A simplified method for generating human inner ear organoids from pluripotent stem cells

Abstract The inner ear detects sound, head movements, and gravity using specialized epithelial cells and neurons. Decreased function in these cells can lead to hearing loss and dizziness. Inner ear disorders impact millions worldwide; however, current therapeutic options are limited. While animal models are a powerful system to assess auditory and vestibular dysfunction, in vitro inner ear models are gaining importance in translational research. Here, we provide a stepwise approach for generating inner ear organoids (IEOs), which contain supporting cells, hair cells, and neurons. Our differentiation regimen, using defined medium components and diluted extracellular matrix proteins, guides a 3D spheroid of pluripotent stem cells into otic progenitor cells by mimicking the environmental cues that occur during fetal development. Control of the TGF and BMP pathways early in the culture, promotes patterning of the spheroid, with an outer layer of surface ectoderm and an inner core of neuroectoderm. Later, FGF activation and BMP inhibition induce placode formation in the outer layer and neural crest cell migration from the core. These two cell lineages co-develop into otic vesicle-like structures surrounded by a layer of mesenchymal, neuronal, and glial cells that can be maintained in culture for over 100 days. The IEOs described in this protocol are a promising tool for otology research.