Engineering High-Fidelity Human Cochlear Organoids

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PROJECT SUMMARY Loss of sensory hair cells and/or innervating neurons in the cochlea causes irreversible hearing loss in humans. However, progress on research for realizing biological restoration of hearing has been hampered due to the paucity of human cochlear tissues. My laboratory recently developed a novel organoid system to generate inner ear sensory epithelia containing functional sensory hair cells from aggregates of human pluripotent stem cells in 3D culture. While these first-generation organoids are a valuable tool for studying human inner ear development, they only generate hair cells with structural and functional properties of native vestibular hair cells and fail to produce any cochlear cell types. Another limitation with our original system is the lack of central nervous system components. To overcome these limitations, we aim at developing a next- generation human microphysiological system that more faithfully recapitulates development of the auditory periphery and brainstem. In Aim 1, we will carry out genetic programming and a small-scale CRISPR screen to increase the number of outer hair cells arising in cochlear organoids. The identity of derived hair cells will be validated by single-cell electrophysiology, electron microscopy and single-cell RNA-sequencing. Additionally, maturation of derived hair cells will be promoted by thyroid hormone treatments. In Aim 2, we will establish novel human cochlear-hindbrain assembloids and assess afferent neural circuit development in these assembloids. In Aim 3, we will develop dynamic/tunable hydrogels and test if introducing a spatial gradient of stiffness in the microenvironment during organoid formation can affect tissue patterning or cellular differentiation. Spatial gradients of signaling molecules will be also introduced in hydrogels to test if the dorso- ventral axis formation can be recapitulated during organoid differentiation. The outcome of this study will provide a paradigm-changing approach for studying normal and pathological development of cochlear hair cells and their ascending neural circuits.