Literatura académica sobre el tema "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<br>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.<br>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<br>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.<br>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.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>Eskitis Institute for Cell and Molecular Therapies<br>Science, Environment, Engineering and Technology<br>Full Text

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<br>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