Generation of inner ear sensory cells from human ES cells toward a cure for deafness
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.
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.
The main goals of our research are to establish an experimental protocol for coaxing embryonic stem cells into inner ear sensory cells (sensory hair cells). This work has implications on future treatment of hearing loss and balance disorders, for which no current treatment exist. We began with using mouse embryonic stem cells to explore specific experimental conditions that lead to cell differentiation in direction of the inner ear. In the last funding period, we were able to increase the efficacy of this procedure by approximately 10-fold. At the same time, we were able to cut the time needed for a guidance experiment from 2-3 months to 8 days. We have now begun testing whether human embryonic stem cells are also able to follow the same guidance protocol and we found that human cells can also coaxed toward the inner ear lineage with a slightly modified protocol. The efficiency with human cells is comparable to the efficiency experienced with the mouse cells. The time needed for initial differentiation of the human cells into early inner ear is not very much different from the time needed for differentiating mouse cells, but we still need to verify this preliminary result.
In addition to in vitro guidance tests, we have begun with assessing the in vivo potential of mouse and human inner ear precursor cells, which were derived from mouse and human embryonic stem cells. Toward this goal, we have begun to establish a coculture system in which we culture the mouse and human cells inside the developing inner ear of chicken embryos. Before starting with the more complicated in ovo experiments, we decided to first establish a culture system in vitro, which will allow us to perform more pilot tests in a more controlled cell culture environment. Establishment and initial assays in this regard have been conducted in the past funding period and we are optimistic that we will be able to assess the potential of mouse and human embryonic stem cell-derived inner ear cell types using the established in vitro tests in the upcoming funding period.
In the last year, we have achieved a major milestone by showing that it is possible to generate functional inner ear sensory cells (hair cells) from mouse embryonic stem cells using a guidance protocol that was completely conducted in vitro (in cell culture). The goal of this project is to achieve the same result with human cells at the end of the funding period. We are on track with this endeavor, but we have encountered a number of quite substantial roadblocks. The major roadblock is that human cells appear to require much more time to differentiate into mature inner ear cell types when compared with mouse cells. We are currently working on a protocol that allows us to provide a sustained stimulation of the cell signaling pathways that are needed to keep human cells on track to develop along the otic (inner ear) pathway. We have learned a lot about how human embryonic stem cells react when exposed to various signaling environments and we have made the discovery that the embryonic stem cells are able to differentiate relatively quickly into early embryonic cells (lineages), but that the development of later cell types (i.e. organogenesis), does require quite some time.
The overarching goal of this grant was to develop an experimental protocol for coaxing human embryonic stem cells to develop into inner ear sensory hair cells. This work has implications on future treatment of hearing loss and balance disorders, for which no current treatment exist. Generation of sensory hair cells and their accompanying so-called supporting cells is a complex endeavor because these cells develop from an embryonic structure called the otic placode. Generation of placodal cells is quite complex and appears not to be a default pathway, which can be explored for generation of myocytes or neural cells. Another aggravating factor was that human cells appear to require a different guidance protocol than mouse ESCs.
With 4 years of CIRM funding, we were able to overcome most of the obstacles that we encountered along the way and we are now at a point where we are able to generate human hair cell-like cells in the culture dish. These cells express appropriate marker proteins and they display the cytomorphological specializations that you would expect to find in a sensory hair cell. Still, more experiments are needed to ensure that the cells function properly. These analyses are complicated and they require an additional manipulation of human ESCs (adding a transgenic reporter). This work is still ongoing and it is required for publishing the results of the study.