Organotypic Cocultures of Human Pluripotent Stem Cell Derived-Neurons with Mammalian Inner Ear Hair Cells and Cochlear Nucleus Slices.

Tomoko Hyakumura,Bryony A Nayagam,Mirella Dottori,Karina Needham,Stuart Mcdougall,Sue Finch

doi:10.1155/2019/8419493

Abstract

Stem cells have been touted as a source of potential replacement neurons for inner ear degeneration for almost two decades now; yet to date, there are few studies describing the use of human pluripotent stem cells (hPSCs) for this purpose. If stem cell therapies are to be used clinically, it is critical to validate the usefulness of hPSC lines in vitro and in vivo. Here, we present the first quantitative evidence that differentiated hPSC-derived neurons that innervate both the inner ear hair cells and cochlear nucleus neurons in coculture, with significantly more new synaptic contacts formed on target cell types. Nascent contacts between stem cells and hair cells were immunopositive for both synapsin I and VGLUT1, closely resembling expression of these puncta in endogenous postnatal auditory neurons and control cocultures. When hPSCs were cocultured with cochlear nucleus brainstem slice, significantly greater numbers of VGLUT1 puncta were observed in comparison to slice alone. New VGLUT1 puncta in cocultures with cochlear nucleus slice were not significantly different in size, only in quantity. This experimentation describes new coculture models for assessing auditory regeneration using well-characterised hPSC-derived neurons and highlights useful methods to quantify the extent of innervation on different cell types in the inner ear and brainstem.

Highlights

The hair cells and auditory neurons of the inner ear work cooperatively to convey acoustic information to the brain
We report for the first time the growth of human pluripotent stem cells (hPSCs)-derived sensory neurons toward postnatal inner ear hair cells and cochlear nucleus neurons in organotypic coculture with one and two other cell/tissue types
Human PSC-derived sensory neurons (SC) were cocultured with auditory HC-only explants from P1-3 rats (Figure 1(d)) fixed after 12 days in vitro. They were cultured in the absence of any other cell types (Supplementary Figure 3)

Summary

Introduction

The hair cells and auditory neurons of the inner ear work cooperatively to convey acoustic information to the brain. A number of studies have investigated in vivo stem cell replacement therapy for hearing loss over the past two decades, and these have been extensively reviewed in recent literature [1, 2]. These studies have demonstrated that both mouse and human embryonic stem cells are capable of surviving within the host cochlea for an extended period of time without eliciting a severe host immune response [3,4,5]. Transplanted stem cell-derived neurons have been shown to innervate the sensory hair cells

Methods

Results

Conclusion