Rabbit vomeronasal organ-derivered cells have mesenchymal profile and neuronal commitment / Las células derivadas del órgano vomeronasal del conejo tienen perfil mesenquimatoso y compromiso neuronal

SUMMARY: The vomeronasal organ (VNO) is an accessory organ involved on the olfactory pathway, that detects pheromones and emits signals in order to modulate social and reproductive behavior. The VNO stem cells replace neurons throughout life. The aim of this study was to isolate and characterize cells derived from the vomeronasal organ from New Zealand rabbits. Five male rabbits with 120 days were used for cell isolation and culture. Results: VNO-derived cells presented labelling for proliferation (PCNA), undifferentiated profile (Nanog), neuronal (GFAP), mesenchymal stem cells (CD73, CD90 and CD105 and Stro-1). Also, presence of cytoskeletal (Vimentin, b-tubulin and CK-18) and absence of hematopoietic markers (CD34, CD117 and CD45) both by immunofluorescence and flow cytometry. By PCR it was possible to verify the expression of some undifferentiated profile (Oct-4), neuronal (Nestin) and mesenchymal (CD73, CD105 and Vimentin) genes. Functionally, VNO-derived cells differentiate in vitro into adipocytes, osteocytes and chondrocytes, and presented no tumorigenic potential when injected to Balb/c nu/nu mice. In conclusion, the rabbit VNO-derived cells have a profile that could be supportive to VNO olfactory/neuroreceptor epithelium by delivering factors to epithelial turnover or even by differentiation into epithelial cells to replacement of commissural epithelium.

RESUMEN: El órgano vomeronasal (OVN) es un órgano accesorio de la vía olfatoria, que detecta feromonas y emite señales que afectan la modulación del comportamiento social y reproductivo. Las células madre OVN reemplazan las neuronas durante toda la vida. El objetivo de este estudio fue aislar y caracterizar células derivadas del órgano vomeronasal de conejos raza Nueva Zelanda. Para el aislamiento y el cultivo celular se utilizaron cinco conejos machos con una edad de 120 días. Las células del OVN presentaron etiquetado para la proliferación (PCNA), un perfil indiferenciado (Nanog), neuronal (GFAP), células madre mesenquimales (CD73, CD90 y CD105 y Stro-1). Además, se ob- servó presencia de citoesqueleto (Vimentina, β-tubulina y CK-18) y ausencia de marcadores hematopoyéticos (CD34, CD117 y CD45) tanto por inmunofluorescencia como por citometría de flujo. Me- diante PCR fue posible verificar la expresión de algunos genes de perfil indiferenciado (Oct-4), neuronal (Nestin) y mesenquimatoso (CD73, CD105 y Vimentin). Las células derivadas del OVN se diferencian in vitro en adipocitos, osteocitos y condrocitos, y no presentan un potencial tumorigénico al ser infiltrados en ratones Balb / c nu / nu. En conclusión, las células derivadas de OVN de conejo tienen un perfil que podría ser compatible con el epitelio olfatorio / neurorreceptor de OVN transmitiendo factores al recambio epitelial o incluso mediante la diferenciación en células epiteliales para reemplazar el epitelio comisural.

Olfactory epithelium as an infinitive source of neural stem cells for derivation of inner ear hair cells / El epitelio olfatorio como fuente infinita de células madre neurales para la derivación de los vestibulocitos del oído interno

Surgical techniques for treatment of sensory neural hearing loss (SNHL) have unpredictable outcomes and in recent years cell therapy investigated for treatment of SNHL. Olfactory epithelium proceed neurogenesis during life time and provide an easily accessible source of neural stem cells. So the aim of this study was isolating neural stem cells from olfactory epithelium of rat and differentiation of these cells into hair cells of inner ear in vitro. The epithelium tissue of olfactory mucosa of rats were removed and digested by collagenase H. The digested tissue was cultured in flasks in suspension forms to create spheres. Spheres were passaged and from passage 2 spheres selected for differentiation. At this stage cells of spheres isolated from each other and placed in flask containing defined differentiation medium. Cells at this stage cultured in adhesive form. Immunohistochemistry and RT-PCR were used for neural stem cells and hair cells identification. Spheres formed from olfactory epithelium culture and immunohistochemistry revealed that cells of spheres from passage one and two expressed the neural stem cells markers. After culture of isolated cells in differentiation medium, the morphology of cells begun to change. The cells presented neural cells projections and after 10 days the projections elongated more and interact to each other in multi layers. RT-PCR and immunohistochemistry revealed that differentiated cells expressed hair cells specific genes. In this study we showed that neural stem cells of olfactory epithelium can differentiate into hair cells of inner ear and therefore can be used for treatment of SNHL.

Las técnicas quirúrgicas para el tratamiento de la pérdida auditiva neural sensorial (PANS) tienen resultados impredecibles y en los últimos años la terapia celular ha sido investigada para su tratamiento. El epitelio olfatorio se forma durante la neurogénesis y proporciona una fuente fácilmente accesible de células madre neurales. El objetivo de este estudio fue aislar las células madre neurales del epitelio olfativo de la rata y la diferenciación de estas células en vestibulocitos del oído interno in vitro. Se retiró el tejido del epitelio de la mucosa olfatoria de ratas y fue digerido con colagenasa H. El tejido se cultivó en forma de suspensión para crear esferas. Se seleccionaron dos esferas para la diferenciación. En esta fase, las células de esferas fueron aisladas unas de otras y colocadas en un medio de diferenciación definido. Células en esta etapa fueron cultivadas en forma adhesiva. Inmunohistoquímica y RT-PCR se utilizó para las células madre neurales y la identificación de células ciliadas. Las esferas formadas a partir del cultivo del epitelio olfatorio y la inmunohistoquímica revelaron que las células de esferas en etapas uno y dos expresaban los marcadores de células madre neurales. Se observaron cambios en la morfología de las células después del cultivo de células aisladas. RT-PCR e inmunohistoquímica revelaron que las células diferenciadas expresaron células específicas de gen de vestibulocitos. Se observó que las células madre neuronales de epitelio olfatorio puede diferenciarse en células en forma de cabello del oído interno y por lo tanto puede ser utilizado para el tratamiento de PANS.

Rabbit olfactory stem cells. Isolation protocol and characterization

PURPOSE: To describe a new technique for isolation of a mesenchymal stem cells (MSCs) population from the olfactory mucosa in rabbits. METHODS: Olfactory stem cells (OSCs) were retrieved from under the cribriform plate of the Ethmoid bone. Several assays were accomplished to characterize the cell population and attest its viability in vitro. The cells were submitted to flow cytometry with the antibodies CD34, CD45, CD73, CD79, CD90 and CD105 and also they were induced to differentiate in three lineages. Functional evaluation involved analysis of in vitro growth behavior, colony forming unit like fibroblasts (CFU-f) and cryopreservation response. Further transduction with Green Fluorescent Protein (GFP) was also performed. RESULTS: The OSCs showed mesenchymal features, as positive response to CD34, CD73 and CD90 antibodies and plasticity. Additionally, these cells have high proliferated rate, and they could be cultured through many passages and kept the ability to proliferate and differentiate after cryopreservation. The positive response to the transduction signalizes the possibility of cellular tracking in vivo. This is a desirable feature in case those cells are used for pre-clinical trials. CONCLUSION: The cells harvested were mesenchymal stem cells and the technique described is therefore efficient for rabbit olfactory stem cells isolation.

Dinâmica dos precursores celulares do epitélio olfatório de cães sem raça definida: um estudo imunohistoquímico e ultra-estrutural / The dynamic of precursor of the olfactory epithelium of mongrel dogs: an immunohistochemical and ultrastructural study

O epitélio olfatório apresenta um mecanismo de diferenciação em que células-tronco dão origem a células progenitoras amplificadoras, as quais expressam um gene pró-neural denominado Mammalian Achaete Scute Homolog 1 (Mash1). Estas células podem se diferenciar em receptores olfatórios. O epitélio olfatório de cães sem raça definida (3 machos de um ano e 2 fêmeas de três de idade) foi analisado por imunolocalização do antígeno nuclear de proliferação celular (PCNA) e por microscopia eletrônica de transmissão. Verificou-se marcação positiva para PCNA em células do epitélio olfatório, particularmente acima da linha da membrana basal. A ultra-estrutura do epitélio olfatório revelou células adjacentes à lâmina basal, cuja eletrodensidade assemelha-se àquelas presentes no epitélio de sustentação, reforçando a idéia da renovação das células de sustentação e dos neurônios olfatórios locais. O epitélio olfatório é composto células basais, comprometidas com sua renovação, caracterizadas através da intensa atividade mitótica, identificada pela reação positiva ao PCNA. Estes resultados sugerem que há reposição das células sustentaculares locais e do sistema através de mecanismos semelhantes.

Olfactory epithelium presents a mechanism of differentiation where stem cells give arise to amplifying progenitor cell which express Mammalian Achaete Scute Homolog 1 (Mash1). These cells can be differentiated into olfactory receptors. An immunolocalization study and ultrastructural analysis by transmission electron microscopy of olfactory epithelium of mongrel dogs were made using 3 males (one year old) and 2 females (three years old). Labeled cells with positive staining by Proliferating cell nuclear antigen (PCNA) were observed in specific areas of the olfactory epithelium, especially above the basal membrane. The ultrastructure revealed cells adjacent to the basal membrane with morphology resembling sustentacular cells, supporting the idea of renewal of sustentacular and olfactory sensorial cells. Olfactory epithelium contains basal cells committed to self-renewal, characterized by high metabolic activity, identified by positive reaction to PCNA. These results suggested the renewal of sustentacular and sensorial olfactory cells through the same pathway.

Role of nitric oxide during neurogenesis in the olfactory epithelium

In mammals, neurogenesis continues during adulthood in restricted places of the nervous system, namely the subventricular zone, the dentate gyms and the olfactory epithelium. A dual role of the second messenger nitric oxide has been reported in such places, either promoting or inhibiting proliferation of neuronal precursors depending on the cellular signal implicated. In this review the regulation of adult olfactory epithelium neurogenesis by nitric oxide is discussed.