Hearing impairment is the most common sensory deficit, the most common occupational disease, and the third most prevalent chronic disability of mankind, resulting in an enormous socio-economic impact. It is caused by the irreversible death of cochlear hair cells. Hair cell regeneration does not occur naturally in the mammalian cochlea, nor has it been reproducibly achieved in an experimental setting. This proposed collaborative project between California and collaborative funding partner research laboratories represents the first systematic plan to overcome the prevailing bottlenecks that impede the development of translational approaches toward novel human cell-based treatments for hearing loss.
The most prevalent translational bottleneck hampering translational approaches toward curing hearing loss is the lack of purity of otic progenitor cells generated from human ES and iPS cells. The bottleneck is 1) that this heterogeneous progenitor cell pool contains tumorigenic cells, which hampers the use of the cells in in vivo repair studies. Likewise, 2) the presence of non-defined cell types renders the cell population inadequate for bioassay development, such as the development of high-throughput assays for compounds with ototoxic, but also otoprotective or otoregenerative efficacy.
Ototoxic drugs are drugs such as aminoglycoside antibiotics or cisplatin, which can cause irreversible hearing loss (drug side effects). Our work will make it possible to develop ototoxicity tests with human cells; currently such tests do not exist.
Otoprotective drugs prevent hair cell loss by counteracting ototoxicity. Such drugs (when discovered) could prevent hearing loss caused by, for example, loud noise, certain infections, ototoxic drugs, and perhaps even sudden hearing loss and age-related hearing degeneration. No human cell-based tests exist for otoprotection.
Otoregenerative drugs directly address curing hearing loss and the identification of drug candidates with otoregenerative potential opens the door for developing a cure for hearing loss. No human cell-based tests exist for otoregeneration.
Our research goals are 1) to identify novel biomarkers that are present on human ES cell-generated otic progenitor cells and to use these biomarkers to purify progenitor cells with a) high capability to generate human inner ear cells and b) no tumorigenic potential. Successful development of this novel technology will remove the bottleneck described above. 2) We will demonstrate that the purified otic progenitors are useful in bioassays for ototoxicity, otoprotection, and otoregeneration.
Statement of Benefit to California:
This proposed collaborative project between California- and collaborative funding partner research laboratories represents the first systematic plan to overcome the prevailing bottlenecks that impede the development of translational approaches toward novel human cell-based treatments for hearing loss.
Hearing loss affects one in 1,000 newborns and the same number of children lose their hearing before puberty. In adults, hearing loss can happen as a result of drug side effects (ototoxicity), loud noise exposure, certain infections, sudden hearing loss, or the effects of aging. About 3.5 million Californians have disabling hearing loss (affecting both ears); worldwide, more than 350 million people are affected.
Beside the obvious medical benefit, we expect that our proposed research will lead to the development of novel techniques that can directly be adopted by existing California biotech companies. We anticipate that, in the long run, this new technology will lead to new jobs in research & development. If the new tools lead to discovery of novel treatments, they will have a long lasting effect on the California-based biotech industry including jobs, increased tax income for the state, and overall maintaining the status of California as worldwide hub for technology and innovation.
The goal of this application is to advance therapeutic approaches for irreversible hearing loss caused by the death of the sensory cells (hair cells) of the auditory portion of the inner ear (cochlea). The applicants propose to develop a protocol for the purification of otic (ear) progenitor cells derived from human embryonic stem cells (hESC), and to develop a human inner ear model in the dish. The goal of Aim 1 is to discover surface markers suitable for purification of otic progenitors from mixed hESC-derived cultures. Once purified, the progenitors are then differentiated into hair cells and supporting cells as an in vitro model of the human inner ear. In Aim 2, the applicants plan to use these cells to develop and validate an in vitro ototoxicity assay (hearing loss in a dish), followed by a screen of existing drugs for their potentially toxic effects in this assay. The goal of Aim 3 is to develop bioassays for otoprotection and otoregeneration. In these assays, hESC-derived otic cells will be challenged with ototoxic compounds, followed by a screen for small molecules that either protect the hair cells or that cause their regeneration from remaining hair cells or from supporting cells. In parallel, animal in vivo models and putative otic stem cells isolated from native human inner ear will be used in validation studies.
Hearing loss represents a major and poorly addressed medical need. A critical roadblock to translational research on cochlear hair cells and their progenitors is the present inability to obtain them in large enough numbers and in pure form. Therefore, reviewers expressed considerable enthusiasm about pluripotent stem cells as a potential source of functional otic cells, especially since this promising approach is not widely pursued in the inner ear field. Reviewers judged the rationale for otic marker identification and bioassay development to be sound yet only moderately innovative. Some argued that the need for progress in treating hearing loss outweighs the desire for high innovation, and reviewers agreed that successful execution of the proposal would likely have a significant impact on advancing regenerative medicine approaches for hearing loss.
Major concerns regarding feasibility dampened reviewers’ enthusiasm for this project. Although the preliminary data and a recent high profile publication clearly demonstrate the applicant’s ability to derive cells of the otic lineage from mouse pluripotent stem cells, the preliminary data shown on human cells was considered sparse, especially in light of several years of previous CIRM funding dedicated to working out the hESC-based protocol. The technical demands that must be met in Aim 1 in order to develop a sufficiently robust hESC-based otic cell system required for the proposed bioassays in Aims 2&3 are formidable. Although the applicant reports success with otic cell purification in mouse, the reviewers considered the level of enrichment achieved to be moderate. Some reviewers thought that the proposed strategies for marker identification appear well conceived, whereas others cautioned that the comparison of mixed populations of hESC-derived cells is not likely to enable discovery of new reagents for purifying the desired progenitors. Furthermore, although the applicants allude to ongoing improvements in culture conditions for otic progenitors, in part with the aid of unspecified extracellular matrix components, they did not address how they will achieve the production of sufficiently large numbers of cells. Reviewers remained unconvinced that the applicants will be able to generate enough hESC-derived otic cells of high enough purity to carry out the desired screens. Beyond this limitation, reviewers believed that the preliminary data supported the feasibility of the proposed validation and ototoxicity screens. Some reviewers appreciated the inclusion of known ototoxic compounds for validation of the inner ear in the dish model, but others criticized the lack of rationale for the chosen screening parameters. For example, using a single dose of test compounds will likely lead to some false readouts, and the relationship between the EC50 for known ototoxic compounds with the dose chosen for testing of library compounds is not explained.
The principal investigator (PI) is a well-established leader with an impressive publication record and a high level of grant support in the cell biology of cochlear hair cells and their progenitors. Experience with screening for compound discovery is a major plus. Based on the experience and track record, reviewers were convinced of the PI’s capacity to oversee a project of this scope. Team members at the applicant institution appear well qualified, and the supporting environment is exemplary. The collaborative group also has a very high level of expertise and an excellent track record of first-rate publications. The reviewers considered the strengths of the two groups as being complementary, and there is clear evidence of a well-conceived, ongoing collaboration.
In summary, reviewers strongly endorsed the notion of establishing a hearing loss in a dish model, and lauded the assembled team’s superb qualifications and track record in the field of inner ear biology. However, they equally strongly expressed doubt regarding the feasibility of this proposal. Despite many years of experience in the mouse system and considerable previous effort to establish a hESC-based system, preliminary data did not convince reviewers that difficulties with hESC-based approaches would be overcome in a timely manner. They did therefore not recommend this application for funding.