# Modeling Inner Ear Differentiation with Pluripotent Stem Cells

> **NIH NIH R01** · INDIANA UNIVERSITY INDIANAPOLIS · 2021 · $573,417

## Abstract

PROJECT SUMMARY
Human inner ear tissues, sensory cells in particular, are scarce for experimentation, since biopsy is not a
standard procedure for patients with profound hearing loss or balance disorders. To circumvent this challenge,
we recently established a defined 3D culture system to efficiently generate human inner ear sensory epithelia
from aggregates of human pluripotent stem cells. These so-called “human inner ear organoids” harbor a layer
of tightly packed supporting cells and hair cells that are innervated by sensory neurons. Based on our initial
characterization, these human stem cell-derived hair cells exhibit structural, biochemical and functional
properties comparable to those of native sensory hair cells. The primary goal of this application is to define the
temporal progression, transcriptional pathways, structural changes and protein-protein interactions during
sensory cell differentiation in the human inner ear organoid. In Aim 1, we will test how PAX2-positive otic
progenitors give rise to different cell types in the inner ear. Using a combination of single-cell RNA-seq, ChIP-
seq and lineage-tracing analyses, we will determine developmental trajectories of gene expression, lineage
specification and transcriptional networks essential for specification of hair cells and sensory neurons in the
human inner ear. In Aim 2, we will elucidate the transcriptional pathways distinctive for vestibular vs. cochlear
specification and determine biochemical and structural properties of hair cells derived from ventralized otic
progenitors. In Aim 3, we will define temporal progression of hair cell differentiation (e.g. hair bundle and
ribbon synapse development) in human inner ear organoids at both light and electron microscopic levels.
Additionally, using a combination of single-cell electrophysiology and optogenetics, we will test whether human
stem cell-derived hair cells make functional synaptic connections with sensory neurons that are concomitantly
arising in culture. Moreover, using yeast two-hybrid screening, we will identify novel protein-protein
interactions essential for hair bundle formation. By accomplishing these aims, we will not only advance our
understanding of the biology of human inner ear development, but also establish a defined and scalable
human model system with which to investigate pathogenesis of various forms of hereditary inner ear disorders
and identify compounds with the potential of regenerating hair cells in humans.

## Key facts

- **NIH application ID:** 10144982
- **Project number:** 5R01DC013294-08
- **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS
- **Principal Investigator:** Eri Hashino
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $573,417
- **Award type:** 5
- **Project period:** 2014-03-01 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10144982

## Citation

> US National Institutes of Health, RePORTER application 10144982, Modeling Inner Ear Differentiation with Pluripotent Stem Cells (5R01DC013294-08). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10144982. Licensed CC0.

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