Modeling Inner Ear Differentiation with Pluripotent Stem Cells

项目总结

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.