Figure 9

Neurospheres can be produced from glial cells isolated from adult auditory nerve.

(a–g) Glial cell enrichment using immunomagnetic sorting with a glial cell surface maker P75. After cell sorting, a majority of the cells were identified as glial cells by their spindle-shaped morphology in differential interference contrast (DIC) view (e) and positive staining with Sox10 antibody (arrows) (g), whereas only a small portion of cells displayed the glial morphological characteristics (a) and stained positively with Sox10 antibody in non-sorted cell preparations (c). P75 immmunoreactivity (green) appeared on the surface of Schwann cells in the peripheral auditory nerve (d). Auditory nerve axons were labeled with neurofilament (NF, red) and cell nuclei were counterstained with Dapi (blue). White arrowheads indicate Sox10⁻ cells with large oval-shaped nuclei. (i,j) Neurospheres generated from glial enriched fractions. Image in (j) showed an enlarged view of a neurosphere. (k) A majority of the cells from the non-glial enriched fraction were attached the bottom of the culture dish and failed to generate neurosphere. (l) Comparison of the neurosphere formation capability between the glial enriched and non-glial enriched fractions. Mean number of neurospheres ± S.E.M was obtained from glial enriched fractions (n = 12) and non-glial enriched fractions (n = 4). Asterisks indicate statistically significant differences between the two groups (Student’s unpaired t-test, p < 0.05). (m–p) Neurosphere cells generated from glial enriched fractions stained positively with TuJ1 (red) and Sox10 (green) under neural differentiation conditions. Percentages of Sox10⁺ and TuJ1⁺ cells were tabulated from 4 individual cultures with glial enriched cell populations prepared from adult mouse auditory nerves. Scale bars, 25 μm in (c,d,g,o).