Fig. 3
Differentiation of otic progenitors into hair cell-like cells and spiral ganglion neuron (SGN)-like cells. a Cells derived from otic epithelial progenitors (OEPs) expressed hair cell markers BRN3C, MYO7A, and ATOH1. Uptake of FM1–43 was detected. Scale bars = 20 μm. b Percentage of double-positive cells: 49.42 ± 15.49% of cells expressing both BRN3C and MYO7A, 46.42 ± 8.9% of cells positive for BRN3C and ATOH1, and 28.26 ± 9.2% of cells double-labeled with FM1–43 and MYO7A (n = 6, mean ± SD). c After 3 weeks of culture on mitotically inactivated chicken utricle stromal cells, the appearance of apical projections reminiscent of a hair bundle on the surface of induced cells was detected by scanning electron microscopy (SEM). Scale bar = 1.0 μm. d Upregulated expressions of hair cell markers ATOH1, BRN3C, MYO7A, and ESPIN detected by gene expression analysis in respective cells compared to that in OEPs. e Outward K+ (IK), inward K+ (IK1) and inward Ca2+ (Ica) currents recorded from induced hair cell-like cells. Currents were elicited by 10 voltage steps from the holding potential (left and right panel: −84 mV; middle panel: −64 mV). Peak current voltages are also indicated. The current density (pA/pF) of IK, IK1, and ICa was 6.22 ± 0.90, −4.78 ± 0.44, and −5.40 ± 1.29, respectively (n = 8, mean ± SD). f SGN-like cells derived from otic neural progenitors (ONPs) were positive for NF200, β-Tubulin III (TUJ1), and BRN3A (POU4F1). g Percentage of double-positive cells: 92.8 ± 2.6% and 90.79 ± 3.0% of induced cells were double positive for TUJ1 and BRN3A, and NF200 and BRN3A, respectively (n = 4, mean ± SD). h Expression of neuronal markers NTRK2, BRN3A, NEUROD1, and ISLET1 detected by reverse transcription-polymerase chain reaction (RT-PCR). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference