A cóclea humana adulta é reconhecidamente incapaz de se regenerar de modo significativo na prática clínica após uma lesão. Tal fato se explica pela perda da capacidade de proliferação de suas células sensoriais após o período neonatal. Em diferentes espécies de mamíferos neonatos, foi demonstrada a capacidade de proliferação das células sensoriais cocleares através de ensaios de formação de esferas e da presença de marcadores para células-tronco, como o ABCG2. Neste estudo, foram utilizadas cócleas humanas adultas para ensaio de formação de esferas e para a pesquisa do marcador de células-tronco ABCG2 através de citometria de fluxo. As cócleas foram obtidas de pacientes portadores de schwannomas vestibulares submetidos a procedimento cirúrgico para ressecção do tumor via translabiríntica e doadores de órgãos em morte encefálica. Foi possível observar formação de esferas in vitro e identificar o marcador ABCG2 através de citometria de fluxo nas células dissociadas do tecido coclear / The human cochlea is, allegedly, unable to regenerate after trauma due to the loss of the proliferation capacity after the neonatal period. It has been demonstrated in different neonatal mammal species that sensorial cochlear cells retain their proliferation capacity in the neonatal period using sphere-formation assays and stem cell markers such as ABCG2. In the present work, human adult cochleas have been used for sphereformation assay and flow cytometric identification of ABCG2. Human adult cochleas have been removed either from patients undergoing vestibular schwannoma resection via translabyrinthine approach or brain-dead organ donors. There have been identified sphere formation in vitro and the stem cell marker ABCG2 in flow cytometry analysis after cochlear dissociation

A cóclea humana adulta é reconhecidamente incapaz de se regenerar de modo significativo na prática clínica após uma lesão. Tal fato se explica pela perda da capacidade de proliferação de suas células sensoriais após o período neonatal. Em diferentes espécies de mamíferos neonatos, foi demonstrada a capacidade de proliferação das células sensoriais cocleares através de ensaios de formação de esferas e da presença de marcadores para células-tronco, como o ABCG2. Neste estudo, foram utilizadas cócleas humanas adultas para ensaio de formação de esferas e para a pesquisa do marcador de células-tronco ABCG2 através de citometria de fluxo. As cócleas foram obtidas de pacientes portadores de schwannomas vestibulares submetidos a procedimento cirúrgico para ressecção do tumor via translabiríntica e doadores de órgãos em morte encefálica. Foi possível observar formação de esferas in vitro e identificar o marcador ABCG2 através de citometria de fluxo nas células dissociadas do tecido coclear / The human cochlea is, allegedly, unable to regenerate after trauma due to the loss of the proliferation capacity after the neonatal period. It has been demonstrated in different neonatal mammal species that sensorial cochlear cells retain their proliferation capacity in the neonatal period using sphere-formation assays and stem cell markers such as ABCG2. In the present work, human adult cochleas have been used for sphereformation assay and flow cytometric identification of ABCG2. Human adult cochleas have been removed either from patients undergoing vestibular schwannoma resection via translabyrinthine approach or brain-dead organ donors. There have been identified sphere formation in vitro and the stem cell marker ABCG2 in flow cytometry analysis after cochlear dissociation

In the inner and outer hair cells (OHCs) of the inner ear, an unconventional myosin 15a localizes at the tips of mechanosensory stereocilia and plays an important role in forming and maintaining their normal structure. A missense mutation makes the motor domain of myosin 15a dysfunctional and is responsible for the congenital deafness DFNB3 in humans and deafness and vestibular defects in Shaker-2 (Sh2) mouse model. All hair cells of homozygous Shaker-2 mice (Myo15sh2/sh2) have abnormally short stereocilia, but, only stereocilia of Myo15sh2/sh2OHCs start to degenerate after the first few days of postnatal development and lose filamentous tip links between stereocilia that are crucial for mechanotransduction. The exact mechanisms of this degeneration are unknown even though they may underlie DFNB3 deafness in humans. We hypothesize that structural abnormalities in Myo15sh2/sh2 OHCs may alter the mechanical forces applied to the mechano-electrical transduction (MET) channels resulting in abnormal ionic homeostasis, which may lead to eventual degeneration of Myo15sh2/sh2 OHCs. Therefore, we investigated the ionic conductances and integrity of mechanotransduction apparatus in Myo15sh2/sh2 OHCs. Surprisingly, we found that myosin 15a-deficiency is associated not only with structural abnormalities of OHC stereocilia but also with alterations of voltage-gated ion conductances.

No description available.

La destruction des cellules ciliées de la cochlée entraine des surdités sensorielles. Chez les mammifères ces cellules ne se régénèrent pas et les déficits auditifs occasionnés sont définitifs. Aucune thérapie visant à remplacer les cellules ciliées détruites n'est actuellement proposée.L'objectif de cette thèse est de contribuer au développement d'une thérapie cellulaire basée sur la greffe de cellules souches / progénitrices cochléaires et destinée à promouvoir la régénération des cellules ciliées.Au cours de nos travaux, nous avons isolé une population de cellules souches cochléaires chez des souris néonatales appartenant à la « side population » (Savary et al. 2007). Nous avons également montré, par des expériences de perte et de gain de fonction in vitro, que la voie de signalisation Notch est nécessaire pour l'auto-renouvellement et la différenciation de ces cellules (Savary et al., 2008). Des lignées de souris transgéniques exprimant la GFP sous le promoteur de la GFAP et de la Nestine nous ont permis de suivre l'expression de ces marqueurs de cellules souches dans des cochlées de souris P3 et adultes. En étudiant l'expression combinée d'autres marqueurs comme Sox2 et Abcg2, nous avons montré que les cellules progénitrices cochléaires sont réparties différemment chez les souris néonatales et les souris adultes (Smeti, Savary et al 2010).Nos expériences préliminaires de transplantation in vitro dans un modèle murin de surdité génétique humaine de type DFNA15 démontrent que les cellules souches / progénitrices greffées sont capables d'intégrer l'épithélium sensoriel lésé et de se différencier en cellules exprimant un marqueur de cellules ciliées. / The destruction of cochlear hair cells causes sensory deafness. In Mammals these cells do not regenerate and damages are irreversible. Currently, there is no proposed therapy to replace the destroyed hair cells.The focus of this thesis is to develop a novel cell therapy based on transplantation of cochlear progenitor cells in order to promote regeneration of hair cells.We first isolated a population of cochlear stem cells from neonatal mice by using the side population analysis technique (Savary et al. 2007). Then, we showed, by in vitro loss and gain of function experiments, that the Notch signaling pathway is necessary for cellular self-renewal and differentiation (Savary et al., 2008).Transgenic mice strains expressing GFP under the control of GFAP and Nestin promotors allowed us to monitor the expression of these markers of stem cells in the P3 and adult mice cochleae. By studying the combined expression of other stem cells markers such as Sox2 and ABCG2, we showed that the niches of cochlear progenitor cells are differently distributed in neonatal and adult mice (Smati, Savary et al 2010).Our preliminary in vitro transplantation experiments in a mouse model that mimics human genetic deafness DFNA15 show that the transplanted stem / progenitor cells are able to migrate to the lesion site, to integrate the damaged sensory epithelium and to differentiate into cells expressing a marker of hair cells.

No description available.

No description available.