Cochlear Sox2+ Glial Cells Are Potent Progenitors for Spiral Ganglion Neuron Reprogramming Induced by Small Molecules

Abstract

In the mammalian cochlea, spiral ganglion neurons (SGNs) relay the acoustic information to the central auditory circuits. Degeneration of SGNs is a major cause of sensorineural hearing loss and severely affects the effectiveness of cochlear implant therapy. Cochlear glial cells are able to form spheres and differentiate into neurons in vitro. However, the identity of these progenitor cells is elusive, and it is unclear how to differentiate these cells toward functional SGNs. In this study, we found that Sox2+ subpopulation of cochlear glial cells preserves high potency of neuronal differentiation. Interestingly, Sox2 expression was downregulated during neuronal differentiation and Sox2 overexpression paradoxically inhibited neuronal differentiation. Our data suggest that Sox2+ glial cells are potent SGN progenitor cells, a phenotype independent of Sox2 expression. Furthermore, we identified a combination of small molecules that not only promoted neuronal differentiation of Sox2– glial cells, but also removed glial cell identity and promoted the maturation of the induced neurons (iNs) toward SGN fate. In summary, we identified Sox2+ glial subpopulation with high neuronal potency and small molecules inducing neuronal differentiation toward SGNs.

WNT Activation and TGFβ-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro

“…For instance, glial cells expressing PLP1 and SOX2 can be efficiently converted into new auditory neurons following overexpression of genetic factors, including the transcription factors Ascl1 and NeuroD1 [ 39 ], Neurog1 and NeuroD1 [ 37 ] and the RNA binding protein lin-28 [ 38 ]. Such a conversion of spiral ganglion glial cells to auditory neurons has also been demonstrated following pharmacological reprogramming [ 40 ]. However, a direct conversion (i.e., trans-differentiation) is also possible, thereby, raising the problem of an exhaustion of the supporting cell pool.…”

“…The WNT agonist CHIR99021, in combination with other pharmacological treatments, has also been shown to promote the conversion of SOX2 expressing glial cells to auditory neurons [ 40 ]; however, its potential to promote ANPG expansion has been poorly studied. In our hands, stimulating the WNT pathway was able to enhance ANPGs growth, however, not to a level sufficient to achieve significant passaging and amplification.…”

Regulation of Spiral Ganglion Neuron Regeneration as a Therapeutic Strategy in Sensorineural Hearing Loss

“…Transcriptome profiling of cells at different stages of SGNs differentiation, including exogenous stem cell-derived and endogenous glial cell-derived SGNs regeneration, could provide useful information about the molecular processes involved. Most recently, the emerging cell transcriptome analysis such as single-cell RNA sequencing and bulk RNA-sequencing have been applied in identifying the subtypes of SGNs and inner ear glial cells, characterizing the dynamic expression pattern of SGN genes, and analyzing the transcriptome of the induced neurons after regeneration (Shrestha et al, 2018 ; Sun et al, 2018 ; Li C. et al, 2020 ; Chen et al, 2021 ; Tasdemir-Yilmaz et al, 2021 ). For example, Li C. et al ( 2020 ) compared the transcriptomes of SGNs and two other inner ear cell types, HCs and glia to identify genes that were expressed specifically in SGNs within the cochlea and exhibited either constant (e.g., Scrt2) or dynamic (e.g., Celf4) expression patterns.…”

“…These genes might represent promising candidate regulators of SGN cell-fate determination and/or differentiation. Chen et al ( 2021 ) performed transcriptomic analysis of the glial cells-derived SGNs under different stimuli conditions in vitro and found that the small molecules cocktail FIBCL treatment promoted the newborn SGNs maturation. Despite the transcriptome analysis of the inner ear cells still facing some challenges such as a limited number of cells obtained from the cochleae, the lower cell viability, etc.…”

Active role of glia‐like supporting cells in the organ of Corti: Membrane proteins and their roles in hearing

“…The passive K + conductance channels, which give rise to very low membrane resistance and linear current–voltage (I–V) relationship, are unique features that define mature astrocytes in the brain (Bae et al, 2020; Mi Hwang et al, 2014; Zhou et al, 2021). GLSs also participate in the maturation, proliferation, protection, and regeneration of cochlear cells (Chen, Huang, et al, 2021; Ramírez‐Camacho et al, 2006) like glial cells in the brain. When hair cells are damaged, they are extruded from the epithelium, phagocytosed by surrounding GLSs and nearby GLSs fill in the gap, leaving a cross‐shaped F‐actin scar (Bird et al, 2010; Raphael & Altschuler, 1991; Wagner & Shin, 2019).…”