From Otic Induction to Hair Cell Production: Pax2^EGFPCell Line Illuminates Key Stages of Development in Mouse Inner Ear Organoid Model

Abstract

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2 organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2 facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.

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

“…Several factors must be considered when establishing an organoid. Pax2 [52] or Fbxo2 [53] can induce organoid formation to model otic induction, which is a pivotal developmental event when the preplacodal tissue adopts otic fate.…”

Potential of Gene and Cell Therapy for Inner Ear Hair Cells

“…In majority of these protocols, the successful differentiation rate was relatively low, only 1 or 2% of total cells were differentiated into the target cells [ 77 ]. Recent approaches that are adopting 3D culture technique successfully differentiated hair cells from embryonic stem cell, both rodent and human [ 76 , 80 – 83 ]. In this section we are trying to explain the differentiation protocol by adopting well established 3D culture technique in prior publication [ 81 ].…”

“…Although the differentiation of stem cells to HCs seems to be well established, based on literature reports, a careful step by step reevaluation of the differentiation process is needed to allow its optimization and to minimize potential complications when the method is clinically applied. There are efforts to reduce the steps and to increase the efficiency for the differentiation process by gene modulation [ 76 , 77 ]. Despite early stage of technology to generate the inner ear organoid, these approaches might shed light on design of a therapeutic modality for clinical stem cell application for hearing loss patients.…”

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

“…It is less clear what drives the differences in outcomes in these protocols (Koehler et al, 2017). The generation of reporter lines to track lineage differentiation would lead to improved protocols, with higher efficiency and yield (Hartman et al, 2018;Koehler et al, 2017;Schaefer et al, 2018). The rapidly evolving field of gene editing will surely lead to further implementation of these tools (Nie and Hashino, 2017).…”