Bar chart showing the percentage of highly double positive cells at the FGF 75^th percentile threshold (n=3; mean + s.e.m).

a and b. Morphology of an OEP colony. Bar is 100 μm. In all remaining panels, bar is 50 μm. c and d. Show the partial lifting of OEPs when treated with a short, mild trypsin incubation. e and f. Typical morphology of ONPs, showing cytoplasmic projections. g. Side-by-side ONP and OEP colonies, double-labeled for PAX8 and SOX2. h. ONP colony labeled for PAX8 and NESTIN.

a. Mid-modiolar section of a transplanted cochlea showing the location of the newly formed, ectopic ganglion. b. Detail of the ganglion showing neuronal differentiation by TuJ1 staining (red). Neural fibers project from the ganglion towards the organ of Corti (c and d, arrows), passing through the Rosenthal’s canal (c and d, asterisk). e. New neuronal bodies (arrows) are also found in the Rosenthal’s canal (asterisk). f. Ectopic ganglion at the base of the modiolus, projecting TuJ1⁺ fibers centrally, towards the internal auditory meatus. g. RFP⁺ fibers (arrowheads) approaching the inner hair cells and expressing GluA2 (green), primarily concentrated in postsynaptic densities (PSDs) around the basal pole of IHCs (arrow). Dotted lines show the positions of the IHCs. Fibers (including PSDs) were also positive for NKAα3 (purple), a marker of afferent terminals. Nine out of ten animals analyzed had fibers contacting the IHC, while the three animals labeled for GluA2, were positive. h, i. RFP⁺ fibers in the cochlear nucleus, expressing synaptophysin (green, arrows). In (h), the fiber branches and surrounds the cell, with morphology highly reminiscent of the maturing endbulb of Held. j. SGN density 10 weeks after transplantation. Conditions compared are cochleae treated with ouabain and sham operated versus those with ouabain and transplanted with ONPs. Density was significantly increased (p<0.01) from 112.5±11.9 (n=3; mean + s.e.m) to 546.4±30.6 (n=8). As a reference, the density of the control, untreated cochleae was 1,743±71.5 TuJ1⁺ cells mm⁻². Scale bars for a-f are 100 μm and for g-i are 50 μm.

a. Evolution of the mean ABR thresholds (click) obtained in the transplanted animals (n=18; mean ± s.e.m) compared to the controls (n=8). b. Trace ABR showing the abolition of waves after ouabain treatment (AO) and the restoration of the complexes 10 weeks posttransplanation (PT). c. Graph showing the mean auditory threshold shift reduction obtained by the transplantation (transplanted 28.6±3.6 dB; n=18 vs 53±1.7 dB; n=8 in the control, p 0.0002; mean + s.e.m). d. Comparison of the wave ii-iii amplitudes obtained by tone ABRs. A general trend of enhanced amplitudes was obtained across all frequencies tested, being significantly different from the untransplanted controls at 22, 26 and 30 kHz. Amplitudes before ouabain (BO) were equivalent between the transplanted (n=6) and untransplanted animals (n=5; mean ± s.e.m). e. Latencies of wave ii-iii complexes were, in general, comparable before ouabain and after transplantation. Only at 30 kHz, a significant delay was observed (BO: 4.58±0.2 ms, n=6; PT: 5.9±0.4 ms, n=5; p<0.05; mean ± s.e.m). f. A significant correlation was observed between the mean density of TuJ1/GFP positive cells and the ABR thresholds (n=8; p<0.05).