A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.

L'infection congénitale par le cytomégalovirus humain (HCMV) est la première cause de séquelles acquises du système nerveux central (CNS). Elle est responsable de surdités neurosensorielles, de paralysies cérébrales ou d'anomalies neuro-développementales graves (0,1% des naissances) telles que des microcéphalies ou des anomalies de gyration. Pour étudier les effets de l'infection par le HCMV sur le développement cérébral, nous utilisons des cellules souches neurales (NSC) humaines dérivées de cellules souches embryonnaires (ES), ainsi que des coupes histologiques de cerveaux fœtaux infectés. Notre travail a porté sur l'analyse des conséquences de l'infection sur un facteur de transcription essentiel lors du développement cérébral, le Peroxisome Proliferator-Activated Receptor gamma (PPARg). Nous avons démontré que l'infection par le HCMV diminuait la neuronogénèse, en association avec une augmentation des niveaux d'expression et d'activité de PPARg. En accord avec ces résultats, nous avons montré que le niveau d'expression de l'acide 9-hydroxyoctadecadienoique (9-HODE), un agoniste connu de PPARg était augmenté dans les NSC infectées. En outre, l'ajout de 9-HODE dans les NSC reproduit l'effet de l'infection sur PPARg conduisant à une augmentation du nombre de cellules positives pour l'antigène viral IE parmi les NSC infectées. De plus, nous avons démontré que : (1) l'activation pharmacologique ou l'expression ectopique de PPARg suffisent pour perturber la neuronogénèse de NSC non infectées ; (2) le traitement de NSC non infectées par le 9-HODE diminue la différenciation des NSC ; (3) le traitement de NSC infectées par du T0070907, un inhibiteur de PPARg restaure un taux normal de différenciation. Le rôle crucial de PPARg dans les pathologies fœtales liées à l'infection a été souligné par la mise en évidence de sa translocation nucléaire au sein des zones germinatives de cerveaux fœtaux infectés congénitalement par le HCMV (N=20), mais pas dans les cas contrôles. Nous avons également identifié un des gènes cibles de PPARg dans le cerveau infecté: LIS1, le gène de la lissencéphalie classique, dont l'expression est également augmentée dans les NSC infectées, de façon dépendante de l'activité de PPARg. Nous avons mis en évidence que l'expression de LIS1 était augmentée de façon massive dans les cerveaux fœtaux infectés congénitalement par le HCMV (N=6) par rapport aux cas contrôles (N=3). Ceci pourrait jouer un rôle central dans la physiopathologie, car il est connu que toute perturbation de l'expression de LIS1 conduit à des anomalies importantes de la migration neurale et au développement d'un phénotype dit "lissencephaly-like". L'ensemble de nos données révèle le rôle clé de PPARg dans la neuronogénèse et la pathophysiologie de l'infection congénitale par le HCMV. Elles ouvrent la voie à une meilleure compréhension des mécanismes régissant les phénotypes pathologiques, notamment concernant le rôle de LIS1 dans les anomalies de la migration neurale. / Congenital infection by human cytomegalovirus (HCMV) is a leading cause of permanent sequelae of the central nervous system, including sensorineural deafness, cerebral palsies or devastating neurodevelopmental abnormalities (0.1 % of all births). To gain insight on the impact of HCMV on neuronal development, we used both neural stem cells from human embryonic stem cells (NSC) and brain sections from infected fetuses. We investigated the outcome of infection on Peroxisome Proliferator-Activated Receptor gamma (PPARg, a transcription factor critical in the developing brain. We observed that HCMV infection dramatically impaired the rate of neuronogenesis and strongly increased PPARg levels and activity. Consistent with these findings, levels of 9-hydroxyoctadecadienoic acid (9-HODE), a known PPARg agonist, were significantly increased in infected NSCs. Likewise, exposure of uninfected NSCs to 9-HODE recapitulated the effect of infection on PPARg activity. It also increased the rate of cells expressing the IE antigen in HCMV-infected NSCs. Further, we demonstrated that (1) pharmacological activation of ectopically expressed PPARg was sufficient to induce impaired neuronogenesis of uninfected NSCs, (2) treatment of uninfected NSCs with 9-HODE impaired NSC differentiation and (3) treatment of HCMV infected NSCs with the PPARg inhibitor T0070907 restored a normal rate of differentiation. The role of PPARg in the disease phenotype was strongly supported by the immunodetection of nuclear PPARg in brain germinative zones of congenitally infected fetuses (N=20), but not in control samples. We also identified LIS1 as one of the target genes for PPAR??in the infected brain. Levels of LIS1, the gene of classical lissencephaly, were strongly increased in infected NSC, presumably resulting from increased PPAR? activity. The relevance of this finding was further supported by our demonstration of a massive increase in the immunodetection in LIS1 fetal brains congenitally infected with HCMV (N = 6), relative to control cases (N = 3). Indeed, it is well known that overexpression of LIS1 is responsible for significant abnormalities of neural migration and development of a lissencephaly-like phenotype. Altogether, our findings reveal a key role for PPARg in neurogenesis and in the pathophysiology of HCMV congenital infection. They also pave the way to the identification of PPARg gene targets in the infected brain.

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Un certain nombre de régions du cerveau des vertébrés, y compris chez l’homme, continuent d’être le siège de l’ajout de nouveaux neurones à l’âge adulte. Ces nouveaux neurones sont produits à partir de cellules spécialisées, appelées cellules souches neurales (CSN). Celles-ci sont capables de s’auto-renouveler et sont principalement trouvées dans un état d’arrêt transitoire du cycle cellulaire que l’on appelle quiescence. A l’heure actuelle, les mécanismes cellulaires et moléculaires permettant aux CSN de trouver un équilibre entre maintien et différentiation, ainsi que les règles générales gouvernant l’évolution de leur population, ne sont que partiellement compris. A l’échelle moléculaire, plusieurs facteurs et voies de signalisation apparaissent déterminants pour l’homéostasie des CSN. Notamment, la voie de signalisation du récepteur Notch s’avère essentielle pour maintenir à la fois l’état de quiescence et le caractère souche des CSN. Il demeure néanmoins inconnu si la signalisation Notch affecte ces deux propriétés de manière indépendante ou non. A l’échelle cellulaire, la plupart des modèles actuels suggèrent que les CSN se divisent rarement et principalement de manière asymétrique. Cette dernière propriété permettrait aux CSN de se perpétuer tout en donnant naissance à des cellules filles déterminées à se différencier en neurones. Le pallium du poisson-zèbre abrite une population particulièrement importante de CSN, que l’on appelle glies radiaires (GR), et qui possèdent les mêmes caractéristiques fondamentales que leurs homologues chez les mammifères. Notre laboratoire avait précédemment démontré que le récepteur Notch3 était nécessaire au maintien de la quiescence des GR. Le travail présenté dans ce manuscrit se décompose en deux études complémentaires dont les objectifs respectifs étaient: (1) d’améliorer notre compréhension du rôle de la voie de signalisation Notch3 dans l’homéostasie des GR et (2) d’étudier les schémas de divisions adoptés par les GR afin de maintenir leur nombre sur une longue durée. Dans la première étude, nous démontrons que le rôle de la signalisation Notch3 s’étend au-delà du simple contrôle de la quiescence des GR en contribuant également au maintien de leur caractère souche par l’intermédiaire de son gène cible hey1. Un point important de cette découverte est que l’action du facteur Hey1 sur le caractère souche des GR apparaît indépendante du rôle de Notch3 dans le maintien de leur quiescence. Dans la seconde étude, nous avons réalisé une analyse clonale du devenir des GR exprimant le gène her4.1. Ceci nous a permis de mettre en évidence que leurs choix entre différentiation, amplification et auto-renouvellement apparaissent stochastiques, mais équilibrés, ce qui leur permet de maintenir leur population dans le temps. De façon très intéressante, nous avons aussi observé que le nombre total de GR du pallium augmente au cours de la vie, ce qui, au regard du comportement homéostatique de la population de GR exprimant her4.1, nous amène à proposer que la zone neurogénique du pallium est organisée selon une hiérarchie dans laquelle une population inconnue de progéniteurs produit continuellement de nouvelles GR, qui ensuite se maintiennent grâce à un équilibre probabiliste entre leurs différents lignages. / New neurons continue to be added into discrete brain regions of most adult vertebrate species, including humans. Adult born neurons arise from precursor cells, called neural stem cells (NSCs), endowed with self-renewal potential and mostly found in a state of reversible cell cycle arrest, named quiescence. Currently, the molecular, cellular and population rules allowing NSC to balance maintenance and differentiation remain incompletely understood. At the single cell level, several factors and signalling pathways were demonstrated to be essential for NSC homeostasis. Among them, the Notch signalling pathway is critically involved in the control of NSC quiescence and stemness. However, whether these two properties represent molecularly distinct or overlapping outputs of the Notch signalling pathway remains unknown. At the cellular level, current models state that NSCs divide rarely and mostly asymmetrically, allowing both self-renewal and the generation of a more committed progeny that ultimately exits the cell cycle and fulfils neuronal differentiation. The adult zebrafish pallium harbours NSCs, called radial glia (RG), which share with their mammalian counterparts the same basic properties. Previously, our laboratory demonstrated that Notch3 was necessary to maintain RG quiescence. Here, in two different and complementary works, we took advantage of the widespread neurogenic ventricular zone (VZ) of the adult zebrafish pallium to (1) explore further the role of Notch3 signalling in RG homeostasis and (2) investigate the division pattern and dynamics allowing the RG population to be maintained on the long run. In the first study, we demonstrate that the role of Notch3 signalling extends beyond the simple maintenance of RG quiescence and that Notch3 also contributes to RG stemness. By overlapping the transcriptomic profiles of both notch3 mutant RG and adult pallial VZ progenitors, we identified different sets of Notch3 target genes potentially responsible for its pleitropic effect in RG. Notably, we show that the Notch3 signalling contribution to RG stemness critically relies on the transcriptional activation of its canonical target gene hey1 and this, independently of Notch3 action on RG quiescence. In the second study, we performed a quantitative analysis of the fates of individual her4.1(Hes5)-expressing RG. We demonstrate that these cells adopt balanced stochastic fates, which allows their population to reach homeostasis. We also report that the overall RG population of the zebrafish pallium continues to grow during adulthood and that this expansion is very likely driven by a yet undefined upstream population of progenitors. As a consequence, we propose that the adult zebrafish is organised into a hierarchy of progenitors dominated by an unknown population that fuels the ongoing production of an intrinsically homeostatic population of RG which, itself, follows neutral drift dynamics.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23464号 / 医博第4771号 / 新制||医||1053(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 林 康紀, 教授 伊佐 正, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Parkinson's disease (PD) is the second most common neurodegenerative disease. With the rapid global increase in population aging, neurodegenerative diseases are considered a primary threat to human health. As dopaminergic neuronal cell death and dysfunction are the main pathogenic mechanisms of PD, neural stem cell (NSC) replacement therapy has been identified as a potentially effective and indeed ideal therapeutic strategy. However, current in vitro stem cell culture methods, which require various chemical growth factors (GFs), are unsafe and relatively inefficient. To solve this problem, we developed two strategies for enhancing the proliferation and differentiation of NSCs in vitro based on extracellular nanomatrices and natural active ingredients. First, we developed novel nanomatrices comprising biomaterials used for promoting NSCs proliferation and differentiation without requiring additional GFs. We developed two types of inorganic sculptured extracellular nanomatrices comprising SiO2 (iSECnMs) which deposited by glancing angle deposition (GLAD). The physiological properties of nanomatrices mediate the activation of multiple bio-signaling pathways. Accordingly, iSECnMs, especially those sculptured in zigzag forms, can significantly promote the proliferation and specific neuronal phenotypic differentiation of NSCs without requiring additional GFs. The differentiated neurons survived well in vivo and achieved outstanding therapeutic effects in a rat model of 6-OHDA-induced parkinsonism. Second, 20(S)-protopanaxadiol (PPD) and oleanolic acid (OA), two crucial active ingredients derived from ginseng, significantly enhanced the proliferation and neuronal differentiation of NSCs through activating Wnt/GSK-3β/β-catenin pathway. This research is expected to promote significant developments in the induction of NSCs and provide insights into stem cell therapies for PD without undesirable prognoses

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19230号 / 医博第4029号 / 新制||医||1011(附属図書館) / 32229 / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 斎藤 通紀, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Human neurosphere stem cells offer promising potential for the treatment of neurodegenerative diseases. Their well characterized multi-potency of differentiating into neurons, astrocytes, and oligodendrocytes when exposed to the optimum exogenous growth factors make them an exciting area of study (38). Finding novel endogenous methods of modulating stem cell metabolism will allow for the safer treatment of various brain disorders (34). In this experiment, melatonin, N-acetylserotonin, L-tryptophan, and L-DOPA are added in three different concentrations to neurospheres suspended in HNSC/GBM media with less than optimal concentrations of exogenous epidermal growth factor (EGF) and fibroblast growth factor (FGF). The alamarBlue assay (resazurin) was chosen as the most suitable assay for measuring neurosphere metabolism. Metabolic neural stem cells would cause the greatest reduction of the oxidized alamarBlue reagent (resazurin?resorufin), which was detected by a fluorescent plate reader (39-41). The percent reduction in alamarBlue was calculated for all four molecules at three different concentrations and compared to controls without any molecule. Our results illustrate that there was no statistically significant difference at p<0.05 between the biological molecules and the control group except for two exceptions (labeled with asterisks on figures 3 and 5) L-DOPA at a 40 micromolar concentration after 4 hours of incubation and melatonin at a 40 micromolar concentration after 52 hours of incubation.