The data show that 6 days after placing neomycin in the perilymph, neither hair cells nor differentiated supporting cells can be found. The rapid and devastating effect of this ototoxic regimen on hair cells has been described in the past (Jyung et al., 1989
; Zappia et al., 1989
). In this study, we show an equally devastating effect on supporting cells, such that the epithelium replacing the organ of Corti is flat and neuronal fibers are absent. We also show that this transformation has negative implications for adenovirus mediated therapeutic treatments. Adenovirus transduction of the tissue is substantial but less robust than in differentiated supporting cells. Forced expression of Atoh1
in the flat epithelium does not lead to appearance of new hair cells.
The mechanism inducing transformation of differentiated supporting cells to an undifferentiated flat epithelium after hair cell removal is not clear. If kanamycin and ethacrynic acid are used to eliminate hair cells, supporting cells can remain differentiated for up to 10 weeks in the guinea pig model (Izumikawa et al., 2005
) and perhaps longer. Presence of differentiated supporting cells in deaf ears with no hair cells was shown using other models (Sugawara et al., 2005
). Together, these data suggest that the loss of hair cells is not sufficient to cause the loss of differentiated supporting cells. It may be that the supporting cells, themselves, are sensitive to extremely ototoxic aminoglycosides such as neomycin. Furthermore, this might not be the only reason for flattening of the auditory epithelium. In some experimental conditions, supporting cells become flat a long time after the ototoxic insult is stopped, suggesting that secondary degeneration may take place (Forge et al., 1998
), which might be attributed to a mechanism that is distinct from the direct effects of the ototoxic drugs. Thus, several factors may determine the survival or degeneration of non-sensory cells in the auditory epithelium. Our results suggest that future reparative approaches will be more successful when differentiated supporting cells remain in the tissue, and therefore it will be important to understand the transition to the flat epithelium and design ways to prevent it.
Phenotypic (immunocytochemical) characterization of the flat epithelium will help to determine the identity of these cells and facilitate attempts to manipulate these cells for therapeutic purposes. Several proteins have already been detected in this epithelium. For instance, these cells connect to each other with tight junctions, as determined by the presence of ZO-1 immunoreactivity, and express the supporting cell marker S-100 (Kim et al., 2007
). More complete molecular characterization will help determine whether these cells are de-differentiated supporting cells of the organ of Corti (such as Deiters, pillar or Hensen cells), and/or cells that have migrated from the flanking areas such as the inner or outer sulcus.
In birds, ototoxic lesions that deplete hair cells, leave behind differentiated supporting cells and regeneration occurs spontaneously (Cotanche, 1999
; Stone et al., 2000
; Stone et al., 1998
). A more severe lesion that also influences the supporting cells has been accomplished with noise (Cotanche et al., 1995
). In such cases, non-sensory cells that flank the basilar papilla can migrate into the sensory epithelium, as may be the case in the flat epithelium. These cells then become the therapeutic target for regenerative attempts or insertion of stem cells. Previous data show that cell migration is indeed possible in the auditory epithelium in mammals (Forge et al., 1998
Better understanding of the biology of the flat epithelium will assist in advancing tasks such as integration of stem cells, enhancement of neuronal survival or induction of transdifferentiation to the hair cell phenotype. In mammals, the flat epithelium can undergo a robust proliferative phase (Kim et al., 2007
). This may be of help for designing therapies and inserting genes or stem cells. More work is necessary to characterize the origin of these cells, their general biology and their amenability to taking up external molecules or vectors for gene delivery. It will be important to identify surface receptors on these cells that will allow the design of gene transfer vectors that will have specificity to these cells. It will also be necessary to determine if these cells are heterogenous in their origin and characteristics, as tentatively suggested by their pattern of transduction with adenovirus, with only a sub-population showing positive transgene expression.
The inability of Atoh1
to induce transdifferentiation of non-sensory cells to new hair cells may be related to the state of differentiation of the epithelium. Specifically, the fate of developing cells that express Atoh1
(in the inner ear and elsewhere) depends on the context determined by previous developmental gene expression in each of these tissues. The expression of Atoh1
is usually a final step in differentiation. Thus, forced expression of Atoh1
would be expected to exert different developmental outcomes depending on the developmental history of the cell. The current findings show that the flat epithelium fails to undergo transdifferentiation following forced expression of Atoh1
and suggest that the flat epithelium has regressed to a very early state of differentiation and no longer present the commitment to the hair cell or supporting cell phenotype. Based on the lack of response to Atoh1,
it can be assumed that the flat epithelium is less differentiated than cells in the vicinity of the organ of Corti, such as interdental cells, where forced Atoh1
expression induces ectopic hair cell formation (Kawamoto et al., 2003
; Minoda et al., 2007
These findings are important for conceptual and practical therapeutic approaches for hair cell regeneration. When considering transdifferentiation therapy, rebuilding the auditory epithelium may need to begin with inducing forced expression of early developmental genes, perhaps as early as otocyst specific genes that induce formation of the sensory areas, the epithelial ridges of the developing cochlear epithelium. Once expression of these early developmental genes recreates that epithelial ridge, Atoh1 over-expression may be used as a final stage for inducing generation of new hair cells.
Better understanding and ability to manipulate the flat epithelium may also be of help for enhancing cochlear implant procedures. The present data show a complete lack of hair cells along with a near complete loss of nerve fibers in the auditory epithelium. However, we found that some nerves continued to meander in the epithelium despite the lack of hair cells, possibly looking for a target. Similar finding were reported before in other models (Bohne et al., 1992
; Strominger et al., 1995
). The maintained ability of neurons to meander in the deafened epithelium, which appears to occur after several etiologies for hair cells loss, is important for the feasibility of innervating therapeutically-placed new hair cells or stem cells. However, the degree of neural survival may depend on the state of the supporting cells in the auditory epithelium (Sugawara et al., 2005
). It would be important to characterize this correlation in human temporal bones, where deafferented spiral ganglion neurons tend to survive to a larger extent than in lab animals.
In conclusion, our data confirm that the neomycin model is an efficient method for creating a flat epithelium in the cochlea. We demonstrate that adenovirus is a useful gene carrier into the flat epithelium and Atoh1 does not induce transdifferentiation of the flat epithelium into new hair cells. As such, it is necessary to design ways to prevent degeneration of supporting cells in ears depleted of hair cells, and to direct specific therapies in cases where the flat epithelium does occur.