Abstract
BackgroundCulturing otospheres from dissociated organ of Corti is an appropriate starting point aiming at the development of cell therapy for hair cell loss. Although guinea pigs have been widely used as an excellent experimental model for studying the biology of the inner ear, the mouse cochlea has been more suitable for yielding otospheres in vitro. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy.MethodsOrgans of Corti were surgically isolated from P3 guinea pig or mouse cochlea, dissociated and cultivated under non-adherent conditions. Cultures were maintained in serum-free DMEM:F12 medium, supplemented with epidermal growth factor (EGF) plus either basic fibroblast growth factor (bFGF) or transforming growth factor alpha (TGFα). Immunofluorescence assays were conducted for phenotype characterization.ResultsThe TGFα group presented a number of spheres significantly higher than the bFGF group. Although mouse cultures yielded more cells per sphere than guinea pig cultures, sox2 and nestin distributed similarly in otosphere cells from both organisms. We present evidence that otospheres retain properties of inner ear progenitor cells such as self-renewal, proliferation, and differentiation into hair cells or supporting cells.ConclusionsDissociated guinea pig cochlea produced otospheres in vitro, expressing sox2 and nestin similarly to mouse otospheres. Our data is supporting evidence for the presence of inner ear progenitor cells in the postnatal guinea pig. However, there is limited viability for these cells in neonatal guinea pig cochlea when compared to the differentiation potential observed for the mouse organ of Corti at the same developmental stage.
Highlights
The sense of hearing, one of the five primary senses, is mediated through a complex sensory system that allows the perception and reaction to a huge variety of sound stimuli
New hair cells seem to originate from supporting cells that reenter the cell cycle and subsequently divide asymmetrically; or they may arise after transdifferentiation from supporting cells of the vestibular system, but not from cochlea[10,11]
We found a significant difference between groups regarding the number of sphere when data was combined for both animals, with more spheres observed in the transforming growth factor alpha (TGFa) group (23.3 ± 8.5) than in the basic fibroblast growth factor (bFGF) group (9 ± 1, p = 0.044, Student’s t-test)
Summary
The sense of hearing, one of the five primary senses, is mediated through a complex sensory system that allows the perception and reaction to a huge variety of sound stimuli. The auditory system comprises a highly specialized sensory epithelium, the organ of Corti It contains mechanosensory hair cells as the primary transducers of auditory stimuli, and supporting cells that provide a structural and physiological supporting epithelium. It has been reported that supporting cell proliferation and hair cell regeneration spontaneously occurs in vitro after aminoglycoside ototoxicity in the vestibular sensory epithelia of adult mammals, including guinea pigs and humans[8,9]. In these instances, new hair cells seem to originate from supporting cells that reenter the cell cycle and subsequently divide asymmetrically; or they may arise after transdifferentiation from supporting cells of the vestibular system, but not from cochlea[10,11]. The aim of this study was to compare conditions and outcomes of otosphere suspension cultures from dissociated organ of Corti of either mouse or guinea pig at postnatal day three (P3), and to evaluate the guinea pig as a potential cochlea donor for preclinical cell therapy
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