Morphological differentiation of tau–green fluorescent protein embryonic stem cells into neurons after co-culture with auditory brain stem slices

Most types of congenital and acquired hearing loss are caused by loss of sensory hair cells in the inner ear and their respective afferent neurons. Replacement of spiral ganglion neurons (SGN) would therefore be one prioritized step in an attempt to restore sensory neuronal hearing loss. To initiate an SGN repair paradigm we previously transplanted embryonic neuronal tissue and stem cells (SC) into the inner ear in vivo. The results illustrated good survival of the implant. One such repair, however, would not have any clinical significance unless central connections from the implanted SGN could be established. For the purpose of evaluating the effects of cell transplantation on cochlear nucleus (CN) neurons we have established organotypic brain stem (BS) cultures containing the CN. At present we have used in vitro techniques to study the survival and differentiation of tau–green fluorescent protein (GFP) mouse embryonic stem cells (MESC) as a mono- or co-culture with BS slices. For the co-culture, 300 μm thick auditory BS slices encompassing the CN were prepared from postnatal Sprague–Dawley rats. The slices were propagated using the membrane interface method and the CN neurons labeled with DiI. After 5±2 days in culture a tau-GFP MESC suspension was deposited next to CN in the BS slice. Following deposition the MESC migrated towards the CN. One and two weeks after transplantation the co-cultures were fixed and immunostained with antibodies raised against neuroprogenitor, neuronal, glial and synaptic vesicle protein markers. Our experiments with the tau-GFP MESC and auditory BS co-cultures show a significant MESC survival but also differentiation into neuronal cells. The findings illustrate the significance of SC and auditory BS co-cultures regarding survival, migration, neuronal differentiation and connections.