Orientador: Fernanda da Cruz Landim / Resumo: As pesquisas envolvendo a biologia das células-tronco abordam um amplo espectro de fenômenos, que vão desde o nível tecidual e celular, até o seu uso em terapias celulares. Esta crescente atenção sugere que é necessário estudar conceitos básicos da biologia das células-tronco para compreender completamente os processos de diferenciação funcional. Desta forma, o instrumento da reprogramação celular por meio da manipulação gênica fornece subsídios para melhor compreender os processos de renovação e diferenciação que constituem as características fundamentais das células-tronco. A obtenção dessas células em medicina veterinária visa validar diversos modelos experimentais domésticos, como o equino, na busca de novos fármacos e terapias alternativas para reabilitação. Uma série de estudos, porém, ainda são necessários para que tais aplicações sejam viáveis, uma vez que os mecanismos fundamentais das técnicas empregadas ainda não estão totalmente elucidados. Embora a reprogramação celular por meio de vetores virais tenha sido relatada com sucesso em diversas espécies animais, outras técnicas também podem ser empregadas, como o uso de transposons, sequências de DNAs capazes de se movimentar de uma região para outra no genoma de uma célula. Não se tem conhecimento de qual o melhor tipo celular a ser utilizado, e nem tão pouco qual a metodologia de reprogramação mais eficiente. Sabe-se que o cordão umbilical possui uma reserva rica em células-tronco mesenquimais, as quais por serem mu... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Researches on the biology of stem cells cover a broad spectrum of phenomena, ranging from tissue and cellular level, to their use in cell therapy. This growing attention suggests that is necessary to study basic concepts of stem cells organization in order to fully understand the functional differentiation processes. Thus, the cell reprogramming through gene manipulation provides grants to better understand the processes of renewal and differentiation which are the essential characteristics of stem cells. Obtaining these cells in veterinary medicine aims to validate various household experimental models, such as horses, on the search for new drugs and alternative therapies for rehabilitation. However, a number of studies is still necessary for such applications to be feasible, since the fundamental mechanisms of techniques employed are not fully elucidated yet. Although cell reprogramming using viral plasmid has been reported with success in several animal species, other techniques may also be employed, such use transposons, this is, DNAs sequences capable of moving from one region to another in the cell genome. The unawereness of what the best cell type to be used, and nor what is the most efficient reprogramming methodology. It is known that the cord has rich reserves mesenchymal stem cells, which are multipotent and can improve the efficiency of obtaining the induced Pluripotent Stem Cells (iPS) compared to the use of fibroblast, inefficient to be reprogrammed. The aim of this study was to obtain iPS through viral transfection and nonviral adult fibroblasts and equine cord cells, aiming to observe which transfection and cell type is more efficient for cell reprogramming. Both cell types was infected with viral vectors and transposons containing the genes OCT-4, SOX-2, c-MYC, and KLF-4; transformed cells were evaluated for morphology, immunocytochemistry... (Complete abstract click electronic access below) / Doutor

Identifying interventions, such as drugs, that can counteract the effects of aging is crucial due to the complex nature of the aging process, which involves multiple biological processes. By targeting these processes, interventions have the potential to promote healthy aging. Utilizing pairs of drugs that exhibit synergistic effects becomes particularly effective as they can simultaneously impact multiple pathways associated with aging and reprogramming, enhancing their anti-aging potential. The Panacea (predicting anti-aging combinations from expression analysis) framework was developed to facilitate the discovery of such drug combinations. Deep generative models were incorporated into the Panacea framework to effectively capture complex patterns in gene expression data, leveraging their non-linear nature for an accurate representation of relationships and interactions. This makes them ideal for predicting drug combinations. The trained models, using the CMap dataset, demonstrated an improved performance to predict the effect of drugs. The age effect of these drug combinations was evaluated using an age-predictive model, revealing that synergistic anti-aging combinations mainly comprised reprogramming (the process of transforming one type of cell into another by altering its gene expression and properties), apoptosis (programmed cell death mechanism), and chemotherapy drugs, while pro-aging combinations involved cellular growth-limiting, longevity-extending, and chemotherapy drugs. These results emphasize the capability of deep generative models in predicting potent drug combinations for anti-aging and anti-cancer interventions.

Acute myeloid leukemia (AML) is a fatal cancer of the human hematopoietic system characterized by the rapid accumulation of non-functional, immature hematopoietic cells in the bone marrow (BM) and peripheral blood (PB) of affected patients. Limited sources of safe hematopoietic stem/progenitor cells (HSPCs) for transplantation and incomplete mechanistic understandings of disease initiation, progression and maintenance have impeded advances in therapy required for improvement of long-term AML patient survival rates. Toward addressing these unmet clinical needs, the ability to generate induced pluripotent stem cells (iPSCs) from human somatic cells may provide platforms from which to develop patient-specific (autologous) cell-based therapies and disease models. However, the ability to generate iPSCs from human AML patient somatic cells had not been investigated prior to this dissertation. Accordingly, I hypothesized that cellular reprogramming of human AML patient somatic cells to iPSCs is possible and will enable derivation of autologous sources of normal and dysfunctional hematopoietic progenitor cells (HPCs). I first postulated that reprogramming AML patient fibroblasts (AML Fibs) to pluripotency would provide a novel source of normal autologous HPCs. Our findings revealed that AML patient-specific iPSCs devoid of leukemia-associated aberrations found in the matched bone marrow (BM) could be generated from AML Fibs, and demonstrated that this cellular platform allowed for the derivation of healthy HPCs capable of normal differentiation to mature myeloid lineages in vitro. During the tenure of these experiments we also redefined conventional reprogramming methods by discovering that OCT4 transcription factor delivery combined with culture in pluripotent-supportive media was minimally sufficient to induce pluripotency in AML and normal Fibs. Toward capturing and modeling the molecular heterogeneity observed across human AML samples in vitro, we next asked whether reprogramming of AML patient leukemic cells would enable generation of iPSCs and derivative HPCs that recapitulated dysfunctional differentiation features of primary disease. Our results demonstrated that conventional reprogramming conditions were insufficient to induce pluripotency in leukemic cells, but that generation of AML iPSCs could be reproducibly achieved in one AML sample when reprogramming conditions were modified. These AML iPSCs and their derivative HPCs harboured and expressed the leukemia-associated aberration found in the BM leukemic cells and similarly possessed dysfunctional differentiation capacities. Together, this body of works provides the proof of principle that cellular reprogramming can be applied on a personalized basis to generate normal and dysfunctional HPCs from AML patient somatic cells. These foundational findings should motivate additional studies aimed at developing iPSC-based cell therapies and disease models toward improving AML patient quality of life and long-term survival rates. / Thesis / Doctor of Philosophy (PhD)

Biotecnologias reprodutivas como a produção in vitro de embriões e a transferência de núcleo apresentam grande potencial de aplicação na medicina veterinária seja para a correção de infertilidades, para o aumento na eficiência da produção animal ou mesmo para um melhor entendimento sobre os mecanismos envolvidos no desenvolvimento embrionário inicial. Porém, manipulações in vitro de gametas ou embriões levam a alterações na regulação epigenética, podendo causar altas taxas de anormalidades no desenvolvimento e no nascimento de indivíduos derivados. A geração de um modelo de indução da pluripotência in vitro, ou seja, a geração de células iPS (do inglês induced pluripotent stem cells) possibilitou estudar o processo de reprogramação in vitro de maneira robusta e precisa. Os genes OCT4 e SOX2 são fundamentais no processo de aquisição e manutenção da pluripotência celular, e recentemente foi reportado que a ação destes dois fatores exerce grande influência sobre a regulação de alguns genes imprinted, em especial, no locus H19/IGF2, sabidamente importantes para o desenvolvimento normal do embrião e de sua placenta. Este estudo propõe a geração de um modelo experimental in vitro onde os fatores em questão sejam estudados, juntos ou em combinação, quanto à sua influência na regulação do imprinting genômico. Para tal, três linhagens de fibroblastos fetais bovinos (bFF1, bFF2 e bFF3) foram transduzidas com vetores lentivirais contendo cDNAs de OCT4 ou SOX2 humanos. Os fibroblastos foram analisados através de citometria e as células positivas foram separadas e recuperadas (sorted). Os fibroblastos expressando OCT4, SOX2, ambos (OCT4 + SOX2), nenhum (controle) juntamente com um controle recuperado (não sorted) não transgênico (total de cinco tratamentos) foram investigados quanto à expressão de genes relacionados à pluripotência e expressão de genes imprinted, bem como a manutenção dos padrões de metilação do DNA no locus H19/IGF2. Além disso, estas células foram submetidas à reprogramação in vitro e produção de células iPS. A indução à pluripotência foi realizada através da transdução dos fibroblastos com o vetor policistrônico contendo o cDNAs murino ou humano dos fatores de transcrição OCT4, SOX2, c-MYC e KLF4 (OSMK, vetor STEMCCA). Os resultados da análise de fluorescência por citometria de fluxo foram, em média, de 40,4% para OCT4, 6,1% para SOX2 e 0,63% para OCT4 + SOX2. A bFF1 foi a única linhagem a apresentar uma recuperação pós-sorting, o que possibilitou sua utilização para a indução da pluripotência. De maneira interessante, as células que não passaram pela citometria geraram colónias de células iPS, enquanto que os demais grupos não. A quantificação de transcritos por qRT-PCR mostrou que a expressão de OCT4 e de SOX2 estava aumentada nos respectivos grupos, a expressão do gene H19 mostrou-se aumentada no grupo controle que passou pelo procedimento de sorting e a expressão do gene imprinted IGF2R não variou entre os grupos. Já a análise preliminar da manutenção do padrão de metilação de DNA na DMR do locus H19/IGF2 mostrou que o grupo controle sorted apresentou uma leve diferença no padrão de metilação quando comparada aos outros grupos. Neste estudo, portanto, o procedimento de separação e recuperação celular por citometria de fluxo celular, aliado ao elevado número de repiques celulares durante o cultivo prolongado pode ter levado a um efeito prejudicial sobre a eficiência de reprogramação in vitro / Reproductive biotechniques such as in vitro embryo production and somatic cell nuclear transfer may greatly contribute for fertility improvements, to enhance animal production or else to contribute to a better understanding of the underlying mechanism involved during initial embryonic development. However, in vitro manipulation of gametes or embryos may lead to possible disruptions on epigenetic regulation, causing high developmental abnormalities and decreased healthy calves born at term. The generation of induced pluripotency models (induced pluripotent stem cells, or iPS) made it possible to study the process of in vitro reprogramming in a more solid and precise manner. OCT4 and SOX2 are fundamental genes for the acquisition and maintenance process of cellular pluripotency. Recently, it has been reported that both factors may have a huge influence on the regulation of some imprinted genes, specially at locus H19/IGF2, known to be important for the normal development of embryo and placenta. Therefore, this study aimed to generate an in vitro experimental model where the above transcription factors will be studied together or separately regarding their influence on genomic imprinting regulation. For that, three bovine fetal fibroblasts cell lines (bFF1, bFF2 and bFF3) were transduced with lentiviral vectors containing human OCT4 or SOX2 cDNAs. The fibroblasts were analyzed trough cell cytometry and positive cells were sorted. Fibroblasts expressing OCT4, SOX2, both (OCT4+SOX2), none (control) together with a non-sorted and non-transgenic control (five treatments) were investigated regarding pluripotency and imprinted gene expression, as well maintenance of DNA methylation patterns at H19/IGF2 locus. Further, these cells were also submitted to in vitro induced reprogramming and production of iPS cell colonies. Induction into pluripotency was realized by transducing fibroblasts with polycistronic excisable vector containing the murine or human cDNA of OCT4, SOX2, c-MYC and KLF4 transcription factors (OSMK, STEMCCA vector). The results of fluorescence analysis by flow cytometry were, on average, 40.4% for OCT4, 6.1% for SOX2 and 0,63% for OCT4+SOX2 groups. bFF1 was the only lineage presenting a post-sorting recovery that enabled its use for pluripotency induction. Interestingly, non-sorted cells generated biPS colonies whereas sorted cells (control non transgenic, OCT4, SOX2 and OCT4+SOX2 expressing cells) did not generate biPS cells. The transcript quantification by qRT-PCR showed that OCT4 and SOX2 expression were increased in the respective groups, the expression of H19 gene was increased in the control sorted group and IGF2R expression was not different between groups. Preliminary results of imprinting pattern methylation at H19/IGF2 locus showed that sorted group was slightly different from others. In this study, therefore, analysis and sorting procedure by flow citometry, together with an extended period in culture may have lead to a detrimental effect on in vitro reprogramming efficiency

The blood system consists of two main lineages: myeloid and lymphoid. The myeloid system consists of cells that are part of the innate immune response while the lymphoid system consist of cells that are part of humoral response. These responses protect our bodies from foreign pathogens. Thus, malignancies in these systems often cause complications and mortality. Scientists world wide have been researching alternatives to treat hematologic disorders and have explored induced pluripotent stem cells (iPSCs) and the conversion of one cell type to another. First, iPS cells were generated by overexpression of four transcription factors: Oct4, Sox2, Klf4 an cMyc. These cells closely resemble embryonic stem cells (ESCs) at the molecular and cellular level. However, the efficiency of cell conversion is less than 0.1%. In addition, many iPS colonies can arise from the same culture, but each has a different molecular signature and potential. Identifying the appropriate iPS cell lines to use for patient specific therapy is crucial. Here we demonstrate that our system is highly efficient in generating iPS cell lines, and cell lines with silent transgenes are most efficient in differentiating to different cell types . Second, we are interested in generating hematopoietic stem cells (HSCs) from fibroblasts directly, without going through the pluripotent state, to increase efficiency and to avoid complications associated with a stem cell intermediate. However, a robust hematopoietic reporter system remains elusive. There are multiple hematopoietic reporter candidates, but we demonstrate that the CD45 gene was the most promising. CD45 is expressed early during hematopoiesis on the surface of HSCs; and as HSCs differentiate CD45 levels increase. Furthermore, the CD45 reporter is only active in hematopoietic cells. We were able to confirm the utility of the CD45 reporter using an in vitro and an in vivo murine model. In conclusion, The goal of this research was to expand the knowledge of stem cell reprogramming, specifically the reprogramming of iPS cells. Furthermore, it is our desire that the CD45 reporter system will undergo further validation and find utility in clinical and cell therapy environments.

Les cellules immunitaires proviennent d'un ensemble commun de cellules souches hématopoïétiques qui se différencient hiérarchiquement en lignées myéloïdes et lymphoïdes. Ce processus est étroitement régulé par un réseau entrelacé de facteurs de transcription et de régulateurs épigénétiques, qui contrôlent l'activation et la répression des gènes impliqués. Les travaux récents sur la reprogrammation cellulaire ont montré que certaines protéines peuvent reprogrammer des cellules différenciées, comme le facteur de transcription C/EBPa qui peut induire la trans-differenciation de cellules B en macrophages. De plus, une courte induction de Cebpa suivie de l’expression des quatre facteurs de transcription Oct4-Sox2-Klf4-cMyc permet une reprogrammation extrêmement rapide en cellules pluripotentes. Afin de déchiffrer le réseau de régulation moléculaire contrôlant la spécification et la reprogrammation des cellules immunitaires, j’ai combiné différentes méthodes à haut débit pour analyser l’expression des gènes et leur régulation épigénétique, et ce au court de la reprogrammation des cellules B. J’ai découvert des interactions entre différents facteurs de transcription, au niveau des régions régulatrices de gènes des différents programmes génétiques impliqués (lymphoide, myeloide et pluripotence), et j’ai identifié des facteurs régulant l’état de la chromatine également impliqués dans la reprogrammation (notamment Lsd1, Hdac1, Brd4 et Tet2). Enfin, J’ai intégré ces données dans un modèle dynamique du réseau moléculaire régulant la spécification des cellules B et des macrophages à partir de progéniteurs multipotents. J’ai utilisé à la fois des méthodes analytiques (analyse des états stables) et des simulations (simulations logiques asynchrones, chaînes de Markov à temps continu) pour étudier in silico la différenciation et la reprogrammation cellulaire. Ces analyses ont révélés des régulations transcriptionelles encore inconnues, que nous avons pu confirmer expérimentalement. Nous avons ainsi obtenu une meilleure compréhension des circuits de régulation contrôlant le destin cellulaire. / Immune cells arise from a common set of hematopoietic stem cells, which differentiate hierarchically into the myeloid and lymphoid lineages. This process is tightly regulated by an intertwined network of transcription and epigenetic factors, which control both the activation and repression of gene programs, to ensure cell commitment. However, recent work on cellular reprogramminghas shown that the ectopic expression of some specific factors can enforce the trans-differentiation of committed cells. The transcription factor C/EBPa can induce the reprogramming of B-cells into macrophages. Furthermore, a pulse of Cebpa expression in B cells followed by the expression of the four transcription factors Oct4-Sox2-Klf4-cMyc leads to an extremely fast and efficient reprogramming into induced pluripotent stem cells. Despite the many data we have on the molecular mechanisms by which specific genes are regulated, we are still lacking a global understanding of the interplay between these factors and how theycontrol cell fate. In order to decipher the molecular regulatory network controlling immune cell specification and their reprogramming, I have combined a variety of high-throughput methods to measure changes in gene expression and epigenetic regulation during B cells reprogramming. I have revealed the interplay between different transcription factors at enhancers regulating genes of the different programs (B cells, macrophages and pluripotent cells) and identified epigenetic regulators forming complexes and controlling enhancers activities (such as Lsd1, Hdac1, Brd4 and Tet2) and consequently regulating cell fate. Finally, I integrated these data together with published data, in a computational model of the regulatory network controlling the specification of B-cells and macrophages from multipotent progenitors. I used both analytic tools (stable states analysis) and simulations (logical asynchronous simulations, continuous time Markov chains) to study in silico differentiation and reprogramming.These analyses have revealed previouslyunknown transcriptional regulations, which weconfirmed experimentally, and allowed us to get abetter understanding of the regulatory circuitscontrolling cell fate commitment.

Biotecnologias reprodutivas como a produção in vitro de embriões e a transferência de núcleo apresentam grande potencial de aplicação na medicina veterinária seja para a correção de infertilidades, para o aumento na eficiência da produção animal ou mesmo para um melhor entendimento sobre os mecanismos envolvidos no desenvolvimento embrionário inicial. Porém, manipulações in vitro de gametas ou embriões levam a alterações na regulação epigenética, podendo causar altas taxas de anormalidades no desenvolvimento e no nascimento de indivíduos derivados. A geração de um modelo de indução da pluripotência in vitro, ou seja, a geração de células iPS (do inglês induced pluripotent stem cells) possibilitou estudar o processo de reprogramação in vitro de maneira robusta e precisa. Os genes OCT4 e SOX2 são fundamentais no processo de aquisição e manutenção da pluripotência celular, e recentemente foi reportado que a ação destes dois fatores exerce grande influência sobre a regulação de alguns genes imprinted, em especial, no locus H19/IGF2, sabidamente importantes para o desenvolvimento normal do embrião e de sua placenta. Este estudo propõe a geração de um modelo experimental in vitro onde os fatores em questão sejam estudados, juntos ou em combinação, quanto à sua influência na regulação do imprinting genômico. Para tal, três linhagens de fibroblastos fetais bovinos (bFF1, bFF2 e bFF3) foram transduzidas com vetores lentivirais contendo cDNAs de OCT4 ou SOX2 humanos. Os fibroblastos foram analisados através de citometria e as células positivas foram separadas e recuperadas (sorted). Os fibroblastos expressando OCT4, SOX2, ambos (OCT4 + SOX2), nenhum (controle) juntamente com um controle recuperado (não sorted) não transgênico (total de cinco tratamentos) foram investigados quanto à expressão de genes relacionados à pluripotência e expressão de genes imprinted, bem como a manutenção dos padrões de metilação do DNA no locus H19/IGF2. Além disso, estas células foram submetidas à reprogramação in vitro e produção de células iPS. A indução à pluripotência foi realizada através da transdução dos fibroblastos com o vetor policistrônico contendo o cDNAs murino ou humano dos fatores de transcrição OCT4, SOX2, c-MYC e KLF4 (OSMK, vetor STEMCCA). Os resultados da análise de fluorescência por citometria de fluxo foram, em média, de 40,4% para OCT4, 6,1% para SOX2 e 0,63% para OCT4 + SOX2. A bFF1 foi a única linhagem a apresentar uma recuperação pós-sorting, o que possibilitou sua utilização para a indução da pluripotência. De maneira interessante, as células que não passaram pela citometria geraram colónias de células iPS, enquanto que os demais grupos não. A quantificação de transcritos por qRT-PCR mostrou que a expressão de OCT4 e de SOX2 estava aumentada nos respectivos grupos, a expressão do gene H19 mostrou-se aumentada no grupo controle que passou pelo procedimento de sorting e a expressão do gene imprinted IGF2R não variou entre os grupos. Já a análise preliminar da manutenção do padrão de metilação de DNA na DMR do locus H19/IGF2 mostrou que o grupo controle sorted apresentou uma leve diferença no padrão de metilação quando comparada aos outros grupos. Neste estudo, portanto, o procedimento de separação e recuperação celular por citometria de fluxo celular, aliado ao elevado número de repiques celulares durante o cultivo prolongado pode ter levado a um efeito prejudicial sobre a eficiência de reprogramação in vitro / Reproductive biotechniques such as in vitro embryo production and somatic cell nuclear transfer may greatly contribute for fertility improvements, to enhance animal production or else to contribute to a better understanding of the underlying mechanism involved during initial embryonic development. However, in vitro manipulation of gametes or embryos may lead to possible disruptions on epigenetic regulation, causing high developmental abnormalities and decreased healthy calves born at term. The generation of induced pluripotency models (induced pluripotent stem cells, or iPS) made it possible to study the process of in vitro reprogramming in a more solid and precise manner. OCT4 and SOX2 are fundamental genes for the acquisition and maintenance process of cellular pluripotency. Recently, it has been reported that both factors may have a huge influence on the regulation of some imprinted genes, specially at locus H19/IGF2, known to be important for the normal development of embryo and placenta. Therefore, this study aimed to generate an in vitro experimental model where the above transcription factors will be studied together or separately regarding their influence on genomic imprinting regulation. For that, three bovine fetal fibroblasts cell lines (bFF1, bFF2 and bFF3) were transduced with lentiviral vectors containing human OCT4 or SOX2 cDNAs. The fibroblasts were analyzed trough cell cytometry and positive cells were sorted. Fibroblasts expressing OCT4, SOX2, both (OCT4+SOX2), none (control) together with a non-sorted and non-transgenic control (five treatments) were investigated regarding pluripotency and imprinted gene expression, as well maintenance of DNA methylation patterns at H19/IGF2 locus. Further, these cells were also submitted to in vitro induced reprogramming and production of iPS cell colonies. Induction into pluripotency was realized by transducing fibroblasts with polycistronic excisable vector containing the murine or human cDNA of OCT4, SOX2, c-MYC and KLF4 transcription factors (OSMK, STEMCCA vector). The results of fluorescence analysis by flow cytometry were, on average, 40.4% for OCT4, 6.1% for SOX2 and 0,63% for OCT4+SOX2 groups. bFF1 was the only lineage presenting a post-sorting recovery that enabled its use for pluripotency induction. Interestingly, non-sorted cells generated biPS colonies whereas sorted cells (control non transgenic, OCT4, SOX2 and OCT4+SOX2 expressing cells) did not generate biPS cells. The transcript quantification by qRT-PCR showed that OCT4 and SOX2 expression were increased in the respective groups, the expression of H19 gene was increased in the control sorted group and IGF2R expression was not different between groups. Preliminary results of imprinting pattern methylation at H19/IGF2 locus showed that sorted group was slightly different from others. In this study, therefore, analysis and sorting procedure by flow citometry, together with an extended period in culture may have lead to a detrimental effect on in vitro reprogramming efficiency

En dehors du cadre pathologique, la notion de temps est la base essentielle dans le processus de vieillissement de l’organisme et des systèmes associés. Ces derniers vont progressivement présenter un déclin de leur(s) fonction(s) où de nombreux mécanismes complexes vont intervenir à différents niveaux. Parmi les premiers constats proposés, le vieillissement est la conséquence d’un processus inéluctable, d’une succession d’agressions au niveau cellulaires qui pourraient être réparées voire évitées, ouvrant ainsi la voie à de futures études qui permettront à terme de proposer une explication détaillée, complète et claire de ce processus. Pour exemple, les travaux que nous avons menés tentent d’apporter un élément de réponse afin d’établir un lien entre sénescence et vieillissement, avec pour base de générer par reprogrammation cellulaire des cellules hiPSCs à partir de cellules issues de biopsies de patients jeunes et âgées, sénescentes ou prolifératives. Outre la caractérisation de leur état pluripotent comparable à celui des cellules souches embryonnaires, nous avons également mis en évidence après une différenciation spécifique en fibroblastes, que les caractéristiques cellulaires de ces fibroblastes présentaient un effacement des marques du vieillissement, signe d’une plasticité cellulaire possible au cours du vieillissement. A présent, étendre une telle étude au modèle tissulaire cutané par la mise en place de protocoles de différenciation dans le lignage épidermique permettra à l’avenir de mieux comprendre pour mieux appréhender les pathologiques associées au vieillissement, et ainsi pouvoir offrir aux patients une médecine appropriée et concrète. / Beside the pathological context, time is the essential basis in the aging process of the body and associated systems. These will gradually introduce a decline in function(s) where many complex mechanisms will take part at different levels. Among proposed findings, aging is the result of an inevitable process, a succession of cellular stress that could be prevented or repaired, opening the way for future studies that will eventually offer an explanation detailed, clear and complete which occur. For example, our work provide a response element to establish a link between senescence and aging, based on the generation of hiPSCs by cellular reprogramming of cells from biopsies of young, older, senescent and proliferating cells patients. Further characterization of their pluripotent state comparable to that of human embryonic stem cells, we have also showed by a specific differentiation into fibroblasts, that cellular characteristics of those fibroblasts had erased aging features. Next step, is to extend such study in cutaneous tissue model by the introduction of differentiation protocols in the epidermal lineage which will able us to better understand aging-associated diseases, and thus bring the ability to propose an appropriate and cocnrete medicine to aged patients.

Direkte Zellreprogrammierung basiert auf Transkriptionsfaktoren (TFs), die die Identität bestimmter Zelltypen induzieren.Diese TF vermittelte Zellreprogrammierung ist in den meisten Zelltypen häufig durch hemmende Mechanismen limitiert.Um solche Barrieren in C. elegans zu identifizieren, verwendeten wir den zuvor charakterisierten Zinkfinger-TF CHE-1, der erforderlich ist, um glutamaterge ASE-Neuronen zu induzieren.In dieser Studie haben wir eine mögliche Barriere für die Reprogrammierung von Keimzellen zu Neuronen identifiziert und charakterisiert:die NAD+ abhängige mitochondriale Isocitratdehydrogenase 3 (IDH3).Der RNAi-Knockdown ergab in Kombination mit ektopischer Expression von CHE-1 einen konsistenten Phänotyp hinsichtlich der Expression des neuronalen ASE- Reporters in der Keimbahn. Wir konnten feststellen,dass IDH3-Knockdowns zu globalen Veränderungen der repressiven Histonmodifikationen führen.Mittels genetischer Untersuchungen identifizierten wir Mitglieder der Jumonji-Proteine sowie die a-KG-abhängigen Histon-Demethylasen, die an diesem Reprogammierungsphänotyp beteiligt sind. Durch Massenspektrometrie und weiterführenden genetische Untersuchungen haben wir festgestellt, dass Zellen bei IDH3-Mangel eine sogenannte Glutamin-Anaplerose verwenden, um den a-KG-Spiegel wieder aufzufüllen und somit einen teilweise aktiven Zitronensäurezyklus beizubehalten.Des Weiteren sind diese Prozesse erforderlich, damit die TF vermittelte Zellreprogrammierung stattfinden kann.Wir haben außerdem festgestellt, dass Signale von Zellen der somatischen Gonade diesen durch IDH3-Mangel vermittelten Zellreprogrammierungsprozess von Keimzellen ermöglichen.Daher ist anzunehmen, dass der Reprogrammierungsphänotyp in der Keimbahn nicht gewebsautonom ist. Zusammengefasst identifiziert diese Studie die Rolle der evolutionär konservierten Isocitrat-Dehydrogenase 3 (IDH3) bei der Aufrechterhaltung der Zellidentität und damit auch als Barriere für die Zellreprogrammierung. / Direct reprogramming makes use of transcription factors (TFs) that induce the identity of specific cell types. These TFs often are restricted in most cell types by inhibitory mechanisms. In order to identify these barriers in C. elegans, we used the previously described zinc-finger TF CHE-1 that is required to induce the glutamatergic ASE neuron fate. In this study, we identified and characterized a candidate barrier for reprogramming germ cells into neurons, the NAD+ dependent mitochondrial isocitrate dehydrogenase 3 (IDH3). RNAi knockdown of alpha (IDHA-1) or gamma (IDHG-1) subunit of this complex gave a consistent and reliable phenotype of the expression of ASE reporter in the germ line upon ectopic expression of CHE- 1. This study shows that idha-1 depletion-mediated reprogramming of germ cells to neurons is partially repressed in animals that lack the hypoxia-induced factor, TF HIF-1. It has been shown that mitochondrial dynamics change during differentiation. This suggests that disturbing mitochondrial function may feed-back to chromatin thus altering gene expression and allowing reprogramming. We were able to identify that knock down of idha-1 leads to global histone modification changes; and by performing a genetic screen we identified members of the Jumonji proteins, the a-KG dependent histone demethylases, involved in this conversion phenotype. By performing Mass Spectrometry and genetic screens, we have identified that cells utilize glutamine anaplerosis to replenish a-KG levels and display a partially active citric acid cycle upon IDH3 depletion; and these processes are required for the TF- mediated reprogramming to occur. Furthermore, the IDH3 depletion-mediated germ cell reprogramming is not tissue autonomous. We identified that signals from the somatic gonad enable the reprogramming process. Overall, this study identifies the role of the conserved Isocitrate Dehydrogenase 3 in cell fate safeguarding and thus as a barrier to reprogramming.

Die Entschlüsselung der Mechanismen zur Regulierung der Zellidentität im Kontext der zellulären Reprogrammierung ist von zentraler Bedeutung für die Entwicklung von Strategien, die die Qualität und Sicherheit reprogrammierter Zellen für medizinische Anwendungen gewährleisten. Die Bedeutung der verschiedenen Regulationswege und die Art und Weise, wie die ursprüngliche Zellidentität verloren geht, während die neue Zellidentität durch Reprogrammierung etabliert wird, sind noch nicht vollständig verstanden. Um diese Fragen zu klären, haben wir ein neuartiges System entwickelt, in dem Coelomozyten (CCs), die in C. elegans endocytische und hepatische Funktionen haben, durch Überexpression des GATA-Transkriptionsfaktors (TF) ELT-7 bzw. des ZNF-Transkriptionsfaktors (TF) CHE-1, sowohl in darm-, als auch in neuronenartige Zellen umprogrammiert werden können. Wir haben einen RNAi-Screen mit 732 Chromatinregulatoren durchgeführt, um neue Enhancer/Suppressor-Pathways zu identifizieren, die an der direkten Reprogrammierung von CCs beteiligt sind. Dabei konnten wir zeigen, dass die Deletion von Effektorproteinargonauten und von Komponenten des nuklearen RNAi-Pathways die Reprogrammierung von CCs in Neuronen oder Darmzellen unterdrückt. Argonaut NRDE-3, das aus dem Zytoplasma in den Zellkern wandert, zeigte bei seiner Deletion die stärkste Unterdrückung der Reprogrammierung. Die Ergebnisse deuten darauf hin, dass die nukleäre RNAi-Maschinerie für die direkte zelluläre in vivo Reprogrammierung erforderlich sein könnte. Darüber hinaus haben wir ATAC-seq in FACs-sortierten CCs durchgeführt, um die Chromatinlandschaft während der CC-Reprogrammierung zu untersuchen. Darüber hinaus haben wir ein menschliches Transdifferenzierungsmodell etabliert, um die Rolle der nuklearen RNAi-Maschinerie und der zahlreichen konservierten Reprogrammierungsfaktoren, die in C. elegans während der direkten Reprogrammierung in vivo identifiziert wurden, zu erforschen. / Dissecting cell fate regulatory mechanisms in the context of cellular reprogramming is central to developing strategies that ensure the quality and safety of reprogrammed cells for medical applications. The importance of different regulatory pathways and how the original cell fate is shut down while establishing the new cell fate during reprogramming are not fully understood. To address these questions, we developed a novel system where coelomocytes (CCs), which have scavenging and hepatic function in C. elegans, can reprogram into both intestinal- and neuronal-like cells upon overexpression of GATA-type transcription factor (TF) ELT-7 and ZNF-type TF CHE-1, respectively. We performed an RNAi screen consisting of 732 chromatin regulators/remodelers to identify novel enhancer/suppressor pathways involved in the direct reprogramming of CCs. We showed that depletion of effector protein Argonauts and the nuclear RNAi pathway components suppresses CC reprogramming into either neurons or intestinal cells. Specifically, the core member Argonaut NRDE-3, which translocates from the cytoplasm to the nucleus, showed the most robust suppression in reprogramming upon its depletion. These findings suggest that nuclear RNAi machinery might be required for in vivo direct cellular reprogramming. Moreover, we also performed the ATAC-seq in FACs-sorted CCs to uncover accessibility in chromatin states during CC reprogramming. Furthermore, we established a human transdifferentiation model to reveal the role of nuclear RNAi machinery and the numerous conserved reprogramming factors identified in C. elegans during in vivo direct reprogramming.