# Tympanic membrane progenitor cells in homeostasis in injury

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2023 · $561,561

## Abstract

Project Summary
Hearing in mammals is dependent upon the ability to efficiently conduct sound vibrations from
the environment to the inner ear. This conduction apparatus includes the auricle, external
auditory canal, tympanic membrane (TM), middle ear space, and ossicular chain. Although a
great deal of research has been directed to the biophysical properties of the ear less is known
about the cellular composition of these structures and how the diverse cells that make up these
structures are formed, maintained, and interact in pathological states. The TM has three layers:
an outer layer of stratified squamous epithelium, a middle layer of connective tissue, and an
inner layer of mucosal epithelium. It is unknown how many different cell types are present in
each of these layers, where the stem or progenitor cell populations of these layers reside, or the
dynamics of how these layers are maintained. Classic dye studies indicated that the outer
epithelium of the TM migrates radially outward from the malleal attachment to the TM. We and
others have shown that within the TM the vast majority of the proliferation is occurring near the
malleus, and that cells then migrate radially outward. This implies at least two populations: a
stem/progenitor population near the malleus, and a progeny population within the radial portions
of the TM. We will first dissociate normal and injured TMs from mice and humans, sort cells,
and perform single cell RNA sequencing analysis combined with nearest-neighbor clustering
analysis using a novel algorithm, CellfindR, in order to identify the cellular subpopulations within
the TM and to predict lineage relationships between them. We will confirm these populations by
using immunofluorescent staining of mouse and human TMs. We will then perform pulse-chase
labelling experiments using EdU as well as lineage tracing with genetically modified mice to
validate the lineage hierarchies predicted by the psuedotime analysis, and definitively identify
the stem and progenitor populations of the TM during perforation repair. Finally, we will perturb
the PDGFR and BMP signalling pathway in defined populations of the TM using inducible and
cell specific knockout mice in vivo as well as defined small molecule inhibitors in a novel ex vivo
air-liquid interface model of explanted mouse and human TMs in order to define the
mechanisms by which these populations differentiate and are maintained and to provide a
therapeutic proof of concept. We hope that once we gain a deeper understanding of the
functional cellular architecture and physiology within the TM, we can then learn how these
processes go awry in and create better biological and surgical treatments for disorders of the
TM.

## Key facts

- **NIH application ID:** 10599161
- **Project number:** 5R01DC018076-04
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Aaron Tward
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $561,561
- **Award type:** 5
- **Project period:** 2020-04-25 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10599161

## Citation

> US National Institutes of Health, RePORTER application 10599161, Tympanic membrane progenitor cells in homeostasis in injury (5R01DC018076-04). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10599161. Licensed CC0.

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