Дисертації з теми "Olfactory Ectomesenchymal Stem Cell"

Les métalloproteases matricielles (MMPs) appartiennent à une famille d'endopéptidases dépendantes du zinc, présentent sous forme secrétée ou membranaire (MT-MMP) et qui jouent un rôle fondamental dans la signalisation cellulaire. L'activité des MMPs est régulée par leur inhibiteurs endogènes, les inhibiteurs tissulaires des MMPs (TIMPs). Le système MMP/TIMP régule les interactions cellule-cellule et cellule-matrice extra cellulaire et module la motilité cellulaire par clivage protéolytique des composants de la matrice extra cellulaire aussi bien lors de processus physiologiques que dans des situations pathologiques.Dans un premier temps, nous avons mis en évidence le rôle de TIMP-1 dans la modulation de la croissance neuritique et la morphologie neuronale, via l'inhibition de MMP-2 et non de MMP-9. souches de la muqueuse olfactive (OE-MSCs). Nous montrons dans cette étude que les gélatinases MMP-2 et MMP-9 ainsi que la MMP membranaire MT1-MMP, sont impliquées dans la migration des OE-MSCs. Nous montrons également que les gélatinases sont probablement impliquées dans les propriétés neurotrophiques des OE-MSCs et des cellules engainantes olfactives.L'ensemble de ces résultats apporte de nouveaux éléments fondamentaux, dans la compréhension du rôle du système MMP/TIMP dans les processus post-lésionnels qui ont lieu au sein du système nerveux central
The matrix metalloproteinases (MMPs) belong to a growing family of Zn2+-dependent endopeptidases, secreted or membrane-bound (MT-MMP), which play a fundamental role in the cell signalling. The activity of the MMPs is regulated by their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs). The MMP / TIMP system regulates the cell-cell and cell-extracellular matrix interactions and modulates the cellular motility through the cleavage of protein components of the extracellular matrix, as well during physiological and pathological conditions.Our results suggest that TIMP-1 is implicated in the modulation of the neurite outgrowth and morphology of cortical neurons through the inhibition at least in part, of MMP-2 and not MMP-9. Afterward, we study of the system MMP / TIMP in the migration of the stem cells of olfactory ectomesenchymal stem cells (OE-MSCs). We show that gelatinases MMP-2 and MMP-9 as well as MT1-MMP, are involved in OE-MSCs migration. We also show that gelatinases are probably involved in neurotrophic properties of the OE-MSCs and olfactory ensheathing cells.Altogether, these results provide new evidences on the role of MMP/TIMP system in central nervous system post-lesional processes

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.

Thesis (Honors paper)--Florida State University, 2008.
Advisor: Cathy W. Levenson, PhD., Florida State University, College of Human Sciences, Dept. of Nutrition, Food and Exercise Sciences. Includes bibliographical references.

Among all the possible sources of mesenchymal stem cells, adipose tissue and olfactory mucosa have raised great interest and have become some of the most investigated sources. Adipose tissue-derived mesenchymal stem cells and the fat itself as a source of human adipose derived stem cells, represent one of the major fields of research in regenerative medicine. A great advantage is represented by the minimal invasive and high accessibility to adipose tissue and its ready availability. In the present study, hADSCs were isolated from the adipose tissue donated by several patient and have been investigated and characterized through different technical approaches, such as flow cytometry and immunocytochemistry. These hADSCs reproducibly fulfill the general definition of MSCs by both phenotypic and differentiation capabilities criteria, showing also the expression of neural markers, as observed by confocal microscope analysis. Lipoaspirated adipose tissue showed positivity to ß-tubulin III that was also maintained in lipoaspirate-derived hADSCs. A population of stem cells retaining typical characteristics of surface markers of classical adipose tissue stem cells and MSC was obtained when adipose tissue was subjected to culture in vitro, either by processing through centrifugation or by direct plating without enzymatic digestion with collagenase. Flow cytometry analyses showed that hADSCs expressed classical mesenchymal markers such as CD44, CD73, CD90, CD105 and CD166, while endothelial (CD31, CD34, CD144, CD146) and hematopoietic (CD45, CD133) markers were much less represented. Also the ability to give rise to tissue of mesenchymal origins, such as osteoblastic and adipogenic lineages, were present in hADSCs. In addition, the immunofluorescence staining indicated the expression of neural stem markers in hADSCs which consequently co-expressed nestin, β-tubulin III and glial GFAP. We have also characterized human olfactory ensheathing stem cells. Olfactory mucosa is specialized tissue inside the nasal cavity involved in olfactory perception and capable of lifelong regeneration throughout adulthood. Multipotent stem cells obtained from it offer the possibility of promoting regeneration and reconstruction in regenerative medicine, being readily accessible with minimal invasive techniques, capable of expansion in vitro and retaining broadly potent differentiative capacity as stem cell progenitors. Among the several members of the olfactory mucosa, Olfactory Ensheathing Cells (OECs) are well known to be useful in repairing the nervous system. By following our method, cells can be easily isolated and maintained in TCM, and their cultivation in large flasks allowed obtaining rich cultures of OECs in 2 weeks. Cell cycle analysis showed that the majority of cells are in G0/G1 phase, while just a lesser part is in S/G2 phase. In our growth conditions, no chromosomal abnormalities were observed also at high culture passage (p14). Live morphology of obtained cells showed a fibroblast-like phenotype and the immunohistochemical analyses showed the expression of beta-Tubulin III, Vimentin, Nestin, Glial Fibrillary Acidic Protein and Microtubule-Associated Protein 2. By FACS analysis we demonstrated that OECs are positive to typical surface mesenchymal markers (CD44, CD73, CD90, CD105, CD146 and CD166). As expected, some endothelial (CD31, CD34) and hematopoietic (CD45) markers were very few represented, while some others (CD56, CD144, CD146, CD133) are partially found. These cells also express genes that constitute the core circuitry of self-renewal such as SOX2, NANOG and OCT4 and the stemness marker CD133. OECs incubated with serum-free medium, normally used for the formation of neurospheres, spontaneously formed large spheroids reaching a mean diameter of 100 μm in 10 days of culture. Immunofluorescence of specific proteins showed that spheres were positive to markers such as Nestin, Vimentin, TUJ-1, MAP2 and GFAP. In conclusion, our method allows the quickly and easily hADSCs and hOESCs isolation from human adipose tissue and nasal biopsies. The obtained cells can be cultured without altering their mesenchymal properties, suggesting the pluripotency nature of these cells and that they are a reliable source for regenerative medicine.

The Olfactory Epithelium (OE) is a specialized epithelial tissue inside the nasal cavity that is involved in the smell sensation. The OE maintains neuroregeneration, i.e. producing new olfactory sensory neurons, throughout the adult life via neural stem cell self-renewal, proliferation, neuronal differentiation and maturation. The neural stem cell niche regulates stem cell self-renewal and proliferation, and consists of stem cells, blood vessels and multiple extracellular matrix proteins (ECMs). ECMs regulate stem cell adhesion, proliferation, differentiation and migration via integrins. One of the main mediators of intracellular integrin signaling is the Focal Adhesion Kinase (FAK). Our previous studies found that FAK inhibition increased cell proliferation in adult mouse olfactory epithelium (OE) via up-regulation of Ciliary Neurotrophic Factor (CNTF). Now we continue to test whether FAK inhibition increases neuroregeneration through CNTF in the adult mouse OE using BrdU-chase pulse method. Adult male and female C57BL/6, CNTF wildtype and CNTF knockout (lack the CNTF gene) mice were systemically injected with PBS or FAK inhibitor (FAK14) for 3 days. During these 3 days, BrdU was injected into mice 4 h following PBS or FAK on each day. BrdU acts as a thymidine analog and is incorporated into DNA during DNA syntheses. Using immunohistochemistry with anti-BrdU antibody, BrdU+ cells can be visualized in the tissue. The BrdU+ cells are the ones who are replicating during the time frame when BrdU was given. 20 days after last BrdU injection, we fixed the mice via cardiac perfusion. The whole heads of mice was decalcified with EDTA and then frozen cross head sections including OE were cut using cryostat and mounted onto slides. The OE sections were then stained with anti-BrdU antibody followed by FITC-conjugated secondary antibody. The BrdU+ cells in the OE were counted in three sections (both left and right sides) per mouse and normalized to linear length of OE basement membrane. The results of the experiment showed that FAK 14 significantly increased BrdU+ stem cells and olfactory sensory neurons in the OE of C57BL/6 and CNTF wildtype mice but not knockout mice, indicating that FAK inhibition promotes olfactory stem cell self-renewal and neuroregeneration via CNTF. Collectively, this data indicates that FAK normally inhibits OE neuroregeneration by inhibiting CNTF expression and identifies the OE is a good model to study neuroregenerative mechanisms in the CNS.

Mestrado em Biologia Molecular e Celular
Comprehending the mechanisms that make lifelong neurogenesis possible has a clear interest for the better understanding of the basic principles that govern cellular and molecular interactions in the nervous system, as well as a relevant clinical interest. The limited ability of the central nervous system to generate new neurons in order to replace those that have been lost is a formidable obstacle to recovery from neuronal damage caused by injury or neurodegenerative disease. The olfactory system (OS) is an ideal system to study the process of neuronal recovery after injury, as it is known for its lifelong capacity to replenish cells lost during natural turnover, as well as its remarkable ability to regenerate after severe lesion. The olfactory epithelium (OE) shows neurogenesis throughout life. Newly differentiated olfactory receptor neurons (ORNs) are continuously reintegrated into an existing circuitry to maintain the sense of smell. The aim of this thesis is to describe the morphological and functional alterations that occur over time in the OS of larval Xenopus laevis, after transection of the olfactory nerve (ON). Results obtained using immunohistochemistry essays, as well as sensory neuron labeling and calcium imaging techniques, indicate that ORN cell death reaches its peak 48 hours after transection, and that proliferating stem cells found in the basal cell layer of the OE are quickly upregulated after lesion. Supporting cells seem to maintain both morphological and functional integrity after transection of the ON. The OE recovers its original morphological structure 1 week after transection, at which time the first axons reach the olfactory bulb (OB) and begin the process of reinnervation. Spontaneous activity of mitral/tufted cells occurs in the OB during the first weeks after transection but no odor-induced activity is observed. After 3-4 weeks glomerular responses were observed in some animals upon application of stimulus, but the response and glomerular morphology are clearly altered as compared to control. After 6-7 weeks responses seem to have fully recovered, indicating that the OS of larval X. laevis recovers morphologically and functionally 6-7 weeks after ON transection.
O estudo dos mecanismos responsáveis pela neuro-regeneração tem um marcado interesse para a compreensão dos princípios básicos que governam as interações celulares e moleculares no sistema nervoso, bem como um interesse clínico relevante. A limitada capacidade do sistema nervoso central para dar origem a novos neurónios é um obstáculo formidável para a recuperação do sistema após lesão neuronal ou doença neurodegenerativa. O sistema olfativo é um sistema ideal para o estudo do processo de recuperação após lesão neuronal, pois é conhecido no mundo científico pela sua capacidade contínua e vitalícia para repor células perdidas durante a renovação celular natural, bem como a sua notável capacidade para regenerar após uma lesão grave. O epitélio olfativo apresenta a capacidade para dar origem a novos neurónios ao longo de toda a vida. Neurónios sensoriais olfativos diferenciados são continuamente reintegrados num circuito já existente, mantendo assim o sentido do olfato. O objetivo desta tese é descrever as alterações morfológicas e funcionais que ocorrem ao longo do tempo no sistema olfativo de Xenopus laevis em estado larvar, após o corte do nervo olfativo. Os resultados obtidos através do uso de ensaios de imunohistoquímica, bem como técnicas de marcação neuronal sensorial e de imagiologia de cálcio, indicam que a morte celular na população de neurónios sensoriais olfativos atinge o seu máximo 48 horas após a lesão, e que células estaminais encontradas na camada basal do epitélio olfativo são positivamente reguladas após lesão e proliferam rapidamente. Células de suporte parecem manter tanto a integridade morfológica como funcional após o corte do nervo olfativo. O epitélio olfativo recupera a sua estrutura morfológica inicial 1 semana após a lesão, momento em que os primeiros axónios atingem o bolbo olfativo e começam o processo de reintegração. Ocorre atividade espontânea das células mitrais/tufados do bolbo olfativo durante as primeiras semanas após a lesão, mas nenhuma atividade induzida por estímulo com odor foi observada. Depois de 3-4 semanas, atividade glomerular foi observada em alguns animais após a aplicação de estímulos, mas a resposta e morfologia glomerular foram claramente alteradas em relação ao controlo. Depois de 6-7 semanas as respostas parecem ter recuperado totalmente, indicando que o sistema olfativo de X. laevis em estado larvar recupera morfológica e funcionalmente 6-7 semanas após o corte do nervo olfativo.

The olfactory mucosa has been shown to be a site where continual regeneration takes place throughout adulthood, making this system relatively unique with great potential in respect to autologous cell graft therapies. The olfactory mucosa is readily accessible with no deleterious effects on olfaction if biopsied. The presence of olfactory ensheathing cells (OECs), as well as a stem cell to facilitate the regeneration of this tissue, has been previously described. The first aim of this study was to further characterise olfactory ensheathing cells in rat and human and to gain some insights into their biology in vitro and in vivo, with the hope of applying this knowledge to clinical therapies employing autologous cell grafts. The second aim was to isolate stem cells in the form of neurospheres from the adult olfactory lamina propria, to elucidate their differentiation potential into both neural and non neural lineages, and to subsequently graft these cells into animal models of spinal cord injury to assess their behaviour in vivo in regards to survival, proliferation, and integration into the host spinal cord. Recovery of locomotor function due to axon regeneration across the lesion site following grafting was also assessed. Glial cells isolated from the adult olfactory lamina propria were identified as OECs in vitro and in vivo using the immunological markers GFAP, S100, and p75NTR. Neurotrophin growth factors were shown to promote their proliferation and purification in vitro from both rat and human. Immunocytochemistry and PCR were used to confirm the expression of neurotrophin trk receptors A, B, and C, as well as NT3, NGF and BDNF by OECs in rat and human. NT3 was shown to purify adult human OECs in vitro. Through the use of EGF and FGF2, it was shown that the olfactory lamina was capable of generating neurospheres consisting of a heterogeneous cell population, containing fully differentiated cells, progenitor cells, as well as undifferentiated stem cells indentified by the markers nestin and c-kit. Using growth factors, single or in combination, as well as tissue conditioned medium, olfactory stem cells within these neurospheres were induced to differentiate into the neural lineage, namely neurons, astrocytes, and oligodendrocytes. Non neural differentiation into liver, skeletal muscle, and cardiac cells was induced in vitro by cell-cell contact between neurospheres and frozen rat tissue sections.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Eskitis Institute for Cell and Molecular Therapies
Science, Environment, Engineering and Technology
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Precursores neurais originados na zona subventricular (ZSV) de algumas espécies animais possuem uma rota de migração neuronal destinada ao bulbo olfatório principal (BOP), onde os neuroblastos migrantes se diferenciam em interneurônios. Esta corrente migratória é mantida na idade adulta. A compreensão de como se organiza na idade fetal é essencial para a compreensão geral e estabelecimento de novas terapias celulares. O objetivo deste estudo é caracterizar a composição celular e organização morfológica da ZSV e da corrente rostral migratória (CRM) em encéfalos de fetos caninos. A ZSV, CRM e BOP foram obtidos de fetos caninos de aproximadamente 57 dias de idade gestacional. O tecido foi analisado através de coloração de Nissl, método de imunohistoquímica de dupla marcação com duplacortina (DCX), fator de transcrição SOX2, proteína glial fibrilar ácida (GFAP), calbindina (CALB), calretinina (CALR) e tirosina-hidroxilase (TH). Foram feitas a análise relativa da expressão da imunorreatividade e análise quantitativa de colocalização celular, além do método de microscopia eletrônica de transmissão. Os resultados mostram que a ZSV dorsal possui células imunorreativas (ir) para o DCX ao longo da parede ventricular, dispostas tangencialmente e fileiras de células SOX2-ir foram encontradas na mesma orientação. A imunorreatividade de GFAP foi mais forte na ZSV dorsal e as células possuem fibras dirigidas tangencialmente adjacentes ao ventrículo lateral e fibras orientadas radialmente em direção ao córtex. A CRM de feto de cão tem início na ZSV anterior e segue caudalmente ao redor da cabeça do núcleo caudado e desce na vertical até se curvar rostralmente em direção ao BOP onde termina na camada de células granulares (CCG). A CRM tem aparência homogênea e densa e possui células positivas para o DCX nas porções iniciais e para SOX2 e GFAP por toda a extensão. Não houve células positivas para CALB, CALR e TH em nenhuma região da ZSV e CRM. No BOP, os resultados mostraram que a camada glomerular (CG) possui células imunorreativas a CALR, TH, SOX2 e GFAP. Na camada plexiforme externa (CPE) houve células imunorreativas a CALB, CALR, SOX2 e GFAP e na CCG, houve células imunorreativas a CALR, SOX2 e GFAP. Na análise de colocalização, foram encontrados na CG neurônios CALR que colocalizam com células SOX2 e uma baixa colocalização de neurônios TH e células SOX2. Na CPE, foi observado um baixo número de colocalização de neurônios CALR e CALB e na CCG, as células SOX2 colocalizam com os neurônios CALR. As conclusões mostram que o feto de cão possui uma CRM em direção BOP, com imunorreatividade celular para DCX, SOX2 e GFAP na ZSV e CRM e para CALB, CALR, TH, SOX2 e GFAP nas principais camadas do BOP
Neural precursors originated in the subventricular zone (SVZ) of some animal species have a migration route destined for main olfactory bulb (MOB), where migrants neuroblasts differentiate into olfactory interneurons. This migratory stream is maintained in adulthood. Understanding how it is organized in fetal age is essential for general understanding and establishment of new cell therapies. The aim of this study is characterize the cellular composition and morphological organization of the SVZ and rostral migratory stream (RMS) of brains of canine fetuses. The SVZ, RMS and MOB was obtained from canine fetuses of the approximately 57 gestacional days-old. The tissue was analyzed by Nissl staining and by immunohistochemical methods for double labelling with doublecortin (DCX), transcription factor SOX2, glial fibrillary acid protein (GFAP), calbindin (CALB), calretinin (CALR) and tyrosinehydroxylase (TH). Semiquantitative analysis of immunoreactivity and quantitative analysis of colocalization were realized, besides ultrastructural analysis by electron microscopy. The results show that in dorsal SVZ, DCX immunoreactive cells were found along the ventricular wall, arranged tangentially and lines of SOX2 cells were also found in the same orientation. The GFAP immunostaining is stronger in dorsal SVZ with tangentially directed fibers near the lateral ventricle and radially oriented fibers toward the cortex. The RMS of dog fetus begins at anterior SVZ and follows caudally around the head of the caudate nucleus and vertically descends to bend rostrally into the MOB, where it ends in the granular cell layer (GCL).The RMS have SOX2 positive cells on entire length, showing a homogeneous appearance and high cell density. There is no positive CALB cells or CALR in any region of the SVZ and RMS. The results of the MOB show that the glomerular layer (GL) there were cells immunoreactive to CALR, TH, SOX2 and GFAP. In the external plexiforme layer (EPL) there were immunoreactive cells for CALR, CALB, SOX2 and GFAP and, the GCL, the prevalence is higher for CALR neurons, SOX2-ir and GFAP-ir cells. In colocalization analysis, they were found a some CALR positive neurons in GL that colabeled with SOX2 cells and a low colocalization of TH neurons and SOX2 cells. In EPL, was observed a low colocalization number of CALR and CALB neurons and in GCL, SOX2 cells colabeled with CALR neurons. The conclusions show that the dog fetus has a RMS directed to the MOB, with cellular immunoreactivity for DCX, SOX2 and GFAP in the ZSV and RMS and cellular immunoreactivity for SOX2 CALB, CALR, TH and GFAP in main olfactory bulb layers

As células-tronco do epitélio olfatório possuem a capacidade de diferenciação, regeneração de neurônios olfatórios e atuam no processo de mielinização das fibras nervosas. O objetivo desse trabalho foi verificar o potencial terapêutico de células-tronco do epitélio olfatório de ratos Wistar em terapia celular em coelhos submetidos à lesão medular. Foram utilizados ratos com idade de 2 meses e coelhos da raça nova Zelândia obtidos no Biotério do Departamento de Patologia da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. Os fragmentos do epitélio olfatório dos ratos foram tratados e colocadas em cultivo em DMEM-F12 suplementado. Foram testadas nos coelhos nova Zelândia 4 tipos de lesão medular, hemisecção dorsal e ventral e secção lateral e total. Foi realizado teste de potencial teratogênico em camundongos NUDE e para terapia foram transplantadas 5x105 células em 4 coelhos submetidos a lesão medular ventral. A morfologia predominante das células em cultivo era fibroblastóide. Os ensaios de curva de crescimento e tendência a formação de colônia demonstraram que as condições de cultivo destas células estavam adequadas. Foi encontrada positividade para Vimentina, Oct-4, GFAP, OMP, Nanog, Citoqueratina-18 e Beta tubulina em imunofluorescência. Em citometria de fluxo foi encontrada negatividade para CD 113, CD 117 e Stro-1 e positividade para Vimentina, Nanog e OMP. Em análise por imunohistoquíca verificou-se marcação positiva para Vimentina, OMP, GFAP e Nanog. Verificou-se marcação positiva no material coletado por retrovírus GFP. Houve melhora clínica nos animais avaliados 21 dias após o transplante de células com retorno de alguns reflexos como o de propriocepção consciente e colocação tátil, o mesmo apresentou ainda reflexo de pedalagem. Conclui-se que o melhor modelo para indução da lesão medular em coelhos é a hemisecção ventral da medula e que as células-tronco olfatórias de ratos Wistar possuem grande potencial terapêutico em animais submetidos à lesão medular.
Stem cells from olfactory epithelium are capable of differentiation, regeneration of olfactory neurons and act in the myelination process of nerve fibers. The aim of this study was to verify the therapeutic potential of stem cells from the olfactory epithelium of Wistar rats in cell therapy in rabbits subjected to spinal cord injury. Rats were aged 2 months and New Zealand rabbits obtained in the bioterio of the Animal Pathology Department, Faculty of Veterinary Medicine, at University of Sao Paulo. Samples of the olfactory epithelium of rats were treated and placed in culture in DMEM-F12 supplemented. Four types of spinal cord injury were tested in New Zealand rabbits: hemisecction dorsal and ventral, total and lateral section. The teratogenic potential test was made in NUDE mices. For therapy were transplanted 5x105 cells in 4 rabbits subjected to spinal cord injury in the ventral region. The cells showed predominant fibroblastoid morphology. Tests for growth curve and colony formation demonstrated that culture conditions in these cells were suitable for the development of these cells. In the immunofluorescence analyses the cells showed positive reaction for vimentin, Oct-4, GFAP, OMP, Nanog, cytokeratin-18 and beta tubulin. In flow cytometry analyses was found negative reaction for CD 113, CD 117 and Stro-1 and positive reaction for vimentin, Nanog and OMP. In immunohistochemistry analysis were observed positive reaction for vimentin, OMP, GFAP and Nanog. Positive labeling was showed on the collected material for GFP. Clinical improvement occurred in the animals evaluated 21 days after cell transplantation. With some reflections about how the placement of conscious proprioception and tactile, it also presented a reflection of pedaling. After this, we concluded that the better model for induction of spinal cord injury in rabbits is the hemisecction ventral and the olfactory stem cells of Wistar rats showed a great therapeutic potential in animals subjected to spinal cord injury.

Neuronal differentiation encompasses an elaborate developmental program which until recently was difficult to study in vitro. The advent of several cell lines able to differentiate in culture proved to be the turning point for gaining an understanding of molecular neuroscience. In particular the olfactory epithelium provides an attractive tool with which to investigate fundamental questions relating to neuronal differentiation, as it displays a unique capacity to regenerate and to retain a neurogenetic potential from its genesis and throughout adult life. The coordinated regulation of gene expression is fundamental to the control of neuronal differentiation. In order to reveal active processes at the molecular level and to dissect key components of molecular pathways, differential gene expression studies provide a foundation for the elucidation of dynamic molecular mechanisms. This thesis identified genes involved in neuronal differentiation by utilising a clonal olfactory receptor neuronal cell line (OLF442). Gene expression levels were identified using differential display and oligonucleotide array technology before and after serum deprivation. Differential display revealed two kinases whose expression levels were elevated during the differentiation of OLF442, identified as focal adhesion kinase (FAK) related non-kinase (FRNK) and mammalian ste20 like (MST)2 kinase. Furthermore, analysis of the oligonucleotide array data confirmed the expression of genes involved in altering presentation of extracellular matrix molecules, in mediating cytoskeletal rearrangements, and in ceasing the cell cycle, supporting the use of OLF442 as a model for studying differentiation. The differentiation of OLF442 results from the synchronisation of multiple transduction cascades and cellular responses as evidenced by the microarray data. A protein that can synchronise such signalling is the non-receptor protein tyrosine kinase, FAK. Thus the finding of the endogenous FAK inhibitor FRNK by differential display was intriguing as there was no difference in the expression level of FAK induced by differentiation, contrasting that of FRNK. This induced FRNK expression was derived autonomously as it was not responsive to the caspase-3 inhibitor, DEVD-CHO. This is particularly pertinent since the primary role of FRNK is to act as an inhibitor of FAK by competing with its substrates and reducing the phosphorylation of both FAK and its associated proteins. Differential display also revealed the upregulation of another kinase, which had 90% homology with rat MST2 kinase within the 3' UTR. Both mouse MST2 kinase (sequence submitted to GenBank, accession number AY058922) and the closely related family member MST1 kinase were sequenced and cloned. Moreover, evidence to support an autonomously expressed carboxyl-terminal domain of MST2 kinase is presented in Chapter 3 and provides a unique way in which MST2 may regulate its own activity. To further understand the role of MST in neuronal differentiation, a series of stable OLF442 transfections (with mutant and wild-type MST constructs) were carried out. MST was localised with cytoplasmic structures that may represent actin stress fibres, indicating a potential cytoskeletal role during neuronal differentiation. This indicated that MST1 may play a role in the morphological processes involved in neuronal differentiation. The identification of two kinases by differential display provided the motivation to understand the cellular context of OLF442 and to determine the phosphorylation status of the mitogen-activated protein kinase (MAPK) signalling cascades. Differentiation of OLF442 induced high-level phosphorylation of a putative B-Raf isoform, MEK2 and ERK1/2. Interestingly, there was a switch between preferential phosphorylation of MEK1 in undifferentiated OLF442 to preferential phosphorylation of MEK2 following differentiation. SAPK/JNK was also phosphorylated, as was the transcription factor c-Jun, which is a common substrate of both the ERK and SAPK/JNK signalling modules. The mapping of the cellular context of differentiating OLF442 has identified a promising model of a novel MAPK module. This consists of FAK signalling through Rap1 to ERK providing sustained activation, which is buffered or terminated by the expression of the endogenous FAK inhibitor FRNK. Furthermore, MST kinase could potentially play a role in regulating the cytoskeletal re-arrangements that are necessary for neuronal differentiation. MST kinase may signal transiently via the SAPK pathway to provide concomitant activation of c-Jun that is required for neuronal differentiation. An understanding of the gene expression pattern of the normal neuronal differentiation program allows a greater understanding of potential developmental aberrations. This could provide an opportunity for therapies to be conceived, while understanding the complexity of neuronal determination could also provide opportunities for stem cell transplantation.

Neuronal differentiation encompasses an elaborate developmental program which until recently was difficult to study in vitro. The advent of several cell lines able to differentiate in culture proved to be the turning point for gaining an understanding of molecular neuroscience. In particular the olfactory epithelium provides an attractive tool with which to investigate fundamental questions relating to neuronal differentiation, as it displays a unique capacity to regenerate and to retain a neurogenetic potential from its genesis and throughout adult life. The coordinated regulation of gene expression is fundamental to the control of neuronal differentiation. In order to reveal active processes at the molecular level and to dissect key components of molecular pathways, differential gene expression studies provide a foundation for the elucidation of dynamic molecular mechanisms. This thesis identified genes involved in neuronal differentiation by utilising a clonal olfactory receptor neuronal cell line (OLF442). Gene expression levels were identified using differential display and oligonucleotide array technology before and after serum deprivation. Differential display revealed two kinases whose expression levels were elevated during the differentiation of OLF442, identified as focal adhesion kinase (FAK) related non-kinase (FRNK) and mammalian ste20 like (MST)2 kinase. Furthermore, analysis of the oligonucleotide array data confirmed the expression of genes involved in altering presentation of extracellular matrix molecules, in mediating cytoskeletal rearrangements, and in ceasing the cell cycle, supporting the use of OLF442 as a model for studying differentiation. The differentiation of OLF442 results from the synchronisation of multiple transduction cascades and cellular responses as evidenced by the microarray data. A protein that can synchronise such signalling is the non-receptor protein tyrosine kinase, FAK. Thus the finding of the endogenous FAK inhibitor FRNK by differential display was intriguing as there was no difference in the expression level of FAK induced by differentiation, contrasting that of FRNK. This induced FRNK expression was derived autonomously as it was not responsive to the caspase-3 inhibitor, DEVD-CHO. This is particularly pertinent since the primary role of FRNK is to act as an inhibitor of FAK by competing with its substrates and reducing the phosphorylation of both FAK and its associated proteins. Differential display also revealed the upregulation of another kinase, which had 90% homology with rat MST2 kinase within the 3 ΠUTR. Both mouse MST2 kinase (sequence submitted to GenBank, accession number AY058922) and the closely related family member MST1 kinase were sequenced and cloned. Moreover, evidence to support an autonomously expressed carboxyl-terminal domain of MST2 kinase is presented in Chapter 3 and provides a unique way in which MST2 may regulate its own activity. To further understand the role of MST in neuronal differentiation, a series of stable OLF442 transfections (with mutant and wild-type MST constructs) were carried out. MST was localised with cytoplasmic structures that may represent actin stress fibres, indicating a potential cytoskeletal role during neuronal differentiation. This indicated that MST1 may play a role in the morphological processes involved in neuronal differentiation. The identification of two kinases by differential display provided the motivation to understand the cellular context of OLF442 and to determine the phosphorylation status of the mitogen-activated protein kinase (MAPK) signalling cascades. Differentiation of OLF442 induced high-level phosphorylation of a putative B-Raf isoform, MEK2 and ERK1/2. Interestingly, there was a switch between preferential phosphorylation of MEK1 in undifferentiated OLF442 to preferential phosphorylation of MEK2 following differentiation. SAPK/JNK was also phosphorylated, as was the transcription factor c-Jun, which is a common substrate of both the ERK and SAPK/JNK signalling modules. The mapping of the cellular context of differentiating OLF442 has identified a promising model of a novel MAPK module. This consists of FAK signalling through Rap1 to ERK providing sustained activation, which is buffered or terminated by the expression of the endogenous FAK inhibitor FRNK. Furthermore, MST kinase could potentially play a role in regulating the cytoskeletal re-arrangements that are necessary for neuronal differentiation. MST kinase may signal transiently via the SAPK pathway to provide concomitant activation of c-Jun that is required for neuronal differentiation. An understanding of the gene expression pattern of the normal neuronal differentiation program allows a greater understanding of potential developmental aberrations. This could provide an opportunity for therapies to be conceived, while understanding the complexity of neuronal determination could also provide opportunities for stem cell transplantation
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text

Les lésions médullaires traumatiques (LMT) conduisent à une atteinte des voies nerveuses sensitives et motrices. Leur taux de mortalité reste très élevé, d'où la nécessité de trouver de nouveaux traitements. Les Cellules Gliales Olfactives (CGOs) représentent un candidat intéressant de par leur fonction au sein du système olfactif primaire. La découverte d'une population de cellule souche neurale bordant le canal central de la moelle spinale (MS) adulte, appelées cellules épendymaires, suscite un nouvel espoir dans le domaine des biothérapies. Ce travail de thèse a permis d'étudier l'effet d'une transplantation de CGOs sue le comportement des cellules résidentes de la moelle spinale et notamment les cellules souches épendymaires qui, en association avec les astrocytes et les péricytes, participent aux mécanismes de guérison des LMT. L'utilisation du modèle murin hFoxJ1-CreERT2::YFP (permettant le suivi spécifique des cellules épendymaires et de leur progénie), a montré que les CGOs augmentaient in vitro le potentiel d'auto-renouvellement des cellules souches de la MS et modifiaient leur voie de différenciation vers un type neural. In vivo, la transplantation de CGOs augmente la prolifération des cellules épendymaires ainsi que leur différenciation en astrocytes hypo-réactifs conduisant à la formation d'un environnement post-lésionnel bénéfique à la survie neuronal et l'établissement d'une neurogenèse. Nos travaux ont montré pour la première fois que la transplantation de CGOs après LMT permettait la génération de nouveaux neurones. Ceci constitue un nouvel espoir dans l'établissement de stratégies thérapeutiques pour le traitement des LMT chez l'Homme
The spinal cord injuries (SCI) lead to the damages of the spinal cord or nerves and often cause permanent changes in body functions leading to the death. Cell therapies have raised great hope for regenerative medicine. Clinical data showed that the olfactory ensheathing cells (OECs) enhanced functional recovery after SCI and could be a very attractive therapeutic approach. Moreover, the discovery of a new endogenous resident stem cell population, lining the central canal of the spinal cord, named ependymal stem cells, represents a new hope for the therapy. This thesis analyzed the role of OECs transplantation, on the behaviour of ependymal stem cells since these cells, together with astrocytes and pericytes significantly contribute to the recovery of SCI. The use of the mouse model hFoxJ1-CreERT2::YFP (allowing to specifically follow the ependymal stem cells ant their progeny) showed that OECs increased in vitro the self-renewal potential of spinal cord stem cells and modified their differentiation pathway towards a neural type. In vivo, OECs transplantation significantly increases the proliferation of ependymal cells and their differenciation into hypo-reactive astrocytes leading to the formation of a beneficial environment to neuronal survival and the neurogenesis establishment. Our results also showed for the first time that OECs transplantation after SCI allows the generation of new neurons by non-ependymal cell-derived progenitors. These results represent a new hope in the establishment of therapeutic strategies for the treatment of SCI in humans

La dysautonomie familiale (FD) est une neuropathie héréditaire provoquée par des mutations au sein du gène IKBKAP, la plus commune d'entre elles induisant un épissage alternatif de l'exon 20 au sein de du pré-ARNm de façon tissu-spécifique. L'épissage aberrant est particulièrement prononcé dans les tissus nerveux, conduisant à la dégénerescence progressive des neurones sensoriels et autonomes. La spécificité de la perte des cellules nerveuses dans la FD est mal comprise, par manque d'un modèle approprié. Afin de mieux comprendre les mécanismes moléculaires de l'épissage des ARNm d'IKBKAP, nous avons utilisé un modèle original : les cellules souches olfactives ecto-mesenchymateuses (hOE-MSC) de patients FD. Les hOE-MSC sont pluripotentes et ont la capacité de se différencier en diverses lignées cellulaires, y compris les neurones et les cellules gliales.Nous avons confirmé la présence du transcrit exempt de l'exon 20 d'IKBKAP dans les hOE-MSC de FD et nous avons observé une expression significativement inférieure de la somme des transcrits IKBKAP chez ces patients, du fait de la dégradation d'une partie des isoforme aberrants. Cette réduction est correlée avec une réduction d'expression de la protéine traduite à partir du transcrit d’IKBKAP possèdant l’exon 20, IKAP/hELP1. Nous avons localisé IKAP/hELP1 dans différents compartiments cellulaires, y compris le noyau, ce qui soutient des rôles multiples de cette protéine. Nous avons confirmé que la kinétine, une cytokinine, améliorait le taux de transcrit incluant l'exon 20 et rétablissait des niveaux normaux d'IKAP/hELP1 dans les hOE-MSC de FD. Par ailleurs, nous avons pu modifier le rapport d'épissage d'IKBKAP en augmentant ou en réduisant le ratio WT (inclusion de l'exon 20) : MU (saut de l'exon 20) respectivement, en produisant des sphères flottantes, ou en engageant les cellules vers une différentiation neurale. Les sphères et les cellules différenciées ont été étudiées au niveau pan-génomique, ce qui a permis d'identifier le développement du système nerveux comme étant le processus le plus affecté chez les FD. De plus, nous soulignons le rôle de la kinétine comme un probable régulateur de facteurs d'épissage contribuant à la restauration d'un épissage correct d'IKBKAP.Les hOE-MSC isolées de patients FD représentent une nouvelle approche pour modéliser la pathologie et mieux comprendre l'expression génétique et les approches thérapeutiques possibles de la FD. En outre, elles offrent une application originale à la compréhension d'autres maladies génétiques neurologiques
Familial dysautonomia (FD) is a hereditary neuropathy caused by mutations in the IKBKAP gene, the most common of which results in variable tissue-specific mRNA splicing with skipping of exon 20. Defective splicing is especially severe in nervous tissue, leading to incomplete development and progressive degeneration of sensory and autonomic neurons. The specificity of neuron loss in FD is poorly understood due to the lack of an appropriate model system. To better understand and modelize the molecular mechanisms of IKBKAP mRNA splicing, we collected human olfactory ecto-mesenchymal stem cells (hOE-MSCs) from FD patients. hOE-MSCs have a pluripotent ability to differentiate into various cell lineages, including neurons and glial cells.We confirmed IKBKAP mRNA alternative splicing in FD hOE-MSCs and observed a significant lower expression of both IKBKAP transcripts and IKAP/hELP1 protein in FD cells resulting from the degradation of the transcript isoform skipping exon 20. We localized IKAP/hELP1 in different cell compartments, including the nucleus, which supports multiple roles for that protein. Moreover, we showed that kinetin improved exon 20 inclusion and restores a normal level of IKAP/hELP1 in FD hOE-MSCs. Furthermore, we were able to modify the IKBKAP splicing ratio in FD hOE-MSCs, increasing or reducing the WT (exon 20 inclusion):MU (exon 20 skipping) ratio respectively, either by producing free-floating spheres, or by inducing cells into neural differentiation. Spheres forming cells and lineage neuroglial progenitors were investigated at the genome-wide level, and we confirmed that nervous system development was the most altered process in FD. More, we highlight kinetin role as a putative regulator of splicing factors which contribute to restore a correct splicing of IKBKAP.hOE-MSCs isolated from FD patients represent a new approach for modeling FD to better understand genetic expression and possible therapeutic approaches. This model could also be applied to other neurological genetic diseases

Les lésions de la moelle spinale constituent un problème de santé public d’une ampleur grandissante. Bien que l’espérance de vie ait été améliorée, les patients médullo-lésés souffrent de certains handicaps entraînant une perte partielle ou complète des fonctions sensorielles et/ou motrices. La moelle spinale lésée entreprend aussitôt une réponse à cette lésion. Chronologiquement, la lésion se divise en deux grandes phases : la phase primaire qui se caractérise par la destruction tissulaire induite par le traumatisme mécanique, suivie d’une destruction cellulaire. Alors que la phase secondaire est la conséquence moléculaire et cellulaire de la phase primaire. Durant plusieurs années, différentes stratégies thérapeutiques ont été proposé principalement la thérapie cellulaire qui a prouvé ses effets bénéfiques dans différents modèles expérimentaux de la lésion , mais de nombreux obstacles sont à prendre en considération tel que, principalement, son caractère invasif. afin de pouvoir l’appliquer chez l’homme d’une manière efficace et reproductible . A la vue de ces contraintes cliniques, nous avons décidé d’explorer un traitement non invasif connu pour ses effets neuroprotecteurs et neurotrophiques dans le SNC ; la stimulation magnétique répétitive trans-spinale (rTSMS). Etonnement, peu d’études ont exploré cette thérapie dans le cadre des LMTs, et rare sont celles qui l’ont utilisé d’une manière focale, c’est à dire directement au niveau du site de la lésion. A ce jour, les mécanismes et les voies sous-jacentes de ces effets dans ce cadre restent toujours inconnus. C’est pourquoi nous avons entrepris de caractériser ces effets dans le cadre de mes travaux de Thèse. En effet, en premier lieu, nous avons évalué les effets de la rTSMS sur la réparation tissulaire, via la modulation de la cicatrice médullaire et de ces différentes composantes in vivo, ainsi que sur la récupération fonctionnelle dans différents paradigmes (aigue et chronique) et à différents âges (juvénile, adulte et vieux) chez des souris WT ayant subi une transsection complète de la moelle spinale. En second lieu, l’objectif était de décrire les mécanismes à l’origine des effets de la rTSMS. Pour ce faire, des analyses protéomiques ont été réalisées, puis nous avons évalué l’effet de la rTSMS sur la réactivité des cellules souches endogènes de la moelle, ainsi que, la contribution de ces dernières dans la mise en place de la cicatrice gliale in vitro et in vivo via un modèle de souris transgénique hFoxJ1-CreER T2 ::tdTomato. L’objectif global était d’étudier, pour la première fois, l’effet de la rTSMS sur la réponse des différentes composantes cellulaires résidentes de la moelle spinale, les mécanismes à l’origine de ces effets, ainsi que la capacité à restaurer les fonctions motrices perdues suite à la lésion médullaire
Spinal cord injury (SCI) leads to a loss of sensitive and motor functions. Currently, there is no therapeutic intervention offering a complete recovery. Here, we report that repetitive trans-spinal magnetic stimulation (rTSMS) can be a noninvasive SCI treatment that enhances tissue repair and functional recovery. Several techniques including immunohistochemical, behavioral, cells cultures, and proteomics have been performed. Moreover, different lesion paradigms, such as acute and chronic phase following SCI in wild-type and transgenic animals at different ages (juvenile, adult, and aged), have been used. We demonstrate that rTSMS modulates the lesion scar by decreasing fibrosis and inflammation and increases proliferation of spinal cord stem cells. Our results demonstrate also that rTSMS decreases demyelination, which contributes to axonal regrowth, neuronal survival, and locomotor recovery after SCI. This research provides evidence that rTSMS induces therapeutic effects in a preclinical rodent model and suggests possible translation to clinical application in humans

Les faibles capacités régénératives intrinsèques du système nerveux central, après la survenue de lésions traumatiques ou l'apparition de maladies neuro-dégénératives, ont orienté les recherches vers des thérapies basées sur l'utilisation de cellules souches dans le but de régénérer le tissu cérébral. Cependant, des limitations éthiques et techniques associées aux cellules souches embryonnaires, fœtales ou neurales chez l'adulte restreignent leur utilisation en clinique humaine. À la recherche d'une source alternative, nous nous sommes intéressés à des cellules souches adultes peu connues, provenant du chorion d'un tissu nerveux périphérique en perpétuel renouvellement : les cellules souches de la lamina propria olfactive, localisée dans la cavité nasale. Ces cellules multipotentes ont été décrites comme un sous-type de cellules souches mésenchymateuses, présentant de fortes capacités prolifératives et neurogéniques. En utilisant un premier modèle murin d'amnésie induit par lésion excito-toxique des hippocampes, nous avons montré que des cellules souches olfactives humaines, greffées dans les zones lésées ou dans le liquide céphalo-rachidien, i) s'installent et adoptent un phénotype neuronal, ii) rétablissent la circulation d'informations au sein des réseaux neuronaux défectueux et iii) permettent une récupération des capacités d'apprentissage et de mémorisation. Suite à ces résultats très encourageants, le premier objectif de cette thèse a été de mieux faire connaître ces cellules souches adultes auprès de la communauté scientifique
The brain displaying poor regenerative capacities, exogenous stem cell-based therapy has been proposed as an attractive strategy to regenerate cerebral tissue after acute injuries or neurodegenerative disorders. However, ethical and technical issues, associated with embryonic, fetal or adult neural stem cells, limit their use in human medicine. In search of alternative candidates, we focused our attention on adult stem cells, located in a peripheral nervous tissue: the nasal stem cells sited in the olfactory lamina propria. These multipotent stem cells have been characterized as a member of the mesenchymal stem cell superfamily, displaying strong proliferative and neurogenic properties. Recently, using a mouse model of amnesia induced by excito-toxic lesions of hippocampal neurons, we demonstrated that olfactory stem cells, grafted in lesioned areas or into the cerebrospinal fluid, i) migrate and differentiate into neuron-like cells, ii) contribute to the restoration of local neuronal networks and iii) promote recovery of learning and memory abilities. In line with these promising results, the first aim of the current thesis was to promote the use of these adult stem cells by the scientific community. For this purpose, we published an article and a book chapter in which we demonstrated that they are suitable for autologous cell therapy in humans. Using an audiovisual document, we showed that these cells i) can be safely obtained in humans, under local anesthesia, without any loss of smell and ii) are easily and quickly amplifiable in vitro

La terapia mediante l’uso di cellule staminali allogeniche è una potenziale alternativa per la cura di malattie infiammatorie e degenerative. Vari gruppi di ricerca hanno messo in luce le proprietà immunomodulatorie di varie cellule staminali, come per esempio le cellule mesenchimali stromali, ma al momento non è stato ancora fatto uno studio comparativo mostrante differenze qualitative e quantitative delle proprietà immunoregolatorie di staminali di diversa origine. Lo scopo del lavoro è stato quindi quello di confrontare le proprietà immunologiche di varie cellule staminali, tra cui cellule mesenchimali stromali di origine midollare, cellule staminali olfattorie, cellule staminali isolate da leptomeningi, e tre tipi di cellule staminali esprimenti il recettore c-Kit, ovvero cellule staminali del fluido amniotico, cellule staminali cardiache e cellule staminali polmonari. Tutte queste tipologie cellulari mostravano caratteristiche immunologiche comuni, come per esempio l’espressione dei marcatori attivatori ICAM-1, VCAM-1, HLA-ABC, e HLA-DR, e quando coltivate con linfociti T, NK e B purificati, erano in grado di regolarne la proliferazione. Il trattamento con citochine infiammatorie riduceva l’immunogenicità delle staminali analizzate nei confronti di linfociti NK rispetto alle cellule controllo, e induceva l’espressione dell’indolamina-2,3 diossigenasi (IDO) responsabile dell’immunosoppressione dei linfociti T. Infine, tutte le cellule staminali analizzate mostravano un effetto anti-apoptotico nei confronti delle cellule effettrici immuni non stimolate. Inoltre, in questo lavoro abbiamo mostrato che l’effetto immunosoppressivo non è una proprietà costitutiva delle cellule staminali, ma è una conseguenza dell’induzione mediata da citochine infiammatorie, effetto precedente mostrato per le cellule mesenchimali stromali. L’immunoregolazione è una proprietà condivisa da vari tipi di cellule staminali, e una caratterizzazione più approfondita di questo meccanismo potrebbe essere fondamentale per il loro uso terapeutico.
Allogeneic stem cell (SC)-based therapy is a promising tool for the treatment of a range of human degenerative and inflammatory diseases. Many reports highlighted the immune modulatory properties of some SC types, such as mesenchymal stromal cells (MSCs), but a comparative study with SCs of different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods employed for MSC characterization to compare the immunological properties of bone marrow (BM)-MSCs, olfactory ectomesenchymal SCs (OE-MSCs), leptomeningeal SCs (LeSCs), and three different c-Kit-positive SC types, that is, amniotic fluid SCs (AFSCs), cardiac SCs (CSCs), and lung SCs (LSCs). We found that all the analyzed human SCs share a common pattern of immunological features, in terms of expression of activation markers ICAM-1, VCAM-1, HLA-ABC, and HLA-DR, modulatory activity toward purified T, B, and NK cells, lower immunogenicity of inflammatory-primed SCs as compared to resting SCs, and indoleamine-2,3-dioxygenase (IDO)-activation as molecular inhibitory pathways, with some SC type-related peculiarities. Moreover, the SC types analyzed exert an anti- apoptotic effect toward not-activated immune effector cells (IECs). In addition, we found that the inhibitory behavior is not a constitutive property of SCs, but is acquired as a consequence of IEC activation, as previously described for MSCs. Thus, immune regulation is a general property of SCs and the characterization of this phenomenon may be useful for a proper therapeutic use of SCs.

In the olfactory epithelium (OE), new olfactory receptor neurons (ORNS) are continually generated throughout mammalian adulthood. Given this substantial neuronal turnover, a stem cell is proposed to reside within the basal compartment of the OE, which generates ORNs on demand when stimulated by changes in its microenvironment. Although previous studies have identified possible candidates for the olfactory stem cell, its exact identity is as yet unknown. We hypothesize that a population o f horizontal basal cells (HBCs), situated upon the basement membrane of the OE, contains stem cells that contribute to olfactory neurogenesis. A major impediment to the study of these cells is the lack of reliable cell surface molecular markers to distinguish them from other OE cell types. By screening a panel of selected clusters of differentiation (CD) antigens, we have identified three new cell surface markers for the HBC population, namely intercellular adhesion molecule -1 (ICAM-1), β₁ integrin and β₄ integrin. Using these markers to characterize the HBC layer following bulbectomy-induced ORN loss, we have provided evidence of stem cell traits in vivo, including proliferative quiescence relative to OE progenitors, response to lesion, and possible molecular heterogeneity within the HBC compartment. In addition, these studies indicate changes in the populational and subcellular distribution of HBC markers upon loss of ORNs, suggesting a role for these adhesion receptors in the regulation of HBC function in addition to highlighting possible molecular similarities to stem cells of other self-renewing tissues. We have developed a method to select for HBCs in vitro using magnetic activated cell sorting (MACS) and by exploiting their expression of ICAM-1. Using in vitro colony-forming analyses, we obtained evidence that the ICAM-1+ population is enriched for progenitor activity. Further, the efficiency of colony formation can be modulated in vitro by growth factors and adhesive substrates. Lastly, immunohistochemical analysis demonstrated that globose basal cell (GBC) progenitors, ORNs and olfactory ensheathing glia (OEGs) are generated by the ICAM-1+ fraction in clonal culture. Based on these results, we conclude that ICAM-1+ HBCs contribute to the progenitor cell compartment, possibly as stem cells, during olfactory neurogenesis and that the function of these cells may be modulated via adhesion and growth factor signaling by components resident within their in vivo microenvironment.

博士
國立臺灣師範大學
生命科學系
107
Postnatal neurogenesis in the dentate gyrus and subventricular zone (SVZ)-olfactory bulb pathway in mammals is regulated by extrinsic and intrinsic factors. Since endogenous neural stem cells (NSCs) in the adult brain have potential to treat neurodegenerative disorders, studying mechanisms regulating postnatal NSCs may provide clinical applications. However, the role of TRIP6, YAP and ginkgolide B in postnatal NSCs remain unclear. TRIP6 belongs to zyxin family of LIM proteins, which have been shown to interact with various proteins to regulate cell proliferation, survival and migration. We find that TRIP6 is expressed by adult NSCs in the SVZ but not migrating neuroblasts. TRIP6 is necessary and sufficient for self-renewal and proliferation of adult NSCs, but inhibits their differentiation. We also find that TRIP6 activates the Notch signaling, a pathway required for NSC self-renewal. Previous studies show that the Hippo pathway regulates cell proliferation and organ size through inhibiting YAP. We find that TRIP6 inhibits the Hippo pathway and activates YAP through PP1A. TRIP6 promotes NSC maintenance and proliferation and inhibits neuronal differentiation through YAP. During differentiation of NSCs, we also find that ginkgolide B promotes neurogenesis through the Wnt pathway. These findings show that YAP acts downstream of TRIP6 to promote adult NSC maintenance, whereas ginkgolide B promotes neurogenesis in the postnatal NSCs.

La neurogenèse persiste à l’âge adulte dans deux régions du système nerveux central (SNC) des mammifères : la zone sous-ventriculaire (SVZ) du cerveau antérieur et la zone sous-granulaire (SGZ) de l’hippocampe. Cette neurogenèse est possible grâce à la capacité de prolifération des cellules souches présentes dans les niches de la SVZ et la SGZ, mais en vieillissant, le cerveau subit une diminution dramatique du nombre de cellules souches neurales adultes (CSNa), une diminution de la prolifération cellulaire et une altération des niches de neurogenèse. Cependant, une importante question reste sans réponse : comment la perte tardive des CSNa est temporellement reliée aux changements de l’activité de prolifération et de la structure de la principale niche de neurogenèse (la SVZ)? Afin d’avoir un aperçu sur les événements initiaux, nous avons examiné les changements des CSNa et de leur niche dans la SVZ entre le jeune âge et l’âge moyen. La niche de la SVZ des souris d’âge moyen (12 mois) subit une réduction de l’expression des marqueurs de plusieurs sous-populations de précurseurs neuraux en comparaison avec les souris jeunes adultes (2 mois). Anatomiquement, cela est associé avec des anomalies cytologiques, incluant une atrophie générale de la SVZ, une perte de la couche de cellules sousépendymaires par endroit et l’accumulation de gouttelettes lipidiques de grande taille dans l’épendyme. Fonctionnellement, ces changements sont corrélés avec une diminution de l’activité de la SVZ et une réduction du nombre de nouveaux neurones arrivant aux bulbes olfactifs. Pour déterminer si les CSNa de la SVZ ont subi des changements visibles, nous avons évalué les paramètres clés des CSNa in vivo et in vitro. La culture cellulaire montre qu’un nombre équivalent de CSNa ayant la capacité de former des neurosphères peut être isolé du cerveau du jeune adulte et d’âge moyen. Cependant, à l’âge moyen, les précurseurs neuraux semblent moins sensibles aux facteurs de croissance durant leur différenciation in vitro. Les CSNa donnent des signes de latence in vivo puisque leur capacité d’incorporation et de rétention du BrdU diminue. Ensemble, ces données démontrent que, tôt dans le processus du vieillissement, les CSNa et leur niche dans la SVZ subissent des changements significatifs, et suggèrent que la perte de CSNa liée au vieillissement est secondaire à ces événements.
Neurogenesis persists throughout the adulthood in two regions of the mammalian central nervous system (SNC): the sub-ventricular zone (SVZ) of the forebrain and the sub-granular zone (SGZ) of the hippocampus. Neurogenesis is possible due to the proliferation capacity of stem cells present within both the SVZ and SGZ niches, but with aging, the forebrain undergoes a drastic reduction in its number of adult neural stem cells (aNSCs), a decrease of cell proliferation and an alteration of the neurogenic niches. However, a key unresolved question remains: how the onset of aNSC loss is temporally related to changes of proliferating activity and to structural alterations within the principal stem cell niche (the SVZ)? To gain insights into the initial events leading to aging-associated aNSC loss, we investigated the changes occurring to aNSCs and the SVZ niche between young adulthood and middle-age. The SVZ niche of middle-aged mice (12-months-old) was found to display reduced expression of markers for multiple neural precursor sub-populations when compared to young adult mice (2-months-old). Anatomically, this was associated with significant cytological aberrations, including an overall atrophy of the SVZ, loss of sub-ependymal cells, and accumulation of large lipid droplets within the ependyma. Functionally, these changes correlated with diminished SVZ activity and reduced number of newly born neurons reaching the principal target tissue: the olfactory bulbs. To determine whether changes were evident at the level of the SVZ stem cells, we evaluated key in vitro and in vivo parameters of aNSCs. Tissue culture experiments showed that equal numbers of neurosphere-forming aNSCs could be isolated from young adult and middle-aged forebrains. However, at middle-age, neural precursors seemed to be less sensitive to growth factors during their in vitro differentiation and displayed signs of increased quiescence in vivo. Collectively, these findings demonstrate that, with early aging, aNCS and their SVZ niche go through significant changes, and suggest that aging-associated aNSC loss is secondary to these events.