Otic Regeneration and Development: Advancement of Stem Cell-Based Methodology for In Vitro Modeling of Mammalian Inner Ear Sensory Epithelia

Abstract

Hearing loss treatments have improved significantly with the advent of cochlear implants and advancement of hearing aids. Still, they fall short of full restoration of function and do not benefit severe cases. An ideal approach to hearing restoration would be replacement of hair cells as loss or dysfunction of these cells is a major cause of sensorineural deafness. Therefore, recent efforts have focused on differentiation of pluripotent embryonic stem cells toward hair cell fate. A relatively novel 3-dimensional organoid method of differentiation has produced results remarkably similar to native hair cells in form and function. However, several challenges remain: Organoid hair cells are immature, vestibular instead of auditory, and low in yield, limiting practical use in the clinic or in the lab.

Our goal was to advance the current state of hair cell regeneration efforts through effective adaptation of developmental signaling cues. In these studies, we investigated parallels between early embryonic inner ear development and derivation of inner ear organoids using cell and molecular biology techniques. Specific experimental questions include the following:

1. Do inner ear organoids recapitulate mechanisms downstream of fibroblast growth factors (FGFs) involved in embryonic otic induction, establishing progenitors with inner ear fate?

2. Are outcomes dependent upon transforming growth factor beta (TGFβ) inhibition used to recapitulate embryonic germ layer patterning?

3. How closely do derived otic vesicles mimic native embryonic vesicles at the transcriptome level?

A fluorescent reporter cell line was used to track differentiation through Pax2 upregulation at the crucial otic induction stage. The results established the utility of this reporter cell line and revealed key parallels with embryogenesis and opportunities for advancing the organoid technology. In a follow-up study, the first differentiation step directing stem cells toward an inner ear lineage—inhibition of TGFβ signaling—was modified. The results demonstrated that this step was dispensable for formation of otic progenitors but necessary for later maturation into organoid epithelia. We performed comparative transcriptome analysis of stem cell-derived otic vesicles treated differentially at this stage and embryonic otic vesicles. From our analysis, targets for further optimization efforts emerged, including retinoic acid signaling and several key otic genes.

Elucidating parallels between organoid differentiation and embryonic development will contribute knowledge necessary to scale organoid production for practical use. In the future, this work may provide suitable models of inner ear development, physiology, and disease for laboratory study and provide replacement cells for clinical treatment.