Le diabète de type 1 (DT1) résulte de la destruction des cellules β productrices d’insuline par le système immunitaire. Cette condition représente un enjeu de santé publique majeur car, malgré les thérapies actuelles, les patients atteints développent trop souvent des complications cardio-vasculaires. Des thérapies alternatives se doivent donc d’être mises au point. Ainsi, diverses approches visent à reprogrammer/différencier (in vitro ou in vivo) différents types cellulaires pancréatiques afin de générer des cellules β (productrices d’insuline) fonctionnelles. Dans ce but, notre laboratoire a notamment montré que les cellules α (productrices de glucagon) embryonnaires peuvent être régénérées et converties en cellules β fonctionnelles par l’expression ectopique du seul gène Pax4 (un gène normalement impliqué dans la spécification embryonnaire du lignage β - (Collombat and Mansouri, 2009)). Dans la première partie de ce travail, nous démontrons que les cellules α à l’âge adulte (Al-Hasani et al., 2013) retiennent leur capacité de régénération et de conversion en cellules β, celles-ci étant fonctionnelles et capable de remplacer plusieurs fois l’ensemble des cellules β du pancréas. Cependant, cette approche transgénique serait difficile à mettre en œuvre chez l’homme. De nombreux cribles furent donc initiées dans le but de trouver des petites molécules/composés chimiques mimant les effets de Pax4. Un composé potentiel, GABA, fut ainsi identifié et caractérisé. / Type 1 diabetes (T1D) results from the destruction of insulin-producing β-cells by the immune system. This condition is a major public health issue because, despite current therapies, patients often develop cardiovascular complications. Therefore alternative therapies need to be developed. Thus, various approaches are designed to reprogram / differentiate (in vitro or in vivo) different pancreatic cell types to generate functional (insulin-producing) β-cells. To this end, our laboratory has shown that especially the embryonic α-cells (producing glucagon) can be regenerated and converted into functional β-cells by the ectopic expression of the Pax4 gene (usually a gene involved in the specification of embryonic lineage β - (Collombat and Mansouri, 2009)). In the first part of this work, we show that α-cells in adulthood (Al-Hasani et al., 2013) retain their capacity for regeneration and conversion into β-cells, the latter being functional and able to replace repeatedly all the β-cells of the pancreas. However, this transgenic approach would be difficult to implement in humans. Many screens were therefore initiated in order to find small molecules / chemical compounds that mimic the effects of Pax4. A potential compound, GABA, was identified and characterized. Our results demonstrate that treatment of WT mice with GABA results in a significant increase in the number and size of the islets (caused by insulin+ cell hyperplasia). By using lineage tracing tools, our results indicate that these "β-like" neo-generated cells are coming from glucagon+ cells.

O Diabetes Mellitus tipo I (DM1) é causado pela destruição auto-imune das células β pancreáticas, encontradas na porção endócrina do pâncreas, constituída pelas ilhotas pancreáticas. As células β são responsáveis pela produção e liberação de insulina, um hormônio que promove a internalização da glicose pelas células. Junto com outros hormônios, a insulina é um dos principais reguladores do nível de glicose sanguinea (glicemia). Uma das terapias utilizadas para o tratamento do DM1 é o transplante de ilhotas pancreáticas. Entretanto, um dos maiores problemas em relação a esta terapia é a falta de massa celular adequada para ser infundida no paciente. Uma tentativa para solucionar este problema, é o desenvolvimento de fontes alternativas de células produtoras de insulina, como as células-tronco, que possuem a capacidade de se diferenciarem em diversos tipos de células, inclusive nas produtoras de insulina. Pax4 é um dos fatores de transcrição responsáveis pela diferenciação de células β , sendo essencial para o apropriado desenvolvimento e maturação destas, constitui um bom candidato para induzir a diferenciação de células-tronco em células produtoras de insulina in vitro. Para introduzir o Pax4 nas células-tronco, sem provocar alterações no genoma das células diferenciadas, em virtude dos potenciais efeitos indesejáveis de vetores que se integram ao genoma celular, recorreu-se às proteínas contendo domínio de transdução (PTDs), que são capazes de carregar a proteína Pax4, através da membrana, diretamente para o interior das células. As PTDs são pequenas sequências peptídicas que permitem a translocação de proteínas através de membranas celulares e sua internalização em células-alvo. Uma das PTDs mais comumente estudadas é a VP22, produto do gene UL49 do Herpes Simplex vírus tipo I. Portanto, a proteína de fusão VP22.Pax4 permitiria que o Pax4 fosse inserido em células-tronco, possibilitando que este fator de transcrição ative a transcrição de certos genes que aumentariam a eficiência de diferenciação das células-tronco em células produtoras de insulina. Para tal, amplificamos e clonamos o cDNA do Pax4 a partir do RNA das células RINm5f de insulinoma murino, construímos o vetor pVP22.Pax4, o qual foi transfectado em células CHO, que passaram a produzir a proteína de fusão VP22.Pax4. Após o tratamento de células-tronco com a proteína de fusão VP22.eGFP e análise por microscopia confocal, comprovamos que a VP22 é capaz de tranduzir a proteína de fusão também neste tipo celular. Portanto, incorporamos a um dos passos do protocolo de diferenciação de células-tronco em células produtoras de insulina, utilizado em nosso laboratório, a co-cultura com células CHO produtoras de VP22.Pax4. Observamos que a introdução do Pax4 leva a formação de um número maior de agregados celulares (clusters) produtores de insulina. Concluímos, então, que a utilização da VP22 como ferramenta para internalização de proteínas em células-tronco é viável e que a adição do Pax4 pode trazer melhorias para protocolos que busquem a produção de células produtoras de insulina. / Diabetes Mellitus type 1 (DM1) is caused by an auto-imunne destruction of the pancreatic β cells, found in the endocrine portion of the pancreas, known as pancreatic islets. These β cells are responsible production and release of insulin, a hormone which promotes glucose internalization by cells. Along with other hormones, insulin is a major regulator of blood glucose levels (glycemia). One of the therapeutical strategies used to treat DM1 is pancreatic islet transplantation. One of the major problem related to this therapy is the lack of adequate cell mass to be infused into the pacients. An attempt to solve this problem is the development of an alternative source of insulin-producing cells by differentiation of stem cells, which display this differentiating potential. Pax4 is one of the transcription factors responsibles for β cell differentiation, being essential for its proper development and maturation, therefore being a good candidate to induce stem cell differentiation into insulin producing cells in vitro. A promising alternative to avoid the alterations of the differentiated cells genome due to its undesirable effects of integrating vectors, but yet allowing the Pax4 to act in diferentiation within the cells are the proteins with a transduction domain (PTDs), which would have the ability to lead the Pax4 protein directly into the cells. The Pax4 could thus act in the nucleus and generate specific transcriptional responses. The PTDs are small peptide sequences which allow translocation of proteins across cell membranes and their internalization into target cells. One of the most commonly studied PTDs is the VP22, a product of the UL49 gene from Herpes Simplex vírus type I. Therefore, the VP22.Pax4 fusion protein would transduce Pax4 into the stem cells, thus allowing the transcription activation of certain genes by Pax4, leading to improvement in the process of stem cells differentiation into insulin-producing cells. To this end, we cloned the Pax4 cDNA from RINm5f murine insulinoma cells, constructed the pVP22.Pax4 vector and transfected this construct into CHO cells, which then produced the VP22.Pax4 fusion protein. Upon verifying that VP22 was also able to transduce proteins into stem cells, by confocal microscopy analysis, after the treatment of these cells with the fusion protein VP22.eGFP, we incorporated the fusion protein VP22.Pax4 to one of the steps of the protocol used for stem cells diferentiation into insulin producing cells in our lab, by co-culturing with CHO cells producing VP22.Pax4. We observed that the addition of Pax4 led to the formation of a higher number of insulin producing cell clusters, therefore we conclude that VP22 may be used as a tool to internalize proteins into stem cells, and that the addition of Pax4 may improves protocols seeking the production of insulin-producing cell

Dans ce travail, nous démontrons que l’expression ectopique de Pax4 dans les cellules glucagon+ adultes induit, indépendamment de l’âge, leur néogenèse et transformation en cellules «bêta-like», ce qui entraîne une hypertrophie des îlots et une néogenèse inattendue des îlots. Par l’utilisation de plusieurs approches de traçage, nous démontrons que la conversion des cellules alpha en cellules «bêta-like» médiée par l’expression de Pax4, induit également la mobilisation de précurseurs situés dans ou à proximité des canaux pancréatiques. Ces cellules ré-expriment le gène développemental Ngn3 et adoptent successivement une identité de cellules glucagon+ puis de cellules «bêta-like», suggérant le réveil des mécanismes embryonnaires. Il et à noter que ces processus sont capables de régénérer la totalité de la masse de cellules bêta après plusieurs séries d’induction chimique du diabète. Ces résultats offrent ainsi des perspectives prometteuses pour concevoir de nouvelles stratégies thérapeutiques et régénératrices dans le contexte du diabète du type I. Dans un deuxième chapitre, ce travail décrit nos résultats d'analyse par puce à ADN de pancréas transgénique d’animaux exprimant conditionnellement le gène Pax4 dans les cellules alpha adultes. Cette approche nous permis d'identifier de potentiels gènes cibles de Pax4, qui pourraient jouer un rôle important dans les processus de régénération de la masse de cellules bêta. L’analyse de la fonction de l’un de ces gènes, le facteur de croissance indépendante 1 (Gfi1) est décrite. / In this work we demonstrate that the inducible misexpression of Pax4 in glucagon+ cells age-independently provokes their conversion into beta-like cells and their glucagon shortage-mediated replacement, this process resulting in islet hypertrophy and in an unexpected islet neogenesis. Taking advantage of several lineage-tracing approaches, we show that, upon Pax4-mediated alpha-to-beta-like cell conversion, pancreatic duct-lining precursor cells are mobilized, re-express the developmental gene Ngn3, and successively adopt a glucagon+ and a beta-like cell identity through a mechanism involving the reawakening of the epithelial-to-mesenchymal transition (EMT). It is worth mentioning, that these processes can repeatedly regenerate the entire beta-cell mass, and thereby reverse several rounds of streptozotocin-mediated chemically-induced Type I diabetes. This approach thereby provides promising perspectives to design novel therapeutic regenerative strategies. Aiming to gain further insight into the molecular mechanisms underlying these regeneration and reprogramming processes, and thereby identify new putative targets of interest, a thorough micro array analysis was performed using pancreata from transgenic mice conditionally misexpressing Pax4 in adult alpha-cells. We thereby identified several promising candidate genes, whose gene expression was significantly altered in induces animals. Among these was Growth factor independent 1 (Gfi1): its expression pattern and putative function in the murine pancreas will be described in this work.

O Diabetes Mellitus tipo I (DM1) é causado pela destruição auto-imune das células β pancreáticas, encontradas na porção endócrina do pâncreas, constituída pelas ilhotas pancreáticas. As células β são responsáveis pela produção e liberação de insulina, um hormônio que promove a internalização da glicose pelas células. Junto com outros hormônios, a insulina é um dos principais reguladores do nível de glicose sanguinea (glicemia). Uma das terapias utilizadas para o tratamento do DM1 é o transplante de ilhotas pancreáticas. Entretanto, um dos maiores problemas em relação a esta terapia é a falta de massa celular adequada para ser infundida no paciente. Uma tentativa para solucionar este problema, é o desenvolvimento de fontes alternativas de células produtoras de insulina, como as células-tronco, que possuem a capacidade de se diferenciarem em diversos tipos de células, inclusive nas produtoras de insulina. Pax4 é um dos fatores de transcrição responsáveis pela diferenciação de células β , sendo essencial para o apropriado desenvolvimento e maturação destas, constitui um bom candidato para induzir a diferenciação de células-tronco em células produtoras de insulina in vitro. Para introduzir o Pax4 nas células-tronco, sem provocar alterações no genoma das células diferenciadas, em virtude dos potenciais efeitos indesejáveis de vetores que se integram ao genoma celular, recorreu-se às proteínas contendo domínio de transdução (PTDs), que são capazes de carregar a proteína Pax4, através da membrana, diretamente para o interior das células. As PTDs são pequenas sequências peptídicas que permitem a translocação de proteínas através de membranas celulares e sua internalização em células-alvo. Uma das PTDs mais comumente estudadas é a VP22, produto do gene UL49 do Herpes Simplex vírus tipo I. Portanto, a proteína de fusão VP22.Pax4 permitiria que o Pax4 fosse inserido em células-tronco, possibilitando que este fator de transcrição ative a transcrição de certos genes que aumentariam a eficiência de diferenciação das células-tronco em células produtoras de insulina. Para tal, amplificamos e clonamos o cDNA do Pax4 a partir do RNA das células RINm5f de insulinoma murino, construímos o vetor pVP22.Pax4, o qual foi transfectado em células CHO, que passaram a produzir a proteína de fusão VP22.Pax4. Após o tratamento de células-tronco com a proteína de fusão VP22.eGFP e análise por microscopia confocal, comprovamos que a VP22 é capaz de tranduzir a proteína de fusão também neste tipo celular. Portanto, incorporamos a um dos passos do protocolo de diferenciação de células-tronco em células produtoras de insulina, utilizado em nosso laboratório, a co-cultura com células CHO produtoras de VP22.Pax4. Observamos que a introdução do Pax4 leva a formação de um número maior de agregados celulares (clusters) produtores de insulina. Concluímos, então, que a utilização da VP22 como ferramenta para internalização de proteínas em células-tronco é viável e que a adição do Pax4 pode trazer melhorias para protocolos que busquem a produção de células produtoras de insulina. / Diabetes Mellitus type 1 (DM1) is caused by an auto-imunne destruction of the pancreatic β cells, found in the endocrine portion of the pancreas, known as pancreatic islets. These β cells are responsible production and release of insulin, a hormone which promotes glucose internalization by cells. Along with other hormones, insulin is a major regulator of blood glucose levels (glycemia). One of the therapeutical strategies used to treat DM1 is pancreatic islet transplantation. One of the major problem related to this therapy is the lack of adequate cell mass to be infused into the pacients. An attempt to solve this problem is the development of an alternative source of insulin-producing cells by differentiation of stem cells, which display this differentiating potential. Pax4 is one of the transcription factors responsibles for β cell differentiation, being essential for its proper development and maturation, therefore being a good candidate to induce stem cell differentiation into insulin producing cells in vitro. A promising alternative to avoid the alterations of the differentiated cells genome due to its undesirable effects of integrating vectors, but yet allowing the Pax4 to act in diferentiation within the cells are the proteins with a transduction domain (PTDs), which would have the ability to lead the Pax4 protein directly into the cells. The Pax4 could thus act in the nucleus and generate specific transcriptional responses. The PTDs are small peptide sequences which allow translocation of proteins across cell membranes and their internalization into target cells. One of the most commonly studied PTDs is the VP22, a product of the UL49 gene from Herpes Simplex vírus type I. Therefore, the VP22.Pax4 fusion protein would transduce Pax4 into the stem cells, thus allowing the transcription activation of certain genes by Pax4, leading to improvement in the process of stem cells differentiation into insulin-producing cells. To this end, we cloned the Pax4 cDNA from RINm5f murine insulinoma cells, constructed the pVP22.Pax4 vector and transfected this construct into CHO cells, which then produced the VP22.Pax4 fusion protein. Upon verifying that VP22 was also able to transduce proteins into stem cells, by confocal microscopy analysis, after the treatment of these cells with the fusion protein VP22.eGFP, we incorporated the fusion protein VP22.Pax4 to one of the steps of the protocol used for stem cells diferentiation into insulin producing cells in our lab, by co-culturing with CHO cells producing VP22.Pax4. We observed that the addition of Pax4 led to the formation of a higher number of insulin producing cell clusters, therefore we conclude that VP22 may be used as a tool to internalize proteins into stem cells, and that the addition of Pax4 may improves protocols seeking the production of insulin-producing cell

Le pancréas est organisé en deux compartiments : le pancréas exocrine et le pancréas endocrine. Ce dernier est constitué d’Îlots de Langerhans composés de 5 types cellulaires : les cellules α, β, δ, ε et PP, synthétisant respectivement le glucagon, l’insuline, la somatostatine, la ghréline et le polypeptide pancréatique. Le diabète de type 1 est une maladie auto-immune entrainant la destruction des cellules β productrices d’insuline, conduisant à une hyperglycémie chronique. Le remplacement des cellules β nécessite de déchiffrer les mécanismes impliqués dans leur genèse au cours du développement. La coopération de plusieurs facteurs de transcription spécifie successivement les cellules progénitrices en cellules pancréatiques, endocrines puis α, β, δ, ε ou PP. Parmi ceux-ci, Arx et Pax4, jouent un rôle essentiel dans la détermination des cellules α/ PP et β/δ, respectivement. Nous avons récemment démontré que l’expression ectopique de Pax4 dans des cellules α adultes entrainait leur régénération et leur conversion en cellules productrices d’insuline. De plus, une augmentation du nombre de cellules δ a également été notée chez ces animaux. Ces cellules n’étant pas accumulées, nous avons suggéré leur possible conversion en cellules β. Ce travail de thèse porte donc sur la génération et la caractérisation d’animaux permettant l’expression ectopique de Pax4 dans les cellules δ. Nous démontrons que l’expression de Pax4 dans ces cellules δ est suffisante pour induire leur conversion en cellules « β-like ». De plus, nous montrons que cette conversion contribue à la réactivation du développement endocrine, conduisant à une hyperplasie des cellules produisant de l'insuline / The pancreas is organized into two compartments: the exocrine and the endocrine pancreas. The latter consists of functional units named islets of Langerhans which contain five cell subtypes, α, β, δ, ε and PP-cells responsible for the secretion of glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide, respectively. Type 1 Diabetes Mellitus is an autoimmune disorder resulting in the loss of insulin-producing β-cells, leading to chronic hyperglycemia. Therefore, approaches aiming at gaining further insight into the molecular mechanisms underlying β-cell (neo)genesis, are of growing interest. A network involving numerous transcription factors was found to progressively specify endodermal progenitors toward the pancreatic, endocrine, and islet cell fates. Among these, Arx and Pax4, were found to exert key roles for the allocation to the α-/PP- and β/δ-cell lineages, respectively. Importantly, we recently showed that adult α-cells can be regenerated and converted into functional β-like cells upon the ectopic expression of Pax4. Surprisingly, an increase of δ-cells was noted in these animals, such cells not accumulating over time. We therefore wonder whether δ-cells could be regenerated and converted into β-like cells. Here, we report the generation and characterization of transgenic animals allowing the misexpression of Pax4 in somatostatin-expressing cells. We demonstrate that the sole ectopic expression of Pax4 in δ-cells is sufficient to induce their conversion into β-like cells. We show that this conversion contributes to the reactivation of endocrine developmental processes, leading to a massive β-like cell hyperplasia