Tissue isolation and cell handling procedure. A Temporal bone with utricle (box). B Cochlea. Positions of a sector of the organ of Corti (small box) and the spiral ganglion (large box) are indicated. C–E Dissected utricular macula, organ of Corti, and spiral ganglion. nGFP-positive hair cells are visualized by green fluorescence. Flow chart, generation of single cell suspension cultures. Images show specimen dissected at 0 h postmortem from 3-week-old (A, B) and newborn (C–E) mice. Scale bars = 300 μm.

Total number of cells per individual utricle, organ of Corti, and spiral ganglion after generation of a single cell suspension and removal of aggregates. Shown are mean values ± SEM, n = 3.

Shapes of spheres derived from utricle (A–C), organ of Corti (D–F), and spiral ganglion (G–I) directly postmortem (0 h) and after 5 and 10 days. Hollow spheres that did not self-renew were found in preparations from the utricle and organ of Corti and are shown in D, E, and F. Hollow spheres were not included in the sphere size measurements shown in Figure 4 and were not used for further experiments. Scale bars = 40 μm.

Differentiation of mature cell types from spheres derived at different postmortem time intervals. (A–B) Hair-cell-like cells, defined by nGFP expression (green) and labeling with antibody to myosin VIIA (red) can be found in differentiated cells from utricle-derived spheres at 0 h and 5 days postmortem. C At 10 days postmortem, utricle-derived spheres failed to attach to the substrate. D Quantitative assessment of the number of nGFP and myosin VIIA double positive cells of the total cell number of differentiated utricle-derived spheres. E–G Occurrence of hair-cell-like cells in organ of Corti-derived spheres at 0 h and 5 and 10 days postmortem. H Quantitative assessment of the number of nGFP and myosin VIIA double positive cells of the total cell number of differentiated organ of Corti-derived spheres. I–K Neurons, defined by expression of β-III tubulin (TuJ, shown in red), and GFAP expressing cells (shown in green) can be found in differentiated cells from spiral ganglion-derived spheres at 0 h and 5 days postmortem, but not at 10 days postmortem. In some instances, we detected very weak expression of TuJ in GFAP-positive cells, which we interpret as a sign of immaturity of these newly generated cells. L Quantitative assessment of the number of TuJ- and GFAP-positive cells of the total cell number of differentiated spiral ganglion-derived spheres. Nuclei are visualized with DAPI (shown in blue); scale bars = 50 μm.

Cross-sections through the inner ear of newborn Math1/nGFP mice at 0 h (A–E) and 10 days postmortem (A′–E′). A, A′ Shown is the utricle with nGFP-positive hair cells (green). Filamentous actin is visualized in red, and the hair bundle protein espin, detected with Cy5-conjugated secondary antibody, is depicted in white. Nuclei are visualized with DAPI (blue). B, B′ Higher magnification of utricular hair cells reveals loss of nuclear confinement of nGFP (green) at 10 days postmortem. Hair bundles are visualized with antibody to espin (blue) and by labeling with rhodamine-conjugated phalloidin (red) to detect filamentous actin. C, C′ Organ of Corti sections and D, D′ higher magnification images of cochlear hair cells. E, E′ In the spiral ganglion, we observed massive loss of β-III tubulin (TuJ)-immunopositive neural cell bodies and their associated neurites (depicted in red). GFAP immunoreactivity (shown in white) was present at 0 h, although not very pronounced, but appeared stronger at 10 days postmortem than at 0 h. Note that the labeling pattern of antibodies to TuJ and to GFAP appears to be altered at 10 days postmortem, possibly due to extensive autolytic processes that probably reveal novel unspecific antigens in the tissue. The apparent positive immunoreactivity of the tectorial membrane is unspecific because it is also detectable in negative controls where the primary antibodies were omitted. nGFP-positive hair cells are shown in green and nuclei are visualized with DAPI (shown in blue). Scale bars = 50 μm in A, A′, C, C′, E, and E′ and 10 μm in B, B′, D, and D′.

Primary spheres from postmortem tissues. A Shown are the number of solid primary spheres per 10⁴ living cells that formed after 7 days in nonadherent serum-free cultures. B Diameters of solid primary spheres at different postmortem time intervals. Shown are mean values ± SEM, n = 3.

Propagation and expansion of spheres from utricle, organ of Corti, and spiral ganglion into the second and third generation. Shown is the increase of second- and third-generation sphere numbers from a single sphere. Shown are mean values ± SEM, n = 3; 30–50 spheres were used for each individual experiment.