Transforming cochlear supporting cells into auditory hair cells for inner ear sensory organ regeneration Open Access

Pan, Alexander Yan (2012)

Permanent URL: https://etd.library.emory.edu/concern/etds/37720d11k?locale=en
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Abstract


The cochlear sensory domain, known as the organ of Corti, within the mammalian
inner ear is composed of sensory hair cells and interdigitating non-sensory supporting
cells. The hair cells are the primary receptor cells responsible for detecting and
transmitting sound signal. Loss of these cells in humans leads to permanent hearing loss,
since mammalian hair cells cannot be regenerated. In contrast, lower vertebrate classes
exhibit spontaneous regeneration of hair cells fol owing damage by proliferation and
conversion of the supporting cells. Atoh1 is a hair cell differentiation factor, and previous
studies have shown that overexpressing Atoh1 can induce the expression of hair cell
markers in cochlear non-sensory cells. However, these cells are usually located outside the
sensory region domain. Furthermore, our previous study suggests that Atoh1 has a very
limited ability to induce hair cell differentiation in non-sensory supporting cells within the
sensory domain in the postnatal mammalian inner ear. In many regenerative species, hair
cell regeneration in the mature cochlea occurs only after the loss of existing hair cells.
Therefore, I hypothesized that the presence of existing hair cells inhibits Atoh1-mediated
conversion of supporting cells in mammals. To test this hypothesis, I first developed an
efficient hair cell ablation protocol involving two different ototoxic aminoglycosidic drugs. I
then induced Atoh1 expression first throughout the cochlear epithelium, then specifically
within the supporting cells. I show that ablating the endogenous hair cells modestly
increases the number of hair cells converted from non-sensory supporting cells. Together,
this data indicates that the in vitro cochlear explant model can be used to efficiently test
various conditions for hair cell regeneration and that the competency of supporting cells
conversion into hair cells may be modulated to achieve hair cell regeneration.

Table of Contents

INTRODUCTION...................................................................................................................................... 1

EXPERIMENTS/METHODS .................................................................................................................. 5
Organotypic culture of postnatal mouse cochleae ............................................................................... 5
Transgenic mouse models ............................................................................................................................. 5
Characterization of ototoxic drug conditions ......................................................................................... 5
Induction of transgenic Atoh1 in transgenic cochlear explant cultures ....................................... 6
Immunohistochemistry and imaging ........................................................................................................ 6
Cell counts and statistical analyses ............................................................................................................ 7

RESULTS ................................................................................................................................................... 8
Neomycin and gentamicin efficiently ablate hair cells in cochlear explants .............................. 8
Inducing overexpression of Atoh1 throughout the cochlear epithelium induces
regeneration of new hair cells .................................................................................................................. 10
Inducing overexpression of Atoh1 specifically in supporting cells leads to new ectopic hair
cells within the sensory region ................................................................................................................. 11

DISCUSSION ........................................................................................................................................... 13
REFERENCES .......................................................................................................................................... 18
ACKNOWLEDGEMENTS ...................................................................................................................... 20
FIGURES .................................................................................................................................................. 21

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