SPIRAL GANGLION NEURONS IN A DISH – VISUALIZING GROWTH FACTOR STIMULATION OF SGN-LIKE CELLS IN OTIC BIOENGINEERED NEURAL ORGANOIDS (OBENO) IN VITRO

In the past decade, harnessing the potential of progenitor cells for regeneration of the auditory nerve evolved as a new approach to the treatment of cochlear neuropathy e.g. the loss of spiral ganglion neurons (SGN). Learning that cochlear implant (CI) performance is correlated with the number of healthy SGN in the cochlea has prompted the hypothesis that replacing SGNs could enhance hearing restoration in this patient population.Therefore we generated otic organoids by patterning human induced pluripotent stem cells (hiPSC) embedded in a collagen hydrogel towards an otic fate. This study explores the potential of neurotrophic factor addition for the development of SGN-like cells in oBENO for auditory nerve regeneration. Utilizing genome engineering of hiPSCs, we generated an SGN reporter line (trSGN) to enable genetic tracing and live imaging of labeled cells during neuronal growth factor stimulation. We applied BDNF (Brain derived neurotrophic factor), NT-3 (Neurotrophin-3) GDNF (Glial cell line-derived neurotrophic factor) and NGF (Nerve growth factor), and selected factor combinations every two days to whole oBENO and 2D hydrogel spheres (days 40–90). Next, we assessed both SGN differentiation and neuronal growth by live-cell imaging and immunofluorescence. Preliminary data show that administration of a single growth factor already exerts a positive effect on neuronal growth, however, the combination of BDNF, NT-3 and NGF or GDNF has demonstrated an even more pronounced effect. These findings suggest that a more targeted cell differentiation could enhance SGN yield, providing a foundation for future research on auditory nerve regeneration in cases of SGN loss.