Single-Cell Transcriptome Analysis of Developing and Regenerating Spiral Ganglion Neurons

Abstract

The spiral ganglion neurons (SGNs) of the cochlea are essential for our ability to hear. SGN loss after exposure to ototoxic drugs or loud noise results in hearing loss. Pluripotent stem cell-derived and endogenous progenitor cell types have the potential to become SGNs and are cellular foundations for replacement therapies. Repurposing transcriptional regulatory networks to promote SGN differentiation from progenitor cells is a strategy for regeneration. Advances in the Fludigm C1 workflow or Drop-seq allow sequencing of single cell transcriptomes to reveal variability between cells. During differentiation, the individual transcriptomes obtained from single-cell RNA-seq can be exploited to identify different cellular states. Pseudotemporal ordering of transcriptomes describes the differentiation trajectory, allows monitoring of transcriptional changes and determines molecular barriers that prevent the progression of progenitors into SGNs. Analysis of single cell transcriptomes will help develop novel strategies for guiding efficient SGN regeneration.

Fig. 1

iMOP derived neurons and denervated cochlear co-cultures. A Hoechst labeled nuclei (magenta) and Tubb3 labeled neurites from SGNs (green) in untreated cochlear explant. B Ouabain treated cochlea lacking Tubb3 labeled SGN neurites. C iMOP derived neuron expressing mCherry (red) with peripheral (P) and central (C) axons along with D Myo7a labeled hair cells (green) with E phalloidin marked hair bundles (cyan). F The merged image shows that the neuronal process terminates near inner hair cells. Magnified micrograph of G a mCherry labeled neurite near the H basolateral portion of a Myo7a and phalloidin labeled hair cell. I Merged image suggests that the neurite terminates at the base of the hair cell body. Psd95 immunofluorescence (red) at the synaptic region between SGNs and hair cells (green) from J untreated cochlear explant, K ouabain treated cochlear explant and L co-culture of oubain denervated cochlear explant containing iMOP-derived neurons.