Bioengineering a human pluripotent stem cell system for studying inner ear development

About This Grant

Human pluripotent stem cells (hPSCs) have been demonstrated to be powerful tools to study human biology and diseases, especially for which human tissue is difficult to access to and biopsy is challenged to obtain. Inner ear is one of organs that is nearly impossible to access without causing damages. Moreover, it is rare to obtain human inner ear biopsy. However, inner ear disorders, including hearing loss and vestibular dysfunction, are one of the most common sensory disorders. Although animal studies have advanced our understanding in hair cells, the underlying cellular processes may differ from those of human. A human cell-based model is therefore needed to progress our understanding of inner ear and the development of therapeutic strategies. Recently, stem cell-derived three-dimensional (3D) inner ear organoids have been reported to mimic certain aspects of inner ear development and disease pathologies in vivo. Despite this potential, there are limitations in the 3D inner ear organoid system to faithfully recapitulate some developmental processes, e.g., morphogen gradients that are critical for proper patterning. Therefore, we first propose to develop an innovative microphysiological system featuring a microfluidic chip that can precisely establish desired morphogen concentration gradients to which large size, 3D organoids are subjected on-chip (Aim 1). We will mediate BMP, WNT, and SHH signaling pathways to model dorso-ventral patterning in otic vesicles as a proof of concept. Results will assist us to understand how to induce the proper dorso-ventral patterning such that a similar strategy can be applied to effectively induce ventralization in the hPSC-based inner ear organoid system. As many inner ear disorders stem from malfunction of or damages in hair cells, we then propose to understand regulatory processes for the hair cell formation with special focus on epigenetic regulatory mechanisms, which is poorly understood, using the hiPSC-derived 3D inner ear organoid system (Aim 2). We propose to generate comprehensive epigenetic and transcriptomic regulatory networks for nature occurring hair cell differentiation and post-damage-induced hair cell regeneration using single cell (sc) muti-‘Omic approaches. Furthermore, we will validate the findings from sc multi-‘Omic approaches by testing the effects of the identified regulatory enhancer regions and genes in hair cell regeneration using CRISPR/dCas9-based assays (Aim 3). This will allow us to mimic epigenetic regulation of gene expression during hair cell formation. This project is not only improving the technology for the organoid system, but also providing the first step towards systematically advancing our knowledge of the role of epigenetics in human hair cell formation and, ultimately, developing therapeutic strategies for damaged hair cells.