# Modeling Inner Ear Differentiation with Pluripotent Stem Cells

> **NIH NIH R01** · INDIANA UNIVERSITY INDIANAPOLIS · 2024 · $652,201

## Abstract

PROJECT SUMMARY
During inner ear development, multipotent otic progenitors give rise to mechanosensitive hair cells through a
series of cell-fate transition points. The transcriptional cascades dictating hair cell specification have not been
defined although a number of transcription factors essential for inner ear development have been identified.
Using a human pluripotent stem cell-derived vestibular organoid system that allows mechanistic investigation
due to its scalability, we performed a trajectory analysis of otic lineage cells using scRNA-seq. Our results
suggest that supporting cells are precursors of hair cells in human vestibular organoids. Additionally, we
identified LHX3 as a candidate hair cell driver gene, whose expression precedes ATOH1, a previously
perceived hair cell driver. Moreover, our transcriptomic analysis between human vestibular and cochlear
organoids revealed that NR2F1 is among the most differentially expressed genes in both otic progenitors and
hair cells in cochlear organoids than in vestibular organoids. Based on these findings, we propose a series of
experiments to define the role for LHX3 and NR2F1 in hair cell and cochlear specification, respectively. In Aim
1, using a longitudinal single-cell multi-omics approach, we will test if supporting cells are primary hair cell
precursors in human cochlear organoids and if LHX3 is a pioneer transcription factor that promotes hair cell
differentiation from supporting cells. To test the pioneer activity of LHX3, we will identify its target genes with
CUT&RUN. We will also test if inducible expression of LHX3 alone or with another transcription factor can
promote hair cell differentiation from supporting cells or prosensory cells. In Aim 2, we will define the mode of
action of NR2F1 during cochlear specification. We will test if NR2F1 occupies overlapping cis-regulatory
regions of target genes with GATA3. Additionally, we will test if the DNA-binding domain of NR2F1 is essential
for cochlear specification and if NR2F1 regulates the timing of cochlear hair cell differentiation. Moreover, we
will test if NR2F1 regulates thyroid hormone signaling. By accomplishing these aims, we will not only advance
our understanding of the biology of human inner ear development, but also develop a strategy to realize hair
cell regeneration in the human inner ear. Additionally, information gained from this study will be integrated into
a synthetic biology and bottom-up engineering approach to recapitulate developmental trajectories and achieve
better engineering control with human inner ear organoids.

## Key facts

- **NIH application ID:** 10998991
- **Project number:** 2R01DC013294-11
- **Recipient organization:** INDIANA UNIVERSITY INDIANAPOLIS
- **Principal Investigator:** Eri Hashino
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $652,201
- **Award type:** 2
- **Project period:** 2014-03-01 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10998991

## Citation

> US National Institutes of Health, RePORTER application 10998991, Modeling Inner Ear Differentiation with Pluripotent Stem Cells (2R01DC013294-11). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/10998991. Licensed CC0.

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