Figure 1 Scanning electron microscope image of hair cells in the normal cochlea of a bird.

Figure 2 Immature hair cells that have emerged after damage to the cochlea of an adult bird. These hair cells will restore hearing. The article that follows will tell the story of how this was discovered.

The Research and Its Implications for Restoration of Hearing in Humans

This description is originally from an interview with Edwin W. Rubel, Ph.D., Virginia Merrill Bloedel Professor of Hearing Science, and Professor in the Departments of Otolaryngology–Head and Neck Surgery, Physiology & Biophysics, and Psychology at the University of Washington. Dr. Rubel and his colleagues discovered hair cell regeneration in birds. Dr. Rubel has published over 200 scientific articles and edited four books on various topics related to development and plasticity of the auditory system.

What Are Hair Cells and What Do They Look Like?

Figure 3- This schematic drawing depicts a cross section through the human temporal bone and shows the structures in the inner ear (right-hand side of drawing) as well as the external and middle ear (left-hand side).

Hair cells are the sensory receptors located within the inner ear (Figures 1,2). Microscopically, hair cells appear as if they have hairs because of tiny structures called stereocilia that extend from their surfaces.

Auditory hair cells are located in the organ of Corti of the cochlea (Figures 3-6), and they are involved in detecting sounds. Auditory hair cells convert sound information into electrical signals that are sent via nerve fibers to the brain and processed. Vestibular hair cells are located in the vestibular (balance) organs of the inner ear (utricle, saccule, ampullae). They detect changes in head position and send this information to the brain via nerve fibers. This information is used to help maintain body posture, eye position and balance. Without auditory or vestibular hair cells, the energy derived from sound waves or gravity is not converted into neural signals, and hearing or balance deficits ensue.

Damage to hair cells can be caused by a number of agents, including loud sound, ototoxic drugs (some antibiotics and anti-tumor drugs), disease, and processes associated with aging (Figure 7a). In humans and other mammals, hair cell damage results in permanent hearing impairments and/or balance disorders.

Figure 4 - A human inner ear. The vestibular portion of the inner ear houses the vestibular end organs (utricle, saccule, ampullae) and is located on the top. The cochlea is on the bottom, and it houses the hearing organ, the organ of Corti.

Figure 5 Section through the cochlea showing that it contains three tubes, the middle one contouring the hair cells and other structures needed for hearing. The names of important structures are given.

Figure 6- This schematic drawing shows a high-magnification view of the mammalian auditory epithelium in the inner ear, also called the organ of Corti. Sensory hair cells are shown in orange, and non-sensory supporting cells are shown in green. The cochlear nerve is depicted as black or red lines connecting to the bases of the hair cells.

How Did Research on Hair Cell Regeneration Get Started?

Restoration of hair cells in mature animals after damage (hair cell regeneration) was not a major topic of research in hearing science until the late 1980s. Before that time, a small amount of research was being done on post-embryonic (after birth) hair cell growth in the vestibular (balance) epithelium of lower vertebrates, but no studies addressed hair cell regeneration in higher vertebrates such as birds and mammals. Scientists thought that hair cell regeneration was impossible in higher vertebrates and that the mature inner ear was incapable of producing new hair cells in response to trauma. Then, in 1986–87, two groups serendipitously discovered that birds can generate new hair cells after birth. The experiments were done by my group (at the time at the University of Virginia and now at this center) and by my friend, Dr. Doug Cotanche, who was initially at the University of Pennsylvania and is now at Harvard University. Both groups were studying the bird cochlea as a model for how hearing develops in mammals and how hair cell damage occurs in humans, because the bird inner ear is simpler to study than the mammalian inner ear. During these experiments, both groups found evidence suggesting that new hair cells were present in the mature bird cochlea after damage.

What Have You Discovered?

We found that birds have the remarkable natural ability to regenerate their inner ear hair cells after damage produced by either loud noises or ototoxic drugs (Figure 7b). This was quite startling to us, as well as to the rest of the scientific community! We quickly went on, to show that there were indeed large numbers of new hair cells that were generated by cell division after the damage had occurred, and that this regeneration occurred in several species of fully mature birds.

At that point, many scientists became interested in this research area, and since then, new information has emerged at a fast and furious pace. For example, the Rubel laboratory quickly showed that hair cell regeneration also occurs in the balance organs of the mature avian inner ear. In addition, Rubel and others performed several experiments to show that the regenerated hair cells in birds are functional—i.e., they restore hearing and balance sensation, both at the level of physiological responses from the brain and at the level of sensory-dependent behavior.

Three major new approaches to studying hair cell regeneration were taken on and are ongoing in the laboratories at the Virginia Merrill Bloedel Hearing Research Center. One approach is to identify the cellular and molecular events that produce hair cell regeneration naturally in the bird. The second approach is to determine whether hair cell regeneration can be induced in mammals by the addition of molecules that are known to stimulate cell division in other tissues of the body. The third is to identify genes that may inhibit cell division in the inner ear, thereby preventing the first steps toward hair cell regeneration.

Figure 7 - The images shown here are surface views of the auditory epithelium from a rat (A) and a chicken (B), before and after hair cell damage. Note the extent of recovery seen in bottom right panel; similar recovery is not seen in mammals. The top two images were generated by Dr. Marc Lenoir, University of Montpellier in France.

Figure 8 - Schematic drawing depicting results from analysis of cell division in cultured mammalian ears.

A big breakthrough in these areas of research came around 1992-93 when Dr. Elizabeth Oesterle and I, as well as other groups, found ways to keep the mature inner ear sensory epithelium (the part of the hearing and balance organs containing the hair cells) alive in tissue culture over a few days or weeks. This discovery enabled us to efficiently study the response of avian and mammalian inner ear cells to various compounds that might stimulate or alter regeneration. We are starting to identify compounds that can stimulate the production of new cells in the inner ear and other molecules that prevent regeneration (Figures 8,9). We hope these approaches will ultimately lead to an understanding of how regeneration occurs in birds, why regeneration is normally prevented in mammals, and how to make it happen in humans.

Figure 9 - Introduction of a growth factor, transforming growth factor alpha (TGFalpha), causes the production of new cells in the vestibular organs of the adult rat. A cell proliferation marker, tritiated thymidine, was administered to allow the identification of dividing cells. Infusion of TGFalpha with insulin directly into the inner ear of adult rats stimulated cell division in the vestibular hair cell epithelium, and new cells (e.g., arrow) are produced.

There have also been several potentially important spin-offs of this research. For example, the development of tissue culture and organ culture methods for studying mature inner ear tissues has led to new approaches to understanding and preventing hair cell loss. These investigations could lead to new therapies for prevention of hearing loss resulting from aging therapeutic drugs or the interaction of such processes.

Is Restoration of Hair Cells Enough to Actually Restore "Normal" Hearing?

From what we know about hair cell regeneration in birds and the development of the inner ear of mammals, we expect that restoration of nearly normal hearing in humans may be possible someday. In birds, nerve cells appear to re-connect with the regenerated hair cells without difficulty. This may be due to the effects of molecules called "trophic factors," which are thought to attract nerve fibers to the hair cells. It appears that, once new hair cells are produced during normal development and during regeneration, they secrete growth factors and thereby attract nerve fibers to connect with them. Once this happens, critical sensory information should be readily transmitted to the brain.

How Did You Confirm Restoration of Hearing in Birds?

We collaborated with researchers in the psychology and physiology departments who have developed behavioral and physiological methods to study hearing and balance in birds. We can precisely determine the animal's lowest thresholds for detection of sound at different frequencies as well as its ability to distinguish one sound from another. All of these abilities are degraded when an animal or human is exposed to intense periods of noise, or large amounts of ototoxic drugs, and during the aging process. These abilities appear to be restored after hair cell regeneration to near-normal levels. We can also look at the ability of birds to reproduce their own songs, or to discriminate between their own songs and other individual songs of their species.

What Are the Implications for Humans?

The most important implication is that now, for the first time in history, there are teams of investigators world-wide examining the possibility that hair cell regeneration can be induced in the mammalian and, more specifically we hope, the human inner ear. The obvious implications for humans are that we may discover how to restore hearing and balance disorders in the next decade or two. Current therapies for hearing impairment (hearing aids and cochlear implants), while useful, cannot fully alleviate hearing deficiencies, and there are virtually no therapies for chronic balance disorders.

Figure 10 - These schematic drawings depict the cellular dynamics that occur during the two different modes of hair cell regeneration in chickens, cell division (A) and direct transdifferentiation (B).

The second challenge is to induce the newly produced immature cells to differentiate into mature hair cells; that is, to develop into what we call a "hair cell phenotype." Development of several features is required for the new cells to restore hearing or balance function. There is also emerging evidence that hair cells in frogs and birds can be regenerated in an altogether different way that does not involve the production of new cells, by a process called direct transdifferentiation or conversion (Figure 7b). During this process, support cells lose their cell-specific characteristics and then develop all the specializations of hair cells without going through a round of cell division. There is some evidence that this may occur to a limited extent in the balance organs of the mammal inner ear and that this process can be induced experimentally in the mature mammalian inner ear.

What Is the Future of This Research?

The most important challenge for the future is to produce new hair cells in humans. Hair cell production takes the successful completion of two relatively independent processes. The first set of events that must be triggered is the division of progenitor cells, also called support cells, that sit next to hair cells and survive the damage. In birds, support cells divide and give rise to new hair cells (Figure 7a). We must be able to stimulate support cells in mammals to divide and make new cells, similar to what occurs during mammalian development and spontaneously in mature birds.

Where Is the Work on Mammals Now?

Figure 11 - Labeling for dividing supporting cells in the organ of Corti is seen in the top two panels in normal mice (left) and in mice with complete knockout of the cell cycle regulatory gene, p27 (right). Mice were 11 days old at the time of analysis. Dividing cells appear as dark brown dots. Note the paucity of dividing cells (dots) on the left and the abundance of dividing cells on the right.

Several recent investigations provide evidence that hair cell regeneration in mammals is attainable. For instance, we can now induce a small amount of cell division in the balance organs of the adult mammalian inner ear in a dish (that is, in culture), as well as in vivo (within a living organism). This has been done in mice, and rats, using growth-promoting molecules. Therefore, we know that it is possible to induce the first stage of hair cell regeneration in the inner ears of mature mammals.

In addition, recent investigations from the laboratories of the Virginia Merrill Bloedel Hearing Research Center and the House Ear Institute in Los Angeles have demonstrated that cells in the organ of Corti (the cochlea) of young and mature mice are capable of cell division. This has been shown in mice that are lacking one of the genes involved in controlling cell division. In these mice, support cells that are normally quiescent continue to divide well after cell division normally stops developmentally (Figure 11). While direct manipulation of this gene in humans is probably not feasible, this is a very promising finding, suggesting that hair cell regeneration is possible in the hearing organ of humans.

Finally, recent experiments in neonatal and adult rodents point to an important function for the gene called Math1 (also called atonal) in triggering direct transdifferentiation of supporting cells into hair cells. Introduction of Math1 into supporting cells in the cochlea or utricle triggers their conversion into hair cells. These studies have been conducted by a number of laboratories, including Genentech, Dr. Yehoash Raphael’s lab at the University of Michigan, as well as our own laboratories (Figure 9). In experiments in the cochlea performed by Dr. Yehoash Raphael’s lab, this conversion appears to partially restore hearing function.

Good News and Bad News

The good news is that this research has triggered many laboratories around the world to attempt to find ways to regenerate hair cells in the inner ear. In 1987, there were only two laboratories studying hair cell regeneration; now there are probably 20 or 30. The bad news is the limitations on funding. Until we find candidate molecules for use in humans, this research will not be adopted by many pharmaceutical companies. This is unfortunate because, once pharmaceutical companies commit to studying regeneration, they will invest heavily in developing human therapies. Until then, the entire cost of this research must be borne by the federal government, private donations, and by private foundations. We are certain that, within five to ten years, we will find out whether it is possible to regenerate hair cells in mammals at levels sufficient to restore hearing and balance function. From there, it could be as little as another ten years until we achieve hair cell regeneration in humans.

Figure 12- Introduction of Math1 into cultured organ of Corti induces formation of extra hair cells in the greater epithelial ridge (GER), a region that normally does not contain hair cells. Panel A is a low power view of the organ of Corti taken from a neonatal mouse where Math1-GFP was introduced into some cells, as indicated by the cells turning green in color. Hair cells are identified by labeling with an antibody to myosin VI, and they are red in color. A higher power view of the same figure (panel B) illustrates cells that show both red and green colors (e.g., arrows). These are cells that have transformed from supporting cells into hair cells as a result of the introduction of Math1.

Eight years ago, somebody said “We will never be able to restore the complexity and intricacies of hair cells in humans or other mammals”. The response then, as it is now, is "You could be right, but without trying, it surely will not happen. If our goal is to actually restore hearing, hair cell regeneration is the only game in town!"

The Hearing Regeneration Initiative: You Can Help Make a Difference!

A broad-based multidisciplinary team of researchers at the Virginia Merrill Bloedel Hearing Research Center has formed to aggressively pursue hair cell regeneration research. Seven principal investigators at the Center (Drs. Bermingham-McDonogh, Gates, Hume, Oesterle, Reh, Rubel, and Stone)are currently working on various aspects of avian and mammalian hair cell regeneration. In addition, we recently joined forces with investigators at other national and international universities. Collectively, these efforts are termed the Hearing Research Initiative . The goals of this initiative are to develop a more collaborative approach to studying cellular and molecular factors regulating hair cell regeneration, and this involves increasing the degree to which scientific ideas, resources, and results are shared among investigators. We believe this approach will significantly hasten progress in hair cell regeneration research.

The Hearing Regeneration Initiative is supported to a large extent by a generous donation from an anonymous private foundation. It is also funded by donations from individuals in the Seattle community and elsewhere. Any individual donation is matched 100% by our private foundation sponsor. For more information, see Support Us . People interested in financially supporting this Hearing Research Initiative are encouraged to contact the Development Office of the University of Washington (206-543-5686) or the Business Manager at the Bloedel Hearing Research Center (206 616-4105 or bloedel@u.washington.edu).