Modulation of Wnt Signaling Enhances Inner Ear Organoid Development in 3D Culture

Abstract

Stem cell-derived inner ear sensory epithelia are a promising source of tissues for treating patients with hearing loss and dizziness. We recently demonstrated how to generate inner ear sensory epithelia, designated as inner ear organoids, from mouse embryonic stem cells (ESCs) in a self-organizing 3D culture. Here we improve the efficiency of this culture system by elucidating how Wnt signaling activity can drive the induction of otic tissue. We found that a carefully timed treatment with the potent Wnt agonist CHIR99021 promotes induction of otic vesicles—a process that was previously self-organized by unknown mechanisms. The resulting otic-like vesicles have a larger lumen size and contain a greater number of Pax8/Pax2-positive otic progenitor cells than organoids derived without the Wnt agonist. Additionally, these otic-like vesicles give rise to large inner ear organoids with hair cells whose morphological, biochemical and functional properties are indistinguishable from those of vestibular hair cells in the postnatal mouse inner ear. We conclude that Wnt signaling plays a similar role during inner ear organoid formation as it does during inner ear development in the embryo.

Hair Follicle Development in Mouse Pluripotent Stem Cell-Derived Skin Organoids

“…Surprisingly, while testing our otic induction method on other cell lines, we noted that an Atoh1/nGFP mESC line (Lumpkin et al, 2003; Oshima et al, 2010) was highly folliculogenic (Figure 2 and Table S1a). As reported previously, this Atoh1/nGFP mESC line rarely produced inner ear organoids without an additional treatment with a GSK3β inhibitor, CHIR99021 (CHIR), on day 8 of differentiation (DeJonge et al, 2016). Without CHIR treatment, however, we found that Atoh1/nGFP cells produced a significantly higher percentage of HF-bearing aggregates than R1 aggregates (Atoh1/nGFP: 83 ± 5% of aggregates with HF-like bulbs per batch, n = 193 organoids, 10 experiments, P < 0.0001, Table S1a).…”

“…Thus, the full-treatment of SB/BMP-FGF/LDN appears to be optimal for HF-bearing skin organoid formation. Moreover, when we treated the aggregates with CHIR on day 8, as used previously to encourage inner ear organoid induction (DeJonge et al, 2016), we found that presumptive HFs formed alongside inner ear organoids (Figure S2C). Consequently, the pulse of GSK3β inhibition does not disrupt HF induction.…”

“…the inner ear and sensory neurons in cranial nerves VII, VIII, and IX; Groves and Fekete, 2012). While the epithelium develops on the aggregate surface, PSCs persist in the aggregate core during the first week of differentiation (DeJonge et al, 2016). In addition, an intermediate tissue layer between the PSC core and the surface ectoderm forms with Brachyury + mesendoderm cells and N-cadherin + SOX2 + neuroectoderm cells (Koehler et al, 2013).…”

In vivo base editing of post-mitotic sensory cells

“…Our work establishes that local delivery of base editor as an RNP complex into the cochlea enables a high degree of specificity both for the target DNA locus and for the target cells in the cochlea. These features are distinct from the delivery of diffusible small molecules 20 , 55 , 56 , 57 , which can perturb the activity of homologous protein targets and have a greater potential to affect the homeostasis of other tissues in vivo. Another key feature of the RNP delivery approach used in this study is that precise genome alterations are made without exposing cells to exogenous DNA or virus, preventing the possibility of random integration of DNA into the host cell genome.…”

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

“…The tissue was then coaxed to differentiate to a placodal fate by downregulation of BMP signaling and stimulation of FGF signaling, based on the known role of FGF in otic fate specification in the embryo (Litsiou et al, 2005;Martin and Groves, 2006). Subsequent studies from the same group have optimized the protocol by inhibiting GSK3β, using CHIR99021 to activate Wnt signaling and to increase otic fate induction (DeJonge et al, 2016;Liu et al, 2016) (Fig. 3A).…”