Hair Cell Regeneration Continues to Elude Scientists

In a University of Maryland lab, psychologist Robert Dooling trains hundreds of small, colorful parakeets, zebra finches, and canaries to chirp on command. In about three weeks, the birds learn to mimic computer-produced sounds. Once the birds' vocalizations match the template, Dooling, who heads the university's comparative psychoacoustics laboratory, rewards them with seed. Dooling isn't interested in producing sweet songs, but rather in understanding what happens when these little creatures lose their hearing. The birds are then exposed to noise, which damages the thousands of hair cells, or sensory cells, located in the inner ear. When undamaged, these cells transmit sound through nerves to the brain. When injured, the birds become deaf and lose their ability to chirp precisely. Dooling and his partner, Brenda Ryals at James Madison University, want to learn if hearing and speech return to normal levels once the hair cells regrow. According to their tests, birds regain their ability to produce precise vocalizations within 28 days, the amount of time that it takes for all the hair cells to regenerate.1 "From our studies, we think that with a new set of hair cells, the ability to hear is largely back, but the world sounds different than it did before," Dooling says. By focusing on the structural and functional consequences of deafness and auditory restoration, Dooling's and Ryals' work indicates how deaf humans will hear once scientists learn to regenerate a human hair cell. It's an effort that has been going on for some time. Editor's Note: This is the second of five articles in a series on some of the most intriguing research occurring on the senses. The first story, on vision (J.F. Wilson, "Gene therapy, stem cells: Prime for vision restoration," The Scientist, 15[18]:17, Sept. 17, 2001), examined how investigators are using gene therapy and stem cell transplantation to move further toward a cure for blindness. A story on taste, scheduled for Oct. 15, will explore an explosion of research in the past few years that has led to the first identification of the sweet and bitter taste receptors, and is beginning to reveal the complex inner workings of the taste cells. Until 1987, most believed that mammalian hearing loss was irreversible. But that year, two labs discovered that birds spontaneously regrow hair cells and regain hearing.2-3 The news offered the first evidence of hearing regeneration in higher vertebrates. It was an important discovery: Hair cell loss is the leading cause of human deafness, and cell replenishment could end more than 70 percent of it and tinnitus, the ringing in the ear that can be caused by hair cell loss, according to the Deafness Research Foundation. Buoyed by the two reports, numerous researchers set off to harness the mechanisms of avian hearing regeneration to coax human hair cells to regrow. But mysteries of the auditory system have stymied researchers, who had expected to achieve regeneration by now. "In the mature mammal inner ear, nobody has shown clearly yet that new hair cells are born by mitotic division after damage. But that's not to say that the work is not progressing well," says Edwin Rubel, a professor in the departments of otolaryngology and physiology and biophysics at the Virginia Merrill Bloedel Hearing Research Center, University of Washington. It was Rubel and Douglas Cotanche, then at the Medical University of South Carolina and now at Children's Hospital in Boston, who authored the nearly simultaneous 1987 reports. Some evidence exists that hair cell regeneration could occur at a low rate in normal adult mammals. But studies indicate that the machinery for regeneration doesn't operate at a sufficient level under normal circumstances to bring effective recovery.4-6 "People have tried various ways to make cells in the inner ear of mammals re-enter the cell cycle and regenerate. And that's pretty much been a failure," says Neil Segil, chief, section on cell growth and differentiation at the House Ear Institute in Los Angeles. So researchers have lately refocused their goals, and now are working on understanding the molecular biology of hair cell development. Their approach is slowly decoding the system of cell cycle control and the cell differentiation process that leads to hair cell development. Mining the Cell Biology of the Embryo "At this stage, we don't even know if cells that give rise to new hair cells even exist in the mature inner ear," Segil says. So he and Andy Groves, a colleague at the House Ear Institute, are working to find these progenitor cells. Based on advances made in identifying stem cells in other parts of the nervous system, Segil and Groves want to isolate a cell population that gives rise to the sensory cells during mouse embryogenesis. Once identified, these cells will become tools to search for markers that will reveal if progenitors actually exist in the mature ear. If they do, their discovery would provide an important target for therapeutic attempts at inducing hair cell regeneration. At the National Institute of Deafness and Other Communication Disorders in Bethesda, Md., neuroscientist Matthew Kelley is also working with inner ear cells from mouse embryos. Looking downstream from the progenitor cells, Kelley is trying to learn the molecular basis for how hair cells differentiate. He's studying transcription factors with a knockout mouse lacking Jagged-2, a gene that plays a role in hair cell regeneration. Mice without Jagged-2 produce more hair cells than normal, he found, and they also don't express other genes known to be involved in the inhibition of hair cell development.7 This suggests that it's possible to regulate hair cell numbers generated in the ear, according to Kelley. By elucidating the necessary transcription factors, he hopes to uncover the molecular pathway that takes an uncommitted progenitor cell and turns it into a hair cell. "In terms of regeneration, this is the pathway that needs to be recapitulated," he says. Electron microscopic image of hair cells viewed from above their hair bundles But even if this occurs, the pathway might not produce enough cells to return hearing. So Jeffrey Corwin, a professor in the departments of otolaryngology and neuroscience at the University of Virginia, is looking intracellularly at these molecular pathways for proliferation, and developing techniques for amplification. Working with tissue cultures of rodent hair cells in vivo, he and his colleagues study the triggers for the cell divisions that underlie regeneration. He's focusing on mammals and hair cells for the vestibular system--those outside the cochlea that control balance--that have shown greater promise for regeneration in humans and rodents and aren't as highly ordered as the cells in the cochlea. These vestibular hair cells, though different, function similarly to cochlear hair cells. Over the past few years, Corwin and colleague Mireille Montcouquiol have pursued promising therapies for driving the supporting cells to divide and proliferate, and they're also searching for what is required to control or stimulate these newly formed cells to become true sensory hair cells.8 "The most challenging question of all is, 'What is causing the sensory cells in mammals to become quiescent soon after birth, and how can that process be suspended?'" Corwin says. His studies with collaborators Rende Gu and Montcouquiol on mammal tissues have shown that the younger the animal, the more likely the sensory cells will proliferate in response to treatment. A flip side of embryo cell cycle research is focused on how these cells die. Cotanche, professor of otology and laryngology at Children's Hospital in Boston, is studying the early and late stages of the cell death pathway in chick cochlear hair cells in vivo. His work has found that hair cells actively control their own death. 9 "This means it is an active process, and that it might be possible to manipulate, or even block, this process from occurring," Cotanche says. Courtesy of Jeffery T. CorwinMammalian hair cell epithelia (cultured from the ear of a young rat) following a 15-minute treatment with the drug Forskolin followed by 72 hours in culture in a medium that contained recombinant human glial growth factor 2 (right image). The control image appears at the left. If it's impossible to manipulate the ear to repair itself, then transplantation could provide the answer. But the inner ear is so mechanistically sensitive that simply physically injecting cells might do more harm than good, researchers say. In addition to cell biologists, some geneticists want to identify genes that encode for hearing. Recent research at the House Ear Institute, at Bloedel, and at other centers shows that cells near the hair cells will re-enter the cell cycle in young mice missing the p27 gene, which helps control cell division, Rubel says.9-11 This finding hints that a similar restriction could prevent production of new hair cells in the mature mammalian inner ear, but, as yet, "there's very little evidence the new cells produced postnatally in neonatal mice lacking the p27 gene turn into surviving hair cells," he says. Drawing Together Research Rubel has proposed a $20 million, five-year initiative to fund expert cell and molecular biology labs studying other problems to take on hair cell regeneration studies. He hopes to use this initiative to create an effort that brings interdisciplinary researchers together to interact and collaborate. Others suggest that what's most important is continuing to apply traditional science in hopes that a breakthrough will occur eventually. No matter the approach, the goal continues to compel--and advance--hearing work. "When you find something as potentially important therapeutically as hair cell regeneration," Rubel says, "you can't ignore it." Jennifer Fisher Wilson (jfwilson@snip.net) is a contributing editor for The Scientist. References 1. R.J. Dooling et al., "Recovery of hearing and vocal behavior after hair-cell regeneration," Proceedings of the National Academy of Sciences (PNAS), 94:14206-10, 1997. 2. D.A. Cotanche, "Regeneration of hair cell stereociliary bundles in the chick cochlea following severe acoustic trauma," Hearing Research, 30:181-96, 1987. 3. R.M. Cruz et al., "Light microscopic evidence of hair cell regeneration after gentamicin toxicity in chick cochlea," Archives of Otolaryngology Head & Neck Surgery, 113:1058-62, 1987. 4. A. Forge et al., "Ultrastructural evidence for hair cell regeneration in the mammalian inner ear," Science, 259:1616-9, 1993. 5. M.E. Warchol, "Regenerative proliferation in inner ear sensory epithelia from adult guinea pigs and humans," Science, 259:1619-22, 1993. 6. P.R. Lambert, "Analysis of small hair bundles in the utricles of mature guinea pigs," American Journal of Otology, 18:637-43, 1997. 7. P.J. Lanford et al., "Notch signalling pathway mediates hair cell development in mammalian cochlea," Nature Genetics, 21:289-92, 1999. 8. M. Montcouquiol, J.T. Corwin, "Brief treatments with forskolin enhance s-phase entry in balance epithelia from the ears of rats," Journal of Neuroscience, 21:974-82, 2001. 9. C. Torchinsky et al., "Regulation of p27Kip1 during gentamicin mediated hair cell death," Journal of Neurocytology, 28:913-24, 1999. 10. H. Lowenheim et al., "Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti," PNAS, 96:4084-8, 1999. 11. P. Chen, "p27 (Kip1) links cell proliferation to morphogenesis in the developing organ of Corti," Development, 126:1581-90, 1999.

Hair Cell Regeneration Continues to Elude Scientists

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