Engineering High-Fidelity Human Cochlear Organoids

SPECIFIC AIMS: Sensorineural hearing loss is a major contributor to global morbidity. The successful development of more targeted approaches such as gene- and cell-based therapies will require a deeper understanding of the cellular and biomolecular mechanisms underlying human hearing, deafness, and inner ear development. Unfortunately, unlike many other organs, the human inner ear cannot be biopsied without causing significant, permanent damage to the inner ear. We recently established the generation of human pluripotent stem cell- derived inner ear organoids, which offer a highly scalable and high-fidelity alternative to existing animal models. Built upon this platform, we aim at developing a next-generation human microphysiological system to recapitulate auditory neural networks using a combination of stem cell technology, genome engineering technology and chemical/tissue engineering approaches. Specific Aim 1: Establish high-fidelity human cochlear organoids. IKZF2 encodes the Zinc finger transcription factor Helios, which is essential for outer hair cell differentiation in mice. However, IKZF2 is undetectable in hair cells of our human cochlear organoids. By CRISPR-mediated inducible expression of IKZF2 specifically in hair cells in conjunction of a small-scale CRISPR screen, we aim at promoting outer hair cell derivation in human cochlear organoids. The identity of derived outer hair cells will be validated by single- cell RNA-sequencing (scRNA-seq), patch-clump recordings and electron microscopy. Aim 1.1. Increase the number of outer hair cells in human cochlear organoids by genetic programming. Aim 1.2. Perform a CRISPR screen to identify additional transcription factors that can improve the programming efficiency. Aim 1.3. Accelerate hair cell maturation in human cochlear organoids. Specific Aim 2: Establish functional cochlear-hindbrain assembloids. We have developed a protocol to derive dorsal hindbrain organoids from human pluripotent stem cells based on published midbrain organoid derivation protocols with major modifications. We will first test the authenticity of these hindbrain organoids with scRNA-seq and immunofluorescence. Additionally, using our established hESC lines with POU4F1-membrane bound tdTomato and ATOH1-eGFP reporter, we will test if specific afferent neural projections can be established in cochlear-hindbrain assembloids and if the extent of these neural projections depends on developmental ages of afferent and/or target neurons. We will also test if excitatory neural circuits are formed in these assembloids using a genetically-encoded Ca2+ sensor. Aim 2.1. Determine the identity of neural populations in dorsal hindbrain organoids. Aim 2.2. Determine the specificity and developmental dependance of afferent neural projections in cochlear-hindbrain assembloids Aim 2.3. Identify and characterize excitatory neural circuits in cochlear-hindbrain assembloids Specific Aim 3: Recapitulate inner ear patterning with dynamic biomimetic hydrogels. The inner is one of the most elaborate organs in the body and evidence suggests that mechanical stimulations may play a role in inner ear patterning and cellular differentiation. By introducing spatio-temporal changes in stiffness using gelatin-based dynamic hydrogels (GelNB-CH), we have engineered custom miclofluidic chambers. Using these chambers, we hope to recapitulating some features of inner ear patterning in human cochlear organoids. Additionally, by introducing signaling molecule gradients, we will test if cochlear and vestibular tissues arise concomitantly in the 3D culture. Aim 3.1. Determine if temporal or spatial changes in mechanical properties in the miroenvironment alter the overall pattern of human cochlear organoids. Aim 3.2. Determine if spatial gradients of signaling molecules in the microenvironment promotes concomitant generation of cochlear and vestibular organoids IMPACT: The high-fidelity human cochlear organoids we aim to establish in this study will serve as the first human in vitro model with which to study human cochlear development and disease. Moreover, the cochlear- hindbrain assembloids that contain both peripheral and central nervous system components will recapitulate auditory afferent neural networks and will serve as a powerful human model to study genetic maladies that affect both peripheral and central auditory systems. These novel human microphysiological systems are scalable, manipulatable and tractable, thus would offer paradigm-shifting platforms to study normal and abnormal development of the human auditory system.