Induction of Functional Hair-Cell-Like Cells from Mouse Cochlear Multipotent Cells

Figure 3

Molecular characterization of inner ear multipotent cells and derived cells differentiating on poly-L-lysine substratum. (a) RT-PCR analysis of the expression of markers specific for hair cells and supporting cells. The ubiquitously expressed GAPDH is shown as reference. (b) Expression of markers specific for hair cells and supporting cells: Myosin VIIA, Brn3c, Espin, and . Spheres: spheres generated from inner ear multipotent cells, 7 d: cells differentiated for 7 days on the poly-L-lysine substratum in vitro, and 14 d: cells differentiated for 14 days on the poly-L-lysine substratum in vitro (as shown in Figure 2(a)). Statistically significant differences between groups are indicated by , , and (). (c) After 5 days of differentiation, Atoh1-positive cells appeared in differentiating sphere-derived cells (red, left). After 2 weeks of differentiation (as shown in Figure 2(a)), hair-cell-like cells appeared in cells derived from spheres. % () of differentiated cells were labeled with an antibody to Myosin VIIA (green, center), and % () of differentiated cells were labeled with an antibody to Espin (green, right). Nuclei were visualized with DAPI. (d) All cells immunopositive for Myosin VIIA also expressed Atoh1. (e) Myosin VIIA-positive cells in stem-cell-derived cell populations coexpressed the stereocilia marker Espin. (f) The Espin-positive cells in stem-cell-derived cell populations coexpressed Brn3c. (g) Conventional fluorescence microscopy showing nonoverlapping expression of (green) and Myosin VIIA (red) in differentiated cells.

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