los estímulos mecánicos y de superficie. En este sentido, los resultados de esta tesis corroboran esa respuesta, ya que, al igual que ocurría con PLAP- 1, POSTN sólo se expresa en la diferenciación en 3D, mientras que no se observa expresión de la misma en la diferenciación en 2D. – En nuestro estudio hemos utilizado un medio de diferenciación osteogénico para la regeneración periodontal, ya que el perfil genético del ligamento periodontal es muy parecido al del hueso. Además, de momento no se ha descrito un medio de diferenciación periodontal específico. Después de 3 semanas de cultivo de las DPPSC en el medio de osteodiferenciación, las imágenes del SEM mostraron un claro incremento en la cantidad de células, similares a los cementoblastos observados en el control positivo de ligamento periodontal. Además, se observó la formación de una matriz extracelular abundante de fibras de colágeno, lo que sugiere que la diferenciación de las DPPSC a ligamento periodontal fue funcional. – Por lo tanto, con los resultados obtenidos en esta tesis doctoral se propone la terapia con DPPSC como un procedimiento alternativo en la medicina regenerativa del ligamento periodontal. Sin embargo, estudios en animales y ensayos clínicos complementarios son necesarios antes de que estas células se puedan aplicar como tratamiento de medicina regenerativa para las enfermedades periodontales.

Continuous C/EBPa expression converts B cells into macrophages while its transient upregulation before the Yamanaka factors yields highly efficient iPSC reprogramming. C/EBP members regulate Il6 pathway genes and IL-6 signaling participates in macrophage differentiation and somatic cell reprogramming. We have explored the possibility that C/EBPa regulates the Il6 pathway during B cell transitions and found that it activates Il6 or Il6ra expression in different subsets. Il6 expression results dispensable for macrophage switch but it impairs pluripotency and trophectodermal genes in iPSC reprogramming. Those signatures firstly arise during preimplantation development with the segregation of ICM and trophectoderm layers in the blastocyst. We detected C/EBPa in 4- to 8-cell mouse embryos and later in the trophectoderm. We also found Il6 and Il6ra being respectively expressed in the trophectoderm and ICM, with IL-6 blockade delaying blastocyst formation and C/EBPa null blastocysts bearing low Il6 levels. Inducible C/EBPa clones of ESCs activate Il6ra as well as trophectoderm and pluripotency programs among different cells. We speculate that C/EBPa could instruct ICM/trophectoderm segregation through IL-6 signaling regulation.
La expresión continuada de C/EBPa convierte células B en macrófagos mientras que su activación transitoria precediendo a los factores de Yamanaka genera una eficiente reprogramación en células iPSC. Miembros C/EBP regulan genes de la vía del Il6 y su señalización participa en la diferenciación de macrófagos y en la reprogramación de células somáticas. Hemos explorado la posibilidad de que C/EBPa regule la vía del Il6 durante estas transiciones en células B y hemos descubierto que C/EBPa activa la expresión de Il6 o Il6ra en diferentes poblaciones. Il6 resulta prescindible para el cambio a macrófagos aunque impide la activación de genes pluripotentes y del trofectodermo durante la reprogramación en iPSCs. Estos genes aparecen por primera vez en el desarrollo preimplantacional con la segregación de las capas de ICM y trofectodermo en el blastocisto. Hemos detectado C/EBPa en embriones de ratón de 4 a 8 células así como más adelante en el trofectodermo. También hemos visto que Il6 y Il6ra se expresan respectivamente en el trofectodermo y la ICM, que el bloqueo de IL-6 retrasa la formación del blastocisto y que embriones sin C/EBPa muestran bajos niveles de Il6. La inducción de C/EBPa en clones de ESCs activa Il6ra así como programas de pluripotencia y trofectodermo en diferentes grupos de células. Especulamos que C/EBPa podría instruir la segregación de ICM y trofectodermo a través de la regulación de la vía del Il6.

Le myélome multiple (MM) est une néoplasie lymphocytaire B qui se caractérise par l'accumulation d'un clone de cellules plasmocytaires tumorales dans la moelle osseuse interagissant de manière étroite avec le microenvironnement. L'étude du profil d'expression génique à l'aide des puces à ADN a permis de préciser l'hétérogénéité de cette pathologie et de découvrir de nouveaux acteurs susceptibles d'avoir un rôle importent dans sa physiopathologie. La surexpression de Oct-3/4, Sox2, c-Myc et KLF4 dans des cellules adultes les reprogramme à l'état de cellules souches. J'ai montré une surexpression significative de 3 de ces 4 gènes – KLF4, MYC, SOX2 - dans le MM. Ces gènes sont également fréquemment surexprimés dans 18 types de cancers sur 40 étudiés. Par ailleurs, leur expression peut y être associé à un mauvais pronostique ou un signe de progression tumorale, dus peut-être à leur capacité à induire des caractéristiques de cellules souches cancéreuses. Nous avons par la suite débuté l'étude de la fonction des protéines codées par ces gènes dans le MM, en commençant par KLF4, facteur de transcription activateur ou répresseur. KLF4 est exprimé dans les plasmocytes (PC) normaux, mais son expression est perdue dans les PC tumoraux (cellules myélomateuses, MMC) de 2 patients sur 3 atteints de MM au diagnostic. Parmi les patients pour lesquels les MMC expriment KLF4, se trouve un groupe de patients à haut risque, le groupe MMSET portant la translocation t(4 ;14). L'utilisation d'un modèle inductible d'expression de KLF4 dans des lignées de MM avec transduction lentivirale, a mis en évidence un arrêt du cycle associé à l'expression de P27/KIP1 pour les lignées avec des mutations inactivatrices de P53, mais également de P21/WAF1 pour des lignées avec P53 sauvage. Cette sortie du cycle due à l'expression de KLF4 pourrait protéger les plasmocytes tumoraux de l'apoptose induite par certaines drogues ciblant le cycle, comme nous l'observons in vitro avec le Melphalan.L'objectif majeur de notre équipe est de comprendre le fonctionnement du PC normal et celui du PC tumoral. Afin d'atteindre cet objectif, il est nécessaire d'obtenir un système efficace permettant d'introduire un gène dans un PC. Nous avons montré que des lentivirus pseudotypés avec des glycoprotéines tronquées (hemagglutinine et fusion) issus du virus de la rougeole, transduisent de façon stable et efficace des PC normaux et tumoraux
Multiple myeloma (MM) is a B-cell neoplasia characterized by the accumulation of a clone of malignant plasma cells in bone marrow closely interacting with its microenvironment.Gene expression profiling using DNA microarrays has clarified the heterogeneity of this disease and has allowed the finding of new actors that may have an important function in MM pathophysiology.Overexpression of Oct-3/4, Sox2, c-Myc and KLF4 in adult cells causes their return to the state of stem cells, commonly called induced pluripotent stem cells (iPS). Our team has shown a significant overexpression of at least one of these four factors in 18 out of 40 cancers studied. Moreover, their expression may be associated with poor prognosis or may be a sign of tumor progression, perhaps due to their ability to induce characteristics of cancer stem cells.We therefore began the study of the function of these genes in MM, starting with KLF4, which can either be an activator or a repressor of transcription, depending on the promoter. KLF4 is expressed in normal plasma cells (PC), but its expression is lost in 2 out of 3 patients with MM at diagnosis. Among patients for whom the PCs express KLF4, is a group of high-risk patients, the MMSET group, bearing the t(4;14) translocation.An inducible model of KLF4's expression in MM cell lines was obtained using lentiviral transduction. Our model revealed a KLF4 induced cycle arrest, associated with the expression of P27/KIP1 when P53 is mutated, but also P21/WAF1 in case of wild type P53. This cell cycle blockade due to the expression of KLF4 could protect malignant plasma cells from the apoptosis induced by certain drugs targeting the cell cycle, as shown by our in vitro observations using melphalan.The main goal of our team is to understand the normal function of PCs and the PC tumor. To achieve this, it is necessary to obtain an effective system for introducing a gene in a given PC. We have shown that lentiviruses pseudotyped with truncated glycoproteins (Hemagglutinin and Fusion) from measles virus, can a stably and efficiently transduce normal and malignant PCs

Deux états d'autorenouvellement des cellules souches pluripotentes (PSCs) ont été définis, à savoir les états naïf et amorcé. De nombreuses différences existent entre ces deux états dont la plus marquante est la capacité unique des PSCs à l'état naïf, de coloniser l'embryon préimplantatoire et former des chimères. L'objectif de mon projet doctoral a été d'étudier la pluripotence chez le lapin. Dans ce cadre, j'ai d'abord entrepris de fabriquer et de caractériser des cellules souches pluripotentes induites (RbiPSCs), puis d'évaluer leur capacité à coloniser l'embryon et à former des chimères. Trois lignées de RbiPSCs dépendantes du FGF2 ont été obtenues par reprogrammation de fibroblastes de lapin. Leur caractérisation moléculaire et fonctionnelle a révélé des caractéristiques mixtes, naïves et amorcées. En revanche, sur le plan fonctionnel, elles sont incapables de coloniser l'embryon de lapin, une caractéristique de la pluripotence amorcée. La seconde partie de mon projet doctoral a consisté à reprogrammer des RbiPSCs vers l'état naïf. Dans ce but, j'ai surexprimé KLF2 et KLF4, deux gènes appartenant au réseau de pluripotence naïf, et utilisé les conditions de culture des PSCs de souris. Les cellules ainsi obtenues présentent un profil d'expression génique plus proche de celui de l'ICM de lapin, dû notamment à la réactivation de marqueurs spécifiques de la pluripotence naïve. Enfin, les cellules ainsi reprogrammées présentent une capacité accrue pour la colonisation de l'embryon préimplantatoire de lapin. Mes travaux constituent le premier exemple de reprogrammation de cellules souches pluripotentes vers l'état naïf chez le lapin. Les cellules ainsi produites ouvrent la voie à la fabrication de chimères somatiques et germinales
Pluripotent stem cells (PSCs) can self-renew at two distinct states, the naive and primed states. Many differences exist between these two states, the most striking is the unique ability of PSCs naïve to colonize the preimplantation embryo and form chimeras. The purpose of my doctoral project was to study pluripotency in rabbits. In this context, I initially manufactured and characterized induced pluripotent stem cells (RbiPSCs) and then evaluated their ability to colonize the embryo and form chimeras. Three RbiPSCs lines were obtained by rabbit fibroblasts reprogramming. Their molecular characterization revealed mixed characteristics, naïve and primed. However, functionally, they are unable to colonize the rabbit embryo, a feature of primed pluripotency. The second part of my doctoral project was to reprogram RbiPSCs to the naïve state. To this end, I have overexpressed Klf2 and Klf4, two genes belonging to the naïve pluripotency network and the mouse PSCs culture conditions. These new cell lines have a gene expression profile closer to that of the rabbit ICM, particularly due to the reactivation of specific markers of naïve pluripotency. Finally, the reverted cells have an increased capacity of colonization of the preimplantation embryo rabbit. My work represents the first example of pluripotent stem cells reprogramming toward the naive state in rabbits. The cells thus produced pave the way for the production of somatic and germline chimeras

The reactivation of the inactive X chromosome has the potential to provide a unique system to study the developmentally induced formation of euchromatin. However, insights into this process were hampered by the lack of adequate systems, which would allow the dissection of the process using high-throughput sequencing techniques. Here I describe the development of a novel induced pluripotent stem cell reprogramming system that allows the isolation of cells poised for X-reactivation, subsequently achieving near-deterministic efficiency of X-reactivation. Utilizing this novel system, we were able to reveal that the reactivation of silenced genes occurs rapidly and can be divided into distinct initiation and completion phases. Similarly, we could show that chromatin opening of the inactive X proceeds in a two-step fashion, initiating in close proximity to previously open regions, and possibly being initiated by pluripotency factors. Finally, we could show that mega-domains and TADs correspond to two different levels of three-dimensional genome organization superimposed on the Xi, independent of gene expression. We conclude that gene expression and chromatin accessibility during X-reactivation share similar kinetics, while genome organization might follow distinct principles.
La reactivación del cromosoma X inactivo tiene el potencial de proporcionar un sistema único para estudiar la formación de eucromatina inducida por el desarrollo. Sin embargo, la comprensión de este proceso se vio obstaculizada por la falta de sistemas adecuados, lo que permitiría la disección del proceso utilizando técnicas de secuenciación de alto rendimiento. Aquí describo el desarrollo de un nuevo sistema de reprogramación de células madre pluripotentes inducidas que permite el aislamiento de células preparadas para la reactivación de X, logrando posteriormente la eficiencia casi determinista de la reactivación de X. Utilizando este novedoso sistema, pudimos revelar que la reactivación de genes silenciados ocurre rápidamente y puede dividirse en distintas fases de iniciación y finalización. Del mismo modo, podríamos mostrar que la apertura de cromatina de la X inactiva se realiza en dos pasos, iniciando en las proximidades de regiones previamente abiertas, y posiblemente iniciada por factores de pluripotencia. Finalmente, podríamos mostrar que los mega-domains y los TADs corresponden a dos niveles diferentes de organización del genoma tridimensional superpuestos en el Xi, independientemente de la expresión génica. Llegamos a la conclusión de que la expresión génica y la accesibilidad a la cromatina durante la reactivación X comparten una cinética similar, mientras que la organización del genoma podría seguir principios distintos.

Les cellules souches embryonnaires de souris (ESCs) et les cellules souches de l'épiblaste (EpiSCs) représentent, respectivement, les états naïf et amorcé de la pluripotence et sont maintenues in vitro par des voies de signalisation spécifiques. De plus les ESCs cultivées dans un milieu sans sérum avec deux inhibiteurs (2i) sont décrites comme étant les plus naïves. Plusieurs études ont suggéré que chaque type de cellules pluripotentes est caractérisé par une organisation différente de l'épigénome. Nous présentons ici une étude comparative de l'état épigénétique et transcriptionnel des séquences satellites répétées péricentromériques (PCH) entre les ESCs (2i et sérum) et EpiSCs. Nous montrons que H3K27me3 au PCH est très dynamique et peut discriminer les ESCs en 2i des autres cellules souches pluripotentes. Alors que la transcription des séquences satellites est élevée dans les ESCs en sérum, elle est plus faible dans ESCs en 2i et encore plus réprimée dans les EpiSCs. La suppression de la méthylation de l'ADN ou d'H3K9me3 dans les ESCs conduit à un dépôt important de H3K27me3 au PCH, mais peu de changements transcriptionnels de ces séquences. En revanche, l'absence d'H3K9me3 dans les EpiSCs n'empêche pas la méthylation de l'ADN au PCH, mais induit la transcription de ces séquences. La conversion in vitro des ESCs en EpiSCs est plus longue que le passage des cellules de l'ICM en épiblaste in vivo. Cette inefficacité ne peut pas être expliquée par une mise en place retardée du nouveau réseau transcriptionnel. Pour conclure notre étude a révélé que les EpiSCs ont perdu de la plasticité par rapport au ESCs sur l'hétérochromatine ainsi que l’euchromatine, comme le montre la réduction des niveaux d'H3K9ac et des domaines bivalents, étant ainsi plus proche épigénétiquement de cellules somatiques que de la pluripotence naïve
Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent naïve and primed pluripotency states, respectively and are maintained in vitro using specific signaling pathways. Furthermore, ESCs cultured in serum-free medium with two inhibitors (2i) are described as being the most naïve. Several studies have suggested that each pluripotent cell type is characterized by a different epigenome organization. Here we present a comparative study of the epigenetic and transcriptional state of centromeric and pericentromeric (CH/PCH) satellite repeats in ESCs (2i and serum ones) and EpiSCs. We show that the pattern of H3K27me3 at PCH is highly dynamic and discriminate 2i-ESCs from the other pluripotent stem cells. Whereas satellites transcription is high in serum-ESCs, it is lower in 2i-ESCs and even more repressed in EpiSCs. Removal of either DNA methylation or H3K9me3 in ESCs leads to enhanced deposition of H3K27me3 but few changes in satellite transcription. By contrast, in EpiSCs removal of H3K9me3 does not prevent DNA methylation at PCH but de-represses the satellite transcription. In vitro conversion from naive to primed pluripotency showed an important delay compared to the in vivo development of ICM cells into post-implantation epiblast. Such inefficiency cannot be explained by a delayed switch to the new transcriptional network. Altogether our study reveals that EpiSCs have lost the chromatin plasticity of ESCs on heterochromatin as well as euchromatin, as shown by the reduction of H3K9ac levels and bivalent domains, thus being closer to somatic cells in terms of epigenetics than naive pluripotency

Le microenvironnement apporte une multitude d'informations aux cellules régulant ainsi leurs fonctions et leur organisation. L'objectif de cette thèse a été de contrôler différents paramètres du microenvironnement cellulaire in vitro afin de moduler l'autorenouvellement et la destinée des cellules souches embryonnaires (CSE).Cette thèse comporte 3 parties. Premièrement, des films de polyélectrolytes multicouches à base de poly(L-lysine) et de hyaluronane ont été utilisés comme substrats modulables. Les propriétés mécaniques ainsi que la chimie des films régulent la proportion de sous-populations de CSE qui reflètent soit le stade masse cellulaire interne, soit le stade épiblaste.Dans un deuxième temps, un équilibre entre l'expression des marqueurs embryonnaires de l'axe proximodistal a été mis en évidence dans la culture de CSE. L'emploi de micro-patrons adhésifs permettant de contrôler la géométrie des colonies a révélé l'importance des contraintes topologiques sur la distribution des cellules exprimant le marqueur proximal Brachyury. Enfin, l'action combinée de BMP2 et de wnt3a mimant l'environnement biochimique du stade tardif de la ligne primitive a permis d'isoler une population pure et très précoce de progéniteurs cardiaques SSEA1+ multipotents
The microenvironment provides stem cells with numerous pieces of information. Biochemical and mechanical cues synergize to regulate cell function and organization. The aim of this PhD thesis was to control specific microenvironmental parameters to modulate embryonic stem cell (ESC) self-renewal and fate.First, poly(L-lysine) and hyaluronan based polyelectrolyte multilayer films were used as tunable substrates. Both mechanical and chemical properties of the films influenced the balance between ESC subpopulations reflecting different embryonic stages (inner cell mass versus epiblast)Second, a dynamic equilibrium was found between the expression of embryonic proximal and distal markers within ESC culture. The uses of micropatterned substrates to control colony shape uncovered a key role for geometrical constraints in the distribution of Brachyury expression.Last, BMP2 was used together with secreted wnt3a to mimic the late streak stage of the embryo and to trigger the differentiation of pluripotent cells towards the cardiogenic mesoderm. Responsive cells could be sorted out based on SSEA1 expression. This purified population represents the earliest ESC derived multipotent cardiac progenitor population identified to date

Contribution de U2AF1, NCBP1 et eIF4A3 dans le contrôle du maintien de la pluripotence et le devenir cellulaire.Les mécanismes de maturation du transcrit primaire peuvent profondément affecter la diversité et la fonction des protéines produites à partir d’un gène unique dans le but de mettre en place un programme complexe impliqué dans le maintien de pluripotence et/ou l’initiation de la différenciation des cellules souches humaines. Les réseaux transcriptionnels régulant la pluripotence et la différenciation ont été intensément étudiés contrairement au rôle de l’épissage alternatif dans ces mécanismes, rôle qui pour le moment reste mal compris et pour lequel il n’existe que très peux d’exemples de groupes de gènes subissant un changement général de variant d’épissage aboutissant à la modification du devenir cellulaire. Notre objectif est d’identifier les composés essentiels du spliceosome qui sont impliqués dans le maintien de la pluripotence et la différenciation précoce dans les trois feuillets embryonnaires et d’explorer leurs rôles dans ces processus. Via l’analyse de données de séquençage d’ARN nous avons identifié plusieurs facteurs d’épissage différentiellement exprimés entre les cellules souches et les trois feuillets embryonnaires. Parmi ces facteurs nous focaliserons notre étude sur les facteurs préférentiellement surexprimés dans les cellules souches, qui par conséquent devraient y jouer un rôle primordial. Les candidats sélectionnés, U2AF1, NCBP1 et eIF4A3 ont été déplétés dans des cellules souches en utilisant un système shRNA inductible puis une analyse de séquençage ARN à haut débit a été effectuée pour comprendre les changements du transcriptome induits par ces déplétions. La déplétion d’U2AF1 entraine un changement majeur de l’expression de gènes impliqués dans le développement alors que la déplétion de NCBP1 et eIF4A3 entraine un changement d’expression de gènes impliqués dans le métabolisme, le remodelage de la chromatine et le développement. Des analyses complémentaires ont permis de mettre en lumière une régulation transcriptionnelle et post-transcriptionnelle des gènes différentiellement exprimés dans les conditions étudiées. L’épissage alternatif a pour ça part été modifié par les trois déplétions de manière individuelle. Un programme d’épissage tissu spécifique a été associé à chaque candidat et les conséquences de chaque programme seront décrites au niveau du contrôle qualité de l’ARNm et de la synthèse protéique.Nos résultats construisent une nouvelle vision concernant le rôle des composés essentiels du spliceosome dans le contrôle du devenir cellulaire à travers la modulation de l’épissage alternatif. Cet apport ajoute une nouvelle variable au contrôle de l’expression des gènes et permettra de mieux comprendre les mécanismes du développement précoce et de la diversité tissulaire
Contribution of U2AF1, NCBP1 and eIF4A3 to the control of pluripotency maintenance and cell fate determination.Alternative pathways for processing the primary transcript can profoundly affect the diversity and function of the protein products that are generated from a single gene to set up complex programs involved in pluripotency and/or differentiation of human Embryonic Stem Cells (hESCs). While transcriptional networks regulating pluripotency and differentiation has been intensively studied, the role of Alternative Splicing (AS) in this process is not yet completely understood and clear examples of concerted switching of multiple genes from one isoform to another have not been demonstrated. Our goal is to identify Core Spliceosomal Factors (CSF), involved in the control of pluripotency maintenance, early differentiation into the three germ layers, and to explore their role in these processes. By RNA-Seq data analysis, we have identified several splicing factors that are differentially expressed between pluripotent stem cells and the three of the germ layers. Among these identified candidates, we focused on the factors that are more highly expressed in pluripotent stem cells, thereby they play a specific role in pluripotency maintenance. The selected candidates, U2AF1, NCBP1 and eIF4A3 were depleted in pluripotent stem cells using inducible shRNA system and RNA-Seq analyzes have been performed to understand transcriptomic changes induced by these depletions. U2AF1 depletion causes a major switch of developmental genes expression, while NCBP1 and eIF4A3 depletions regulate the expression of genes involved in metabolism, chromatin remodeling and development. Further analysis highlighted a transcriptional and post-transcriptional regulation of differentially expressed genes. Alternative Splicing (AS) were shown to be affected by both depletions. A tissue specific AS program was associated to each of the candidates and the consequences of these changes on mRNA quality control and protein synthesis will be described.Our results build a new idea regarding the role of Core Spliceosomal Factors in cell fate control trough the modulation of AS. This knowledge adds a new layer of gene expression control and will allow a better understanding of early development mechanisms and tissue diversity

Thesis (Ph.D.)--Indiana University, Dept. of Mathematics, 2009.
Title from PDF t.p. (viewed on Jul 8, 2010). Source: Dissertation Abstracts International, Volume: 70-10, Section: B, page: 6244. Adviser: Norman Levenberg.

Embryonic stem cells (ES cells) are derived by explantation of the embryonic portion of the pre-implantation embryo into culture. These cells have unique properties which have made them invaluable in study of the function of genes in vivo and of cell differentiation in vitro. They can be grown in culture for extended periods of time in an undifferentiated state and induced to differentiate in vitro. While undifferentiated they can be genetically manipulated. Subsequent reintroduction of these cells into the blastocyst results in the cells being integrated and contributing to all the cells of the animal including the germ line thus leading to designed genetic change. The homology of these cells, however, to their tissue of origin is not unambiguous. The primary aim of this thesis was to apply global transcriptome analysis to investigate the homology of ES cells to the pluripotent compartment of the embryo. Although ES cells can be grown in bulk, the tissue of origin, the embryonic portion of the peri-implantation embryo are small and inaccessible. It was therefore necessary to develop methods which would allow the transcriptome to be amplified without distorting the transcript profile. A linear amplification method proved to give the best result. The best method for fluorescently labelling the cDNA was shown to be enzymatic incorporation of aminoallyl dUTP followed by coupling to monoreactive Cy dyes. With these tools it was then possible to amplify the transcriptome of both colonies of ES cells and the embryonic portion of various peri-implantation embryos and apply the labelled cDNA to microarray slides. Statistical analysis of the results proved that the transcriptome of ES cells most resembles that of the embryonic ectoderm on day 5.5 of development.

Le LIF (Leukemia Inhibitory factor), une cytokine de la famille de l’Interleukine 6, permet le maintien de la pluripotence des cellules souches embryonnaires murines (CSEm) in vitro. Dans le but de comprendre les mécanismes d’action du LIF dans ce modèle d’étude, une analyse sur puces à ADN a été réalisée et a permis d’identifier trois « signatures LIF » : les gènes « Pluri » (pour Pluripotence), dont le niveau d’expression relatif chute suite au retrait de cette cytokine, et deux catégories de gènes « Lifind » (pour LIF induit) dont le niveau d’expression relatif augmente suite à un ajout de LIF après une culture de 24 ou 48 heures sans cette cytokine. Nous avons mis au point des tests fonctionnels permettant d’étudier la fonction des gènes cibles du LIF dans notre modèle d’étude. Ainsi, nous avons mis en évidence le rôle d’un gène « Pluri », Mras/Rras3, une petite GTPase de la famille Ras, dans la régulation de l’expression d’une part de marqueurs de pluripotence, tels que Oct4 et Nanog et d’autre part de marqueurs de différenciation, tels que Lef1 et Fgf5
LIF (Leukemia Inhibitory factor), a cytokine Interleukin 6 family, allows maintaining the pluripotency of murine embryonic stem cells (mESC) in vitro. To understand the mechanisms of action of the LIF in this model, a microarray analysis was conducted and identified three « signatures LIF » : the « Pluri » (for Pluripotency) genes, whose the relative level of expression falls following the withdrawal of this cytokine, and two classes of « Lifind » (for LIF induced) genes, whose the relative expression level increases as a result of LIF addition after a culture of 24 or 48 hours without this cytokine. We have developed functional tests to study the function of the target genes of LIF in our study model. Thus, we have investigated the role of a « Pluri » gene, Mras/Rras3, a small GTPase of the Ras family, in the regulation of the expression on the one hand of markers of pluripotency, such as Oct4 and Nanog, and on the other hand of differentiation markers, such as Lef1 and Fgf5

Embryonic stem (ES) cells are maintained in an undifferentiated state by a gene regulatory network centred on the triumvirate of transcription factors Nanog, Oct4 and Sox2. Genome-wide chromatin immunoprecipitation studies indicate that in many cases target genes contain closely localised binding sites for each of these proteins, as well as additional members of the extended pluripotency transcription factor network. However, the biochemical basis of the interactions between these proteins is largely unknown, as are the mechanisms by which these interactions control ES cell identity. By purifying Nanog from ES cells and identifying co-purified proteins, we determined a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members and RNA processing factors. Validation of interactions was obtained by co-immunoprecipitation of Nanog with putative partners. Sox2 was identified as a robust interacting partner of Nanog and the interaction was investigated further. We show that the interaction is independent of DNA binding and that a region of Nanog known as tryptophan repeat, in which tryptophan is present every 5th residue is necessary and sufficient for the binding of Sox2. Furthermore, mutation of tryptophan residues within the Nanog tryptophan repeat (WR) abolishes the interaction with Sox2. A region of Sox2 known as serine rich region, a triple-repeat motif (S X T/S Y) within a stretch of 21 residues is required for the interaction with Nanog. Mutation of tyrosines to alanine within the three motifs (S X T/S Y) abrogates the Nanog–Sox2 interaction. The disruption of the Nanog-Sox2 interaction results in the alteration of expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines of the motif with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal. Together these data shed light on the extent of the interactions of Nanog with protein partners as well as the biochemical nature of the interaction between Nanog and one of the most important partners Sox2, an interaction crucial for maintaining optimal mouse ES cell self-renewal efficiency.

In this thesis we focus on Dirichlet's problem for the complex Monge-Ampère equation. That is, for a given non-negative Radon measure µ we are interested in the conditions under which there exists a plurisubharmonic function u such that (ddcu)n=µ, where (ddc)n is the complex Monge-Ampère operator. If this function u exists, then can it be chosen with given boundary values? Is this solution uniquely determined within a given class of functions?

La pluripotence est la capacité d'une cellule à s'auto-renouveler et à donner toutes les cellules somatiques ainsi que les cellules germinales. Les cellules pluripotentes peuvent être aussi reprogrammées à partir de cellules somatiques, ouvrant ainsi de nouvelles opportunités pour l'utilisation thérapeutique des cellules souches dans le traitement des maladies dégénératives. La connaissance des mécanismes moléculaires, en particulier des voix de signalisation qui contrôlent la pluripotence, est cruciale pour l'amélioration de notre compréhension de l'embryogenèse précoce et l'utilisation des iPSC (cellules souches pluripotentes induites) dans la médicine régénérative. Ici, je donne la première description de la Nétrine-1 en tant que régulateur de la reprogrammation et de la pluripotence. La Nétrine-1 et ses récepteurs ont été initialement caractérisés dans le système neuronal, mais il a aussi été montré qu'ils étaient exprimés dans différents types cellulaires et impliqués dans divers processus. Dans la première partie, j'ai contribué à explorer comment Nétrine-1 empêche l'apoptose médiée par son récepteur à dépendance DCC (Deleted in Colon Carcinoma) pendant la reprogrammation. Dans la deuxième partie, j'ai disséqué les fonctions et la régulation de cette voie dans le maintien de la pluripotence et dans l'engagement des lignages
Pluripotency is the ability of embryonic epiblast cells to self-renew and to give rise to all somatic cells as well as germ cells. Somatic cells can also be reprogrammed toward pluripotency, opening new avenues for stem cell based therapies in the treatment of degenerative diseases. Deciphering the molecular mechanisms, and in particular signaling pathways that control pluripotency is crucial to improve our understanding of early embryogenesis and the use of iPSC (inducible Pluripotent Stem Cell) in regenerative medicine.Herein, I provide the first description of Netrin-1 as a regulator of reprogramming and pluripotency. Netrin-1 and its receptors are present in many cell types and are engaged in a variety of cellular processes beyond its initial characterization in the neuronal system. In the first part, I contributed to explore how Netrin-1 prevents apoptosis mediated by its dependence receptor DCC (Deleted in Colon Carcinoma) during reprogramming. In the second part, I dissected the functions and regulation of this pathway in pluripotency maintenance and in lineage commitment

Pluripotency is the ability of a cell to differentiate into derivatives of all three somatic lineages and germ cells. In vivo, pluripotent cells exist transiently in the epiblast of the developing embryo and in rare tumour cells. In vitro, pluripotent cells have been isolated and propagated from teratocarcinomas (EC cells), preimplantation epiblast (ES cells) and post-implantation epiblast (EpiSCs). Pluripotency is governed by a gene regulatory network centred on the triumvirate of transcription factors Oct4, Sox2 and Nanog. Interestingly, transcription factors that are important to direct pluripotent cell identity are not all equally distributed throughout the pluripotent cell population. While Oct4 levels are relatively homogeneous, other transcription factors, such as Nanog, are more heterogeneously expressed. Additionally, an increasing body of evidence indicates that extrinsic cues also play a critical role in the establishment and maintenance of pluripotency. Using biochemical and genetic tools in mouse ES cells, the role of FGF signaling and Sox2 levels on heterogeneous Nanog expression was examined. Interference with FGF or ERK activity by genetic ablation or signal inhibition, promoted high, homogenous Nanog expression and enhanced self-renewal. This is consistent with reports showing that similar manipulations reduced the ability of ES cells to commit to differentiation. Moreover, ES cells with reduced Sox2 levels displayed greater heterogeneity for Nanog expression than wild-type ES cells. Pluripotency is lost in the mouse embryo around E8.5, however, the precise timing and mechanism involved in this process has not yet been defined. Here it is shown that pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis, the expression of both Nanog and Oct4 is regionalized. Interestingly, pluripotency tracks the in vivo level of Oct4, this correlation does not hold true for Nanog. However, Nanog expression reports on pluripotent cells. Indeed, ectopic Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitation of moribund pluripotency. Competence to re-activate the pluripotency network upon enforced Oct4 expression is gradually lost with the progression of embryonic development. ES cells and EpiSCs are two distinct pluripotent populations as they show differences in their ability to undergo clonal propagation, re-colonize embryos, growth factor responsiveness, morphology and gene regulatory networks. It is possible to harness this differential growth factor responsiveness to convert ES cells into EpiSCs. Conversely, EpiSC can be reverted back to ES cell pluripotency through the overexpression of a small number of transcription factors. The inter-conversion of ES cells and EpiSCs has been documented, but detailed analyses of the changes that occur during such transitions had not been performed. The current work shows that Nanog levels are critical for the specification of the pluripotent state of the cells. Furthermore, it is shown that orphan nuclear receptor Esrrb is a potent inducer of ES cell pluripotency in EpiSC. Interestingly, Esrrb was able to restore naïve pluripotency in cells genetically depleted of Nanog.

Nanog is a divergent homeodomain protein with the capacity to direct constitutive self-renewal in the absence of otherwise obligatory cytokine stimulation. Nanog is expressed in the early mouse embryo and is essential for the specification of pluripotent cells. However the mechanism by which Nanog governs pluripotency is incompletely understood. In this thesis experiments are presented that further the functional characterisation of Nanog. In the mouse embryo, Nanog is normally down regulated in cells prior to de-lamination and ingression through the primitive streak. To address the consequence of Nanog over-expression in vivo, a revertible Nanog over-expressing cell line has been generated which can be tracked in the embryo. Results show that the modest 2-3 fold increase in Nanog expression does not cause any overt phenotype at this stage and Nanog over-expressing cells can be detected in the mesoderm of mouse embryos. Nanog is shown to exist in multimers in ES cells. The domain mediating multimerisation is identified as a tryptophan repeat motif and the functional consequence of deletion of this domain is investigated. To identify Nanog partner proteins, a biotinylation tagging system in ES cells has been designed, constructed, and implemented. This led to the identification of putative Nanog partner proteins via mass-spectrometry. Two Nanog partner proteins, Esrrb and HDAC2 have been confirmed by co-immunoprecipitation. In addition, the SLQQ motif within the Nanog homeodomain is shown to be the site of interaction between Nanog and Sall4. This SLQQ motif is found at a similar location in only one other homeodomain protein, Oct4.  Consistent with these observations Sall4 is also shown to bind Oct4.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, June 2011.
"June 2011." Cataloged from PDF version of thesis.
Includes bibliographical references.
The process by which the totipotent zygote undergoes development into an adult organism using a single genome is the foundation for epigenetics and cellular differentiation. Somatic cell nuclear transfer experiments (SCNT) provided unequivocal proof of nuclear equivalency between adult somatic cells. However the molecular mechanisms of somatic cell reprogramming have remained largely a mystery. Recent advancements in epigenetic reprogramming by defined factors provide new opportunities to explore factors that regulate induction of pluripotency in somatic cells. Nuclear reprogramming by SCNT occurs in an 'indirect' manner by unidentified components within oocyte cytoplasm and requires the destruction of embryos. The introduction of induced pluripotent stem cells (iPS cells) and 'direct' reprogramming methods created a tractable system to both study of the process in vitro and potentially derive personalized pluripotent stem cells free of the practical and ethical concerns surrounding embryonic stem (ES) cells and SCNT. Herein we study mouse somatic cell reprogramming by defined factors and develop novel tools to compare the induced pluripotent state to the gold standard of pluripotency, ES cells. First, we designed reprogramming vectors that minimize the number of viruses required to generate iPS cells, yielding pluripotent cells with minimal genomic alterations from reprogramming factors. This allowed the creation of transgenic "reprogrammable mouse" strains after gene targeting in ES cells, providing a renewable source of somatic cells that can be induced to pluripotency by addition of a drug. In addition we can easily introduce or mate these strains to study unique genetic variants during reprogramming. Third, we study factors that influence the induced pluripotent state, specifically how to generate pluripotent cells with all properties of embryonic stem cells including derivation of "all iPS cell mice" by tetraploid complementation assays. In contrast to previous reports, we find the majority (- 80%) of iPS cell lines derived from adult somatic cells of varying organs contain the developmental potential of ES cells. This outcome correlated with high expression of Oct4 and Klf4 and low expression of Sox2 and c-Myc during reprogramming. In addition we report that adult mice derived from iPS cells are healthy and do not develop tumors. Together these results suggest in vitro reprogramming to pluripotency by defined factors holds great promise for regenerative medicine.
by Bryce W. Carey.
Ph.D.

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.

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

Les cellules souches cancéreuses (CSC) sont à l’origine de la progression tumorale, des métastases et rechutes tardives. Elles ont été identifiées dans de nombreux cancers, comme le cancer du sein triple négatif (TNBC) et cancers de grade III-IV. Elles sont résistantes aux chimiothérapies et radiothérapie et résident dans une niche immuno-répressive. Cette étude vise à évaluer une stratégie d’immunothérapie qui cible sélectivement les CSC dans le modèle murin 4T1-GFP-Luc mimant le TNBC. Le phénotype/ génotype des mamosphères a été initialement caractérisé. Basée sur l’analyse génomique des CSC, nous avons développé une immunothérapie active associée à des agents immuno-modulateurs. Nous avons mesuré la taille des tumeurs et suivi l’apparition des métastases par bioluminescence. Une étude immunologique et analyse génomique de la tumeur a été réalisée. La combinaison thérapeutique provoque le recrutement dans la tumeur de lymphocytes T (CD4 +, CD8 +) et lymphocytes B par augmentation de CXCL13, une réduction des lymphocytes T reg et cellules myéloïdes suppressives. Cette induction de réponse immunitaire provoque la diminution de la taille de la tumeur et des métastases. Cette nouvelle immunothérapie active de type vaccinale pourra être utilisée en association avec les traitements actuels pour des mesures prophylactiques et curatives dans une grande variété de cancers
Cancer stem cells (CSCs) are responsible for tumor progression, metastases, and late relapses. They have been identified in many cancers, such as triple negative breast cancer (TNBC) and grade III to IV cancers. They are resistant to chemotherapy and radiotherapy and reside in an immuno-repressive niche.This study aims to evaluate a immunotherapy strategy that selectively targets CSCs in the mouse model 4T1-GFP-Luc mimicking TNBC. The phenotype / genotype of mammosphere was initially characterized. Based on genomic analysis of CSC, we have developed an active immunotherapy associated with immunomodulatory agents. We measured the size of tumors and monitored the appearance of metastases by bioluminescence. We performed an immunological study and genomic tumor analysis. The therapeutic combination causes the recruitment of CD4 + and CD8 + T lymphocytes and B lymphocytes with increased CXCL13, the reduction of T reg cells and suppressive myeloid cells in the tumor. This induction of intra-tumor immune response leads to a decrease in tumor size and metastases.This new active immunotherapy can be used in combination with current treatments for prophylactic and curative measures in a wide variety of cancers

Les cellules souches embryonnaires et adultes sont strictement contrôlées et régulées par différents mécanismes comme l’auto-renouvellement, la différentiation et l’apoptose. Les enzymes impliquées dans la modification des histones et les différents statuts de la chromatine seraient responsables de la mise en place, du maintien et de la propagation des différents profils d’expression des gènes mais le mécanisme sous-jacent reste néanmoins mal compris. Dans nos études, nous avons identifié le rôle de Trrap, un cofacteur des histones acétyltransférases dans le maintien de l’auto-renouvellement des cellules souches embryonnaires et adultes. La perte de la moelle épinière et une mortalité croissante sont survenues suite à la délétion conditionnelle du gène Trrap chez la souris. Ceci est dû à la perte des cellules hématopoïétiques progénitrices ainsi que des cellules hématopoïétiques souches par un mécanisme cellulaire autonome. L’analyse des cellules progénitrices, purifiées, de la moelle épinière à permis de révéler que ces anomalies sont associées à l’induction de l’apoptose indépendante de p53 ainsi qu’à la dérégulation des facteurs de transcription Myc. De plus, la délétion conditionnelle de Trrap dans les cellules souches embryonnaires induit la différentiation due au rôle important que Trrap joue dans la régulation du couplage de la méthylation de l’histone H3 aux lysines K4 et K27 appelées « domaines bivalents », le maintien du statut hyperdynamique de la chromatine et la régulation des gènes spécifiques à l’auto-renouvellement. Ceci est cohérent avec l’essentiel rôle de Trrap impliqué dans le mécanisme qui restreint l’induction de l’apoptose ou de la différentiation, ceci selon le type de cellules souches, et favorise le maintien de l’auto-renouvellement. Ces études ont permis d’identifier les différents rôles essentiels que Trrap joue dans le mécanisme qui permet le maintien des cellules souches embryonnaires et adultes ce qui soulève la possibilité que Trrap et les modifications des histones qui contrôlent l’auto-renouvellement pourraient être importants pour le développement et le maintien des cellules souches cancéreuses. Une meilleure compréhension du mécanisme commun qui implique Trrap et les modifications des histones contrôlant les éléments essentiels des cellules souches normales et cancéreuses s’avèrerait essentiel et très bénéfique pour les stratégies de thérapies épigénétiques qui ont pour but d’éradiquer les cellules souches cancéreuses
Embryonic and adult stem cells are tightly controlled and regulated by self-renewal, differentiation and apoptosis. Histone modifiers and chromatin states are believed to govern establishment, maintenance, and propagation of distinct patterns of gene expression in stem cells, however the underlying mechanism remains poorly understood. In our studies, we identified a role for the histone acetyltransferase cofactor Trrap in the maintenance of embryonic stem cells and hematopoietic stem/progenitor cells. Conditional deletion of the Trrap gene in mice resulted in ablation of bone marrow and increased lethality. This was due to the depletion of early hematopoietic progenitors, including hematopoietic stem cells, via a cell-autonomous mechanism. Analysis of purified bone marrow progenitors revealed that these defects are associated with induction of p53-independent apoptosis and deregulation of Myc transcription factors. Moreover, conditional deletion of Trrap in embryonic stem cells was found to results in unscheduled differentiation. This was due to the essential role of Trrap in coupling of H3K4 and H3K27 methylation ("bivalent-domains"), the maintenance of hyperdynamic chromatin state and regulation of the stemness genes, consistent with the essential function of Trrap in the mechanism that restricts apoptosis or differentiation depending on stem cell type and promotes the maintenance of self-renewal. Together, these studies have identified critical roles for Trrap in the mechanism that maintains embryonic and hematopoietic stem cells and raise the possibility that Trrap and histone modifications controlling self-renewal may be important for the development and maintenance of cancer stem cells. Better understanding of a common molecular mechanism involving HATs and histone modifications that controls key features of normal and cancer stem cells may prove highly beneficial for epigenetics-based therapeutic strategies aiming to eradicate cancer stem cells

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2012.
Cataloged from PDF version of thesis.
Includes bibliographical references (p. 83-89).
Somatic cells can be reprogrammed to a pluripotent stem-cell state by ectopic expression of defined proteins. However, existing reprogramming methods take several weeks, suffer from low efficiencies, and most use DNA-based vectors, which carry mutagenesis risks. Here, we describe efficient and rapid reprogramming of human cells using RNA. Within two weeks, fibroblasts from 7 adult patients, including 5 Parkinson's patients aged 53 to 85, formed colonies that exhibited gene expression consistent with pluripotent stem cells. Established lines generated teratomas in vivo, and differentiated into tyrosine hydroxylase-positive neurons in vitro. Genetic analysis using array comparative genomic hybridization with an 8.9kb median probe spacing demonstrated that RNA reprogramming can yield lines free of copy number variations. The very high efficiency of this technique allowed us to reprogram single adult fibroblasts to pluripotency with a 44% success rate (n = 9). Our results suggest that the efficiency and kinetics of reprogramming methods need not be limited by a fundamental stochastic element as has been suggested. Due to the high efficiency, speed, reliability, and integration-free nature of RNA reprogramming, this technique will likely become the method of choice for generating disease and patient-specific pluripotent stem cells.
by Matthew Angel.
Ph.D.

Pluripotent cells have the dual abilities to self-renewal and to differentiate into all three germ layers. Pluripotent cells can be isolated from two different stages of mouse embryogenesis. Embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of the pre-implantation embryo and are considered to be in a naïve state. On the other hand, cells isolated from epiblast of the post-implantation embryo are referred as epiblast stem cells (EpiSC) and are representative of primed pluripotency. ESCs and EpiSCs are distinct from each other in terms of the morphology, the gene regulatory network and the signalling pathways regulating self-renewal. Under certain conditions, ESCs and EpiSCs can be transitioned into each other. However, the mechanism that regulates this transition from naïve to primed pluripotent state remains to be solved. Nanog, Oct4 and Sox2 form the core gene regulatory network of pluripotency. Additionally, the Id protein family is also important in the maintenance of pluripotency in ESCs. Id proteins function by inhibiting the activity of pro-differentiation factors. Tcf15 is identified as one of the targets of Id inhibition in ESCs. Moreover, Tcf15 has been identified as a repression target of Nanog. In this study, to understand the function of Tcf15, the expression of Tcf15 was characterized in differentiating ESCs. The transient upregulation of Tcf15 mRNA and protein was detected at early stages of differentiation before lineage commitment. Furthermore, Tcf15 protein was heterogeneously expressed in differentiating cells. Mutually exclusive expression of Nanog and Tcf15 proteins were demonstrated in both self-renewing and differentiating ESCs. Further characterization of the effect of Nanog on Tcf15 transcription showed that Tcf15 pre-mRNA was downregulated within 20 minute of Nanog induction. A Nanog binding site was identified at +32kb relative to the Tcf15 transcription start site (TSS). Initially, Nanog binding at this region was confirmed by performing ChIP-PCR experiments. Then, this Nanog binding region was further analysed for its enhancer activity related to the Tcf15 gene. Deletion of the Nanog binding region using CRISPR-Cas9 confirmed that this region acts as Tcf15 enhancer; it was shown that this region was required for the activation of Tcf15 transcription during differentiation. Tcf15 induction experiments were performed in order to the check whether Tcf15 affects Nanog transcription. The results indicate that Nanog is not a direct target of Tcf15, but Tcf15 contributes indirectly to the repression of Nanog. Additional analysis with the Tcf15 enhancer deletion cells showed that Tcf15 is not required for efficient downregulation of naïve markers and the upregulation of primed markers. However, the genes related to the regulation of adhesion properties of cells such as Zyc, Itga3 were induced with lower efficiency in the absence of Tcf15 compared to the wild type cells. In summary, I investigated the reciprocal regulation of Tcf15 and Nanog and the role of Tcf15 for the differentiation. My results suggest that Tcf15 is expressed in the cells that have initiated differentiation but are not lineage-committed. Additionally, Tcf15 can contribute to the regulation of adhesion related genes in order to help the epithelisation of the cells required during the differentiation from naïve to the primed pluripotent state. As a conclusion, Nanog is proposed to help to prevent certain aspects of ESCs differentiation by repressing Tcf15.

Different cell types in multi-cellular organisms heritably maintain different gene expression patterns despite carrying the same genome; a phenomenon termed epigenetics. It is widely believed that the packaging state of the genome, known as chromatin structure, carries epigenetic information. How chromatin states are inherited and how chromatin structure changes during development, moreover how different epigenomes, such as chromatin and DNA modifications communicate with each other during these processes are important questions. Accordingly, understanding the mechanisms that govern pluripotency and differentiation requires details of chromatin dynamics. The major goal of my doctoral thesis was to understand the genome wide view of chromatin dynamics in embryonic stem cells. My studies centered on two aspects of chromatin dynamics in mouse embryonic stem cells—localization and function of two antagonistic chromatin regulators and genome-wide histone variant dynamics. In the first part, we examined the roles of several chromatin regulators whose loss affects the pluripotent state of ES cells. We found that two such regulators, Mbd3 and Brg1, control a large number of genes in ES cells via antagonistic effects on promoter nucleosome occupancy. Moreover, we found that both Mbd3 and Brg1 play key roles in the biology of 5-hydroxymethylcytosine (5hmC), a newly identified DNA modification. Mbd3, which was named by homology to known cytosine methyl binding domains, yet does not bind methylcytosine in vitro, co-localized in ES cells with 5hmC. Furthermore, Mbd3 localization was lost in knockdown cells lacking the major 5mC hydroxylase, Tet1. Our results suggest, contrary to current dogma, that 5hmC is more than just an intermediate in cytosine demethylation pathways, that it may regulate genes via the Mbd3/NuRD complex. Finally, we showed that both Mbd3 and Brg1 are themselves required for normal levels of 5hmC in vivo, identifying a feedback loop between 5hmC and Mbd3. Together, our results identified a possible effector for 5hmC, thereby suggesting a functional role for this DNA modification. Moreover, Brg1 and Mbd3 can now be added to the growing list of regulators with opposite effects on ES cell gene expression, suggesting that pairs of antagonistic chromatin binding proteins may be a common phenomenon in ES cell transcription regulation (Yildirim et al., Cell 2011). The second part of my dissertation concerns the dynamics of several histone variants. Seminal studies in the Henikoff lab showed that certain histone variants are replaced throughout the cell cycle, in contrast to the predominant replication-coupled mode of histone assembly. Work in yeast and flies showed that rapid histone turnover occurs at epigenetically-regulated genomic regions, such as chromatin boundary elements or Polycomb/Trithorax binding sites. Notably, promoter regions of actively transcribed genes exhibit rapid turnover, suggesting that histone turnover may have an important role in gene regulation, as higher histone turnover rate would provide higher probability of DNA element exposure and faster erasure of chromatin marks of the replaced histones. In order to extend such studies to a model for pluripotency and differentiation, we developed a system for measuring histone replacement in mouse ES cells. To be able to carry out turnover experiments in ES cells, we generated stable ES cell lines that can be induced to express epitope-tagged histone variants. Our results confirmed that histone turnover patterns are conserved from yeast to mammals and that turnover profiles are histone variant specific. Murine H3.3 turnover is similar to H3.3 turnover in flies, with peaks at the promoters of highly transcribed genes. MacroH2A2, a variant generally linked to gene repression, had a more complex turnover profile. Surprisingly, we found rapid exchange of macroH2A2 occurring around transcription start sites of a number of highly expressed genes. At poorly expressed genes, on the other hand, macroH2A2 localizes upstream or downstream of transcription start sites and is incorporated slowly, either via slow turnover or via replication-coupled incorporation. Finally, we have used those inducible ES cell lines to generate mice, which will enable future studies on tissue-specific histone replacement in vivo. In summary, my thesis work not only significantly extends our understanding of chromatin regulation in general but also provides a more detailed landscape of chromatin structure and regulation in ES cells. Extending these analyses to differentiating cells and in vivo tissue specific dynamics should provide us with a better understanding not only of cell type specific chromatin organization but also improve our ability to program and re-program genomic landscapes in vitro.

Pluripotency is conserved in the major trunk of Vertebrate evolution, but how the gene regulatory network (GRN) that governs it evolved is poorly understood. A central component of this network is the Homeodomain containing transcription factor Nanog. How Nanog evolved is not understood, as Nanog sequences have not been identified in invertebrate genomes. This study provides evidence of Nanog activity encoded in the homeodomain of the invertebrate Vent gene family. The Vent2 gene from Saccoglossus kowalevskii, a model hemichordate, successfully reprogrammed mammalian pre-iPS cells to pluripotency, as demonstrated by the activation of dormant pluripotency genes, and the ability to generate all three primary germ layers. A second property of invertebrate Vents was also characterised in the Vent gene found in Nematostella vectensis, a sea anemone and model for cnidarian development, expression of which was insufficient to activate the endogenous pluripotency network of pre-iPS cells, though it could induce the cells to a XEN-like state that demonstrated up regulation of extra-embryonic markers, and subsequently gained dependence on ERK signalling. A direct comparison between the Saccoglossus and Nematostella Vent homeodomains was used to provide insight into the step-wise changes that appear to have given rise to Nanog activity. Swapping the homeodomains from one CDS to another, to create hybrid molecules, I demonstrated that the respective reprogramming activities of these genes is conserved in the homeodomain. I then identified specific amino acid (AAs) differences in the homeodomains that conferred a Nanog-like capacity for reprogramming to the Nematostella gene. Identification of a Nematostella EsrrB ortholog, which demonstrated reprogramming activity in mammalian pre-iPS cells, suggests wider conservation of pluripotency factors. I therefore propose that an ancient GRN for pluripotent mesoderm evolved in vertebrates to form part of the ground state network.

A fundamental step in embryo development is the transition through pluripotency. There are two known sequential states of pluripotency termed naïve and primed pluripotency. Considerable efforts have been made to characterize these two cell identities using gene expression, protein, metabolic and epigenetic profiling; here we explore an additional regulatory layer, at the level of alternative pre-mRNA splicing (AS). We performed deep coverage RNA sequencing on embryo-derived stem cell lines and identified a regulated AS landscape between the two states involving over 900 splicing events. These events show distinct cis-regulatory features and can also be observed in vivo and in the equivalent human pluripotency states. Differential gene expression analyses helped to identify two regulatory axes that contribute to shape these AS landscapes: the Rbmx/RbmxL2 and the Rbm47/Esrp axes. We further performed a high-throughput screen to test the effect of 1,500 single knockdowns of RNA binding proteins and chromatin modifiers on naïve-to-primed AS switches. This led us to the identification of Qki as an additional master naïve-to-primed AS regulator. Functional assays show that depletion of these splicing regulators affect the differentiation dynamics of embryonic stem cells. Overall, our results provide new insights into the regulatory programs governing naïve and primed pluripotency and the gain of differentiation potential.

Typically, levels of inhibitor of DNA binding 1 (IdI) are high in stem cells and decrease upon senescence or differentiation. Defining how IdI expression is regulated is critical to understanding what happens at the molecular level as cells differentiate during development and age in the adult organism. It has been proposed that zinc finger E-box binding protein (Zeb)l is a repressor of the pluripotency regulator Nanog and that IdI maintains embryonic stem (ES) cell pluripotency by inhibiting Zebl expression. However, ablation of Id1 in mouse ES cells did not alter Zebl protein levels. Somatic cells can be reprogrammed to pluripotency. The initiation of reprogramming without feeder cells was impaired in Id1 -/- and Id3 -/- MEFs. However, no difference was observed between WT and Id1 -/- in the presence of feeder cells. As MEFs were maintained in vitro and approached senescence, Idl protein, but not transcript, decreased. microRNAs are short, non-coding RNAs that inhibit mRNA expression by binding to the 3' untranslated region (UTR), resulting in either degradation or translational repression of the transcript. The 3' UTRs of mouse Idl - Id4 were cloned into a reporter vector. Reporter transcripts were repressed by the Idl 3' UTR, unlike other family members. A 67 base region of the IdI 3' UTR that was necessary for repression contained a predicted miR-381 / miR-539-3p binding site. A single point mutation at this site relieved repression by the Idl 3' UTR in a reporter vector. However, attempts to modulate activity of these microRNAs did not alter repression by the IdI 3' UTR in a reporter vector, or Id l protein levels. The present work has identified a previously unknown regulatory element located in a highly conserved region of the 3' UTR of Idl. Whereas the mechanism of regulation is unclear, tools have been generated that will allow manipulation of this site in a variety of cell lines.

Regeneration is a process that renews damaged or lost cells, tissues, or even of entire body structures, and is a phenomenon which is widespread in the animal kingdom. Urodeles such as newts and salamanders have a remarkable regeneration ability. They can regenerate organs such as gills, lower jaws, retina, appendages like fore- and hind limbs, and also the tail including the spinal cord. The regeneration process requires the use of resident stem cells or somatic cells, which have to be reprogrammed. In both cases the reprogrammed cells are less differentiated, meaning the cell would have the ability to form any kind of fetal or adult cell which rose from the three different germ layers, the ectoderm, mesoderm and endoderm. Artificial reprogramming of differentiated mammalian somatic cell had been reported previously. It was shown that four pluripotency factors, OCT4 (also called POU5f1), SOX2, c-MYC and KLF4 are sufficient to generate an induced pluripotent stem (iPS) cell. It has been shown that some of these factors are also involved in regenerating processes. In newt limb and lens tissue, Sox2, c-Myc and Klf4 mRNA levels were upregulated in the beginning of blastema formation when compared to non-amputated tissue. Oct4 mRNA however, was not detected. During xenopus tail regeneration, Sox2 and c-Myc were expressed, while the xenopus Pou homologs Pou25, Pou60, Pou79, Pou91 were not detected. In regenerating zebrafish fin tissue, Sox2, Pou2, c-Myc and Klf4 mRNA were not upregulated. The mammalian transcription factor OCT4, a class V POU protein, is responsible in maintaining pluripotency in gastrula stage embryos. It was reported that mouse OCT4 is also expressed in the caudal node of embryos having 16 somites. It is further known that progenitors exist in mouse tailbud, which give rise to neural and mesodermal cell lineage. This suggests that the OCT4 expressing cells in caudal node might be a stem cell reservoir. Oct4 was detected in axolotl during embryonic development, and prior to my work we found Oct4 when screening the axolotl blastema cDNA library. In addition, we also identified Pou2, another class V POU gene. Phylogenetic analysis showed a clear distinction of both genes in the axolotl. We determined the mRNA pattern of Pou2 during embryogenesis and compared it to Oct4 mRNA and protein. Both genes are expressed in the primordial germ cells and the pluripotent animal cap region of the embryo. Apart from this similarity, both genes have a different expression pattern in the embryo. We are interested in the involvement of OCT4, POU2, as well as the transcription factor SOX2 in regenerating axolotl spinal cord. We asked whether the cellular pluripotent character conferred by POU factors is limited to mammals or if it is an ancient characteristic of lower vertebrates. To answer the question we performed in vitro and in vivo studies. Hence this thesis is separated into two chapter. By in vitro studies we investigated the pluripotent PouV orthologs from different species. Therefore, we performed reprogramming experiments using mouse or human fibroblasts and transduced them with axolotl Oct4 or Pou2, in combination with human or axolotl Sox2, c-Myc and/or Klf4. The generated iPS cells with the different sets of factors had similar endogenous pluripotency gene expression profiles to embryonic stem cells. Further, iPS cells expressed the pluripotency markers like OCT4, NANOG, SSEA4, TRA1-60 and TRA1-81. Another evaluation of the iPS cells was the formation of embryoid bodies. Immunouorescence staining showed that tissue from all three germ layers was formed after induction. We observed a positive staining for the endoderm marker !-FEROPROTEIN, the mesoderm marker !-SMOOTH MUSCLE ACTIN and the ectoderm marker \"III TUBULIN in the generated cells. This indicated that the iPS cells generated using axolotl Oct4 and Sox2 in combination with mammalian Klf4 and with or without c-Myc, as well as iPS cell generated with axolotl Pou2 and mammalian Sox2 and Klf4 and with or without c-Myc have a pluripotent potential. In addition, the axolotl factors are able to form heterodimers with the mammalian proteins. Furthermore, we compared the reprogramming ability with POU factors from mouse, human, zebrash, medaka and xenopus. We showed that xenopus Pou91, as the only non-mammalian example, is nearly as efficient as mouse and human Oct4 cDNAs in inducing GFP expressing cells. Also axolotl Pou2, axolotl Oct4 and medaka Pou2 showed reprogramming character however at a much lower efficiency. In contrast, zebrash Pou2 is not able to establish iPS cells. This indicates that a reprogramming ability to a pluripotent cell state is an ancient trait of Pou2 and Oct4 homologs. By in vivo studies we investigated the role of Oct4, Pou2 and Sox2 gene expression in regenerating spinal cord tissue. Performed in situ hybridizations and antibody staining studies in the regenerating spinal cord showed that Oct4, Pou2 and Sox2 were expressed during spinal cord regeneration. Knockdown experiments in regenerating spinal cord using morpholino showed that Pou2-morpholino does not have an effect. In contrast, SOX2 was required for spinal cord regeneration but to a lesser extent, than OCT4, which decreased the regenerated length signicantly compared to control. Even though, with Sox2-morpholino we did not observe the phenotype as a significantly shorter regenerated spinal cord, about 45% of SOX2 knocked down cells were not cycling and proliferating anymore. This indicates that axolotl SOX2 has an effect in regeneration. Therefore we wanted to know whether spinal cord cells would also have a pluripotent character in vivo and form other tissue types. Regenerating cells of the spinal cord are only able to form the same cell type and thus they keep their cell memory. However, when we performed transplantations of OCT4/SOX2 expressing spinal cord cells into somite stage embryos, we could show the formation of muscle cells. This shows that the spinal cord cells have the potential to change their fate in an embryonic context, where the normal environment of spinal cord has changed. However, our data do not indicate whether muscle is formed directly from the spinal cord or whether spinal cord cells fuse to developmental myoblasts, a cell type of embryonic progenitors, which give rise to muscle cells. To clearly state whether regenerating OCT4/SOX2 expressing spinal cord cells are pluripotent we have to perform OCT4 knock down in spinal cord and transplant these less proliferating cells into embryos, observing their cell fate.

Conventional or “primed” human embryonic stem cells (hESCs) rely on FGF and TGFβ signalling for self-renewal, and occupy a developmentally advanced state of pluripotency comparable to mouse EpiSCs. Recent reports demonstrate that a naïve state of human pluripotency can be consistently derived either through transient histone deacetylase inhibition mediated resetting of conventional hESCs or via isolation of the inner cell mass. Long-term propagation of this state can be achieved using a cocktail of MEK, GSK3 and PKC inhibition in conjunction with leukaemia inhibitory factor (LIF) supplementation (t2iLGö) and a feeder layer of inactivated mouse embryonic fibroblasts. However, the way in which this signalling environment is interpreted in order to maintain naïve pluripotency remains unclear. I demonstrate a substrate consisting of a high concentration of tissue-derived laminin in combination with t2iLGö is sufficient to replace the feeder layer. Cultures maintained under these conditions are karyotypically normal, maintain a naive pluripotent transcriptional profile and exhibit reduced aberrant expression of mesodermal and endodermal lineage markers. I utilise the increased stringency of this culture system in combination with small molecule inhibitors to examine the roles of FGF, Activin/Nodal and JAK/STAT signalling in human naïve pluripotency. Naïve hESCs proliferate and maintain pluripotency marker expression in the presence of FGF receptor inhibition. In contrast, TGFβ signalling inhibition leads to rapid downregulation of human specific naïve pluripotency marker, KLF17, followed by the eventual collapse of the naïve transcription factor circuitry. Naïve hESCs self-renew in both the absence of LIF and presence of JAK/STAT inhibitors. However, further investigation of JAK/STAT signalling identified the increased potency of Interleukin 6 (IL-6) over LIF to activate the JAK/STAT pathway. Supplemental IL-6 improves colony-forming capacity under self-renewing conditions and attenuates differentiation following inhibitor withdrawal. Furthermore, prolonged activation of IL-6 signalling suppresses expression of GATA2 and GATA3 and upregulates KLF4 transcripts. Finally, I investigate whether ablation of PKCι is sufficient to replace the activity of the PKC inhibitor, Gö6983. Established naïve cultures that are PKCι null continue to express naïve markers and suppress upregulation of lineage makers following withdrawal of Gö6983. Furthermore, ablation of PKCι in conventional ESCs enables the maintenance of NANOG expression and the emergence of KLF17 expression in the absence of Gö6983 during histone deactylase mediated resetting.

Great progress has been made towards the understanding of the molecular mech- anisms driving factor induced somatic cell reprogramming to pluripotency since the discovery by Takahashi and Yamanaka. However this process remains highly stochastic and inef cient. More study is needed in order to achieve a more de- terministic cell fate conversion which could further improve the quality of stem cells generated, essential for prospective therapeutic applications. The work presented here was developed under the premise that natural embryonic devel- opment can serve as a guide to achieve more ef cient pluripotency induction. It was observed that the histone variant H2A.Z, which has a role in pluripotency in embryonic stem cells, is highly expressed in the oocyte and upon over-expression, together with Pou5f1, Sox2, Klf4 and Myc, was able to increase the ef ciency of somatic cell reprogramming to induced pluripotency. A gene co-expression network analysis of somatic cells being reprogrammed identi ed hub genes as- sociated with H2Af.z and chromatin remodelling related genes which could be tested for further improving the reprogramming ef ciency induced by H2Af.z over-expression. Moreover, the study of genetic networks from pre-implantation embryos identi ed preserved genetic circuits also present during the course of reprogramming. The most preserved network modules are associated with the nal stages of pluripotency induction. However the analysis also identi ed a genetic network associated with the zygotic genome activation in the totipotent embryo stage which is also found in a sub-population of pluripotent stem cells characterised by the expression of genes from the Zscan4 family, Tet1, Etv5 and Mga among other genes. This provocative observation led me to hypothesise that during the course of reprogramming the forced activation of hub genes from such network may help improve its ef ciency, possibly by recapitulating the natural embryonic processes which induces totipotency prior to pluripotency. The identi- cation of preserved network modules and its hub genes presented in this work may serve a platform for further reprogramming studies in a quest for improved cocktails of reprogramming factors capable of more ef ciently generating induced pluripotent stem cells.

Central to the control of virtually all cellular activity is the regulation of gene expression. In eukaryotes, this regulation is greatly influenced by chromatin structure, which is itself regulated by numerous chromatin-remodeling complexes. These are typically large protein complexes with interchangeable subunits that allow for highly specialized functions in different cell types. Moreover, additional specificity can be gained through complexes formed from different subunit isoforms. Histone modifications also regulate chromatin by recruiting remodeling complexes to particular genomic regions. In this thesis we characterize MBD3C, an isoform of the Nucleosome Remodeling and Deacetylase (NuRD) complex subunit MBD3. MBD3 is essential for pluripotency and development, but MBD3C appears to be expressed only in embryonic stem cells (ESCs), and whether it forms a distinct NuRD complex, how its expression is regulated, and its precise function(s) remain unknown. We show that MBD3C forms a complete NuRD complex that functions redundantly with the other MBD3 isoforms in ESC gene regulation. Furthermore, MBD3C binds the SET/MLL complex subunit WDR5 through a conserved motif within its unique N-terminal region, and this interaction is necessary for the regulation of >2,000 ESC genes. Together, these findings indicate that ESCs can utilize isoforms of the same protein to achieve similar functions through diverse mechanisms. The second part of this thesis focuses on the role of the histone modification H3.3K56ac in pluripotency and differentiation. Although H3K56ac is well-studied in yeast, in mammalian cells it is far less abundant and its functions are largely unknown. Our data indicate that the H3.3K56R mutant is largely normal for ESC maintenance and loss of pluripotency markers during differentiation, but H3.3K56ac is necessary for proper lineage commitment. Ongoing studies will characterize the H3.3K56Q phospho-mimetic mutant during differentiation, and examine H3.3K56ac function at lineage-specific genes.

Central to the control of virtually all cellular activity is the regulation of gene expression. In eukaryotes, this regulation is greatly influenced by chromatin structure, which is itself regulated by numerous chromatin-remodeling complexes. These are typically large protein complexes with interchangeable subunits that allow for highly specialized functions in different cell types. Moreover, additional specificity can be gained through complexes formed from different subunit isoforms. Histone modifications also regulate chromatin by recruiting remodeling complexes to particular genomic regions. In this thesis we characterize MBD3C, an isoform of the Nucleosome Remodeling and Deacetylase (NuRD) complex subunit MBD3. MBD3 is essential for pluripotency and development, but MBD3C appears to be expressed only in embryonic stem cells (ESCs), and whether it forms a distinct NuRD complex, how its expression is regulated, and its precise function(s) remain unknown. We show that MBD3C forms a complete NuRD complex that functions redundantly with the other MBD3 isoforms in ESC gene regulation. Furthermore, MBD3C binds the SET/MLL complex subunit WDR5 through a conserved motif within its unique N-terminal region, and this interaction is necessary for the regulation of >2,000 ESC genes. Together, these findings indicate that ESCs can utilize isoforms of the same protein to achieve similar functions through diverse mechanisms. The second part of this thesis focuses on the role of the histone modification H3.3K56ac in pluripotency and differentiation. Although H3K56ac is well-studied in yeast, in mammalian cells it is far less abundant and its functions are largely unknown. Our data indicate that the H3.3K56R mutant is largely normal for ESC maintenance and loss of pluripotency markers during differentiation, but H3.3K56ac is necessary for proper lineage commitment. Ongoing studies will characterize the H3.3K56Q phospho-mimetic mutant during differentiation, and examine H3.3K56ac function at lineage-specific genes.

Reprogramming of somatic cells to pluripotency is a topic of extensive research driven by the potential of induced pluripotent cells in in vitro disease modelling, drug screening and regenerative medicine. The pluripotency associated transcription factor Nanog has been subject to a lot of controversy. Initially thought to form an essential trinity with Oct4 and Sox2 to maintain embryonic stem cell (ES) self-renewal, it was shown that actually ES cells can maintain their pluripotency in the absence of Nanog. In vivo however, Nanog is essential to establish the pluripotent epiblast. For Nanog -/somatic cells reprogramming by defined factors, in a culture condition using inhibitors for MEK/ERK and GSK3 (2i) fails to occur. This led to the assumption that acquisition of pluripotency in reprogramming is dependent on Nanog. This observation however, had not been verified extensively in alternative culture conditions. Using neural stem cells as a system to study reprogramming, I investigated the use of Knockout Serum Replacement (KSR) + LIF medium instead and found that in this condition reprogramming of Nanog -/- cells in fact does occur, Nanog is thus not absolutely required for the induction of pluripotency. The addition of 2i to KSR +LIF medium abolished reprogramming of Nanog -/- cells again. I did observe that the efficiency of reprogramming by defined factors is enhanced by constitutive overexpression of Nanog. I also found that addition of constitutively overexpressed Nanog to the generally used reprogramming factors enables reprogramming of somatic cells in a culture condition that does not support pluripotency, however presence of LIF was required. LIF was dispensable for maintenance of the obtained Nanog overexpressing iPS cells, suggesting that it could play a role in the acquisition of pluripotency. I also observed that if LIF is added to 2i medium during reprogramming the number of iPS cell colonies that emerges is enhanced. LIF contributes to maintenance of pluripotency via activation of the JAK/ST AT3 signalling pathway. ST AT3 was found to reprogram post-implantation epiblast derived stem cells (EpiSCs) to nai:ve pluripotency. I showed that for reprogramming of adult neural stem cells, increased activation of JAK/ST AT3 enhances efficiency and acts as a potent reprogramming factor. In addition, I demonstrated that sufficient activation of JAK/ST AT3 can overcome the reprogramming block of pre-iPS cells and obviates a need for further pluripotency culture environment requisites for reprogramming and maintenance of nai:ve pluripotency. I showed that FGF signalling, which promotes the exit of nai:ve pluripotent cells from self-renewal, does not prevent JAK/ST A T3 driven EpiSC conversion to nai:ve pluripotency. Moreover, even in the presence of FGF plus Activin, which instructs and maintains the primed state, I found that JAK/ST AT3 can dominantly enforce a nai:ve pluripotent state to EpiSCs.

Germ cells are the only population of cells in the adult organism shown to retain pluripotency – the ability to differentiate into all the different germ layers of the embryo. This property is shared with embryonic stem cells and tumour-derived embryonal carcinoma cells, and the molecular mechanisms underpinning pluripotency are likely to be similar in all three systems. The core circuitry of transcription factors required to establish and maintain pluripotency is relatively well characterised. However, many of these important transcription factors also regulate a large number of genes involved in processes other than transcription, some of which may also play an important role in maintaining stem cell plasticity. Understanding the entire molecular basis of pluripotency will improve the use of ES cells as a model system for differentiation and development, enhance the therapeutic potential of stem cells and provide insights into the mechanisms of tumourigenesis. Testis Expressed Gene 19 has been identified in screens for genes expressed in spermatogonia, embryonic stem cells, and for targets of translational regulation by the germ cell-specific protein Dazl. The work presented here characterises the expression pattern of Tex19 in the gonads and stem cells, establishing a correlation between the expression of Tex19 and pluripotent potentiality. A human homologue of Tex19 is found to be expressed in stem cells and cancer, and the evolution of the gene family in mammals is investigated. Finally, the function of Tex19 and its relationship with Dazl is also investigated.