Congenital and acquired deafness is a major public health problem affecting more than 36 million American people. Recent breakthroughs in stem cell biology have revealed that a complex sensory organ with all neuronal subtypes can be formed from aggregates of pluripotent stem cells in 3D culture, which seemed remote and futuristic not long ago. Spurred by these seminal studies, we have established a novel 3D culture system to faithfully recapitulate inner ear induction using a combination of small molecule inhibitors and recombinant proteins. We have demonstrated that, by precise temporal control of BMP, TGF? and FGF signaling, stem cell aggregates transform sequentially into non-neural, pre-placodal and otic placode-like epithelia. Remarkably, in a self-guided process, vesicles containing prosensory cells emerge from the presumptive otic placodes and give rise to hair cells bearing stereocilia and a kinocilium. These stem cell-derived hair cells are structurall and biochemically comparable to those in the vestibular epithelia. In this study, we will first optimize our in vitro system in order to appropriately model the formation and differentiation of the entire inner ear structures, including cochlear cell types (Aim 1). We will test whether manipulation of Wnt and Shh signaling pathways alter the relative number of otic progenitor cells and cochlear cell types, respectively, derived from pluripotent stem cells. In addition, by taking advantage of our high-throughput culture system, we will begin to decipher the molecular mechanisms underlying hair cell differentiation (Aim 2). Using ChIP-based biochemical assays, we will test whether expression of prosensory genes is genetically and epigenetically regulated by Pax2 and whether constitutive methylation of a core histone protein increases the number of stem cell-derived otic progenitors giving rise to prosensory cells, and consequently hair cells. Furthermore, we will validate functional properties of these stem cell-derived hair cells and define the identity of hair cell phenotypes (Aim 3). Using a combination of single-cell electrophysiology, optogenetics and high-resolution imaging techniques, we will test whether stem cell-derived hair cells exhibit structural and functional properties of native sensory hair cells in the inner ear and make synaptic connections with sensory neurons. By accomplishing these aims, we will not only advance our understanding of the biology of hair cell development, but also establish a potent model system with which to investigate pathogenesis of various forms of hereditary deafness and balance disorders.

Public Health Relevance

Profound hearing loss is a major health problem affecting over 36 million adults and children in America. Stem cells could potentially be used to replace damaged auditory receptor cells in patients or to study progression of hearing disorders. This study will test whether stem cells can be guided to become functional auditory receptor cells using a novel strategy.

Agency
National Institute of Health (NIH)
Institute
National Institute on Deafness and Other Communication Disorders (NIDCD)
Type
Research Project (R01)
Project #
5R01DC013294-04
Application #
9232135
Study Section
Auditory System Study Section (AUD)
Program Officer
Freeman, Nancy
Project Start
2014-03-01
Project End
2019-02-28
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
4
Fiscal Year
2017
Total Cost
$388,706
Indirect Cost
$108,287
Name
Indiana University-Purdue University at Indianapolis
Department
Otolaryngology
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Nie, Jing; Koehler, Karl R; Hashino, Eri (2017) Directed Differentiation of Mouse Embryonic Stem Cells Into Inner Ear Sensory Epithelia in 3D Culture. Methods Mol Biol 1597:67-83
Nie, Jing; Hashino, Eri (2017) Organoid technologies meet genome engineering. EMBO Rep 18:367-376
Koehler, Karl R; Nie, Jing; Longworth-Mills, Emma et al. (2017) Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells. Nat Biotechnol 35:583-589
DeJonge, Rachel E; Liu, Xiao-Ping; Deig, Christopher R et al. (2016) Modulation of Wnt Signaling Enhances Inner Ear Organoid Development in 3D Culture. PLoS One 11:e0162508
Liu, Xiao-Ping; Koehler, Karl R; Mikosz, Andrew M et al. (2016) Functional development of mechanosensitive hair cells in stem cell-derived organoids parallels native vestibular hair cells. Nat Commun 7:11508
Longworth-Mills, Emma; Koehler, Karl R; Hashino, Eri (2016) Generating Inner Ear Organoids from Mouse Embryonic Stem Cells. Methods Mol Biol 1341:391-406
Shimomura, Atsushi; Patel, Dharmeshkumar; Wilson, Sarah M et al. (2015) Tlx3 promotes glutamatergic neuronal subtype specification through direct interactions with the chromatin modifier CBP. PLoS One 10:e0135060
Koehler, Karl R; Hashino, Eri (2014) 3D mouse embryonic stem cell culture for generating inner ear organoids. Nat Protoc 9:1229-44