A deafened adult cannot recover the ability to hear, because the sensory hair cells of the inner ear don’t regenerate after damage. In two new studies, partially funded by the National Institutes of Health and Hearing Health Foundation, and published in the Proceedings of the National Academy of the Sciences (PNAS), University of Southern California (USC) Stem Cell scientists explain why this is the case and how we might be able to change it.

“In the non-sensory supporting cells of the inner ear, key genes required for conversion to sensory cells are shut off through a process known as ‘epigenetic silencing.’ By studying how the genes are shut off, we begin to understand how we might turn them back on to regenerate hearing,” says John Duc Nguyen, Ph.D., the first author of one of the papers.

Nguyen earned his doctorate in the USC Stem Cell laboratory of Neil Segil, Ph.D., who passed away from pancreatic cancer in 2022. Segil was a member of Hearing Health Foundation’s Hearing Restoration Project.

The second paper explores when and how the ability to form sensory hair cells is gained in the inner ear in the first place and describes two specific genes that could be useful for regenerating hearing in adults. 

“We focused on the genes Sox4 and Sox11 because we found that they are necessary for forming sensory hearing [hair] cells during development,” says the paper’s first author Emily Xizi Wang, Ph.D., who also conducted her research as a doctoral student in the Segil Lab.

Blocking and Unblocking DNA

One important way that genes are shut off or “silenced” involves chemical compounds called methyl groups that bind to DNA and make it inaccessible—the focus of Nguyen’s paper. When the DNA that instructs a cell to become a hair cell is methylated, the cell cannot access these instructions.

Through their experiments with supporting cells extracted from the inner ears of mice, Nguyen and colleagues confirmed that DNA methylation silences genes that promote conversion into hair cells, including the gene Atoh1 that is known to be a master regulator of inner ear development. 

An enzyme called TET can remove methyl groups from the DNA, thus reversing the gene silencing and restoring the capacity of supporting cells to convert into sensory hair cells. Accordingly, when the scientists blocked the activity of TET, the supporting cells retained their DNA methylation and therefore could not convert into hair cells in vitro. 

Intriguingly, in a separate experiment, the researchers tested the extent of gene silencing in supporting cells from a chronically deafened mouse. They found that gene silencing was partially reversed, meaning that the supporting cells had the capacity to respond to signals to transform into hair cells. 

This finding has important implications: The loss of hair cells itself might partially reverse gene silencing in supporting cells in chronically deaf individuals. If so, the supporting cells of chronically deaf individuals might already be naturally primed to convert into hair cells.

Segil’s longtime collaborator Andy Groves, Ph.D., from Baylor College of Medicine served as the paper’s corresponding author. He is also a member of HHF’s Hearing Restoration Project.

Developmental Insights

In the second paper, Wang and colleagues explored when and how, during embryonic development, the progenitor cells of the inner ear gain the ability to form hair cells. 

The scientists pinpointed when progenitor cells acquire this ability: between days 12 and 13.5 of embryonic development in mice. During this window, the progenitor cells acquire the capacity to respond to signals from the master regulator gene Atoh1 that triggers the formation of hair cells later during development.

What primes the progenitor cells to respond to Atoh1 are two additional genes, Sox4 and Sox11, which change the state of these cells. 

In embryonic mice lacking Sox4 and Sox11, progenitor cells in the inner ear failed to develop into hair cells. Specifically, a loss of Sox4 and Sox11 made the cells’ DNA inaccessible—an effect similar to DNA methylation. With their DNA inaccessible, the progenitor cells couldn’t respond to signals from Atoh1.

Conversely, high levels of Sox4 and Sox11 activity stimulated mouse progenitor cells and supporting cells to form hair cells in vitro. 

More promising still, in mice with damaged sensory cells in the inner ear, high levels of Sox4 and Sox11 activity increased the percentage of vestibular (balance) supporting cells that converted into sensory hair cells—from 6 percent to 40 percent.

“We’re excited to continue exploring the mechanisms by which cells in the inner ear gain the ability to differentiate [into] sensory cells during development and how these can be used to promote the recovery of sensory hearing [hair] cells in the mature inner ear,” says Ksenia Gnedeva, Ph.D., the paper’s corresponding author.

Gnedeva completed her postdoctoral training in the Segil Lab and is now an assistant professor in the USC Tina and Rick Caruso Department of Otolaryngology–Head and Neck Surgery, and the department of stem cell biology and regenerative medicine. 

“These two papers are not only great science, but also a clear example of Neil Segil’s enduring legacy as an exceptional mentor to the next generation of stem cell researchers,” adds Gage Crump, Ph.D., a coauthor on both papers and the interim chair of USC’s department of stem cell biology and regenerative medicine at USC’s Keck School of Medicine.

Juan Llamas, Ph.D., from the Segil lab and Tuo Shi from the Crump lab are coauthors of both studies. For Wang and Gnedeva’s paper, additional coauthors include Talon Trecek, Litao Tao, Ph.D. (now at Creighton University and a member of HHF’s Hearing Restoration Project), and Welly Makmura from the Segil lab.

This is adapted from a USC press release. Both studies were supported by the National Institutes of Health (NIH grant RO1 DC015829) and Hearing Health Foundation’s Hearing Restoration Project (HRP). Nguyen and Groves’s study received support from three additional NIH grants (F31 DC018703, T32 HD060549, and RO1 DC014832), and Wang and Gnedeva’s study received support from two additional NIH grants (R21 DC016984 and T32DC009975).

HRP members Andy Groves, Ph.D. (left, top) is a professor and the Vivian L. Smith Endowed Chair in Neuroscience in the departments of neuroscience and molecular and human genetics at Baylor College of Medicine, and a 1996–1997 and 2012 Emerging Research Grants scientist; and Litao Tao, Ph.D., is an assistant professor in the department of biomedical sciences at Creighton University School of Medicine.

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