The first thing most scientists do when studying something is to look at it. A chiropterologist looks at a bat. A geologist looks at the Earth. But for a scientist interested in the inner ear, it gets trickier. It’s hard to examine an inner ear that is attached to a living animal who needs that ear for hearing and balance without either killing the sensitive cells it houses, destroying the complex ear structure itself, or just throwing the whole system off balance. In these cases, it's easier to grow an ear, or something like one, in the lab and study that instead.
Scientists have been creating organoids out of a variety of tissues such as brains, kidneys, and livers for years (1–3). For external tissues like the ear, however, the field is in its infancy. But in recent years, there’s been a wave of advances in the molecular biology of hearing loss and a burgeoning ability to accurately model the ear’s complex architecture in the lab using stem cells.
Hearing loss is the third most common health challenge for adults, following only heart disease and arthritis. Approximately 1.3 billion people worldwide are affected by it, with genetics, infection, and environmental noise numbering among the most common causes (4). Hearing loss frequently results from damaged hair cells. These highly specialized cells convert the deflections of their hair-like projections in the cochlea into neuronal signaling. Over a person’s lifetime, these cells wear out. Since they do not regenerate, this lifetime of damage to the ear can be permanent (5).
Scientists in the field are creating new ear organoid systems for testing therapeutics for the inner ear. Because of the sophisticated, elaborate architecture of the ear, they are laboring under some of the most difficult biological circumstances the human body has to offer. Over time, they’ve learned that often the best model for studying the ear looks more like a ball of cells.
A ball of cells
When Karl Koehler began graduate school at Indiana University, he was interested in stem cells. “In particular, I was fascinated with sensory development — how we develop these really complicated apparatuses that sense external signals and send those to the brain,” he said. He had a lucky break. “It so happened that the only lab that was taking new students that was focused on sensory development and stem cells was an inner ear lab.”
Koehler joined the lab of Eri Hashino, a trailblazer in the stem cell biology field. When Koehler arrived, scientists all over the world were trying to develop replicable methods for using pluripotent stem cells to create any tissue a researcher desired. Hashino’s group used stem cells to create auditory nerve cells, which are frequently damaged by environmental insults like loud noises and do not grow back. Koehler wanted to go a different way; he wanted to use stem cells to model the architecture of the inner ear itself to help scientists see how diseases of the ear disrupt the flow of information from sound to the ear to the brain.
The brain is a big target; the ear is a bullseye.
- Karl Koehler, Harvard Medical School
“The brain is a big target; the ear is a bullseye,” said Koehler, now a neurobiologist and sensory researcher at Harvard Medical School. But unlike a dart board, the inner ear is not flat. It is a complex, multi-layered, three-dimensional structure. Not appreciating the dimensionality of the ear led to years of failures for stem cell researchers trying to model the ear in vitro or grow hair cells, which convert mechanical stimulation into nerve signals. “Instead of growing the stem cells on a flat dish, which is very much unlike how the cells will normally develop in the embryo, if you take them off the dish and have them in three dimensions, you can give them the degrees of freedom that they need to self organize,” said Koehler.
Taking cues from a research group in Japan using three-dimensional structures to grow retinal organoids, in 2013, Koelher and Hashino grew stem cells in culture with extracellular matrix (ECM) proteins, which acted as a scaffolding for the developing inner ear (6). However, since these are not fully formed auditory systems with an ear canal, a brain, and an auricle — the fleshy satellite dish structure mammals have on their heads that is frequently called “the ear” — it’s difficult to measure how close to real hearing organoids can get.
Nevertheless, the development of synthetic, ear-like models in the lab couldn’t have come at a better time.
“There was recently the passage of legislation, the FDA Modernization Act 2.0, which lays the groundwork for using cell-based assays entirely to validate your new biologic or drug or gene therapy before moving into clinical trials,” said Koehler. “That places more importance on the development and perfection of cell-based systems.”
Ear researchers come in pairs
Like Koehler, Jeffrey Holt and Gwenaelle Géléoc started off interested in the finer details of the nervous system. But nothing in particular led them to study ears. After meeting as young postdoctoral fellows, life’s happenstance showed that they liked working together, and now they run a joint lab at Boston Children’s Hospital.
“I was just fascinated by the sensory cells and how they did this unique job of converting a stimulus of sound into an electrical signal that's transmitted to the brain. And for me at that stage, and even today, the brain is like a big black box,” said Holt. “I wanted to understand how information got in there.”
Holt’s and Géléoc’s groups study ear organoids and develop gene therapies to treat hearing defects. They use a variety of viral vectors to study Usher syndrome, an incurable genetic disorder that results in hearing and sight defects and is among the most common causes of deafblindness worldwide (7).
From that work, their groups had gotten quite skilled at the finer mechanics of creating gene therapies, like delivering oligonucleotides directly to cells in the lab. “I think it was around 2011 when I realized, hey, this stuff actually could work,” said Holt. Instead of relying on an in vitro model of an ear using cells grown in a dish, they injected mice that carried specific Usher syndrome-causative mutations with antisense oligonucleotides (ASO) that can suppress those mutations (8).
“The most startling thing was actually called the ‘startle response,’” said Holt. Like a deaf human, a deaf mouse will not respond to a sudden, unexpected noise. “We can use that as a behavioral assay for a mouse. A deaf mouse doesn't jump no matter how much noise you make. But these mice, once we injected them with our initial viral vectors and played a startling sound, they began to jump. And of course, once they were jumping with a startle, we started jumping for joy,” said Holt.
Holt and Géléoc warned that using organoids and ASOs in the clinic is something very much in the future.
A job for stem cells
Humans are born with approximately 15,000 hair cells that last a lifetime (9). Unlike other parts of the body, the inner ear does not have a native pool of stem cells to draw on, so hair cells do not regenerate if damaged by noise or physical injury.
Although research into delivering stem cells to the ear or even converting neighboring cells into hair cells is ongoing, according to Holt and Géléoc, it’s a difficult ask to stand in for a lost hair cell. Hair cells are not only highly specialized cells with unique villi bundles that project out into the ear like flagpoles, but their physical positioning in space is critical to their function. This might be the most difficult feature to recapitulate. If a new hair cell grows or is added to the ear with incorrect orientation, it won’t function. On top of that, even if a new hair cell is positioned correctly, it may not connect correctly to support structures in the ear such as the tunnel of Corti, which funnels sound waves into the inner ear. Upon hair cell loss, the tunnel of Corti simply collapses into a mound of inconspicuous cells (9). Spackling on stem cells in the hope that they’ll regrow the inner ear is out of reach for the moment.
Every synthetic biologist, inventor, or tinkerer must ask themselves a critical question: is this futuristic invention, an ear organoid, better than solutions that already exist?
“It’s one thing we often don’t talk about, this challenge for us,” said Géléoc. “There are devices available to help patients with hearing loss. Cochlear implants are doing a tremendous job. They're not perfect, but most patients do really well with them.”
“It makes the job even harder, because now we have to do better. If we go to the FDA, we have to convince them that what we're offering is going to be better than cochlear implants or maybe complimentary,” she added.
Start somewhere
Stem cell therapy for hearing problems is likely not the next thing coming to the clinic, but it’s not science fiction either. In fact, some of the earliest work after the discovery of induced pluripotent stem cells (iPSCs) was done to research hearing loss.
“A long-standing problem in our field has been the limitations of access to the material to study proper biochemistry and molecular biology of the ear, particularly in the 1990s when I started with this kind of work,” said Marcelo Rivolta, a neurobiologist and sensory stem cell biologist at the University of Sheffield.
In the early 2000s, Rivolta arrived at the university just as it was setting up its stem cell biology research center, which was the first in the United Kingdom to use the first human embryonic stem cells generated by Jamie Thompson, a stem cell researcher at the University of Wisconsin-Madison. It was the beginning of a whole new world. Once Thompson’s and Shinya Yamanaka’s (currently at the University of California, San Francisco) groups published their methods for creating iPSCs in 2007, Rivolta started to dream. “I was starting to play with the idea of using the cells not only for modeling, but also for cell therapy,” he said.
Several research groups had successfully grafted stem cells into laboratory animal models of deafness, but none improved hearing or neuronal excitement in the ear. Rivolta’s group initially attempted to harvest stem cells from fetal cochleae, one of the few sources of ear-specific stem cells. But these rare and difficult-to-obtain cells usually died after a mere 25 divisions. Instead, by tweaking other researchers’ protocols, Rivolta’s group succeeded in generating and grafting embryonic stem cells into the ears of deafened gerbils (10). The stem cells successfully differentiated into hair cell-like cells and improved neuronal firing thresholds from auditory stimulation in the ear. The work from Rivolta’s group and others at the University of Sheffield led to the biotech company Rinri Therapeutics, which works to produce cell therapies for hearing loss.
Listening for the future
Rivolta is more upbeat than Holt and Géléoc about the ability of stem cells to treat inner ear conditions. What, then, prevents this work from translating to the clinic? For now, according to Rivolta, it’s partially a cell manufacturing concern.
“The manufacturing limitations are to make sure that we are producing the right cells, that we have the right way of identifying the cells, and the right kind of markers,” said Rivolta. “We are working on the delivery, how to access the right places in the ear, and we have made good progress in that sense as well.”
[Koehler] can basically make any new ear in a dish.
- Gwenaelle Géléoc, Boston Children’s Hospital
Holt, Géléoc, and Koehler are teaming up too. Their groups are collaborating to produce a unique tool: inner ear organoids derived directly from patient cells. This project is crucial for testing tailored gene therapies for hearing loss, especially for patients with Usher syndrome. Usher syndrome comes in three different genetic classes and can be caused by mutations in upwards of a dozen different genes (8). On top of that, the ear is at the center of more than just hearing, so successful gene therapies for conditions like Usher syndrome will improve more than the startle response.
Stem cells are a famously finicky material to work with. Creating stem cells from individual patients is a gamble, but the researchers are confident that their experience stemming from the early days of the field will see them through.
“[Koehler] can basically make any new ear in a dish,” said Géléoc.
References
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- Ramachandran, S. D. et al. In Vitro Generation of Functional Liver Organoid-Like Structures Using Adult Human Cells. PLoS ONE 10, e0139345 (2015).
- Basner, M. et al. Auditory and non-auditory effects of noise on health. The Lancet 383, 1325–1332 (2014).
- Wagner, E. L. & Shin, J.-B. Mechanisms of Hair Cell Damage and Repair. Trends in Neurosciences 42, 414–424 (2019).
- Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D. & Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013).
- Geng, R. et al. The Mechanosensory Structure of the Hair Cell Requires Clarin-1, a Protein Encoded by Usher Syndrome III Causative Gene. Journal of Neuroscience 32, 9485–9498 (2012).
- Pan, B. et al. Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c. Nat Biotechnol 35, 264–272 (2017).
- Brigande, J. V. & Heller, S. Quo vadis, hair cell regeneration? Nat Neurosci 12, 679–685 (2009).
- Chen, W. et al. Restoration of auditory evoked responses by human ES-cell-derived otic progenitors. Nature 490, 278–282 (2012).
