Generation and characterization of vestibular inner ear organoids from human pluripotent stem cells

The inner ear has a pivotal role in auditory and vestibular perception. Despite the vast number of individuals worldwide affected by hearing loss and balance disorders, therapeutic options have been largely limited to technological aids. The recent advent of gene therapies for genetic hearing loss in human patients underscores the urgency of developing scalable platforms to investigate a broader spectrum of inner ear disorders. Although animal models are powerful for assessing auditory and vestibular dysfunction, in vitro human inner ear models have shown promise in disease modeling and as platforms for studying developmental biology. Several studies have demonstrated that stem cells can be guided to differentiate into otic progenitor cells by mimicking environmental cues present during normal fetal inner ear development. Here we present a step-by-step approach to creating inner ear organoids (IEOs), which is an extension of our previous method for skin organoid generation, with which it shares foundational methodology and reagents. We used these organoids to elucidate the subtle signaling cues that govern their developmental trajectories. Generating sensory hair cells takes about 40 d, and cultures can be maintained for up to 150 d to allow further development. Moreover, we outline methods for assessing late-stage organoids, including whole-mount imaging of cleared IEOs, vibratome sectioning of live and fixed IEOs and other endpoint analyses, to study inner ear biology. IEOs are ideal for investigating human inner ear development, studying the mechanisms of inner ear disorders and developing therapeutic strategies. This protocol requires proficiency in basic stem cell culture techniques.