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Avaliação do Papel da Via Canônica e Não Canônica de NFB na Manutenção da Pluripotência e na Diferenciação, por Meio da Técnica de Imunoprecipitação de Cromatina / Evaluation of Canonical and Non-Canonical NFB Pathways in the Maintenance of Pluripotency and Differentiation by Chromatin Immunoprecipitation TechniqueHudson Lenormando de Oliveira Bezerra 30 September 2014 (has links)
As células pluripotentes (CPs), em teoria, são capazes de dar origem a todos os mais de 200 tipos de células do organismo. Na natureza, há três tipos de células pluripotentes: células-tronco embrionárias, células germinais embrionárias e células de carcinoma embrionário. As características das CPs têm permitido um importante avanço para a pesquisa básica e apontam uma grande aplicabilidade na medicina regenerativa. No núcleo das CPs existem fatores atuantes responsáveis pela manutenção da identidade pluripotente; dentre eles destacam-se OCT4, NANOG, SOX2, KLF4 e MYC. Muito já se sabe sobre os mecanismos que estes fatores atuam para promover a manutenção da pluripotência celular. Baseados nestes estudos foi possível gerar células de pluripotência induzida (iPSCs). Porém, os mecanismos moleculares que direcionam a indução da pluripotência ainda não estão muito bem esclarecidos. Alguns estudos revelaram que componentes chaves da via NFB estão envolvidos na regulação da pluripotência, bem como na diferenciação e destino celular das células-tronco. Neste estudo, analisamos a participação de componentes da via canônica (RelA e NFB1) e não-canônica (RelB e NFB2) de NFB nos processos de diferenciação e destino celular ou manutenção da pluripotência. Para isto usamos técnicas de PCR quantitativa em Tempo Real (qPCR) e Imunoprecipitação de Cromatina (ChIP) investigando os papéis das vias canônica e não-canônica de NFB na manutenção da pluripotência e diferenciação de CPs, em um modelo de indução de diferenciação celular mediado por ácido trans-retinóico (atRA) em células de carcinoma embrionário NTera-2. Foram avaliadas as ligações dos fatores de transcrição RelA e RelB nas regiões promotoras dos genes OCT4, SOX2, MYC, KLF4 e GFAP e a regulação transcricional associada. Nossos resultados identificaram que as células não tratadas com atRA apresentaram níveis baixos na expressão dos componentes da via canônica de NFB, RelA e NFB1, e GFAP e quando induzidas à diferenciação por atRA durante 4 dias esses níveis se elevaram. Uma situação oposta foi vista nos componentes da via não-canônica de NFB, RelB e NFB2, e na expressão dos fatores de pluripotência OCT4, NANOG, SOX2 e KLF4, que apresentaram níveis de expressão elevados nas células não tratadas com atRA e sofreram redução com a indução da diferenciação celular. O ensaio de ChIP revelou que RelA liga-se nas regiões de regulação dos genes OCT4, SOX2, KLF4, MYC e GFAP apenas quando a célula está em processo de diferenciação, enquanto RelB se apresentou ligado às mesmas regiões tanto nas células indiferenciadas quanto naquelas induzidas à diferenciação por 4 dias. Com estes dados sugerimos que a via canônica de NFB pode estar relacionada com o processo de diferenciação e destino celular através da regulação negativa executada por RelA e NFB1 nos genes responsáveis pela identidade pluripotente das células aqui estudadas enquanto a via não-canônica de NFB, representada pela ativação de RelB e NFKB2, pode participar na manutenção da pluripotência através da regulação positiva destes mesmos fatores. / Human pluripotent stem cells (hPSCs) are able to give rise to all the 200 cell types of the adult organism. In nature, there are three types of hPSCs: embryonic stem cells, germ line stem cells and embryonal carcinoma cells. hPSCs characteristics have allowed a major advance in basic research, and are thought to have great applicability in regenerative medicine. In the nucleus of hPSCs there are transcription factors responsible for the maintenance of their pluripotent identity. OCT4, NANOG, SOX2, KLF4 and MYC are considered the core pluripotency factors in hPSCs. A great deal of knowledge about the mechanisms that promote and maintain pluripotency has been generated. Based on these studies it was possible to generate induced pluripotent stem cells (iPSCs). However, the molecular mechanisms that drive the induction of pluripotency are not fully understood. Some studies have recently indicated that key components of the NFkB may be involved in regulating pluripotency as well as cell differentiation and cell fate. In this study we analyzed the involvement of components of the canonical (RelA and NFB1) and the non-canonical NFB pathways (RelB and NFB2) in the maintenance of pluripotency, differentiation and cell fate processes. The techniques of quantitative real-time PCR (qPCR) and chromatin immunoprecipitation (ChIP) were used to interrogate the roles of the canonical and non-canonical NFB pathways in maintenance of pluripotency and differentiation in a model of cell differentiation induced by all trans-retinoic acid (atRA) on embryonal carcinoma cells NTera-2. The transcription factors RelA and RelB occupancy in the promoter regions of OCT4, SOX2, KLF4, MYC and GFAP, and the transcriptional regulation associated were evaluated. Our results showed that undifferentiated cells exhibited low expression levels of canonical NFB pathway components, RelA and NFB1, while cells induced to differentiate for 4 days exhibited downregulated expression of these factors. In the other hand, the non-canonical NFB pathway components, RelB and NFB2, and the pluripotency factors OCT4, NANOG, SOX2 and KLF4 were expressed in higher levels in undifferentiated cells, and were downregulated upon the differentiation process. ChIP assay revealed that RelA binds to the regulatory regions of OCT4, SOX2, KLF4, MYC, and GFAP only when cells are induced to differentiate, while RelB was found bound to the same regions in both undifferentiated and differentiated cells. This data suggests that the canonical NFB pathway may be associated to differentiation and cell fate processes by downregulation of genes responsible for the pluripotent identity, and that the non-canonical NFB pathway may act in the maintenance of pluripotency through the upregulation of the same factors.
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Estudo dos lipídeos relacionados aos mecanismos reguladores da pluripotência em Células-tronco Pluripotentes Induzidas (iPS) Humanas / Lipids profile changes associated to pluripotency regulatory mechanisms during mesenchymal cells reprogramming to Human Induced Pluripotent Stem cells (iPS)Pedro Ratto Lisboa Pires 02 June 2016 (has links)
A geração de células-tronco pluripotentes induzidas (iPS) a partir de células somáticas demonstrou que células adultas de mamíferos podem ser reprogramadas a um estágio de pluripotência através da inserção de fatores de transcrição embrionários. Esta descoberta tem levantado questões fundamentais sobre os mecanismos, que através destes fatores de transcrição, influenciam epigeneticamente as células e seus potenciais de diferenciação após a reprogramação e um normal desenvolvimento. Componentes lipídicos e lipoprotéicos afetam vários aspectos no comportamento celular durante sua manutenção e diferenciação, podendo afetar diretamente fatores essenciais em processos de reprogramação celular, manutenção da pluripotência e perfil epigenético das células. Nesse sentido, esta tese propôs o estudo da composição lipídica com diferentes abordagens entre células iPS, células-tronco embrionárias (H1) e células fibroblastos (BJ). Foram produzidas três linhagens de células pluripotentes induzidas no modelo humano que foram caracterizadas quanto 1a sua pluripotência e utilizadas, juntamente às linhagens H1 e BJ como modelos para o estudo da composição lipídica proposto. Foram identificadas e estudadas um total de 44 espécies lipídicas das classes PC, PE, PI, SM e PS, e discutidas frente a reprogramação celular e manutenção da pluripotência. Foi identificado um padrão de composição fosfolipídica distinta entre células pluripotentes e não pluripotentes, e especulamos que a presença dessas espécies parecem ter um envolvimento fundamental para a manutenção da pluripotência. Este padrão, mostrou pela análise de componente principal, que durante o processo de reprogramação, alterações na composição lipídica ocorrem de forma com que a pluripotência surge durante a reprogramação, evidenciando alterações lipídicas particulares do estádio da pluripotência, sugerindo uma ligação entre estas alterações na composição lipídica com as alterações metabólicas da própria reprogramação celular. O estudo da quantificação de fosfolipídios entre linhagens celulares pluripotentes e não pluripotentes evidenciaram que existe uma diferença fosfolipídica entre estas linhagens, observamos que as linhagens iPS e H1, do ponto de vista das classes observadas e os fosfolipídios quantificados, são similares entre si e diferentes de células não pluripotentes. É evidente que estas moléculas lipídicas, individualmente, não são capazes de modular processos como a reprogramação celular, entretanto, é de extrema importância o entendimento das mesmas dentro da reprogramação celular e manutenção da pluripotência. Nossos dados sugerem que a composição lipídica de células pluripotentes tem importante papel para o desenvolvimento e evolução do processo de reprogramação celular e o entendimento da manutenção da pluripotência / The generation of induced pluripotent stem cells (iPS) from adult somatic cells has shown that mammalian cells can be reprogrammed to a pluripotent state by the insertion of embryonic transcription factors. This finding has raised questions about the fundamental mechanisms through which these transcription factors epigenetically influence cells, their potential of differentiation after reprogramming and normal development. Lipid and lipoprotein components affect numerous aspects of cell behavior during its maintenance and differentiation, which can directly affect main factors in cell reprogramming processes, maintenance of pluripotency and epigenetic profile of the cells. Thus, this thesis proposed to study, with different approaches, the lipid composition of iPS cells, embryonic stem cells (H1) and fibroblast cells (BJ). Three induced pluripotent cell lines were produced in the human model. They were characterized regarding their pluripotency and used along with H1 and BJ cell lines, as models for the proposed lipid composition study. A total of 44 species of lipid from the classes PC, PE, PI, PS and SM have been identified, studied and discussed regarding cellular reprogramming and maintenance of pluripotency. A different phospholipid composition pattern was observed between pluripotent and non-pluripotent cells, and it is speculated that the presence of these species appears to have a major involvement on the maintenance of pluripotency. This array showed, by the principal component analysis, that during the reprogramming process changes in the lipid composition occur, so that pluripotency takes place during reprogramming, highlighting lipid changes particular of the pluripotency state, suggesting a connection between these changes in lipid composition and the metabolic changes of cell reprogramming. The study of the quantitation of phospholipids from pluripotent and non-pluripotent cell lines indicated a phospholipid difference between these cell lines when considering the observed classes and quantified phospholipid. It was eminent that iPS lines and H1 are similar and differ from non-pluripotent cells. It is clear that these lipid molecules are not individually capable of modulating processes such as cell reprogramming, however, it is extremely important to understand them within cellular reprogramming and maintenance of pluripotency. Our data suggests that the lipid composition of pluripotent cells has important role in the development and evolution of cellular reprogramming process and the understanding the maintenance of pluripotency
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Contribution of U2AF1, NCBP1 and eIF4A3 to the control of pluripotency maintenance and cell fate determination / Contribution de U2AF1, NCBP1 et eIF4A3 dans le contrôle du maintien de la pluripotence et le devenir cellulaireLaaref, Abdelhamid Mahdi 24 November 2017 (has links)
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.
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Développement d'outils pour suivre la différenciation précoce de cellules souches embryonnaires / Establishing tools to investigate and guide early embryonic stem cell differentiationBera, Agata Natalia 11 September 2012 (has links)
Les cellules souches embryonnaires (ES) sont des cellules pluripotentes, capables de s'auto-renouveller indéfiniment dans des conditions de culture appropriées. Cela signifie que ces cellules restent dans un état prolifératif et indifferencié en culture et ont le potentiel de se différencier dans les trois feuillets embryonnaires, à savoir l'ectoderme, le mésoderme et l’endoderme, et leurs dérivés. Cette capacité à se différencier dans tous les types cellulaires, souligne la diffculté à contrôler la différenciation des cellules ES in vitro et à les guider vers un lignage spécifique. Mon projet de thèse porte sur la différenciation des cellules ES murines. Une étape importante du développement embryonnaire est le choix entre l’ectoderme et le mésendoderme. Dans ce but, j'ai développé une lignée ES qui permet de suivre exclusivement l'expression de Brachyury (T) dans le mésendoderme à l'exclusion de la notochorde: la lignée TRepV. Pour cela, jai cloné un fragment de 1 kb du promoteur murin de Brachyuryen amont du rapporteur Venus (YFP). Avant d'utiliser cette lignée, j’ai cherché à la valider. Malheureusement l'expression du rapporteur TRepV ne reproduit pas fidèlement l'expression endogène de T. Une hypothèse est que le fragment de 1kb ne contient pas tous les éléments de régulation de T nécessaires pour expression fidèle in vitro. De manière surprenante, j’ai observé que le rapporteur TRepV est exprimé de façon hétérogène dans les cellules ES non différenciées. Au cours de mon travail de thèse, je me suis intéressée à cette expression hétérogène. J'ai montré que les cellules ES TRepV+ représentent une sous-population distincte des cellules souches, qui peut être maintenue séparément exprimant le rapporteur de manère stable, à la difference d'autres gènes exprimés de manière hétérogène dans les cellules ES. Nous avons trouvé un marqueur d'une population distincte parmi les cellules ES et de nouveaux gènes impliqués dans pluripotence, qui seront abordés dans des études futures. / Embryonic stem cells (ESCs) are a powerful system to investigate developmental processes in vitro, and a promising tool to generate specific cell types for cellular therapies and regenerative medicine. ESCs are self-renewing, pluripotent cells, maintaining a proliferative and undifferentiated state in culture, while retaining the capacity to differentiate into the three embryonic lineages: ectoderm, mesoderm and endoderm, and all their derivatives. Here, I established a primitive streak specific Brachyury/T Reporter ESC line (TRepV) to investigate early ESC differentiation. In contrast to previously published Tknock-in line, we established a transgene T ESCs reporter line, in order to avoid the disruption of the T locus, which may result in a hapoinsuficient phenotype. During the validation process, I observed discrepancies in expression between the TRepV and the endogenous T locus. I followed upon these observations with a more detailed analysis and obtained evidence that T is regulated differently in the ESC system compared to in vivo development. Against expectations, I also observed heterogeneous expression of the TRepV reporter in undifferentiated ESCs. Undifferentiated ESCs were found to be a mix of TRepV+ and TRepV- cells. This finding became the focus of my studies: I found TRepV+ cells represent a distinct population of ESCs with a unique identity. Unlike other heterogeneous ESC populations (such as Stella or Nanog), TRepV+ cells do not interconvert in their fate and represent an explicit, stable subpopulation of ESCs. Finally, I performed a microarray analysis of TRepV+ and TRepV- ESCs and identifed new genes which may be involved in the regulation of self-renewal and pluripotency.
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Regulation of cell fate and cell behaviour during primitive endoderm formation in the early mouse embryoSaiz, Nestor January 2012 (has links)
The preimplantation stages of mammalian development are dedicated to the differentiation of two extraembryonic epithelia, the trophectoderm (TE) and the primitive endoderm (PrE), and their segregation from the pluripotent embryonic lineage, the epiblast. The TE and PrE are responsible for implantation into the uterus and for producing the tissues that will support and pattern the epiblast as it develops into the foetus. PrE and epiblast are formed in a two step process that involves random cell fate specification, mediated by fibroblast growth factor (FGF) signalling, and cell sorting through several mechanisms. In the present work I have addressed aspects of both steps of this process. Chimaera assays showed that epiblast precursors transplanted onto a recipient embryo rarely differentiate into PrE, while PrE precursors are able to switch their identity and become epiblast. Transient stimulation or inhibition of the FGF4-ERK pathway in the chimaeras can modify the behaviour of these cells and restore the plasticity of epiblast precursors. This work shows that epiblast precursors are refractory to differentiation signals, thus ensuring the preservation of the embryonic lineage. I have also found that atypical Protein Kinase C (aPKC) is a marker of PrE cells and that pharmacological inhibition of aPKC impairs the segregation of PrE and epiblast precursors. Furthermore, it affects the survival of PrE cells and can alter the subcellular localisation of the PrE transcription factor GATA4. These data indicate aPKC plays a central role for the sorting of the PrE and epiblast populations and links cell position within the embryo to PrE maturation and survival. Lastly, I have found that aPKC can directly phosphorylate GATA4 in vitro. Knockdown of GATA4 affects cell position within the embryo, whereas aPKC knockdown reduces the number of GATA4-positive cells. These results indicate GATA4 plays an important role in cell sorting during preimplantation development and suggest phosphorylation by aPKC could determine its presence in the nuclei of PrE cells. My work, in the light of the current knowledge, supports a model where the earliest cell fate decisions during mammalian development depend on cellular interactions and not on inherited cell fate determinants. This robust mode of development underlies the plasticity of the preimplantation embryo and ensures the formation of the first mammalian cell lineages, critical for any further progression in mammalian development.
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Etude de la balance pluripotence-differenciation des cellules souches embryonnaires murines sous l'effet du LIF : rôle du gène MRAS / Study of balance pluripotency - differentiation of murine embryonic stem cells under the effect of LIF : Role of MRAS geneMathieu, Marie-Emmanuelle 12 December 2011 (has links)
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.
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Characterization of pluripotency genes in axolotl spinal cord regenerationDuemmler, Annett 25 June 2013 (has links)
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.
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Genetic modification in CPVT patient specific induced pluripotent stem cells with CRISPR/Cas9Zimmermann, Maximilian 02 December 2019 (has links)
No description available.
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Chemical Biology Approaches for the Molecular Recognition of DNA Double Helix / DNA二重らせんの分子認識に関するケミカルバイオロジー研究Abhijit, Saha 23 March 2015 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18807号 / 理博第4065号 / 新制||理||1585(附属図書館) / 31758 / 京都大学大学院理学研究科化学専攻 / (主査)教授 杉山 弘, 教授 三木 邦夫, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM
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Autotaxin-mediated lipid signaling intersects with LIF and BMP signaling to promote the naive pluripotency transcription factor program / Autotaxinによる脂質シグナリングはLIFおよびBMPシグナル伝達経路と交わり、ナイーブ型多能性転写因子プログラムの形成を促進するCody, West Kime 26 March 2018 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21025号 / 医科博第86号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 渡邊 直樹, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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