Tools and Technologies I
Half of all adults will suffer from some degree of hearing loss, by the time they reach retirement age. Both genetic and environmental factors (loud noise, aminoglycoside antibiotics, chemo-therapeutic drugs such as cisplatin) contribute to hearing loss. This age-dependent hearing loss (presbycusis) is most commonly caused by the death of sensory hair cells in the inner ear. Regeneration of lost cochlear sensory hair cells does not occur in mammals, including humans, with the result that age-dependent hearing loss is permanent. However, aside from prosthetics such as hearing aids and the cochlear implant, developing the tools to induce regeneration in the sensory epithelia of the inner ear of humans offers the only hope for the millions of people who suffer from deafness due to hair cell loss. In contrast, other vertebrates, such as birds, can regenerate their hair cells by the stimulated cell division and then differentiation of neighboring cells known as supporting cells. Our previous studies in mice indicate that some supporting cells in the mouse inner ear retain a latent ability to divide and turn into hair cells (White et al., 2006), although under normal circumstances they never do. Thus, these cells represent a target population of cells that may be "manipulated" to regenerate hair cells, or alternatively, that might be a source of adult stem cells that could be transplanted into the damaged inner ear for therapeutic purposes. However, there are several different types of supporting cells that can be recognized morphologically and which serve different functions in the inner ear. Currently, the number of molecular markers for these different sub-populations of supporting cells is very limited, and there are no good ways to physically separate and purify them so that their individual regenerative capacity can be tested. Nor are there ways of growing and expanding the small population of supporting cells for experimentation or therapeutic purposes. In the first and second aims of this proposal, we will develop tools for the molecular identification of these sub-populations of supporting cells, as well as new ways to purify them from the general supporting cell population. In addition, we propose to develop new ways to grow them in culture, so that we can increase the number of available cells for experimental characterization. In parallel, we propose to adapt these newly developed techniques to purify and grow supporting cells from rare human inner ear tissue that is available as a result of special inner ear surgery. In a final aim, we propose to develop genetic tools to permanently mark supporting cells, so that we can follow the fate of these cells and their progeny after their manipulation both in the animal and in our culture dishes. This tool will be crucial for testing the efficacy of any pre-clinical trial involving manipulation or transplantation of progenitors/supporting cells.
Statement of Benefit to California:
Hearing loss affects millions of Californians. Both genetic and environmental factors (such as loud noise, aging, and commonly used drugs such as aminoglycoside antibiotics and chemo-therapeutic drugs such as cisplatin) contribute to hearing loss. All forms of hearing loss, either age-related, as among the baby boomers; noise-induced, as is common in the military and many manufacturing and construction professions; or drug-induced, as is common following chemotherapy for cancer, are most commonly caused by the death of sensory hair cells in the inner ear. Regeneration of lost sensory hair cells does not occur in mammals, including humans, with the result that hearing loss is almost always permanent. Aside from prosthetics such as hearing aids and the cochlear implant, developing the tools to induce regeneration in the sensory epithelia of the inner ear of humans offers the only hope for the millions of people who suffer from deafness due to hair cell loss. According to the National Institutes of Deafness, approximately 1 in 2000 children born in the US, including California, are born with congenital hearing loss, making it the most common birth defect. Babies and children learn language and communication through listening to their parents or caregivers, a process that is disrupted if hearing loss is not detected and corrected. According to the Department of Veterans Affairs, of the 1.3 million service men who have served in Iraq or Afganisitan, 58,000 are currently on disability due to hearing loss and an additional 78,000 due to tinnitus (ringing in the ear), making noise induced hearing loss the #1 disability of returning veterans. Finally, age-related hearing loss -- presbycusis -- is the leading cause of deafness in the population as a whole, with over 50% of adults suffering significant hearing loss by the age of 65 (Davis et al., 2003; Seidman et al., 2002). California's population of people older than 65 will begin a rapid expansion in 2010, and by 2025 will have doubled from the current ~3.5 million older adults, to around 7 million by the year 2025. Half of this elderly popualtion, 3.5 million people, are predicted to experience significant hearing loss, making hearing loss a growing epidemic in the state. Our long-term research goal is the treatment of deafness and balance disorders through the stimulated regeneration of sensory hair cells in the inner ear. The work described here will greatly aid this endeavor by providing tools for the study of hair cell progenitors/stem cells from the inner ear. Based on a comparison to other animals that are able to regenerate their hearing, and our own recently published findings, supporting cells represent the most likely target for future therapeutic manipulations,including the derivation of adult stem cells, to bring about hair cell regeneration in humans.
The overall goal of the research in this proposal is the treatment of deafness and balance disorders through stimulated regeneration of sensory hair cells in the inner ear. The applicant seeks to develop tools to isolate different subpopulations of cochlear supporting cells from the inner ear of mice and humans in order to better understand their differentiation potential. The applicant will use transgenic mouse lines in which different subpopulations of cells in the inner ear are tagged with fluorescent markers, to test the hypothesis that different kinds of progenitor cells exist in the ear’s organ of Corti. The applicant then proposes to screen these isolated subpopulations of supporting cells for endogenous cell surface proteins that will serve as molecular markers and would allow isolation of these cells from humans. In the final part of the proposal, the applicant will generate transgenic mice that contain markers of different cell types, in order to follow the fate and stem cell properties of supporting cells under a variety of experimental conditions. Reviewers were very positive about the research team and their system for developing tools to study regeneration in the inner ear. Identifying the specific cells within the inner ear that have stem cell potential in humans could pave the way for stem cell or regenerative therapy in patients with deafness and balance problems. However, reviewers were unsatisfied with the stem cell aspects of the proposal, and at least one reviewer did not support funding this project given the applicants’ previous data suggesting that a transdifferentiating cell type had already been identified. The tools generated by this proposal could have a significant impact on a major problem, the lack of markers for potential stem cell populations in the inner ear. Transgenic mice that target fluorescent markers (green fluorescent protein, or GFP) to different subpopulations within the cochlear supporting cell population should allow investigators to more clearly define putative stem cell subpopulations. One reviewer commented, however, that no experiments are proposed to study whether the different GFP+ subpopulations that are isolated indeed have stem cell properties, which significantly diminished enthusiasm for the project overall. Another reviewer commented that the team has already identified a subgroup of supporting cells (pillar and/or Hensen’s cells) that have an increased ability to transdifferentiate into neuronal hair cells in cell culture as compared to other cochlear supporting cells. This reviewer commented that the applicant does not present any evidence to suggest that other supporting cells in the inner ear have greater or even comparable potential to transdifferentiate into neurons than pillar/Hensen’s cells do. So although the proposal is of interest for developing therapies to treat deafness, the applicants did not provide a clear rationale for further characterizing these additional supporting cells. Reviewers had positive comments about the application: the proposal is well written and clearly planned, the work is based on high impact factor publications, and human tissue is available for the indicated studies. However, there were concerns regarding feasibility of the research plan. First, the project is very ambitious for the timescale, especially the experiments involved in characterizing human cells. Second, the assumption that gene expression patterns will identify markers suitable for antibody detection in different cell populations should acknowledge that many of the most useful markers detect post transcriptional changes, which will not be identified by microarray as proposed. Third, a key piece of preliminary data, the expression of GFP in the different cell populations in transgenic mice, is not actually shown. Given the availability of these mice as documented in the application, reviewers found this a surprising omission. Finally, experiments to trace the lineage of supporting cells (Aim 3) did not fit in well with the rest of the proposal. The principal investigator (PI) proposes to trace cell lineage under various experimental conditions, but these were not appropriately described in the proposal. The PI on the project is an established investigator well-versed in studies of ear development. The personnel involved are qualified to carry out the proposed studies, but reviewers suggested that they are unlikely to complete all of the proposed experiments in 2 years. The budget was judged to be appropriate. Overall, reviewers felt that in light of the applicant’s previous success with pillar/Hensen’s cells, the rationale for studying additional supporting cells should have been more clearly described, and a proposal to follow up on the findings in pillar/Hensen’s cells would have been more exciting. Furthermore, an effort should have been made to more clearly establish the stem cell properties of the cells to be studied.