Учебники / Auditory Trauma, Protection, and Repair Fay 2008

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can be generated in the avian ear after different types of induced lesions such as those caused by overstimulation, ototoxic drugs, and genetic disease (Corwin and Cotanche 1988; Ryals and Rubel 1988; Tucci and Rubel 1990; Wilkins et al. 2001). Along with the regeneration of hair cells, birds can restore their auditory function (Adler et al. 1993; Niemiec et al. 1994; Marean et al. 1995; Dooling et al. 1997). Interestingly, hair cell regeneration in the avian ear can occur more than once (Niemiec et al. 1994).

The basilar papilla does not contain a clearly identifiable population of basal cells. Nevertheless, after a lesion to the sensory epithelium, the supporting cells of the basilar papilla replicate their DNA and divide near the luminal surface (Raphael 1992, 1993; Hashino and Salvi 1993), then some of the new cells become new hair cells (Cotanche et al. 1994; Stone and Cotanche 1994). When avian supporting cells proliferate, they dedifferentiate, undergo S-phase near the basal lamina, then round up near the luminal surface and divide (Raphael et al. 1994). The importance of this finding is in the fact that supporting cells are differentiated cells with specialized function and structure. As such, their ability to dedifferentiate and produce new hair cells is among the very few examples of transdifferentiation (phenotypic conversion). The transdifferentiation of supporting cells into new hair cells in the avian basilar papilla can be even more drastic when it occurs without mitosis (Adler and Raphael 1996; Roberson et al. 1996).

In addition to the transdifferentiation of supporting cells into new hair cells, there may be additional contributions from surrounding tissues to the repair of the traumatized basilar papilla. Hyalin and cuboidal cells, neighboring the basilar papilla, have been shown to proliferate following lesions to the hair cells (Girod et al. 1989). These cells have also been shown to migrate into the basilar papilla after a severe lesion that depletes hair cells along with the original supporting cells (Cotanche et al. 1995).

2.2 The Molecular Control of Avian Hair

Cell Regeneration

Research on hair cell regeneration in the avian basilar papilla has provided invaluable information about the morphological and physiological outcome of the regenerative process and identified the differentiated supporting cells as the main cellular contributors to the process. More recently, some aspects of the molecular regulation of hair cell regeneration in the basilar papilla have been elucidated (Bermingham-McDonogh et al. 2001; Witte et al. 2001; Warchol 2002; Hawkins et al. 2003; Matsui et al. 2004; Stone et al. 2004). Data obtained from gene expression assays (Hawkins et al. 2003, 2006) combined with other methods will likely facilitate the identification of the signals that initiate the regenerative activity in the supporting cells of the basilar papilla. Given that basal cells (or stem cells) are apparently absent in both the avian and the mammalian ear, it is not clear why birds can and do regenerate hair cells and humans (along with other mammals) do not.

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In searching for the reasons that account for the inability of mammals to regenerate hair cells, it is tempting to consider mostly the nonsensory components of the epithelium, as the nonsensory cells give rise to new hair cells. Nevertheless, important clues can also be gained from studying hair cells, considering that on their demise, these cells might be able to provide a signal that initiates regenerative activity in the supporting cells.

2.3 Limited Regenerative Capability in Mammalian

Vestibular Sensory Epithelia

The morphology of vestibular hair cells and the organization of the balance sense organs have some features that are similar to those of nonmammalian hair cell systems. One of these features is the ability of the vestibular epithelium to generate new hair cells. After a severe ototoxic lesion to the mammalian vestibular epithelium, much like in the cochlea, hair cell regeneration could not be identified (Lindeman 1969; Hawkins and Preston 1975; Meiteles and Raphael 1994). However, after less severe lesions to the vestibular sensory epithelium, a limited number of new hair cells were observed (Forge et al. 1993; Lopez et al. 1998). The mechanism of vestibular hair cell regeneration is based on the transdifferentiation of supporting cells, and the extent of supporting cell proliferation may vary between species (Warchol et al. 1993; Lopez et al. 1998).

While the finding that a small number of stem cells are present in the vestibular organs may account for the regenerative capability in this epithelium (Li et al. 2003a), the signals for regeneration are not well characterized. The vestibular sensory epithelium in mammals could potentially be a useful model for understanding the signals that mediate and regulate the regeneration thanks to the abundance of markers, reagents, and advanced status of whole genome mapping in mammals. However, the low level of regeneration impairs the ability to use the plethora of available resources in a fruitful manner. When the signals initiating and regulating the regeneration of hair cells are identified (in mammals, birds, or other vertebrates), they may be applicable for enhancing vestibular regeneration. This would be extremely beneficial for treating balance disorders due to hair cell loss, for which no treatment is currently available. Clinically, hearing loss can be treated with amplification and/or cochlear implant, while balance disorders still await treatment.

3. Reactivation of Developmental Programs in the Damaged Organ of Corti

3.1 Transdifferentiation: Induced Phenotypic Conversion

There are two main feasible options to introduce new hair cells to replace lost cochlear sensory cells (Fig. 11.1). One option is to tap into the population of nonsensory cells that remain in the damaged cochlea and use them as a

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Figure 11.1. After hair cells (top box) degenerate, only supporting cells remain in the organ of Corti. The three options to generate new hair cells are by transdifferentiation of supporting cells (left), inducing proliferation (middle), or adding progenitor cells such as stem cells (right). Combining proliferation with transdifferentiation may enhance the regenerative response.

source for generating new hair cells. The other option is to introduce cells from external sources, such as stem cells. Both technologies have advantages and disadvantages. Approaches for using the intrinsic cells in the cochlea as a source for generating new hair cells are discussed in this section and the use of stem cells is discussed in Section 4.

The theory and logic behind the attempt to use cochlear nonsensory cells to generate new hair cells are in the similarity of this principle to what birds do spontaneously. One logical and practical way to accomplish such transdifferentiation is by reactivation of the developmental program that regulates formation of hair cells. Many of the genes that are involved in the developmental of the sensory epithelium have been identified and characterized (Torres and Giraldez 1998; Chen et al. 2002; Fekete and Wu 2002; Kelley 2003; Montcouquiol and Kelley 2003; Barald and Kelley 2004; Woods et al. 2004;

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Fritzsch et al. 2006). The spatial and temporal sequence of gene expression has been linked to cell–cell and cell–matrix interactions. Cell cycle regulation in the developing and mature sensory epithelium is of interest because manipulating the genes involved in this regulation may help produce new cells in mature tissues. Hair cells and supporting cells share common progenitors. Therefore, the developmental stages during which these two cell types undergo fate commitment and differentiation are of immediate relevance to designing strategies to induce the transdifferentiation of nonsensory cells into new hair cells.

Because hair cells and supporting cells are clonally related (Fekete 2000; Fekete and Wu 2002), they have both gone through similar stages of developmental signaling and gene expression and are therefore responsive to similar signals. During development, the bHLH gene Atoh1 (formerly Math1) signals cells to choose the fate of hair cells, rather than supporting cells (Bermingham et al. 1999; Zine 2003; Woods et al. 2004). As such, Atoh1 is an excellent candidate gene to attempt transdifferentiation of nonsensory cells into new hair cells. Initial experiments on the overexpression of Atoh1 were done in tissue cultures. Induced expression of this gene in explants of the cochlea resulted in the transdifferentiation of some of the nonsensory cells into extranumerary hair cells (Zheng and Gao 2000; Shou et al. 2003). When Atoh1 was expressed in the normal guinea pig cochlea in vivo, ectopic hair cells were detected (Kawamoto et al. 2003b). Using neuronal-specific staining, it was determined that neurons can find new hair cells even in ectopic locations (Kawamoto et al. 2003b). These results provide evidence for the principle that mature nonsensory cells in the mammalian cochlea retain their competence to respond to gene expression of a hair cell–specific gene (Atoh1) and transdifferentiate into the hair cell phenotype. These experiments paved the way for testing the outcome of Atoh1 gene expression in the mature deafened cochlea.

The technological ability to introduce genes into supporting cells in vivo (discussed later) depended on inoculation of the adenovirus vectors with transgene inserts into the endolymph (Ishimoto et al. 2002). This technology was used to insert Atoh1 into the nonsensory cells of mature deafened guinea pigs in vivo. The deafening was accomplished by using a high concentration of an aminoglycoside antibiotic in combination with a potent diuretic, which resulted in the elimination of most or all of the hair cells in the cochlea. After the bilateral elimination of the hair cells, the Atoh1 expressive adenovirus was inoculated into the left ear. Transgenic expression of Atoh1 in the nonsensory cells that remained in these ears was efficient. In animals tested two months after the inoculation, Ad.Atoh1 induced the generation of a significant number of new hair cells in the organ of Corti and hearing thresholds improved (Izumikawa et al. 2005). The improvement in threshold does not attest to the qualitative features of hearing in these Atoh1-treated animals. Based on the morphology of these cochleae, hearing is likely to be distorted and abnormal.

These studies, along with findings on the role of Atoh1 during inner ear development (Bermingham et al. 1999; Chen et al. 2002; Woods et al. 2004), suggest that Atoh1 functions as a master regulatory gene, which is both necessary

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and sufficient for hair cell development or regeneration. The data provide the proof for the principle that reactivation of developmental programs may be utilized as therapeutic means in mature tissues of the inner ear and elsewhere. Nevertheless, this type of therapy will need to be improved and characterized along several different avenues before it can be applied clinically.

Atoh1 and other genes that induce phenotypic change are not expected to directly enhance proliferation in the auditory epithelium. It remains to be determined if cell proliferation occurs spontaneously as a secondary effect of the induced transdifferentiation. Even if it does, there will be many cases in which a need to increase the population of nonsensory cells will be the first task in the induced regenerative process. Moreover, it is important to test whether inducing cell division will result in some of the progeny differentiating into new hair cells. Therefore, studies looking at the regulation of the cell cycle in the auditory epithelium and attempts to manipulate the cell cycle are important and exciting.

3.2 Inducement of Proliferation in the Inner

Ear Epithelium

The first experiments that showed generation of new cells in the organ of Corti past the normal end of mitosis involved work on the p27Kip1 (p27) gene. p27 inhibits cyclin-dependent kinase-2 (cdk-2) (Sherr and Roberts 1999) and therefore has an antimitogenic role. In the organ of Corti, p27 appears to be responsible for the quiescence of the supporting cells and is selectively expressed in these cells (Lowenheim¨ et al. 1999). During development of the organ of Corti, p27 expression is induced around E13, when cell division of hair cell progenitors stops (Chen and Segil 1999). p27 expression persists at high levels in differentiated supporting cells of the mature organ of Corti. As such, the traumatized ear, in which no hair cells remain, may be an attractive target for blocking p27 (expression or function) in order to induce mitosis in supporting cells. The inner ears of p27 knockout mice display continued cell division into the postnatal period, as well as supernumerary hair cells (Chen and Segil 1999; Lowenheim¨ et al. 1999). In parallel to the continued mitosis and excessive number of hair cells, the p27 knockout mice are severely hearing impaired (Chen and Segil 1999; Kanzaki et al. 2006). The reason for the deafness in these mice is unknown at present.

While p27 is restricted to mature supporting cells, it appears that Ink4 is expressed in hair cells and prevents their reentry into the cell cycle. Ink4d-/- animals display continued mitosis and cell death in the hair cell population (Chen et al. 2003). Like p27 mutations, Ink4d mutations also lead to hearing loss.

Another gene involved in cell cycle regulation in the organ of Corti is the retinoblastoma tumor suppressor gene Rb1. Mutations in Rb1 cause tumors and loss of cell cycle regulation in multiple tissues (Lohmann and Gallie 2004). Disruption of Rb1 in the inner ear was accomplished by crossing floxed Rb1 mice with collagen1A1 (Col1A1)-Cre mice (Sage et al. 2005). The resulting mutant mice lacked inner ear expression of Rb1 and exhibited a large number of

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supernumerary hair cells in the cochlea and the vestibular epithelium. One of the novel and important findings in this study was that hair cells themselves might be able to replicate their DNA and divide in the absence of functional Rb1. The supernumerary hair cells had several phenotypic features of hair cells. Future improvement in the technology to regulate the control of cell proliferation genes via somatic cell–specific methods will enable the use of the knowledge of cell cycle regulation to design clinical approaches to regenerate hair cells.

3.3 Technological Needs for Inducing Therapeutic Transdifferentiation and Proliferation

To design clinically applicable therapies based on the scientific knowledge generated in the laboratory, it is necessary to develop safe and efficient methods for specific inner ear application of such therapies. Much of the practical application of knowledge at the genetic level will depend on the ability to regulate gene expression in a timeand place-specific manner and at the optimal level. Regulating gene expression can involve overexpression of a gene or blocking gene expression. Overexpression can sometimes be accomplished by the use of ligands that bind to extracellular receptors and initiate gene expression cascades. Thus, genes encoding secreted proteins can be introduced to cells in the vicinity of the target cell and the secreted gene product will act in a paracrine fashion as shown for several growth factors genes (Staecker et al. 1998; Yagi et al. 1999; Chen et al. 2001; Kawamoto et al. 2003a).

In other cases, it is necessary to introduce the gene itself into the cell, along with a promoter that can function in the specific cell type. The use of a variety of vehicles and techniques to influence gene expression in the cells of the inner ear has recently been reviewed (Avraham and Raphael 2003; Patel et al. 2004; Crumling and Raphael 2006). One major challenge in manipulating gene expression in the inner ear is the need to deliver therapeutic agents into cells or fluid spaces. This task involves the risk of disrupting the membranous labyrinth. The ideal route of delivery would be oral or systemic via intravenous injection, but the final concentration in the cochlea would be impractically low. In many cases, the blood–ear barrier would prevent the entry of the delivered reagents into the inner ear. Delivery of genes will probably continue to require direct inoculation into the cochlear fluid. Vector inoculation into the perilymph is a minimally invasive method to penetrate the round window using a micropipette (Stover¨ et al. 1999). Alternatively, cochleostomy can be used. However, if the vector solution needs to be introduced into the scala media, the procedure is likely to result in damage to the cochlear tissues (Ishimoto et al. 2002). With the present adenovirus vectors, it is necessary to inoculate into the endolymph to achieve transgene expression in nonsensory cells of the cochlea (Ishimoto et al. 2002). This procedure is technically complicated, leads to excessive variability in the results, and is not easily applicable to clinical use. The future may bring alternative or improved vectors that will accomplish viral delivery of nonsensory epithelial cells via the perilymph. To utilize cell cycle regulatory genes for hair

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cell regeneration therapy, it will be necessary to develop methods to promote temporally and spatially regulated gene expression.

4. Inner Ear Stem Cells

4.1 Progenitor and Stem Cells from Vestibular and Cochlear Tissues

The advent of stem cell biology research has recently provided new insights and technology that allows the direct testing of the hypothesis that inner ear sensory epithelia with regenerative capacity contain a population of progenitor or stem cells with high proliferative capacity. The inner ear sensory epithelia obviously do not contain undifferentiated basal cells, but some residing differentiated supporting cells may have the ability to dedifferentiate and to proliferate. The defining feature of a stem cell is its capacity for long-term self-renewal without losing the ability to spawn differentiating cells (McKay 1997). Particularly, the ability to proliferate without elaborate stimulation has been used routinely to isolate various stem cell populations from different organs. Today, the terms “progenitor cell” and “stem cell” are used very loosely. Progenitor cells are defined here as not fully differentiated cells, which are able to undergo a limited number of mitoses; stem cells are defined as multipotent or pluripotent cells with the capability of long-term self-renewal.

A first indication for the possible existence of inner ear stem cells in the postnatal mammalian inner ear arose from the observation that dissociated cells from the early postnatal rat organ of Corti developed into floating spherical colonies (Malgrange et al. 2002). Hair cells were detectable within these floating colonies after a 2-week culture period. Malgrange and colleagues further demonstrated that these new hair cells arose from dividing progenitors that incorporated the thymidine analog bromo-deoxyuridine during S-phase. They did not demonstrate, however, that individual cells are capable of generating floating colonies and that these spheres can be propagated. In a related study using dissociated cells from adult vestibular sensory epithelia, Li and colleagues (Li et al. 2003a) also found floating spherical colonies. A series of tests demonstrated that these spheres arose from single cells with high proliferative capacity and that it is possible to propagate and to maintain the spheres over many generations; ergo, the sphere-forming cells within adult vestibular epithelia are stem cells. Grafting of spheres derived from murine inner ear vestibular stem cells into the inner ears of chicken embryos showed that murine cells were able to differentiate into hair cells. When grafted at earlier stages, inner ear vestibular stem cells gave rise to many different cell types in organs derived from all three germ layers. Hence, inner ear vestibular stem cells are pluripotent (Li et al. 2003a).

Propagation of inner ear–derived spheres revealed a second characteristic feature: the majority of the 50–100 cells that make up an individual sphere are differentiating progenitors and only 1–3 cells are stem cells, which are able to

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reform new spheres (Li et al. 2003a). This is a problem because with a very limited capacity for expansion, it is difficult to obtain sufficient numbers of stem cells for extensive experiments. Nevertheless, improvements for growing and expanding other stem cell populations have been developed (Svendsen et al. 1998; Sen et al. 2002), and future improvements of inner ear stem cell passaging will lead to the greater availability of these cells.

The initial observation of sphere formation from dissociated early postnatal rat organ of Corti (Malgrange et al. 2002) raised the question of whether these spheres are, like the vestibular spheres, the manifestation of proliferating stem cells. This is indeed the case, as spheres derived from dissociated postnatal mouse organ of Corti can also self-renew for many generations (Oshima et al. 2007). Interestingly, during the second and third postnatal weeks in mice, the organ of Corti loses about 99% of its sphere-forming capacity. It is unlikely that several hundred stem cells disappear from the organ of Corti during this postnatal maturation period. It has been speculated that the loss or reduction of proliferative capacity goes hand-in-hand with the final maturation of supporting cells or of differentiation of greater epithelial ridge cells to inner sulcus cells. This observation is encouraging for potential hair cell regeneration in the adult mammalian organ of Corti because the prospective hair cell progenitor cells have not vanished but are possibly unable to respond to mitogenic stimulation. It is unclear, however, whether any mitogenic substances are increased, or conversely, whether any antimitotic factors are decreased in the mammalian cochlea as a consequence of hair cell loss (Tsue et al. 1994).

Within the cochlea, progenitor cells or stem cells are not limited to the organ of Corti. Rask-Anderson and colleagues (Rask-Andersen et al. 2005) have recently described the isolation and propagation of sphere-forming cells from adult human and guinea pig spiral ganglion. These adult spiral ganglion stem cells display similar characteristics to adult neural stem cells and can differentiate into neurons and glial cell types. It appears that the 6-week-old murine spiral ganglion harbors only a few stem cells with sphere-forming characteristics, and sphere-forming stem cells from mice older than 6 weeks occur only occasionally, which is too rare to be reliably quantifiable without processing large numbers of inner ears (Oshima et al. 2007). When both studies are taken into account, one can nevertheless hypothesize that spiral ganglion stem cells exist in older mammals and that they are more readily detectable in individual spiral ganglia specimens from guinea pigs or humans because the larger ganglia in these mammals contain more total cells than the murine ganglia. Other important cochlear tissues are located in the stria vascularis. It appears that cells with proliferative capacity are also detectable in the postnatal stria of young postnatal mice (Oshima et al. 2007). Nevertheless, all stem cell populations of the murine cochlea seem to diminish substantially during the initial postnatal period, which is in stark contrast to the vestibular stem cell populations, which appear to be maintained, albeit at low numbers, throughout life.

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4.2 Capacity of Inner Ear Stem Cells to Differentiate into Hair Cells

Sphere-forming stem cells can be propagated as important constituents of floating colonies in serum-free medium containing growth factors (Li et al. 2003a). Withdrawal of the growth factors and attachment of the spheres leads to patches of differentiating cells. Cells that express multiple hair cell markers become obvious after 10–14 days of differentiation, but only in populations derived from either vestibular or organ of Corti sensory epithelium–derived spheres (Li et al. 2003a; Oshima et al. 2007). Differentiating cells from spheres derived from the stria vascularis and from the spiral ganglion do not normally contain hair cell marker–positive cells. Neurons and glial cell types, on the other hand, can be found not only in populations derived from spiral ganglion spheres, but also in cells derived from vestibular cells and organ of Corti sensory epithelia.

These observations suggest that there are substantial differences among different populations of sphere-forming stem cells and that hair cells spontaneously differentiate in vitro only from sphere populations derived from inner ear sensory epithelia. This potential for generating hair cells in vivo became observable after grafting of spheres derived from murine vestibular sensory epithelia into the developing inner ears of chicken embryos at a developmental point before formation of sensory epithelia. After development continued for several days, hair cell marker–positive cells were found integrated into the maturing sensory epithelia (Li et al. 2003a). It remains to be demonstrated whether inner ear stem cells derived from vestibular sensory epithelia or the organ of Corti have the capacity to replace lost hair cells in mammalian cochleae.

4.3 Capacity of Other Non–inner Ear Stem Cells to Differentiate into Inner Ear Cell Types

Sphere-forming neural stem cells, isolated from the embryonic mouse brain, have been used for grafting experiments in the neomycin-treated cochleae of 4-week- old mice. Although the majority of the grafted cells differentiated into neurons and glia, a few hair cell marker–positive cells were found in vestibular sensory epithelia (Tateya et al. 2003). While it appears plausible that stem cells isolated from ectodermally derived organs are best suited to replace lost hair cells, other stem cell types may have features that make them uniquely suitable for use in a therapeutic situation. Bone marrow–derived stem cells, for example, have been found to survive after transplantation into cochleae and to differentiate into neuronal and glial marker–expressing cells (Naito et al. 2004), which suggests that autologous bone marrow grafts could potentially be used to replace lost spiral ganglion neurons. In vitro guidance of mesenchymal stem cells from the bone marrow with a combination of Sonic hedgehog and retinoic acid appears to enhance greatly the expression of neuronal markers and enables the bone marrow–derived cells to grow neurites toward hair cells in coculture experiments (Kondo et al. 2005). Expression of Atoh1, a key transcription factor for hair cell

Table 11.1. Challenges to effective cochlear treatment.

Challenge	Description	Outlook
Cell delivery	Gaining access to the cochlea without causing additional	Refinement of surgical skills. Systematic
	damage. This is particularly difficult in small animal	comparison of different routes of cell
	models such as the mouse.	administration.
Cell homing	Attachment and integration of the grafted cells into the	The correct progenitor cell type may have the
	damaged organ of Corti, not at random locations.	intrinsic capacity for homing. Otherwise,
		engineering of cells with appropriate surface
		receptors.
Functional integration/reinnervation	The cells have to take the place of lost hair cells and become	There are indications that ectopically placed
	afferently and efferently innervated. Stimulation of the	hair cells in the cochlea are innervated
	replacement cells has to evoke action potentials in the	(Kawamoto et al. 2003b)
	auditory nerve.
Generation of different hair cell subtypes	Generation of “generic” hair cells with the ability to attract	The factors that control development of
	and to synaptically connect with afferent nerve fibers is the	different hair cell subtypes are largely
	primary goal. Outer hair cell equivalents are probably	unknown. There is some speculation that the
	necessary to restore functionality completely.	local environment is able to influence the
		subtype of replacement hair cells.
Functional integration/placement	Proper orientation of stereociliary bundles of replacement hair	The grafted cells may be able to receive
	cells and positioning in context with the tectorial	guidance cues from the local environment.
	membrane. Physical connection of the stereociliary bundles	Refinement of the functional integration is
	of outer hair cell equivalents with the tectorial membrane.	perhaps a secondary goal and not necessary
		for initial, but incomplete, functional
		recovery.
Long-term survival	Regenerated hair cells need to survive for an extended period.	No experimental data yet. Need for autologous
		transplants or suppression of the immune
		response.
Mitotic quiescence	Terminal differentiation of all grafted cells.	Cell sorting before transplantation may be
		required.

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