RESUMO
Green fluorescent protein (GFP) has been used extensively to label cells in vitro and to track them following their transplantation in vivo. During our studies using the mouse embryonic stem cell line R1 B5-EGFP, we observed variable levels of fluorescence intensity of the GFP within these transfected cells. The variable fluorescence of this protein coupled with the innately autofluorescent nature of several structures within the cochlea collectively made the in vivo identification of these transplanted stem cells difficult. We have modified previously published protocols to enable the discrimination of an authentic GFP signal from autofluorescence in the adult guinea pig cochlea using fluorescence-based immunohistochemistry. The protocol described can also be used to label tissues of the cochlea using a chromogen, such as 3,3'-diaminobenzidine tetrahydrochloride (DAB). Moreover, the described method gives excellent preservation of structural morphology making the tissues useful for both morphological and quantitative studies in combination with robust immunohistochemistry in the adult guinea pig cochlea.
Assuntos
Cóclea/metabolismo , Fluorescência , Imuno-Histoquímica/métodos , Inclusão do Tecido/métodos , Animais , Células Cultivadas , Cóclea/anatomia & histologia , Cóclea/cirurgia , Embrião de Mamíferos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cobaias , Camundongos , Camundongos Transgênicos , Transplante de Células-Tronco/métodos , Tretinoína/farmacologiaRESUMO
Sensory hair cells in the mammalian cochlea are sensitive to many insults including loud noise, ototoxic drugs, and ageing. Damage to these hair cells results in deafness and sets in place a number of irreversible changes that eventually result in the progressive degeneration of auditory neurons, the target cells of the cochlear implant. Techniques designed to preserve the density and integrity of auditory neurons in the deafened cochlea are envisaged to provide improved outcomes for cochlear implant recipients. This review examines the potential of embryonic stem cells to generate new neurons for the deafened mammalian cochlea, including the directed differentiation of stem cells toward a sensory neural lineage and the engraftment of exogenous stem cells into the deafened auditory system. Although still in its infancy the aim of this therapy is to restore a critical number of auditory neurons, thereby improving the benefits derived from a cochlear implant.
Assuntos
Nervo Coclear/citologia , Nervo Coclear/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/transplante , Regeneração Nervosa/fisiologia , Animais , Vias Auditivas/citologia , Vias Auditivas/fisiologia , Células-Tronco Embrionárias/fisiologia , HumanosRESUMO
The low regenerative capacity of the hair cells of the mammalian inner ear is a major obstacle for functional recovery following sensorineural hearing loss. A potential treatment is to replace damaged tissue by transplantation of stem cells. To test this approach, undifferentiated and partially differentiated mouse embryonic stem (ES) cells were delivered into the scala media of the deafened guinea pig cochlea. Transplanted cells survived in the scala media for a postoperative period of at least nine weeks, evidenced by histochemical and direct fluorescent detection of enhanced green fluorescent protein (EGFP). Transplanted cells were discovered near the spiral ligament and stria vascularis in the endolymph fluid of the scala media. In some cases, cells were observed close to the damaged organ of Corti structure. There was no evidence of significant immunological rejection of the implanted ES cells despite the absence of immunosuppression. Our surgical approach allowed efficient delivery of ES cells to the scala media while preserving the delicate structures of the cochlea. This is the first report of the survival of partially differentiated ES cells in the scala media of the mammalian cochlea, and it provides support for the potential of cell-based therapies for sensorineural hearing impairment.
Assuntos
Cóclea/embriologia , Embrião de Mamíferos/citologia , Células-Tronco/fisiologia , Animais , Diferenciação Celular , Sobrevivência Celular , Células Cultivadas , Potenciais Evocados Auditivos do Tronco Encefálico , Cobaias , Tolerância Imunológica , Imuno-Histoquímica , Camundongos , Células-Tronco/citologiaRESUMO
Cochlear implants are dependent on functionally viable spiral ganglion neurons (SGNs) - the primary auditory neurons of the inner ear. Cell-based therapies are being used experimentally in an attempt to rescue SGNs from deafness-induced degeneration or to generate new neurons. The success of these therapies will be dependent on the development of surgical techniques designed to ensure precise cell placement while minimizing surgical trauma, adverse tissue reaction and cell dispersal. Using 24 normal adult guinea pigs we assessed three surgical procedures for cell delivery into the cochlea: (i) a cochleostomy into the scala tympani (ST); (ii) direct access to Rosenthal's canal - the site of the SGN soma - via a localized fracture of the osseous spiral lamina (RC); and (iii) direct access to the auditory nerve via a translabyrinthine surgical approach (TL). Half the cohort had surgery alone while the other half had surgery combined with the delivery of biocompatible microspheres designed to model implanted cells. Following a four week survival period the inflammatory response and SGN survival were measured for each cohort and the location of microspheres were determined. We observed a wide variability across the three surgical approaches examined, including the extent of the inflammatory tissue response (TL>>RC> or =ST) and the survival of SGNs (ST>RC>>TL). Importantly, microspheres were effectively retained at the implant site after all three surgical approaches. Direct access to Rosenthal's canal offered the most promising surgical approach to the SGNs, although the technique must be further refined to reduce the localized trauma associated with the procedure.