Neuronal Differentiation and Extensive Migration of Human Neural Precursor Cells following Co-Culture with Rat Auditory Brainstem Slices

Abstract

Congenital or acquired hearing loss is often associated with a progressive degeneration of the auditory nerve (AN) in the inner ear. The AN is composed of processes and axons of the bipolar spiral ganglion neurons (SGN), forming the connection between the hair cells in the inner ear cochlea and the cochlear nuclei (CN) in the brainstem (BS). Therefore, replacement of SGNs for restoring the AN to improve hearing function in patients who receive a cochlear implantation or have severe AN malfunctions is an attractive idea. A human neural precursor cell (HNPC) is an appropriate donor cell to investigate, as it can be isolated and expanded in vitro with maintained potential to form neurons and glia. We recently developed a post-natal rodent in vitro auditory BS slice culture model including the CN and the central part of the AN for initial studies of candidate cells. Here we characterized the survival, distribution, phenotypic differentiation, and integration capacity of HNPCs into the auditory circuitry in vitro. HNPC aggregates (spheres) were deposited adjacent to or on top of the BS slices or as a monoculture (control). The results demonstrate that co-cultured HNPCs compared to monocultures (1) survive better, (2) distribute over a larger area, (3) to a larger extent and in a shorter time-frame form mature neuronal and glial phenotypes. HNPC showed the ability to extend neurites into host tissue. Our findings suggest that the HNPC-BS slice co-culture is appropriate for further investigations on the integration capacity of HNPCs into the auditory circuitry.

Cochlear nucleus whole mount explants promote the differentiation of neuronal stem cells from the cochlear nucleus in co-culture experiments

“…In addition cells were positive for GFAP (45%) and ß-III Tubulin (50%), but these cells were mainly adjacent or connected to the neurosphere. Similar to the other publication no estimation of the total number of cells was reported (Novozhilova et al, 2013). Only one study displayed numbers of surviving cells after in vivo transplantation into the auditory nerve.…”

“…Similar to what has been demonstrated for transplanted neuronal precursor cells in the auditory brainstem (Novozhilova et al, 2013), endogenous NSCs in the cochlear nucleus should have the capacity to differentiate into different types of neurons and glia which could restore the cellular gaps that arise in this brainstem nucleus after hearing loss occurs. Therefore, this paper examined the neurogenic environment of the cochlear nucleus and the differentiation potential induced by brain tissue of its NSCs into various cells types of neuronal origin.…”

“…Several transplantation and co-culture studies of exogenous stem cells have been performed in the auditory brainstem displaying good integration of the cells (GlavaskiJoksimovic et al, 2008;Jiao et al, 2011;Kaiser et al, 2014;Novozhilova et al, 2013). However since the cochlear nucleus is located near the fourth ventricle, neurosurgical approaches are difficult and only performed in the rare case of vestibular schwannoma surgery and/or auditory brainstem implantation (Matthies et al, 2013).…”

Tailor-Made Electrospun Culture Scaffolds Control Human Neural Progenitor Cell Behavior—Studies on Cellular Migration and Phenotypic Differentiation

“…These cells respond to physical cues, with increased cell survival and graft-host integration in vivo by using a nanofiber gel as supporting scaffold [31] [32]. Also, chemical cues affect behavior of such cells, as we presented in studies using a co-culture with explanted brain tissue [29], a specific cocktail for dopaminergic neuron differentiation [25], or by different culture media [30]. NB: In all previous studies, we have used routine planar culture dishes as "base-line" control for, e.g., survival-and phenotypic differentiation capacity.…”

“…In further studies it is of great interest to in detail investigate the functional development of the HNPC-derived neurons, by studying e.g. expression of markers for late neuronal differentiation, such as MAP-2 and neurofilament as well as for synaptic formation (e.g., the markers synaptophysin and SV2 [29]. Studies of electrical activity are also needed, and Bourke at al previously reported maintained electrical activity over time in rodent CNS-derived neurons, using patch-clamp techniques [8].…”