Feasibility of treating hearing disorders with stem cells: update.

Abstract

The recent isolation of adult stem cells from the mouse utricle that have the capacity to differentiate into cells from all three germ layers--and more importantly, into inner ear hair cells--offers a viable option for the treatment of hearth gloss. The finding that embryonic stem cells are also capable of differentiating into hair cells further expands the possibility, that we may someday develop restorative treatment of sensorineural hearing loss.

Introduction

Treatment of hearing Joss continues to pose a major challenge to the otolaryngologist, as more than 30% of adults beyond 6 5 years of age have a debilitating hearing disorder. This figure continues to increase in tandem with rising life expectancies. The pervasiveness of hearing loss not only increases the financial burden on society but also causes significant morbidity in affected patients.

More than 80% of all cases of hearing loss can be attributed to the degeneration and death of sensory hair cells and their associated spiral ganglion neurons. (2) The loss of hair cells in mammals is usually permanent. Hair cell loss is caused by genetic mutations, autoimmune disease, ototoxic medications (e.g., aminnglycosides), exposure to excessive noise, and aging. (2,3) The irreversibility of permanent hearing loss is a consequence of the cochlea's inability to repair or regenerate hair cells. The currently available therapeutic options amplification devices and electrical stimulation of the auditory nerve--are reserved for patients with severe to profound hearing loss, and they do not address the issue of hair cell regeneration.

Search for an ideal treatment

An ideal treatment for permanent hearing loss would be based on the regeneration or replacement of damaged hair cells and would be directed toward the site of the lesion--that is, the organ of Corti. However, in the experimental mammalian model, attempts to regenerate damaged inner ear hair cells by genetic means have resulted in only limited success. (4-6) Identification of efficient methods to initiate regeneration remains a major challenge.

An alternative to regeneration is hair cell replacement, using cells that are capable of differentiating into functional hair cells. We know that nonmammalian vertebrates have the capacity for hair cell regeneration through the proliferation of specialized cells called stem cells that are thought to reside in the sensory epithelium of the ear. (7,8) Jones and Corwin have proposed that a subpopulation of cells localized in the supporting cell layer in mammals may be progenitor cells that are responsible for limited hair cell regeneration in vestibular organs such as the utricle. (9) Indeed, adult stem cells were recently found in the sensory epithelium of the mouse utricle, and they are the likely source of the progenitor cells responsible for hair cell regeneration in this organ. (10,ll)

Adult stem cells

A stem cell is a unique type of cell that has the ability to renew itself over long periods of time. Pluripotential stem cells can also differentiate into various kinds of specialized cells when they are appropriately stimulated. (12)

An adult stem cell is an undifferentiated cell that is found in specialized tissue and has the capacity to produce the specialized cell types from the tissue of origin. (12) In the Eaton-Peabody Laboratory at the Massachusetts Eye and Ear Infirmary, Li et al have successfully isolated stem cells from the sensory epithelium of the adult mouse utricle, (10) They have developed a technique to routinely isolate and propagate these stem cells in vitro. This is accomplished by dissecting the utricular sensory epithelium in these mice. The tissue is then dissociated into individual cells that are maintained in a serum-free medium supplemented with growth factors, including epidermal growth factor, insulin-like growth factor-1, and basic fibroblast growth factor. After 8 days in culture, a small number of these cells show a distinct potential for forming floating colonies of cells called spheres (figure 1). (13)

[FIGURE 1 OMITED]

Spheres are clonal colonies generated from a single stem cell. They exhibit strong mitotic activity, which can be visualized by incorporating the thymidine analog BrdU (5-bromo-2'-deoxyuridine). Spheres are made up primarily of pluripotential progenitor cells that are capable of differentiating into cell types from the three germ layers. in particular, the progenitor cells display an ability to differentiate into cells that express several different markers characteristic of hair cells (figure 2). (14,15)

[FIGURE 2 OMITED]

Once the in vitro experiments were completed, the next step was to investigate the in vivo behavior of these stem cell-derived progenitor cells. This involved grafting the utricular mouse-derived progenitor cells into the developing ears (otic vesicles) of embryonic chickens. The results of these animal experiments corroborated the in vitro findings. The cells were successfully integrated into the developing chicken ear, and they gave rise to cells characteristic of hair cells. The success of this experiment was confirmed when the cells expressed immunologic markers indicative of hair cells. (14,15) The sphere-derived cells were then transplanted into the amniotic cavity of the embryonic chicks prior to gastrulation to determine their pluripotentiality. The transplanted sphere-derived cells did indeed give rise to cells from all three germ layers.

Embryonic stem cells

Another source of inner ear progenitor cells are embryonic stem cells, which are derived from the inner cell mass of the mouse blastocyst (figure 3). (16-18) Having established appropriate growth conditions for the directed differentiation of adult stem cells toward hair cells, Li et al attempted to determine if embryonic stem cells could also differentiate into hair-cell-like cells. (10) This was done by initiating the differentiation of embryonic stem cells into aggregates called embryoid bodies.

[FIGURE 3 OMITED]

Following the formation of these bodies, the cell population was enriched with specific growth factors to form progenitor cells that expressed genes indicative of the developing inner ear. To determine if these cells could give rise to differentiated inner ear cell types, growth factors were withdrawn and culture was continued in a defined medium. This process resulted in the differentiation of a population of cells that expressed markers characteristic of hair cells, illustrating how embryonic stem cells can be differentiated into hair cells in a stepwise fashion (figure 3). (19)

The future

Li et al are the first to report the generation of hair cells from stem cells. Their findings could have significant implications for the hearing-impaired, and they might further the possibility of developing a "cure" for some forms of hearing loss. Their research will also help us better understand the molecular nature and development of mammalian hair cells.

Physiologic characterization of stem cell-derived hair cells with recovery of neural synapses will be the obvious next step on what will be a long road toward developing clinically applicable techniques for the treatment of hearing loss with stem cells. But the discovery of this novel means of creating hair cells from stem cells may shorten our journey.

It is exciting to speculate on the impact that these findings will have on the future of clinical treatment of inner ear disorders. At the moment, it seems futuristic to envision therapy with progenitor cell grafts that can survive without proliferation, integrate at appropriate sites, receive innervation, and perform well enough to restore basic mechanoelectrical transduction. But it is conceivable that these dreams will eventually be realized.

References

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From the Department of Otolaryngology and the Program in Neuroscionce, Harvard Medical School, Boston, and the Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Boston.

Reprint requests: C. Eduardo Corrales, MD, Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, 243 Charles St., Boston, MA 02114. Phone: [phone]: fax: [phone]; e-mail: [e-mail]