hair cell regeneration

Single-Cell RNA Sequencing Reveals More Clues for Hair Cell Regeneration

By Mark E. Lush, Ph.D., and Daniel C. Diaz

Sensorineural hearing loss in mammals can often be attributed to damage or destruction of the delicate hair cells located within the inner ear. The microscopic hairlike projections on the surface of these cells are the key structure responsible for converting sound waves to electrical signals that travel to the brain through the auditory nerve. Unlike mammals, other vertebrates such as fish, birds, and reptiles routinely regenerate sensory hair cells during homeostasis and following injury. By studying the genetic program of hair cell regeneration in nonmammalian vertebrate organisms, researchers may discover therapeutic targets for treating hearing loss in humans.

The lateral line is a sensory system that allows aquatic vertebrates to orient themselves by detecting water motion. The lateral line organs (neuromasts), distributed on the head and along the body, contain approximately 60 cells, composed of central sensory hair cells surrounded by support cells and an outer ring of mantle cells. Using single-cell RNA sequencing, we combined some of the less well-defined clusters and identified major neuromast cell types, shown in this illustration, ranging from support cells to mature sensory hair cells. Credit: The lab of Tatiana Piotrowski, Ph.D., Stowers Institute for Medical Research, Kansas City

The lateral line is a sensory system that allows aquatic vertebrates to orient themselves by detecting water motion. The lateral line organs (neuromasts), distributed on the head and along the body, contain approximately 60 cells, composed of central sensory hair cells surrounded by support cells and an outer ring of mantle cells. Using single-cell RNA sequencing, we combined some of the less well-defined clusters and identified major neuromast cell types, shown in this illustration, ranging from support cells to mature sensory hair cells. Credit: The lab of Tatiana Piotrowski, Ph.D., Stowers Institute for Medical Research, Kansas City

One such organism, the zebrafish, has emerged as a powerful model for studying sensory hair cell regeneration. Like other fish, zebrafish contain a network of sensory hair cells throughout their body to detect changes in water movement. The hair cells are located in small organs in the skin called neuromasts, which also contains cell types that are remarkably similar to those found in the mammalian inner ear. To study the genetic program of hair cell regeneration in zebrafish, we sequenced the RNA of individual cells within neuromasts, allowing us to classify cell types based on their gene expression signature. This included cells transitioning from support cells to fully mature sensory hair cells, thereby identifying new genes that are expressed during hair cell development. In addition, we characterized the role of the growth factor fgf3, and found that it acts to inhibit hair cell progenitor proliferation. Our results were published in the journal eLife on Jan. 25, 2019. Future work will examine the function of these genes in sensory hair cell regeneration.

HRP_logo for web.png

Mark E. Lush, Ph.D., and Daniel C. Diaz both work in the lab of Tatjana Piotrowski, Ph.D., at Stowers Institute for Medical Research in Kansas City. Piotrowski is a member of the Hearing Restoration Project, which helped fund this study.

Empower the Hearing Restoration Project's life-changing research. If you are able, please make a contribution today.

 
 
Print Friendly and PDF

On a Data-Driven Mission

By Peter G. Barr-Gillespie, Ph.D.

The annual meeting of Hearing Health Foundation’s (HHF) Hearing Restoration Project was held in Seattle November 11-12, 2018. We used this meeting to update one another on recent progress on HHF-funded projects, discuss in detail the implications of new data, evaluate the directions of ongoing projects, and plan for the next funding period.

As you may recall, in November 2016 the Hearing Restoration Project (HRP) made a deliberate turn toward funding only the highest-impact science that our group leads the world in researching—we have termed this the “Seattle Plan.” We therefore devoted a substantial portion of our efforts to cross-species comparisons that contrast molecular responses to inner ear sensory hair cell damage in species that regenerate their hair cells, especially chickens and fish, with responses in mice, which like other mammals do not regenerate their hair cells. We also have been examining the “epigenetic” structure of key genes in the mouse, as one hypothesis is that epigenetic modifications of the DNA—that is, the inactivation of genes through chemical changes to the DNA—causes mouse (and human) cells of the cochlea to no longer respond to hair cell damage by regenerating hair cells.

Avian and mammal supporting cell subtypes differ, but Stefan Heller, Ph.D., and team are investigating if an evolutionary homogenous equivalent exists in the organ of Corti, and if this knowledge could be used for hair cell regeneration. Credit: Chris Gralapp / Otolaryngology Head and Neck Surgery (OHNS) - Stanford University School of Medicine

Avian and mammal supporting cell subtypes differ, but Stefan Heller, Ph.D., and team are investigating if an evolutionary homogenous equivalent exists in the organ of Corti, and if this knowledge could be used for hair cell regeneration. Credit: Chris Gralapp / Otolaryngology Head and Neck Surgery (OHNS) - Stanford University School of Medicine

I am happy to report that progress over the past two years on these two major projects has been outstanding. For the cross-species comparisons, Stefan Heller, Ph.D., and Tatjana Piotrowski, Ph.D., reported on single cell analysis of, respectively, chick and fish hair cell organs responding to damage. Using single cell analysis—isolating hundreds to thousands of individual cells and quantifying all of the protein-assembly messages they express—we can determine the molecular pathways by which hair cells are formed during development and regeneration. This approach has always been promising, but this year we have begun to reap the expected benefits, as those projects have given us an unprecedented view of hair cell formation.

The epigenetics project overseen by Neil Segil, Ph.D., has now reached maturity, and using the voluminous data acquired over the past several years his lab has shown how supporting cells (from which we intend to regenerate hair cells) change the epigenetic modification of their DNA so they no longer are able to switch on key genes used for turning them into hair cells. A topic of great interest at the meeting was that of genetic reprogramming: Can we use genes (like transcription factors, proteins that control the transfer of genetic information) or small molecules (which often can be taken orally and still reach their targets) to overcome the epigenetic modification and push supporting cells to turn into hair cells? Preliminary results from Segil’s lab and from others in the field make us optimistic that the reprogramming approach will eventually be part of a regeneration strategy.

We also heard from Seth Ament, Ph.D., a bioinformatics expert we recently recruited to the HRP to explicitly compare our various datasets and find the common threads between them. Ament has used gene expression data from the chick, fish, and mouse, as well as the epigenetic data from the mouse, to hypothesize which genes may be important for hair cell regeneration. As a systems biology specialist, Ament brings a fresh eye to the field of auditory science and has not only identified some of the genes we expected to be important, but new ones as well. His success nicely justifies our cross-species approach, and the bioinformatics comparisons that he has been able to achieve in his initial HRP project have been impressive.

Finally, two other Seattle Plan projects have gone well, including our data-sharing platform called the gEAR (gene Expression Analysis Resource), developed by Ronna Hertzano, M.D., Ph.D., which allows us to analyze our data privately but also to efficiently share data with the public. In addition, John Brigande, Ph.D., reported on his project developing mouse models for testing interesting new genes; his group will be adding several powerful models in the year to come.

The excitement at the meeting extended to our future plans. We agreed that the Seattle Plan was the still the proper course, and we eagerly anticipate more data and results to come from our consortium of researchers. We are truly getting a clearer picture of hair cell regeneration due to the HRP’s efforts. That said, there is a long way to go; our efforts show us how surprisingly intricate biology is, despite knowing from the start that systems like the inner ear are remarkably complex. Nature always has surprises for us, by turns dashing treasured hypotheses while revealing unexpected mechanisms. The HRP is most definitely on track for success, and all of us in the HRP sincerely thank you for your continued support.

barr-gillespie.jpg


HRP scientific director Peter G. Barr-Gillespie, Ph.D., is a professor of otolaryngology at the Oregon Hearing Research Center, a senior scientist at the Vollum Institute, and the interim senior vice president for research, all at Oregon Health & Science University. For more, see hhf.org/hrp.

Print Friendly and PDF

The Hearing Restoration Project: Update on the Seattle Plan and More

By Peter G. Barr-Gillespie, Ph.D.

Hearing Health Foundation launched the Hearing Restoration Project (HRP) to understand how to regenerate inner ear sensory cells in humans to restore hearing. These sensory hair cells detect and turn sound waves into electrical impulses that are sent to the brain for decoding. Once hair cells are damaged or die, hearing is impaired, but in most species, such as birds and fish, hair cells spontaneously regrow and hearing is restored.

The overarching principle of the HRP consortium is cross-discipline collaboration: open sharing of data and ideas. By having almost immediate access to one another’s data, HRP scientists are able to perform follow-up experiments much faster, rather than having to wait years until data is published.

Regenerated hair cells from chicken auditory organs, with the cell body, nucleus and hair bundle labeled with various colored markers. Image courtesy of Jennifer Stone, Ph.D.

Regenerated hair cells from chicken auditory organs, with the cell body, nucleus and hair bundle labeled with various colored markers. Image courtesy of Jennifer Stone, Ph.D.

You may remember that two years ago, we changed how we develop our projects. We decided together on a group of four projects—the “Seattle Plan”—that are the most fundamental to the consortium’s progress. These projects, which grew out of previous HRP projects, have now been funded for two years, and considerable progress has been made. We have also funded several other projects that have bubbled up out of new observations and capabilities, and they have added considerably to our knowledge base. With this in mind, I am pleased to share with you the latest updates for our 2018–19 projects.

SEATTLE PLAN PROJECTS

Transcriptome changes in single chick cells
Stefan Heller, Ph.D.

  • Found that all “tall” hair cells are exclusively regenerated mitotically in this animal model.

  • Compiled evidence for different supporting cell subtypes.

  • Obtained good quality single cell RNA sequencing (scRNA-seq) data and are in the process of evolving an analysis strategy for the baseline cell types (control group). Identified about 50 novel marker genes for hair cells, supporting cells, and homogene cells, including subgroups.

  • Developed a strategy to finish all scRNA-seq using a novel peeling technique and latest generation library construction methods.

  •  Established two methods for multi-color in situ hybridization (PLISH, proximity ligation in situ hybridization) and SGA (sequential genomic analysis) for spatial and temporal mRNA expression validation.

Epigenetics of the mouse inner ear
Michael Lovett, Ph.D., David Raible, Ph.D., Neil Segil, Ph,D., Jennifer Stone, Ph.D.

  • Completed epigenetic, chromatin structure, and RNA-seq datasets for FACS-purified cochlear hair cells and supporting cells from postnatal day 1 and postnatal day 6 mice, and provision of these data sets to the gEAR (gene Expression Analysis Resource portal) for mounting on their webpage through EpiViz for access by the HRP consortium.

  • Established a webpage (EarCode) so that HRP consortium members can access the current data directly through a University of California, Santa Cruz, genome browser.

  • Discovered maintenance of the transcriptionally silent state of the hair cell gene regulatory network in perinatal supporting cells is dependent on a combination of H3K27me3 and active H3k27-deacetylation, and that during transdifferentiation, these epigenetic marks are modified to an active state.



Mouse functional testing
John Brigande, Ph.D.

  • Defined in vitro and in vivo model systems to interrogate genome editing efficacy using CRISPR/Cas9.

Implementing the gEAR for data sharing within the HRP
Ronna Hertzano, M.D., Ph.D.

  • Added scRNA-seq workbench for easy sharing and viewing of scRNA-seq data. Such data, which are now driving the field forward, have been particularly difficult to share

  • Created additional public datasets to improve data sharing.

  • Completely rewrote the gEAR backbone to be updated to the latest technologies, allowing the portal to now to handle a much larger number of datasets and users.

  • Performed hands-on gEAR workshops at the Association for Research in Otolaryngology and the Gordon Research Conference, increasing the number of users with accounts to greater than 300.


Single Cell RNA-seq of homeostatic neuromasts
Tatjana Piotrowski, Ph.D.

  • Optimized protocols for fluorescent-activated cell sorting and scRNA-seq; obtained high quality scRNA-seq transcriptome results from 1,400 neuromast cells; clustered all cells into seven groups; and performed analyses to align the cells along developmental time, providing a temporal readout of gene expressions during hair cell development.

OTHER PROJECTS

Integrated systems biology of hearing restoration
Seth Ament, Ph.D.

  • Discovered 29 novel risk loci for age-related hearing difficulty through new analyses of genome-wide association studies of multiple hearing-related traits in the U.K. Biobank (comprising 330,000 people), and predicted the causal genes and variants at these loci through integration with transcriptomics and epigenomics data from HRP consortium members.

  • Generated scRNA-seq of 9,472 cells in the neonatal mouse cochlea and utricle (postnatal days 2 and 7).

  • Conducted systems biology analyses that integrate multiple HRP datasets to characterize gene regulatory networks and predict driver genes associated with the development and regeneration of hair cells. These analyses utilize scRNA-seq of sensory epithelial cells in mouse, chicken, and zebrafish hearing and vestibular organs, as well as epigenomic data (ATAC-seq) from hair cells, support cells, and non-epithelial cells in the mouse cochlea.


Comparison of three reprogramming cocktails
Andy Groves, Ph.D.

  • Created and validated transgenic mouse lines expressing three different combinations of reprogramming transcription factors.

  • Demonstrated these lines can produce new hair cell–like cells in the undamaged and damaged cochlea of the immature mouse.

  • Compiled preliminary data showing Atoh1 and Gfi1 genes can create ectopic hair cells in the adult mouse cochlea.


Signaling molecules controlling avian auditory hair cell regeneration
Jennifer Stone, Ph.D.

  • Identified four molecular pathways (FGF, BMP, VEGF, and Wnt) that control hair cell regeneration in the bird auditory organ. These pathways were identified in Phase I (gene discovery) as being transcriptionally dynamic in birds, fish, and mice during regeneration, which indicated they may be universal regulators of hair cell regeneration.

  • Determined that the Notch signaling pathway (a powerful inhibitor of stem cells) also blocks supporting cell division in the chicken auditory organ after damage. This discovery shows that Notch is a negative regulator of regeneration, conserved in birds, fish, and mice.

  • Identified signaling molecules in birds that are correlated with either mitotic or non-mitotic modes of hair cell regeneration, and are now exploring how these signaling molecules interact to determine which mode of regeneration occurs. Since mammals only exhibit non-mitotic regeneration, we are particularly interested in determining how this mode is controlled.

UP NEXT

We look forward to our annual meeting, which will be held in Seattle in November. There we will discuss and integrate these data to develop our plans for our 2019–20 projects.

barr-gillespie.jpg

As always we are very grateful for the donations we receive to fund this groundbreaking research to find better treatments for hearing loss and related conditions. Every dollar counts, and we sincerely thank our supporters.

HRP scientific director Peter G. Barr-Gillespie, Ph.D., is a professor of otolaryngology at the Oregon Hearing Research Center, a senior scientist at the Vollum Institute, and the interim senior vice president for research, all at Oregon Health & Science University. For more, see hhf.org/hrp.

 

Empower the Hearing Restoration Project's life-changing research. If you are able, please make a contribution today.

 
 
Print Friendly and PDF

A Birthday Wish

By Beth and Jeremy Hochheiser

In Beth’s words

Our son Jeremy recently turned 29, and on his Facebook page for his birthday he asked friends and family to donate to Hearing Health Foundation (HHF) to help reach his fundraising goal, which he exceeded.

Jeremy introduced us to HHF when he discovered its commitment to hair cell regeneration. He has a profound hearing loss and has been using hearing aids successfully since childhood.

We did not realize there was a problem with Jeremy’s hearing until he was 14 months old, mainly because he had been making sounds like a typical baby. Even as a baby, Jeremy had an infectious belly laugh and was always very attentive to what was going on around him.

But when we discussed a potential hearing issue with our pediatrician, he didn’t seem concerned. It was only after we went to see an audiologist that we finally got a diagnosis of profound congenital bilateral sensorineural hearing loss.

A Birthday Wish - Beth and Jeremy.jpg

Throughout Jeremy’s childhood we visited audiologists and doctors regularly. We found an otolaryngologist in New York City who specialized in hearing disabilities, and we kept up with the latest technology in hearing aids to understand options for Jeremy. As he got older, we always encouraged him to ask people to repeat themselves if he couldn’t hear or understand them.

Since Jeremy was diagnosed with quite a bit of residual hearing, the audiologist suggested an unconventional means of learning speech, the auditory-verbal approach introduced by Helen Beebe. Children learn to use the hearing they still have by being bombarded with speech consistently. I read books and introduced pictures by sitting next to Jeremy, not facing him, so that he could learn how to repeat words and speech sounds by hearing them rather than watching my face and lip-reading (speech-reading).

As a result, Jeremy’s speech developed as a typical hearing child’s would—by listening and repeating. We as his parents knew that developing Jeremy’s hearing to its fullest potential was imperative—the “if you don’t use it, you’ll lose it” theory—and that lip-reading could come later, as a supplement.

With his hearing aids Jeremy thrived, learned to play piano at age 7, and was even presented with an award at Carnegie Hall. He excelled at school and showed us that his hearing was not an issue or a factor that would get in the way of his education or competitive spirit. The natural belly laugh he had as an infant translated into a great sense of humor and positive outlook.

As a kid Jeremy liked to place one of his hearing aids on his tummy and say, “Mom, I’m so hungry, I can hear my stomach growling!” We’re so proud of him and his studies, the many activities he has thrown himself into, his thriving career, and his own family and baby to come.

In Jeremy’s words


Be Bold


My advice to anyone with hearing loss (and their loved ones) is to be bold, brave, and up front about your hearing. Accept it and wear it proudly, otherwise others may misinterpret who you really are, or even bully you. Hearing loss isn’t who you are.

I also tell those new to hearing loss to never stop using your mind. Your brain is your most powerful tool. I play chess and compete in Brazilian jiu jitsu, plus I love being a software engineer and doing math, exploring art, and enjoying nature. I love to learn. Being fully engaged keeps your brain active and fights off feeling down from hearing challenges.

Your Voice Matters

I remember being afraid to ask others to repeat themselves, but as I got older I learned to ask, even if I had to do it more than once. In this way, I show I am involved and can contribute meaningfully to the conversation. I earn respect for that. Your voice and opinions really do matter. What definitely won’t work is to hold back.

I consider myself outgoing and social, and sometimes when I ask someone to repeat themselves it can break the flow of the conversation, or cause frustration in a new acquaintance who doesn't understand. But it is infinitely more frustrating if you can’t fully participate.

New Challenges

My wife Lauren has typical hearing and sees me as a typical hearing person. But when I am tired and my hearing is down, I have to ask her to repeat herself or let me see her lips. Then when I am less tired and my hearing is better, I get frustrated again if Lauren is still making accommodations for me that I don’t think I need—and that can frustrate her, constantly having to switch! I love my wife for going with the flow and understanding what I need to hear.

Managing Hearing Loss

I find that there are days where I can understand what people are saying without looking, and then sometimes I have to rely on speech-reading. People have said to me, “You can hear better with your glasses on!”

The company I work at now has wonderful benefits and accommodations for their employees with needs. They hold meetings throughout the year among those who have requested accommodations in order to foster an inclusive environment, and they are proactive about making sure I have everything I need. It’s been very welcoming.

How I Discovered HHF

When I was in middle school, my parents and I went to a support group, and I met a couple of kids my age who also have a hearing loss. One of them happened to end up working for HHF, Laura Friedman (HHF’s former communications and programs manager).

I also followed news on the development of hair cell regeneration in the inner ear. I am encouraged about HHF’s Hearing Restoration Project consortium bringing together multiple labs and scientists. All of this is why I wanted to do my part to raise money for research toward the cure.

Beth Hochheiser lives in New Jersey, and Jeremy Hochheiser lives in Pennsylvania. HHF sincerely thanks the Hochheisers and their family and friends for their support.

Print Friendly and PDF