T lymphocytes play a critical role in adaptive immunity by eliciting and regulating specific immune responses against viral and bacterial pathogens. The development of T cells occurs within the highly specialized thymus and follows a defined set of stage-specific differentiation steps. However, the molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. This was in part due to the inability to obtain substantial numbers of T-lineage cells from hybrid/human fetal thymic organ culture (FTOC) and the inability to recapitulate human T-lymphopoiesis using other systems. To address the molecular and cellular events occurring during early human T-lymphopoiesis, human umbilical cord-blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T-lineage utilizing OP9-DL1 stromal cells. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. In addition, this Thesis demonstrates that in vitro-generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice. In addition, two distinct progenitor subsets, CD34+CD45RA+CD7++CD5-CD1a- (proT1) and CD34+CD45RA+CD7++CD5+CD1a- (proT2), were identified with proT2 cells showing a 3-fold enhanced engrafting capacity than the proT1 subset. As proT2 cells exhibit superior engrafting capacity, these cells were tested for their ability to enhance T cell generation following hematopoietic stem cell transplant (HSCT). We observe that when HSCs are coinjected with proT2 cells, a dramatic improvement in HSC-derived T-lymphopoiesis is observed. This Thesis demonstrates that in vitro-derived proT2 cells reorganize the thymus stromal compartment of the host NOD/SCID/γcnull mouse compared to the highly disorganized cortical and medullary compartments in mice not receiving proT cells. This alteration in thymic architecture likely favours the recruitment of BM derived progenitors. Lastly, we address whether functional CD8 T cells can be generated in vitro using hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells and indeed these cells were capable of proliferating, and secreting effector molecules typical of cytotoxic T cells. Taken together, the ability to generate proT cells and mature T cells from Notch-ligand cultures offers a new tool to study human T cell development.

Rubella virus (RUBV) is a significant human pathogen. RUBV infection takes an enormous toll due to congenital rubella syndrome (CRS), a constellation of birth defects including blindness, hearing defects and mental retardation. Little is known about RUBV-induced teratogenesis due to the absence of useful models. This research is now enabled by the availability of human embryonic stem cells (hESCs) and hESC-derived precursor cell lines. Human neural progenitor cells (hNPCs) serve as a particularly relevant model due to the symptoms and complications of CRS related to neural system development. The overarching question addressed in this dissertation is: what is the mechanism underlying the development of neurological abnormalities seen in CRS? In this context, we investigated the cellular responses of hNPCs to RUBV infection comprehensively by: 1) assessing susceptibility of the cells to RUBV infection; 2) analyzing the effect of infection on cell proliferation; and 3) examining the impact of RUBV infection on differentiation of hNPCs into neuronal and astroglial lineages . We found that hNPCs are susceptible to RUBV infection and that the percentage of infected cells closely mimics CRS in which few cells harbor virus. The virus was able to persist in culture for up to one month without significant alteration of cell morphology and stemness marker expression. In addition, RUBV infection moderately attenuated the proliferation of undifferentiated hNPCs by triggering cell cycle arrest, but not apoptosis or other cell death events commonly seen upon virus infection. This lack of apoptosis appeared to be due in part to virus-induced anti-apoptotic suppression. Interestingly, the virus only had a marginal effect on the induction of cell differentiation into both neuronal and astroglial phenotypes. In fact, RUBV infection promoted terminal differentiation of the culture due to depletion of precursor cells. With differentiation, viral replication was suppressed. We thus propose a model for RUBV-induced neurological defects in which the virus acts by depleting precursor cell pools. The results of this study provide clues for elucidating the mechanisms of RUBV teratogenicity at the cellular level and serves as a potential reference study for elucidating mechanisms of teratogenesis induced by other infectious agents.

Rubella virus (RUBV) is a significant human pathogen. RUBV infection takes an enormous toll due to congenital rubella syndrome (CRS), a constellation of birth defects including blindness, hearing defects and mental retardation. Little is known about RUBV-induced teratogenesis due to the absence of useful models. This research is now enabled by the availability of human embryonic stem cells (hESCs) and hESC-derived precursor cell lines. Human neural progenitor cells (hNPCs) serve as a particularly relevant model due to the symptoms and complications of CRS related to neural system development. The overarching question addressed in this dissertation is: what is the mechanism underlying the development of neurological abnormalities seen in CRS? In this context, we investigated the cellular responses of hNPCs to RUBV infection comprehensively by: 1) assessing susceptibility of the cells to RUBV infection; 2) analyzing the effect of infection on cell proliferation; and 3) examining the impact of RUBV infection on differentiation of hNPCs into neuronal and astroglial lineages . We found that hNPCs are susceptible to RUBV infection and that the percentage of infected cells closely mimics CRS in which few cells harbor virus. The virus was able to persist in culture for up to one month without significant alteration of cell morphology and stemness marker expression. In addition, RUBV infection moderately attenuated the proliferation of undifferentiated hNPCs by triggering cell cycle arrest, but not apoptosis or other cell death events commonly seen upon virus infection. This lack of apoptosis appeared to be due in part to virus-induced anti-apoptotic suppression. Interestingly, the virus only had a marginal effect on the induction of cell differentiation into both neuronal and astroglial phenotypes. In fact, RUBV infection promoted terminal differentiation of the culture due to depletion of precursor cells. With differentiation, viral replication was suppressed. We thus propose a model for RUBV-induced neurological defects in which the virus acts by depleting precursor cell pools. The results of this study provide clues for elucidating the mechanisms of RUBV teratogenicity at the cellular level and serves as a potential reference study for elucidating mechanisms of teratogenesis induced by other infectious agents.

Description of the cell cycle features of neural progenitors during late stages of neurogenesis in a gyrencephalic mammal, the ferret.

INTRODUCCIÓN: La esclerosis múltiple (EM) es una enfermedad autoinmune mediada por células CD4+ autorreactivas frente a antígenos mielínicos severamente discapacitante que afecta fundamentalmente a una población joven y para la que no se dispone de ningún tratamiento capaz de detener la progresión de la discapacidad. Gracias a los resultados experimentales con modelos animales que demuestran que el trasplante autólogo es capaz de conseguir la remisión de la enfermedad autoinmune al igual que el alogénico, pero con mucha menor toxicidad, el trasplante autólogo de progenitores hematopoyéticos (TAPH) se ha erigido en los últimos años como una opción viable para el tratamiento de enfermedades autoinmunes en humanos. Las bases racionales del TAPH en la EM se fundamentan en la ablación del sistema inmune y la erradicación de las células autorreactivas, y la posterior reconstitución de un nuevo sistema inmune tolerante con los autoantígenos. HIPÓTESIS Y OBJETIVOS: La tolerancia inmune tras el TAPH debería conllevar un descenso en la actividad inflamatoria inmunomediada con una mejoría en el curso clínico y radiológico de los pacientes asociado a un descenso de marcadores biológicos de actividad inflamatoria implicados en la patogenia de la EM. Para ello, analizamos la evolución de los niveles de MMP-9, de BDNF, la reconstitución inmune, y el perfil de secreción de citocinas. Asimismo, estudiamos la influencia del polimorfismo funcional BDNF-Val66Met en la capacidad de secreción de BDNF por las células inmunes, así como en la susceptibilidad a padecer EM o en la severidad de la misma.RESULTADOS: 14 pacientes con EM agresiva resistente a la terapia convencional fueron sometidos a un TAPH. La mortalidad del procedimiento fue del 0% y no se produjo ningún evento tóxico grave. Tras una mediana de seguimiento de 3 años se produjo un gran descenso en la tasa de brotes y la probabilidad de permanecer libre de progresión de la enfermedad fue del 85,7%. Se observó la desaparición de las lesiones captantes de contraste en RM y un notable descenso de la carga lesional asociado a una atrofia del cuerpo calloso secundaria a la resolución del edema de las lesiones cerebrales. El TAPH reduce los niveles séricos y de expresión de la MMP-9 con un descenso en el cociente MMP-9/TIMP-1 de actividad proteolítica. El TAPH produce una lenta recuperación de los linfocitos CD4+ con una inversión del cociente de CD4+/CD8+ asociado a un desplazamiento de respuesta inflamatoria hacia un perfil Th2. El TAPH desciende los niveles de BDNF sin evidencia de correlación con las medidas de atrofia cerebral. La distribución de frecuencias alélicas y genotípicas fue similar entre EM y controles, ni se asoció con ningún parámetro de severidad de la EM. La presencia del alelo met no se asocia a un descenso en los niveles de BDNF. CONCLUSIONES: El TAPH en la EM tiene una toxicidad aceptable, reduce la actividad inflamatoria de la enfermedad y parece retrasar la progresión de la discapacidad. Reduce la carga proteolítica asociada a MMP-9/TIMP-1 pudiendo contribuir a la reducción de la actividad de la enfermedad. El TAPH tiene un efecto inmunodepresor e inmunomodulador prolongado como muestra la inversión del cociente CD4+/CD8+ y el desplazamiento de la respuesta inflamatoria hacia un perfil Th2. El descenso en los niveles de BDNF parece influir en la atrofia cerebral de la EM. El polimorfismo BDNF-Val66Met no incrementa la susceptibilidad ni la severidad de la enfermedad, ni disminuye la capacidad de las células inmunes de secretar BDNF.

Les maladies cardiovasculaires sont l’une des principale causes de mortalité dans le monde, engendrant le décès de plus de 17 millions de personnes par an. Ce chiffre éloquent augmentera jusqu’à atteindre selon l’OMS 23,4 millions de décès en 2030. Ces maladies sont associées à un rétrécissement de la lumière des vaisseaux sanguins qui peut entrainer une occlusion partielle ou complète du vaisseau. Le traitement le plus souvent utilisé est un traitement chirurgical visant à créer un pont qui va contourner la section obstruée, ou une section lésée.Actuellement, les conduits les plus utilisés pour les greffes sont les vaisseaux autologues, à savoir la veine saphène ou l’artère thoracique interne. Seulement, ces substituts ne peuvent être utilisés en remplacement que s’ils sont sains. L’alternative aux vaisseaux autologue est l’utilisation de substituts synthétiques. Due à un certain manque de biocompatibilité de ces greffons synthétiques, après quelques années seulement, un phénomène de thrombose s’installe, en cause ; l’absence de cellules endothéliales (CEs) qui recouvrent l’intérieur du substitut.Le point clé réside ici dans la fabrication d’un matériau capable de fournir au CEs un environnement favorable à leur adhésion et leur prolifération pour permettre la génération d’un endothélium à l’intérieur d’un substitut synthétique. In vivo, les cellules capables de coloniser de tels matériaux sont les cellules progénitrices endothéliales, ces cellules sont capables de se différencier en cellules endothéliales matures et possèdent une capacité de prolifération supérieure aux cellules matures. Elles sont capables de réparer les vaisseaux et pourront donc être ciblées afin d’être recrutées in situ et ainsi endothélialiser le biomatériau.C’est dans ce contexte que nous avons choisi de modifier de façon chimique la surface d’un matériau model, un film de PET avec quatre principes actifs innovants sélectionnés pour leur capacité à induire l’adhésion des cellules ou leur différentiation pour permettre la régénération d’un endothélium à la surface du matériau.Ce projet a permis dans un premier temps de mettre au point un protocole pour greffer des principes actifs de façon covalente avec une densité reproductible et de façon microstructurée en utilisant la photolithographie. Ici, les peptides GRGDS et GHM ont été greffés pour améliorer l’adhésion des cellules, le dernier étant spécifique aux cellules endothéliales progénitrices. Le peptide SFLLRN et la sitagliptine ont été greffés pour induire ou accélérer la différenciation des EPCs en CEs matures. Toutes les surfaces ont été caractérisées pour valider le greffage covalent et connaitre la densité de molécules bioactives greffée.D’autre part avec une caractérisation approfondie des EPCs issues du sang de cordon ombilical, certains gènes caractéristiques des cellules souches et endothéliales ont été suivis par immunofluorescence et RT-qPCR pour déterminer leur état de différenciation. Ce travail n’aura été possible qu’après avoir déterminé quels gènes de références nous pouvions utiliser pour étudier le phénotype de trois types cellulaires à savoir, les cellules mononuclées CD34+, les EPCs et des CEs matures (extraites de la veine saphène). [...] En conclusion générale, ce projet prouve que la modification de surfaces des substituts avec des molécules bioactives est indispensable pour rendre le matériau attractif et pour régénérer un endothélium à la surface de celui-ci. Ce travail nous a aidé souligner l’importance de comprendre le comportement des EPCs et leur cinétique de différenciation pour leur utilisation en ingénierie vasculaire. / Cardiovascular disease is one of the leading causes of death in the world, killing more than 17 million people a year. This eloquent figure will increase to 23.4 million deaths in 2030, according to the WHO. These diseases are associated with a narrowing of the lumen of the blood vessels that may cause partial or complete occlusion of the vessel. The treatment most often used is a surgical treatment designed to create a bridge that will bypass the obstructed section or an injured section.Currently, the most used conduits for transplants are autologous vessels, namely the saphenous vein or the internal thoracic artery. Only these substitutes can only be used as a replacement if they are healthy. The alternative to autologous vessels is the use of synthetic substitutes. Due to a certain lack of biocompatibility of these synthetic grafts, after only a few years, a phenomenon of thrombosis sets in; the absence of endothelial cells (ECs) that cover the interior of the substitute.The key point here lies in the manufacture of a material capable of providing the ECs with a favorable environment for their adhesion and proliferation to allow the generation of an endothelium within a synthetic substitute. In vivo, cells capable of colonizing such materials are endothelial progenitor cells, these cells are capable of differentiating into mature endothelial cells and possess a higher proliferation capacity than mature cells. They are able to repair the vessels and can, therefore, be targeted to be recruited in situ and thus endothelialize the biomaterial.It is in this context that we have chosen to chemically modify the surface of a model material, a PET film with four innovative active ingredients selected for their ability to induce cell adhesion or differentiation to allow regeneration. an endothelium on the surface of the material.This project has initially made it possible to develop a protocol for grafting active ingredients covalently with a reproducible density and in a microstructured manner using photolithography. Here, the GRGDS and GHM peptides were grafted to enhance cell adhesion, the latter being specific to endothelial progenitor cells. The SFLLRN peptide and sitagliptin have been grafted to induce or accelerate the differentiation of EPCs into mature ECs. All surfaces have been characterized to validate covalent grafting and to know the density of grafted bioactive molecules.On the other hand, with a thorough characterization of EPCs from umbilical cord blood, some characteristic genes of stem and endothelial cells were followed by immunofluorescence and RT-qPCR to determine their state of differentiation. This work will have been possible only after determining which reference genes we could use to study the phenotype of three cell types namely, CD34 + mononuclear cells, EPCs and mature ECs (saphenous vein extract). [...] As a general conclusion, this project proves that surface modification of substitutes with bioactive molecules is essential to make the material attractive and to regenerate an endothelium on the surface of it. This work has helped us emphasize the importance of understanding the behavior of EPCs and their kinetics of differentiation for their use in vascular engineering.

Submitted by Helmut Patrocinio (hell.kenn@gmail.com) on 2017-11-10T16:29:31Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Marcos_Romualdo_Costa_TESE.pdf: 3272089 bytes, checksum: aaf244c49f569e8c070d41f2727a24c6 (MD5) / Approved for entry into archive by Ismael Pereira (ismael@neuro.ufrn.br) on 2017-11-10T17:31:16Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Marcos_Romualdo_Costa_TESE.pdf: 3272089 bytes, checksum: aaf244c49f569e8c070d41f2727a24c6 (MD5) / Made available in DSpace on 2017-11-10T17:31:31Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Marcos_Romualdo_Costa_TESE.pdf: 3272089 bytes, checksum: aaf244c49f569e8c070d41f2727a24c6 (MD5) Previous issue date: 2006 / Ao longo do desenvolvimento, as c?lulas neuroepiteliais do telenc?falo dividemse originando progenitores respons?veis pela gera??o sequencial dos diferentes tipos de neur?nios, astr?citos e oligodendr?citos do c?rtex cerebral. At? o presente, os progenitores telencef?licos estariam localizados nas zonas ventricular (ZV) e subventricular (ZSV). Sua posi??o ao longo dos eixos dorsoventral e rostro-caudal ? relacionada com territ?rios g?nicos e tipos celulares espec?ficos. Desta forma, observa-se a gera??o de neur?nios corticais glutamat?rgicos ou GABA?rgicos na ZV e ZSV do telenc?falo dorsal e ventral de roedores, respectivamente. Neste trabalho investigamos o potencial proliferativo in vivo e in vitro da zona marginal (ZM), conhecida por possuir neur?nios migrat?rios e diferenciados durante a corticog?nese. Determinamos o fen?tipo de c?lulas proliferativas da ZM e atrav?s de an?lise clonal utilizando infec??o por retrovirus contendo o gene para GFP (prote?na flourescente verde) acompanhamos as linhagens derivadas destes progenitores in vitro. C?lulas proliferativas in vivo foram marcadas atrav?s da administra??o do BrdU (bromodeoxiuridina, marcador da fase S do ciclo celular), combinada a ensaios imunohistoqu?micos para a identifica??o deste ant?geno e da forma fosforilada da histona 3 (expressa no final da fase G2 e durante a fase M do ciclo celular). Identificamos c?lulas proliferativas na ZM de camundongos a partir do dia embrinon?rio 14 (E14 - logo ap?s a divis?o da pr?-placa quando a ZM se torna distingu?vel) e por toda a corticog?nese com um aumento na proporc?o de c?lulas proliferativas de ~tr?s vezes em E18. As c?lulas proliferativas na ZM n?o expressam Pax6 ou Tbr2, fatores transcricionais caracter?sticos dos precursores da ZV e ZSV respectivamente. Ao longo da corticog?nese, esta popula??o precursora apresenta um padr?o de express?o do fator transcricional Olig2 seguindo um gradiente l?tero-medial, de modo que no per?odo perinatal todas as c?lulas proliferativas na zona marginal expressam o gene olig2. A an?lise das linhagens clonais geradas a partir destes precursores revelou um elevado potencial gliog?nico (~70% de clones gliais puros) quando comparado a ZV /ZSV (3,3%). Al?m disso, a ZM apresentou um significativo potencial neurog?nico, originando cerca de 30% de clones contendo neur?nios. Mostramos que os clones gliais puros da ZM s?o significativamente maiores que os da ZV. Conclu?mos, portanto, que a ZM dorsal ? um nicho neurog?nico e gliog?nico no c?rtex cerebral em desenvolvimento apresentando c?lulas proliferativas in vivo e in vitro com caracter?sticas fenot?picas distintas dos progenitores da ZV e ZSV. Atrav?s de estudos de linhagem clonal in vitro, demonstramos diferentes comportamentos proliferativos e potenciais neuro-gliog?nicos das c?lulas isoladas da ZM e da ZV/ZSV, indicando a exist?ncia de um novo tipo de progenitor no telenc?falo. / During development, telencephalic neuroepithelial cells proliferate and give rise to progenitors, which are responsible for the sequential generation of different types of neurons, astrocytes and oligodendrocytes in the cerebral cortex. To date, telencephalic progenitors would be located in the ventricular (VZ) and subventricular (SVZ) zones. Their position along the rostro-caudal and dorsoventral axis is related to gene expression territories and the generation of specific cell types, such that dorsal telencephalic VZ/ZVZ generates glutamatergic neurons and ventral VZ/ZVZ GABAergic neurons. In this work we investigated the in vivo and in vitro proliferative potential of the marginal zone (MZ) described to harbor migrating and differentiating neurons during corticogenesis. We determined the phenotype of MZ proliferative cells and by clonal analysis with infection by GFP (green fluorescent protein) containing retroviruses we followed the lineages derived from the progenitors in vitro. Proliferative cells in vivo were labeled by BrdU (bromodeoxyuridine, S phase cell cycle marker) combined to immunohistochemistry for the identification of BrdU antigen and the phosphorylated form of H3 ?histone (expressed at the end of G2 and during M phase of the cell cycle). We identified proliferative cells in mice MZ from embryonic day (E)14 (just after preplate division when MZ becomes distinguishable) and through all corticogenesis with a three fold increase in E18. Proliferative cells in the MZ do not express Pax6 or Tbr2, transcriptional factors typical of VZ and SVZ precursors respectively. During corticogenesis, this precursor population displays a latero-medial gradient of expression of Olig2, such that perinatally, all proliferative cells in the MZ express Olig2. Clonal lineage analysis from these precursors revealed a high gliogenic potential (~70% pure glial clones) when compared to VZ/SVZ (2,3%). Furthermore, MZ displays neurogenic potential since 30% of all clones contained neurons identified by class III ?-tubulin immunolabeling. Here we show that pure glial clones in the MZ are significantly larger than those generated by VZ. Concluding, the dorsal MZ is a neurogenic and gliogenic niche in the developing cerebral cortex containing proliferative cells with distinct phenotypic characteristics from the VZ and SVZ. By clonal lineage analysis in vitro, we demonstrated different proliferative behaviors and neuro-gliogenic potential from cells isolated from the MZ and VZ/SVZ indicating a novel type of progenitor in the cerebral cortex.

No description available.

The heart arises from two sources of mesoderm progenitors, the first (FHF) and the second heart field (SHF) progenitors. Mesp1 has been proposed to mark the most primitive multipotent cardiac progenitors (MCPs) common for both heart fields. However, it remains unclear whether at the single cell level, Mesp1 progenitors represent a common progenitor for the FHF and SHF. Using mosaic tracing and inducible clonal analysis with a multicolor reporter strategy, we investigated the contribution of Mesp1 cardiovascular progenitors in a temporally controlled manner during the early gastrulation. Our data indicated that the myocardium derives from ~250 Mesp1 expressing cardiac progenitors born during gastrulation. Temporal analysis of clonally labeled Mesp1 cells revealed the existence of temporally distinct populations of Mesp1 progenitors that are restricted to either the FHF or the SHF. FHF progenitors were unipotent, while SHF progenitors, were either uni- or bipotent. Microarray and single cell RT-PCR analysis of Mesp1 progenitors revealed the existence of molecularly distinct populations of Mesp1 progenitors, consistent with their lineage and regional contribution. Moreover biophysical analysis of clonal data revealed that, despite arising at different time points and contributing to different heart regions, the temporally distinct cardiac progenitors present very similar clonal dynamics. Altogether, these results provide insights into the number of cardiac progenitors and their mode of growth. Moreover they provide evidence that heart development arises from distinct populations of unipotent and bipotent cardiac progenitors expressing Mesp1 independently at different time points during gastrulation. Our data reveal that the regional segregation and lineage restriction of cardiac progenitors occurs very early during embryonic development. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

L’identification de signaux impliqués dans la formation des cellules endocrines pancréatiques permettra d’apporter de nouvelles connaissances sur les mécanismes régissant la différenciation des cellules endocrines et de nouvelles applications dans le domaine de la génération de cellules sécrétrices d’insuline afin de traiter des patients atteints de diabète. C’est dans ce contexte que nous avons étudié l’expression et la fonction du récepteur membranaire Glial cell line derived neurotrophic factor Family Receptor α 3 (Gfrα3) et de son ligand Artemin au cours du développement du pancréas et dans le pancréas adulte chez la souris. Des techniques de PCR quantitatives, d’hybridations in situ et d’immunofluorescences nous ont permis de caractériser l’expression de Gfrα3, d’Artn mais aussi d’autres ligands et récepteurs de cette famille. Nous avons aussi utilisé un modèle de souris perte de fonction et générer un modèle de souris transgénique sur exprimant Artn dans le pancréas afin d’étudier la fonction de la voie de signalisation Artn/Gfrα3 dans le pancréas. Nous avons ainsi découvert que le récepteur Gfrα3 est exprimé au cours du développement du pancréas par les progéniteurs endocrines, les cellules insulines-positives et glucagon-positives ainsi que les cellules embryonnaires neuronales. Au stade adulte, Gfrα3 n’est pas exprimé par les cellules à insuline et est exprimé par quelques cellules à glucagon. A ce stade, son expression est aussi observée au niveau des cellules gliales, ainsi que des neurones du système nerveux sympathique et parasympathique. Les différentes expériences de perte et de gain de fonction réalisées afin de comprendre le rôle pancréatique de Gfrα3, ont révélé que ce récepteur n’est pas essentiel à la différenciation et au maintien des cellules endocrines, ni à la formation et au maintien de l’innervation endocrine. En conclusion, nous avons découvert et caractérisé un nouveau marqueur de surface exprimé dans les cellules endocrines pancréatiques en développement. Cette caractéristique pourrait s’avérer utile pour purifier par cytométrie en flux et étudier des sous populations cellulaires générées au cours des protocoles de différentiation in vitro visant à générer de nouvelles cellules sécrétrices d’insuline pour une thérapie cellulaire du diabète. / The generation of therapeutic ß-cell from human embryonic stem cells relies on the identification of growth factors that faithfully mimic pancreatic ß-cell development in vitro. In this context, the aim of the study was to determine the expression and function of a novel endocrine progenitor surface marker, the Glial cell line derived neurotrophic factor receptor α3 (Gfrα3) and its ligand Artemin in islet cell development and function.RT-PCR, In situ hybridization and immunochemistry were used to characterize the expression of Gfra3 and Artn mRNAs and proteins as well as of other members of the GDNF receptor and ligand family. We used Gfra3-deficient mice to study Gfrα3 function and generated a transgenic mice over expressing Artn in the embryonic pancreas to study Artn function. We found that Gfrα3 is expressed at the surface of a subset of Ngn3-positive endocrine progenitors as well as of embryonic α- and ß-cells, while Artn is found in the pancreatic mesenchyme. Adult ß-cell lack Gfrα3, but rare ß cell express the receptor. Gfrα3 is also found in parasympathetic and sympathetic intra islets neurons as well as in glial cell in the embryonic and adult pancreas. The loss of Gfrα3 or overexpression of Artn has no impact on Ngn3-and islet cell formation and maintenance in the embryo. Islet organisation and innervation as well as glucose homeostasis is normal in Gfrα3-deficient mice. Our data show that Gfrα3 is dispensable for islet cell differentiation and innervation suggesting functional redundancy. Gfrα3 could be instrumental as a surface marker for antibody–mediated sorting and characterization of relevant cell population during islet cell differentiation.