Sensory Development in Human Cochlear Organoids

PROJECT SUMMARY This application is in response to the NIDCD Program Announcement PAS-24-058: Advancing Hearing and Balance Research Using Auditory and Vestibular Organoids. Mechanosensitive hair cells in the cochlea are essential for hearing, but are vulnerable to damage by genetic mutations and environmental insults, resulting in irreversible sensorineural hearing loss. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells and innervating sensory neurons. We recently established a new organoid system that recapitulates cochlear differentiation. These so called human cochlear organoids contain functional hair cells, supporting cells and sensory-like neurons. However, the low efficiency of sensory cell derivation and the lack of mature hair cells and sensory neurons are major limitations for broad pre-clinical applications. We propose a series of experiments to better understand the genetic regulatory network underlying sensory differentiation in human cochlear organoids and improve the efficiency of hair cell differentiation and maturation. In Aim 1, to define the developmental trajectories of key sensory cell types, we will perform a longitudinal scATAC-seq & scRNA-seq multi-omics analysis of otic lineage cells isolated from human cochlear organoids at various time points. Additionally, we will develop a new bioinformatics analytical tool tailored for better integration of temporal multi-modal data by adopting the graph-based neural networks. In Aim 2, we will test if expanding intermediate otic cell populations by genetic programming can increase the number of hair cells arising in human cochlear organoids. Our analysis of longitudinal scRNA-seq data with RNA velocity has identified OTX2 and KLF5 as top candidate driver genes for intermediate otic cells and nonsensory cells, respectively. We will perform CUT&RUN to identify direct target genes for OTX2 in otic progenitors. Additionally, we will test if inducible expression of OTX2 or rapid degradation of KLF5 increases the number of otic intermediate cell populations, leading to a larger number of hair cells and supporting cells in human cochlear organoids. In Aim 3, we will test if augmentation of thyroid hormone signaling accelerates maturation of hair cells and sensory neurons as well as pre-/post-synaptic specification, using a combination of transcriptomic profiling and morphometric analyses. The results obtained from the proposed experiments will advance our holistic understanding of cell fate specification in the human cochlea, offer an improved computational tool for analyzing multi-modal biological data and establish a valuable human in vitro model system for preclinical applications on sensorineural hearing loss.