Survival, Migration, and Differentiation of Sox1–GFP Embryonic Stem Cells in Coculture with an Auditory Brainstem Slice Preparation

Abstract

The poor regeneration capability of the mammalian hearing organ has initiated different approaches to enhance its functionality after injury. To evaluate a potential neuronal repair paradigm in the inner ear and cochlear nerve we have previously used embryonic neuronal tissue and stem cells for implantation in vivo and in vitro. At present, we have used in vitro techniques to study the survival and differentiation of Sox1-green fluorescent protein (GFP) mouse embryonic stem (ES) cells as a monoculture or as a coculture with rat auditory brainstem slices. For the coculture, 300 microm-thick brainstem slices encompassing the cochlear nucleus and cochlear nerve were prepared from postnatal SD rats. The slices were propagated using the membrane interface method and the cochlear nuclei were prelabeled with DiI. After some days in culture a suspension of Sox1 cells was deposited next to the brainstem slice. Following deposition Sox1 cells migrated toward the brainstem and onto the cochlear nucleus. GFP was not detectable in undifferentiated ES cells but became evident during neural differentiation. Up to 2 weeks after transplantation the cocultures were fixed. The undifferentiated cells were evaluated with antibodies against progenitor cells whereas the differentiated cells were determined with neuronal and glial markers. The morphological and immunohistochemical data indicated that Sox1 cells in monoculture differentiated into a higher percentage of glial cells than neurons. However, when a coculture was used a significantly lower percentage of Sox1 cells differentiated into glial cells. The results demonstrate that a coculture of Sox1 cells and auditory brainstem present a useful model to study stem cell differentiation.

Acute death of astrocytes in blast-exposed rat organotypic hippocampal slice cultures

“…At the time of fixation, OHCs were about 100 μm thick as they get thinner after a few days in culture [93–95]. Immunostaining with a polyclonal rabbit antibody against GFAP (Dako, Carpinteria, CA, USA Cat# Z0334, RRID: AB_2314535) was conducted directly on the insert membrane, which was cut out around the OHCs [34, 94, 96]. Additionally, OHCs that were pretreated with either Annexin V conjugated to Alexa 488 (ThermoFisher Scientific) or 10,000 kDa Dextran (Dex10) conjugated to Alexa 488 (ThermoFisher Scientific), as described below, were gently peeled from the membrane insert and immunostained against GFAP (Dako) using the free-floating sections method [97].…”

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

“…The BS slices in our model maintain their three-dimensional organization for up to five weeks in culture, and, thus serve as a controlled organotypic system where various experimental approaches for AN reconstruction can be evaluated, including pharmacological treatments and a cellular SGN replacement therapy [42]. We have reported that mouse ESCs survive well and have an increased neuronal differentiation when co-cultured with the BS slice as compared to in monoculture [40], [41] …”

BDNF Increases Survival and Neuronal Differentiation of Human Neural Precursor Cells Cotransplanted with a Nanofiber Gel to the Auditory Nerve in a Rat Model of Neuronal Damage

“…These can be not only derived from embryonic stem cells (ESCs) but also found within the adult nervous system [9]. In vitro , we have demonstrated the capability of several different cell types to integrate into the cochlear nucleus, being the second order neuron in the auditory neuronal pathway [10–12]. Furthermore, in a previous in vivo study we demonstrated good survival and differentiation of mouse tau-green fluorescent protein (GFP) embryonic stem cells transplanted to the AN in deafened rats [13].…”