Hearing loss is the leading birth defect in the United States with ~3 children in 1,000 born with partial to profound compromise of auditory function. Debilitating hearing loss is estimated to affect ~4% of people under 45 years of age, and 34% of those 65 years or over. A major cause of why acquired hearing loss is permanent in mammals lies in the incapacity of the sensory epithelia of the inner ear to replace damaged mechanoreceptor cells, or hair cells. Sensory hair cells are mechanoreceptors that transduce fluid movements generated by sound into electrochemical signals interpretable by the brain. Degeneration and death of hair cells is causal in >80% of individuals with hearing loss In this grant application, we propose to explore, in comparative manner, the potential of at least five human ESC lines to develop into hair cells. We strive to use recently derived human embryonic stem cells for this purpose to avoid problems caused by potential chromosomal abnormalities and nonhuman or viral contaminants, which greatly restrict the use of these stem cells and render their derivatives unacceptable for in vivo studies. Federal funding cannot be used for research with these embryonic stem cell lines. The most exciting long-term goal of the proposed experimentation is to provide an abundant source of human inner ear progenitor cells that can be tapped in the future to routinely create human hair cells for in vitro and in vivo experiments and for clinical studies aimed to repair damaged ears. Having access to human hair cells in vitro offers, for the first time, the opportunity for detailed cell-biological studies of this cell type. We envision that human ESC-derived inner ear progenitor cells can be used to screen for drugs that lead to increased hair cell differentiation. Equally exiting with regard to possible clinical applications are studies aimed at differentiating functional human hair cells in vitro, in organ culture, and in vivo after transplantation of the cells into the cochleae of deaf animal models and potentially into human patients. In the more distant future, we envisage that our proposed research will result in novel treatment strategies to cure deafness and potentially other inner ear diseases such as tinnitus caused by malfunctioning sensory hair cells, and vestibular disorders.
Statement of Benefit to California:
Hearing loss affects about 30 million Americans and consequently about 3 million Californians suffer from debilitating hearing problems, making this condition one of the most common chronic disorders. Degeneration and death of hair cells, and potentially their associated spiral ganglion neurons, is causal in >80% of individuals with hearing loss. The functional replacement of hair cells represents the ultimate treatment modality for deafness. Clinically, the functionality of lost hair cells can be partially restored by electrical stimulation of the auditory nerve achieved with implantation of electronic devices; for example cochlear implants can provide a subset of suitable deaf patients with a form of treatment to improve hearing. In the long-term and for the benefit of patients not suitable for existing treatment, other avenues of therapy need to be explored, for example stimulation of hair cell regeneration after damage. It has recently been shown that it is possible to generate hair cells from mouse embryonic stem cells and the herein proposed experiments aim to extend this research toward generating human hair cells from embryonic stem cells. Having devised a way to coax human embryonic stem cells into hair cells via an intermediate cell type, the inner ear progenitor cell will be a major advance for developing novel treatment strategies to cure deafness and possibly other inner ear disorders. Beside the immediate and obvious benefit for patients, we envision that technological advances that are applicable to millions of patients alone in California, but even more worldwide, bears an enormous commercial potential. Californians could consequently benefit possibly from the first biological treatments for hearing loss offered through local hospitals and the State of California could possibly benefit from local commercialization of novel biotechnology that has a global demand.
SYNOPSIS: The applicant proposes to develop a stepwise guidance protocol to grow hair cells from hESCs by systematic testing of growth factors. The goal is to provide an abundant source of human inner ear progenitor cells. The researchers propose to do this systematically through steps: induction, selection, expansion, and differentiation. They also propose to assess the potential of these cells by transplantation in otic vesicles of chicken and mouse embryos. The PI holds several NIH grants to study adult inner ear sensory epithelial cells, including one to derive inner ear progenitors from mouse ESC, one to isolate stem cells from adult human ear stem cells. He also holds a Johnson & Johnson Focused Giving grant to find a drug that induces cell regeneration in mammalian ear and a McKnight Foundation grant to evaluate the regenerative potential of adult mouse inner ear stem cells. IMPACT AND SIGNIFICANCE: Hearing loss can be a consequence of birth defects or acquired in middle age; it is a relatively common medical condition, yet there are few therapeutic options. The proposal outlines a series of experiments to investigate the potential of human embryonic stem cells to differentiate into inner ear sensory cells. Although there is some preliminary data from Dr. Heller's lab on murine embryonic and tissue-derived stem cells to differentiate as hair cells and neurons, little to nothing is known about the potential of human ES cells, especially those established more recently. The techniques and approaches proposed are rather routine. As the authors point out, an abundant assay cell line for high throughput screens could also facilitate identification of new drugs to target inner ear differentiation and function. Because hearing and balance disorders are very common, the availability of stem cells for therapeutic transplantation could potentially have a profound effect on rehabilitation options. QUALITY OF THE RESEARCH PLAN: One of the most challenging aspects of this work is recognition of progenitor cells. The applicant has substantial experience with a wide variety of tools to identify human inner ear cell types. He has tested, for example, 20 different RT-PCR primer pairs on adult tissue extracted during inner ear surgery, as well as on spheres that were formed from stem cells that reside in the adult vestibular sensory epithelia. These promise to be very useful tools for guiding cells through differentiation steps. The proposal is well constructed and previous work by the PI, as well as other labs, has identified a number of key regulatory growth factors that control the differentiation of inner ear progenitor cells and murine embryonic stem cells into more mature inner ear cell types including hair cells and neurons. Complementary in vitro and in vivo approaches are described to establish conditions to guide hES cells to differentiate into inner ear cell types. The analysis techniques, reagents and transplantation protocols are well-developed in Dr. Heller's lab and should not limit progress towards the goals of the proposal. The investigators have provided preliminary data of how they will use information gleaned from mouse studies and apply this to human ES cells using either various growth factors (e.g. EGF, FGF, IGF) and transcription factors or information from gene array studies. Whether this approach will work for human ES cells is unclear but the protocol is well planned and designed to attempt to translate that information. The researchers hope to utilize information gained on differentiation to generate an in vitro human ear, inner ear progenitors which can be then transplanted in various animals and ultimately create culture models to observe appropriate final differentiation. This objective is extremely well designed, clear, with clear end points and goals, and the likelihood of success of either defining the pathways for differentiation or engraftment is good. Considerable valuable information will be gained through the use of human ES cells by the end of this four year proposal. STRENGTHS: The proposal addresses a prevalent, important human disability with limited treatment options. The investigator has a respectable track record (publications and funding) with a great deal of experience in the field of adult human inner ear progenitors and mouse embryonic stem cells. He is a leader in research directed at the use of tissue-derived and embryonic stem cells for regeneration of the inner ear. Since his laboratory is focused on reparative interventions to ameliorate deafness by using stem cells, the part of the application that proposes to assess transplanted cells is very strong. The PI's was the first laboratory to show that murine embryonic stem cells can be differentiated in a stepwise fashion into inner ear progenitor cells, proof that work of this kind may be feasible. Most of the reagents and techniques required for success of this proposal are already established in this lab. The researchers have a great deal of experience handling, growing, and identifying pluripotent stem cells in adults. A collaboration with Dr. Ricci adds depth to the proposal given his extensive background in auditory and cellular physiology for assessment of the degree of functional maturation of relevant cell types. Dr. Heller's other support description indicates that he does not currently have funding to extend this work to the characterization of human embryonic stem cells. The aims are well designed, the preliminary data on candidate transcription factors are clear, and methods are in place for in vitro and in vivo engraftment including re-innervation of organ of corti and early evidence of inner ear progenitor cells from human ES cells. WEAKNESSES: The work is not mechanistic or imaginative in its approach. It is mostly empirical testing of a limited set of factors that are known to play a role in differentiation and expansion of the human embryonic stem cells. Because the number of growth factors and variables that can be tested is enormous, it is not clear that the proposed work will yield the best approach, particularly since there is no clear theoretical framework to guide the manipulation of the cells. Other investigators are using large-scale screening methods and other approaches. Of concern is the assumption that the important or relevant growth factors to induce hES to differentiate into otic progenitors are limited to the relatively small number that are outlined in the proposal. Aside from the Genechip approach, no systematic approach to identify novel factors or combinations of factors is proposed. Heterogeneity of the population of cells may be a significant problem if only a small subpopulation of hair cell or neuron precursors are present. An appropriate experimental design is essential to derive meaningful data under these circumstances. Although the applicant suggests a FACS approach to enrich for desired precursors based on newly identified markers, success is not guaranteed. The proposal also describes experiments where hES otic progenitor cells will be grafted into chick and mouse otic vesicles in ovo and in vitro. These are potentially very powerful approaches to assess differentiation potential. The proposal suggests several markers for the recipient tissue (chick or mouse), but perhaps more important is the ability to reliably identify donor transplanted cells. Preliminary data should show specific labeling of human ES cells in the context chicken and mouse. The nuclear markers mentioned are not fully described or shown. Several other options would include prelabeling hES with trackable fluorescent dyes or a lentivirus-driven GFP. Another concern is the possibility that generation of hair cells will be a very low yield phenomenon, and there may be problems related to chromosomal aberrations associated with ES lines. The authors are aware of limitations. Collaboration with others is limited, and it is not at all clear that the work will yield a better understanding of human embryonic stem cell biology. DISCUSSION: The work proposed is not very mechanistic or imaginative in its approach to expanding hESCs but this is a worthwhile grant and one of the stronger ones aiming at this approach. While the proposal lacks originality that could yield new insights into the ways to differentiate to inner ear cells, this work promises to bring mESC information to hESC. The applicant has listed very few collaborators. The description of how the experiments will be carried out has holes and needs more specifics, but the PI is given the benefit of the doubt based on his track record.