Generating inner ear organoids containing putative cochlear hair cells from human pluripotent stem cells

Abstract

In view of the prevalence of sensorineural hearing defects in an ageing population, the development of protocols to generate cochlear hair cells and their associated sensory neurons as tools to further our understanding of inner ear development are highly desirable. We report herein a robust protocol for the generation of both vestibular and cochlear hair cells from human pluripotent stem cells which represents an advance over currently available methods that have been reported to generate vestibular hair cells only. Generating otic organoids from human pluripotent stem cells using a three-dimensional culture system, we show formation of both types of sensory hair cells bearing stereociliary bundles with active mechano-sensory ion channels. These cells share many morphological characteristics with their in vivo counterparts during embryonic development of the cochlear and vestibular organs and moreover demonstrate electrophysiological activity detected through single-cell patch clamping. Collectively these data represent an advance in our ability to generate cells of an otic lineage and will be useful for building models of the sensory regions of the cochlea and vestibule.

Application of Mesenchymal Stem Cell Therapy and Inner Ear Regeneration for Hearing Loss: A Review

“…Several protocols have been established to help direct iPSCs into the hair cells and neurons, which have several properties similar to those of their native counterparts. The efficiency, reproducibility and scalability of these protocols are enhanced by incorporating knowledge on inner ear development [58,59].…”

“…These cells have morphological characteristics similar to those of their in vivo counterparts during the embryonic development of cochlear and vestibular organs. Moreover, they present with electrophysiological activity detected via single-cell patch clamping [58]. In addition, the use of human disease models in vitro via the genetic manipulation of iPSCs is feasible [59].…”

Usher Syndrome: Genetics and Molecular Links of Hearing Loss and Directions for Therapy

“…The first inner ear organoid system was developed by Koehler et al using mouse ESCs, which was later adapted to use human ESCs or iPSC (Koehler et al, 2017). Several groups have published protocols inducing inner ear organoids from aggregates of mouse ESCs (Koehler and Hashino, 2014;Liu et al, 2016;Longworth-Mills et al, 2016;Nie et al, 2017) and human PSCs (Jeong et al, 2018). Single-cell electrophysiology tests identified functional vestibular hair cells in inner ear organoids (Jeong et al, 2018).…”

“…Several groups have published protocols inducing inner ear organoids from aggregates of mouse ESCs (Koehler and Hashino, 2014;Liu et al, 2016;Longworth-Mills et al, 2016;Nie et al, 2017) and human PSCs (Jeong et al, 2018). Single-cell electrophysiology tests identified functional vestibular hair cells in inner ear organoids (Jeong et al, 2018). Numerous improved protocols have been described on including the modulation of signaling pathways, like Wnt, Sonic Hedgehog, and Fgf (Lahlou et al, 2018), addition of extracellular matrix scaffolds (e.g., Matrigel; Koehler et al, 2013Koehler et al, , 2017; and addition of bone morphogenetic protein (BMP), transforming growth factor beta (TGFβ), and Wnt antagonists (Zhou et al, 2015).…”

Inner ear organoids: new tools to understand neurosensory cell development, degeneration and regeneration

“…3A). The same protocol has been successfully translated to hESCs and human induced pluripotent stem cells (hIPSCs), by modifying the timing to match human fetal development (Jeong et al, 2018;Koehler et al, 2017;Munnamalai and Fekete, 2017). While in the murine system the first hair cells appear at 2-3 weeks (15-21 days) in vitro, differentiation is extended to 10 weeks (70 days) for human cells (Fig.…”