Stem cells are crucial players during development, homeostasis and tissue regeneration and their interactions with the surrounding microenvironment are key to regulate stem cell fate. The skull's stem cell niches reside in the fibrous joints that connect flat bones of the skull. In the embryo, bone and sutures develop in concert to form a complex, multi-facted structure that requires interaction with multiple differentiating cell types to maintain balance between growth and differentiation. Disruption of this balance drives changes in size and shape of skull bones and can severely impact quality of life. Cranial sutures, often seen as simple extracellular matrix-rich structures bridging the rigid plates of the skull, are major actors in craniofacial morphogenesis of as they harmonize bone growth with expansion of the developing brain and participate in providing osteoblasts during repair. The complexity of the extracellular environment and the important role for sutures in skeletal development makes these niches a compelling structure to investigate how interactions with the surrounding microenvironment can modulate stem cells fate. The key role of sutures in development is highlighted by the numerous severe dysmorphisms arising from failure to maintain suture patency. The ability of the suture to respond to brain growth or trauma and the dysmophisms presented by patients with defective sutures is mediated by both biochemical and mechanical cues but the cell biology of these niches remains elusive, especially during their development. In particular, few studies have shed light on the underlying cellular behaviors behind microenvironmental regulation of cranial suture stem cell fate and what role mechanical inputs play in the establishment of this niche. In my thesis, I addressed gaps in our understanding of suture biology by characterizing the suture stem cell niche microenvironment and exploring how cell-ECM interactions serve as regulators of suture stem cell fate. Making use of various microscopy and analytical techniques I first characterized the composition of the microenvironment in a developing suture niche, such as organization of ECM, cytoskeleton and nuclear morphologies. My work builds on an incomplete transcriptional understanding of suture cell development, such that specific genetic markers are rarely useful for identifying distinct suture cell populations during its morphogenesis. By applying shape description tools to parse suture cells and test whether shape correlates to cell identity, we concluded that suture nuclei are distinct and less spherical than those of other cranial tissues. Using 'global' markers such as nuclear stains, I have also identified physical distinctions between suture nuclei and neighboring tissues, indicating that cell shape is an integral part of midline suture identity and can be used to explore coordination of fate choice and morphogenesis in this enigmatic structure. In addition, I present evidence that supports that maturation of extracellular matrix begins during early stages of suture development. In particular, embryonic midline sutures express high levels of fibrillary collagen, which contributes to the formation of a complex extracellular environment that provides the suture with physical properties distinct from those of developing bones. My work shows the presence of cell-ECM and cell-cell adhesions in the developing midline sutures, as well as a complex actin cytoskeleton that is, in part, mediated by physical stresses resultant from underlying brain expansion. Secondly, I aimed to address how perturbations in ECM composition can affect cell specification. To investigate the importance of ECM maturation in regulating suture cell fate I inhibited the function of lysyl oxidase, a collagen crosslinker, during embryonic development. Disruption of collagen crosslinking altered expression of collagen and ECM receptor encoding genes. In addition, this inhibition induced changes in the shape and size of collagen fibers in the embryonic midline suture and decreased tissue bulk stiffness relative to WT. These abnormal properties of the ECM impact tissue delineation in the cranial mesenchyme through nuclear shape analyses. This might be explained by observed changes in the composition of the nuclear envelop of suture cells as we find altered lamin concentration and localization upon lysyl oxidase inhibition. The work developed during myPhD steps away from the traditional genetic approaches used to study the embryonic suture and provides the first in-depth analysis of the physical properties of the developing midline suture at stages preceding known establishment of the niche. The various methods and analyses applied reveal a complex organization of embryonic suture ECM and its tight relationship with shape and fate in this tissue. This work serves as a foundation for future studies that can explore the mechanisms through which ECM regulates fate and development of the suture niche, and potentially skeletal development more generally.

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La division cellulaire asymétrique (DCA) consiste en une division pendant laquelle des déterminants cellulaires sont distribués préférentiellement dans une des deux cellules filles. Par l’action de ces déterminants, la DCA générera donc deux cellules filles différentes. Ainsi, la DCA est importante pour générer la diversité cellulaire et pour maintenir l’homéostasie de certaines cellules souches. Pour induire une répartition asymétrique des déterminants cellulaires, le positionnement du fuseau mitotique doit être très bien contrôlé. Fréquemment ceci génère deux cellules filles de tailles différentes, car le fuseau mitotique n’est pas centré pendant la mitose, ce qui induit un positionnement asymétrique du sillon de clivage. Bien qu’un complexe impliquant des GTPases hétérotrimériques et des protéines liant les microtubules au cortex ait été impliqué directement dans le positionnement du fuseau mitotique, le mécanisme exact induisant le positionnement asymétrique du fuseau durant la DCA n'est pas encore compris. Des études récentes suggèrent qu’une régulation asymétrique du cytosquelette d’actine pourrait être responsable de ce positionnement asymétrique du faisceau mitotique. Donc, nous émettons l'hypothèse que des contractions asymétriques d’actine pendant la division cellulaire pourraient déplacer le fuseau mitotique et le sillon de clivage pour créer une asymétrie cellulaire. Nos résultats préliminaires ont démontré que le blebbing cortical, qui est une indication de tension corticale et de contraction, se produit préférentiellement dans la moitié antérieure de cellule précurseur d’organes sensoriels (SOP) pendant le stage de télophase. Nos données soutiennent l'idée que les petites GTPases de la famille Rho pourraient être impliqués dans la régulation du fuseau mitotique et ainsi contrôler la DCA des SOP. Les paramètres expérimentaux développés pour cette thèse, pour étudier la régulation de l’orientation et le positionnement du fuseau mitotique, ouvrirons de nouvelles avenues pour contrôler ce processus, ce qui pourrait être utile pour freiner la progression de cellules cancéreuses. Les résultats préliminaires de ce projet proposeront une manière dont les petites GTPases de la famille Rho peuvent être impliqués dans le contrôle de la division cellulaire asymétrique in vivo dans les SOP. Les modèles théoriques qui sont expliqués dans cette étude pourront servir à améliorer les méthodes quantitatives de biologie cellulaire de la DCA. / Asymmetric cell division (ACD) consists in a cellular division during which specific cell fate determinants are distributed preferentially in one daughter cell, which then differentiate from its sibling. Hence, ACD is important to generate cell diversity and is used to regulate stem cells homeostasis. For proper asymmetric distribution of cell fate determinants, the positioning of the mitotic spindle has to be tightly controlled. Frequently, this induces a cell size asymmetry, since the spindle is then not centered during mitosis, leading to an asymmetric positioning of the cleavage furrow. Although small small GTPases have been shown to act directly on the spindle, the exact mechanism controlling spindle positioning during ACD is not understood. Recent studies suggest that an independent, yet uncharacterized pathway is involved in spindle positioning, which is likely to involve an asymmetric regulation of the actin cytoskeleton. Indeed, actin enables spindle anchoring to the cortex. Hence we hypothesize that asymmetric actin contractions during cytokinesis might displace the mitotic spindle and the cleavage furrow, leading to cell size asymmetry. Interestingly, from our preliminary results we observed that cortical blebbing, which is a read-out of cortical tension/contraction, preferentially occurs on the anterior side of the dividing sensory organ precursor (SOP) cells at telophase. Our preliminary data support the idea that Rho small GTPases might be implicated in regulation of the mitotic spindle hence controlling asymmetric cell division of SOP cells. The experimental settings developed for this thesis, for studying regulation of the mitotic spindle orientation and positioning will serve as proof of concept of how geneticist and biochemist experts could design ways to control such process by different means in cancerous cells. The preliminary results from this project open novel insights on how the Rho small GTPases might be implicated in controlling asymmetric cell division hence their dynamics in vivo of such process during SOP development. Furthermore, the assays and the theoretical model developed in this study can be used as background that could serve to design improved quantitative experimental methods for cell biology synchronizing sub-networks of ACD mechanism.

Le cancer de l’ovaire (COv) est le cancer gynécologique le plus létal chez la femme et les traitements existants, chirurgie et chimiothérapie, ont peu évolué au cours des dernières décennies. Nous proposons que la compréhension des différents destins cellulaires tels que la sénescence que peuvent choisir les cellules du cancer de l’ovaire en réponse à la chimiothérapie pourrait conduire à de nouvelles opportunités thérapeutiques. La sénescence cellulaire a été largement associée à l’activité de la protéine TP53, qui est mutée dans plus de 90% des cas de cancer de l’ovaire séreux de haut grade (COv-SHG), la forme la plus commune de la maladie. Dans nos travaux, à partir d’échantillons dérivés de patientes, nous montrons que les cultures primaires du cancer de l’ovaire séreux de haut grade exposées au stress ou à des drogues utilisées en chimiothérapie entrent en senescence grâce à l’activité d’un isoforme du gène CDKN2A (p16INK4A). Dans ces cellules, nous avons évalué les caractéristiques fondamentales de la sénescence cellulaire tels que les altérations morphologiques, l’activité béta galactosidase associée à la sénescence, les dommages à l’ADN, l’arrêt du cycle cellulaire et le phénotype sécrétoire associé à la sénescence. En utilisant des micromatrices tissulaires construites à partir d’échantillons humains de COv-SHG pré- et post-chimiothérapie, accompagnées de leurs données cliniques, nous avons quantifié des marqueurs de sénescence incluant une diminution de la prolifération cellulaire quelques semaines après chimiothérapie. De façon intéressante, l’expression de p16INK4A dans les échantillons de COv-SHG prétraitement corrèle avec une survie prolongée des patientes suite au traitement. Ceci suggère ainsi pour la première fois un impact biologique bénéfique pour la présence de cellules cancéreuses qui sont capable d’activer la sénescence, particulièrement pour le traitement du cancer de l’ovaire. Dans le but de complémenter les thérapies actuelles avec des approches de manipulation pharmacologique de la sénescence, nos résultats suggèrent qu’il serait important de déterminer l’impact positif ou négatif de la sénescence induite par la thérapie sur la progression de la maladie et la survie, pour chaque type de cancer de façon indépendante. / Human ovarian cancer (OvCa) is the deadliest gynecologic malignancy and existing surgical/chemotherapeutic treatment options have been relatively static for decades. We propose that understanding OvCa cell fate decisions taken in response to chemotherapy could guide new therapeutic opportunities. Damage-induced cellular senescence is often associated with TP53 activity, which is heavily mutated in high grade serous (HGS) OvCa (>90%), the most common form of this disease. Here, using patient derived tissues, we show that primary HGS-OvCa cultures predominantly trigger CDKN2A- associated (p16INK4A isoform) senescence following exposure to stress or chemotherapy. Key senescence hallmarks including altered morphology, senescence-associated-Betagalactosidase, DNA damage, cell cycle arrest and the senescence-associated secretory phenotype were evaluated and detected in damaged cells. Using tissue microarrays built from pre- and post-treatment human HGS-OvC tissue samples with accompanying clinical data, we quantified post-treatment hallmarks of senescence including reduced cell proliferation weeks after chemotherapy. Importantly, p16INK4A expression in pretreatment HGS-OvC samples correlated with increased patients survival, suggesting for the first time that senescence-competence in human cancer cells may have a beneficial impact on treatment outcomes for patients. In order to guide the potential improvement of existing human therapies via pharmacological senescence manipulation, our results suggests that it is important to determine for many types of human cancer whether treatment-induced senescence positively or negatively impacts disease progression and patient survival.

Das Ziel dieser Arbeit war, die funktionellen Charkteristika von humanen antigenerfahrenen CD4-T-Zellen in Relation zur Expression von IL-7Ra und CCR7 zu untersuchen. Im Rahmen dessen wurden zwei verschiedene experimentelle Ansätze wurden gewählt: Zur Analyse von Populationen, die während einer Primärantwort auftreten, wurden antigenerfahrene CD4-T-Zellen in vitro mit TSST-beladenen dendritischen Zellen stimuliert. Der zweite Ansatz bestand darin, zirkulierende antigenerfahrene CD4-T-Zellen entsprechend ihrer CCR7- und IL7R-Expression zu isolieren, um die heterogenen zirkulierenden T-Zellen zu untersuchen. Die Experimente zeigen, daß IL7RhiCCR7+ T-Zellen Charakteristika zentraler Gedächtniszellen besitzen. IL7RlowCCR7–identifiziert hingegen Zellen mit Effektor-T-Zellmerkmalen. Dementsprechend wiesen IL7RlowCCR7– T-Zellen ein stark verringertes Zellüberleben auf, waren stark beeinträchtigt in darin in Anwesenheit von homöostatischen Zytokinen zu proliferieren und exprimierten nur wenig IL-2. Im Gegenzug überlebten IL7RhiCCR7+ T-Zellen gut, reagierten auf homöostatische Zytokine, sekretierten IL-2 und expandierten nach antigenspezifischer Stimulation. Interessanterweise erkannten ex vivo isolierte IL7Rlow T-Zellen vornehmlich persistierende Antigene. Dies weist darauf hin, daß diese Zellen chronisch aktiviert sind. In vitro konnte demonstriert werden, daß die funktionelle Ausprägung der den zentralen Gedächtniszellen ähnlichen IL7RhiCCR7+ T-Zellen deutlich von der Stärke der Stimulation während der Generierung der IL7RhiCCR7+ T-Zellen abhängt. Dieser Umstand stützt die Hypothese, daß die Quantität der Signale während der primären Stimulation die Richtung der Zelldifferenzierung bestimmt und ein solcher Mechanismus zur Heterogenität der zentralen Gedächtnis-T-Zellen in vivo beiträgt. Zusammenfassend kann man feststellen, daß, sich die Marker CCR7 und IL7R in Kombination als ein wertvoll zur Identifizierung von CD4-Gedächtnis- und –Effektor-T-Zellen erweisen / The aim of this work was to elucidate the functional characteristics of human antigen-experienced CD4 T cells according to the expression of IL7RAlpha and CCR7. Two different approaches were used: in order to analyze the subsets occurring during a primary response, antigen-experienced CD4 cells were analyzed in vitro after priming with the superantigen TSST. The signal strength of TCR-stimulation during the priming was modulated in order to understand how the levels of stimulation might influence the generation of memory-like T cells. The second approach was to isolate circulating CD4 T cells expressing different combinations of CCR7 and IL7R in order to analyze the heterogeneous pool of antigen-experienced cells found under steady state conditions ex vivo. The results revealed that both in vitro and ex vivo the IL7RhiCCR7+ T cell subset corresponds to cells with TCM characteristics whereas IL7RlowCCR7– identifies cells with effector characteristics. Correspondingly, IL7RlowCCR7–CD4 T cells showed low survival rates, impaired proliferation in the presence of homeostatic cytokines and a low IL-2 production, IL7RhiCCR7+ CD4 T cells survived well, responded to homeostatic cytokines, secreted IL-2 and expanded upon antigenic stimulation. Notably, ex vivo isolated IL7Rlow T cells preferentially recognized under steady state conditions persistent antigens, suggesting that these cells are chronically activated. Finally, it was demonstrated that IL7RhiCCR7+ TCM-like cells generated in vitro acquired different functional properties depending on the strength of stimulation during the priming. This fact supports the concept that the amount of signal received during the priming influences the cell fate decision contributing to the heterogeneity of the TCM pool in vivo. In summary, despite some differences observed between in vitro and ex vivo CD4 T cell subsets, the combination of the markers CCR7 and IL7R is useful to distinguish memory- from effector-like CD4 T cells.

Die vorliegende Arbeit befasst sich mit Aspekten der frühen Entwicklung der Phoronida („Hufeisenwürmer“). An drei Arten wird der Furchungsprozess untersucht (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). Dies erfolgt sowohl mithilfe der 4D-Mikroskopie als auch anhand von immunocytochemischen Markierungen der Mitosespindeln und konfokaler Laser-Scanning-Mikroskopie. Verschiedene morphologische Merkmale des Furchungsprozesses werden quantitativ erfasst und innerhalb sowie zwischen den Arten verglichen. Die Ergebnisse zeigen eine weitgehend übereinstimmende Furchung bei P. pallida und P. muelleri Embryonen: Ab dem dritten Zellzyklus teilen sich die Blastomeren meist schräg – und alternierend dextral und sinistral – zur animal-vegetativ Achse. Dieses Muster zeigt überraschende Übereinstimmungen mit dem Muster der Spiralfurchung. Dies kann als morphologische Unterstützung molekular-phylogenetischer Befunde einer Stellung der Phoronida innerhalb der Spiralia/Lophotrochozoa interpretiert werden. Die Furchung bei P. vancouverensis unterscheidet sich von der Furchung der anderen beiden Arten; sie weist jedoch auch Unterschiede zu einer Radiärfurchung auf. Generell zeigt die Furchung aller drei Arten einen gewissen Grad an Variabilität. Anhand von in-vivo Einzelzellmarkierungen untersucht die Studie darüber hinaus das Schicksal der Blastomeren früher P. pallida Embryonen bis zu späten Gastrulationsstadien. Diese Analysen zeigen, dass die ersten beiden Furchungsteilungen durch die spätere Achse Blastoporus-Apikalplatte, jedoch in keinem konstanten Orientierungsverhältnis zur Ebene der Bilateralsymmetrie der Gastrula verlaufen. Dies unterscheidet sich von der Situation, wie sie von spiralfurchenden Tieren bekannt ist. Die Unterschiede und die beobachtete Variabilität des Furchungsprozesses werden im Licht unterschiedlicher Mechanismen der Spezifizierung von Zellschicksalen und Körperachsen bei verschiedenen Taxa der Spiralia und den Phoronida diskutiert. / This study addresses aspects of the early development of Phoronida (“horseshoe worms”). The cleavage process is analyzed for three species (Phoronis pallida, Phoronis muelleri, Phoronis vancouverensis). These investigations are performed using 4D-microscopy as well as immunocytochemical stainings of the mitotic spindle apparatuses in combination with confocal laser-scanning microscopy. Different morphological features of the cleavage process are quantified and compared within as well as between the species. The results reveal a highly consistent cleavage of P. pallida and P. muelleri embryos: from the third cell cycle onward, the blastomeres divide mostly obliquely – and alternatingly dextral and sinistral – with respect to the animal-vegetal axis. This cleavage pattern shows surprising correspondences to the pattern of spiral cleavage. The finding can be interpreted as morphological support for recent molecule-based phylogenies, which indicate a position of Phoronida within the Spiralia/Lophotrochozoa clade. The cleavage of P. vancouverensis differs from the cleavage in the other two species; however, it also shows differences to a radial cleavage pattern. In all three species, the cleavage process also involves some degree of variability. Furthermore, the study traces the cell fates of early P. pallida embryos up to the state of late gastrulation, by the use of fluorescent in-vivo single cell markings. These analyses reveal that the first two cleavage divisions both pass through the later axis blastopore-apical plate of the gastrula, yet they do not pass in a constant relationship with respect to the later plane of bilateral symmetry. This differs from the situation known from spiral cleaving animals. The differences and the encountered variability of the cleavage process are discussed with respect to different mechanisms of the specification of cell fates and body axes in different taxa of the Spiralia and the Phoronida.

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal. / Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype.

Le glioblastome (GBM) est la tumeur cérébrale primaire la plus agressive comportant une sous-population de cellules souches tumorales (CSG). Elles sont capables d’auto-renouvellement, de prolifération, de différenciation en cellules exprimant les marqueurs neuraux et de trans-différenciation en cellules de types vasculaires. Dans ce contexte, j’ai dérivé et caractérisé plusieurs lignées de CSG à partir de biopsies de patients. Puis j’ai évalué l’impact des peptides thérapeutiques transmembranaires développés au laboratoire, visant les plateformes de récepteurs de neuropiline-1 et de plexine-A1 surexprimées dans les CSG. Les deux peptides diminuent la croissance des CSG in vitro et in vivo. Finalement, j’ai développé un outil génétique fluorescent permettant de suivre le destin des CSG en direct. Basé sur l’expression de 4 rapporteurs fluorescents contrôlés par des promoteurs spécifiques des types cellulaires, il permet d’identifier l’hétérogénéité de ces cellules en différenciation. / The glioblastoma multiforme (GBM) is the most aggressive primary brain tumor and includes a subpopulation of tumoral stem cells (CSG). Those cells can self-renew, proliferate and differentiate by expressing specific neural markers and/or transdifferentiate into vascular-like cells. In this context, my work consisted first to produce and characterize several CSG lines from patient biopsies to constitute a bank of cell lines with different properties. We also evaluated the impact of in house therapeutic transmembrane peptides targeting the neuropilin-1 / plexin-A1 receptor platforms overexpressed in GBM. We thus showed that both targeting peptides decrease the growth of GSC in in vitro and in vivo models. Finally, I developed an inducible mosaic expression system to track the live differentiation of CSG. This system is based on the expression of four different fluorescent reporters controlled by the activity of cell type specific promoters.